losartan-potassium and Hypoxia

The SGLT2-inhibitors significantly reduce heart failure hospitalization and progression to end-stage kidney disease. An increase in hemoglobin/hematocrit is seen with SGLT2i-inhibitor treatment. This increase has been attributed to hemoconcentration resulting from a diuretic effect. In this review, we present evidence suggesting that the hematocrit increase is not due to hemoconcentration, but to an increase in erythropoiesis due to amelioration of hypoxia and more efficient erythropoietin production with SGLT2-inhibitor treatment.. We performed a detailed review of the literature in PubMed for articles describing various mechanisms linking hematocrit increase with SGLT2-inhibitor use to their cardio-renal benefits.. The best predictor of cardio-renal benefits with SGLT2-inhibitors is an increase in hematocrit and hemoglobin. If this hemoconcentration is a results of diuresis, this would be associated with volume contraction and a deterioration in renal function, as seen with long-term diuretic use. This is the opposite of what is seen with the use of SGLT2-inhibitors, which are associated with long-term preservation of renal function. There is now growing evidence that the increase in hematocrit can be attributed to an increase in erythropoiesis due to amelioration of renal hypoxia and more efficient erythropoietin production with SGLT2-inhibitor treatment. Increased erythropoiesis leads to an increase in RBC count which improves myocardial/renal tissue oxygenation and function.. The increase in hematocrit with SGLT2i treatment is not due to hemoconcentration, but to an increase in erythropoiesis due to amelioration of hypoxia and more efficient erythropoietin production with SGLT2i treatment.

Topics: Diuresis; Diuretics; Erythropoiesis; Erythropoietin; Hematocrit; Hemoglobins; Humans; Hypoxia; Sodium-Glucose Transporter 2; Sodium-Glucose Transporter 2 Inhibitors

The production of erythropoietin (EPO), the main regulator of erythroid differentiation, is regulated by hypoxia-inducible factor (HIF). HIF2α seems to be the principal regulator of EPO transcription, but HIF1α and 3α also may have additional influences on erythroid maturation. HIF is also involved in the regulation of iron, an essential component in erythropoiesis. Iron is essential for the organism but is also highly toxic, so its absorption and retention are strictly controlled. HIF also induces the synthesis of proteins involved in iron regulation, thereby ensuring the availability of iron necessary for hematopoiesis. Iron is a major component of hemoglobin and is also involved in erythrocyte differentiation and proliferation and in the regulation of HIF. Renal anemia is a condition in which there is a lack of stimulation of EPO synthesis due to decreased HIF expression. HIF prolyl hydroxylase inhibitors (HIF-PHIs) stabilize HIF and thereby allow it to be potent under normoxic conditions. Therefore, unlike erythropoiesis-stimulating agents, HIF-PHI may enhance iron absorption from the intestinal tract and iron supply from reticuloendothelial macrophages and hepatocytes into the plasma, thus facilitating the availability of iron for hematopoiesis. The only HIF-PHI currently on the market worldwide is roxadustat, but in Japan, five products are available. Clinical studies to date in Japan have also shown that HIF-PHIs not only promote hematopoiesis, but also decrease hepcidin, the main regulator of iron metabolism, and increase the total iron-binding capacity (TIBC), which indicates the iron transport capacity. However, concerns about the systemic effects of HIF-PHIs have not been completely dispelled, warranting further careful monitoring.

Topics: Anemia; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Iron; Procollagen-Proline Dioxygenase; Prolyl Hydroxylases; Prolyl-Hydroxylase Inhibitors; Renal Insufficiency, Chronic

Hypoxia-inducible factor (HIFs) is a new class of drug developed for the management of anemia in chronic kidney disease (CKD) patients. HIFs increase the production of erythropoietin in the kidney and liver, enhance the absorption and utilization of iron, and stimulate the maturation and proliferation of erythroid progenitor cells. Besides, HIFs regulate many physiologic processes by orchestrating the transcription of hundreds of genes. Essential hypertension (HT) is an epidemic worldwide. HIFs play a role in many biological processes involved in the regulation of blood pressure (BP). In the current review, we summarize pre-clinical and clinical studies investigating the relationship between HIFs and BP regulation in patients with CKD, conflicting issues, and discuss future potential strategies.

Topics: Anemia; Erythropoietin; Essential Hypertension; Humans; Hypoxia; Kidney; Renal Insufficiency, Chronic

Renal anemia, a common complication and threat factor of chronic kidney disease (CKD), has long been treated with injectable erythropoietin-stimulating agents (ESAs). As concerns regarding cardiovascular safety and erythropoietin resistance to ESAs have emerged, alternative therapies are urgently needed. Hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI), an oral agent, has been proven to be effective in improving renal anemia. However, the effects of HIF-PHIs on nondialysis-dependent CKD (NDD-CKD) have yet to be supported by updated meta-analyses.. A meta-analysis of clinical randomized controlled trials (RCTs) on HIF-PHI treatment of NDD-CKD patients based on PubMed, EMBASE, and Cochrane databases as of July 16th, 2023, was conducted. The primary outcomes were the level of hemoglobin (Hb) postintervention and the ratio of Hb responses. Most of the analysis was conducted. Twenty-two studies with a total of 7178 subjects in the HIF-PHI group, 3501 subjects in the ESA group and 2533 subjects in the placebo group were enrolled. HIF-PHIs increased the level of Hb and improved iron metabolism but were not inferior to ESAs in terms of safety.. HIF-PHIs may be a convenient and safe alternative to ESAs in patients with NDD-CKD and anemia.

Topics: Anemia; Epoetin Alfa; Erythropoietin; Humans; Hypoxia; Prolyl Hydroxylases; Prolyl-Hydroxylase Inhibitors; Renal Insufficiency, Chronic

Bone marrow (BM) hematopoiesis is tightly regulated process and bone components such as osteoblasts, extracellular matrix, and minerals influence hematopoiesis via regulation of hematopoietic stem cell function. Erythropoietin (EPO) secreted mostly by renal EPO producing (REP) cells which employ the hypoxia-inducible factor (HIF) pathway. When tissue hypoxia occurs, HIFs bind to hypoxia response element in the EPO promoter and induce EPO production. EPO binds to the EPO receptor on red cell progenitors in the BM and triggers expansion of red cell mass. Fibroblast growth factor-23 (FGF23) which is secreted mostly by osteoblasts and less by BM impacts hematopoiesis by influencing EPO production. Reciprocally, increases of EPO (acute or chronic) influence both FG23 production and cleavage resulting in variation of c fragment FGF23 (cFGF23) and intact FGF23 (iFGF23) ratios. As HIFs stimulate EPO production, they indirectly affect FGF23. Direct stimulation of FGF23 synthesis by binding of HIF on FGF23 promoter is also suggested. FGF23 cleavage by furin is another potential mechanism affecting FGF23 levels. Klotho is present in membrane-bound (transmembrane) and free (circulating) forms. Transmembrane klotho is the co-receptor of FGF23 and forms complexes with FGF23 receptors in the membrane surface and required for FGF23 actions. Recent evidence showed that klotho is also associated with EPO and HIF production suggesting a complex relationship between FGF23, klotho, EPO, and HIF. In this review, we have summarized the connections between FGF23, klotho, HIF, and EPO and their reflections to hematopoiesis.

Topics: Erythropoietin; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Glucuronidase; Humans; Hydrolases; Hypoxia

Executive functions, learning, attention, and processing speed are imperative facets of cognitive performance, affected in neuropsychiatric disorders. In clinical studies on different patient groups, recombinant human (rh) erythropoietin (EPO) lastingly improved higher cognition and reduced brain matter loss. Correspondingly, rhEPO treatment of young rodents or EPO receptor (EPOR) overexpression in pyramidal neurons caused remarkable and enduring cognitive improvement, together with enhanced hippocampal long-term potentiation. The 'brain hardware upgrade', underlying these observations, includes an EPO induced ~20% increase in pyramidal neurons and oligodendrocytes in cornu ammonis hippocampi in the absence of elevated DNA synthesis. In parallel, EPO reduces microglia numbers and dampens their activity and metabolism as prerequisites for undisturbed EPO-driven differentiation of pre-existing local neuronal precursors. These processes depend on neuronal and microglial EPOR. This novel mechanism of powerful postnatal neurogenesis, outside the classical neurogenic niches, and on-demand delivery of new cells, paralleled by dendritic spine increase, let us hypothesize a physiological procognitive role of hypoxia-induced endogenous EPO in brain, which we imitate by rhEPO treatment. Here we delineate the brain EPO circle as working model explaining adaptive 'brain hardware upgrade' and improved performance. In this fundamental regulatory circle, neuronal networks, challenged by motor-cognitive tasks, drift into transient 'functional hypoxia', thereby triggering neuronal EPO/EPOR expression.

Topics: Brain; Erythropoietin; Humans; Hypoxia; Neurogenesis; Pyramidal Cells; Recombinant Proteins

The kidney is the main organ that senses changes in systemic oxygen tension, but it is also the key detoxification, transit and excretion site of transition metals (TMs). Pivotal to oxygen sensing are prolyl-hydroxylases (PHDs), which hydroxylate specific residues in hypoxia-inducible factors (HIFs), key transcription factors that orchestrate responses to hypoxia, such as induction of erythropoietin (EPO). The essential TM ion Fe is a key component and regulator of the hypoxia-PHD-HIF-EPO (HPHE) signaling axis, which governs erythropoiesis, angiogenesis, anaerobic metabolism, adaptation, survival and proliferation, and hence cell and body homeostasis. However, inadequate concentrations of essential TMs or entry of non-essential TMs in organisms cause toxicity and disrupt health. Non-essential TMs are toxic because they enter cells and displace essential TMs by ionic and molecular mimicry, e. g. in metalloproteins. Here, we review the molecular mechanisms of HPHE interactions with TMs (Fe, Co, Ni, Cd, Cr, and Pt) as well as their implications in renal physiology, pathophysiology and toxicology. Some TMs, such as Fe and Co, may activate renal HPHE signaling, which may be beneficial under some circumstances, for example, by mitigating renal injuries from other causes, but may also promote pathologies, such as renal cancer development and metastasis. Yet some other TMs appear to disrupt renal HPHE signaling, contributing to the complex picture of TM (nephro-)toxicity. Strikingly, despite a wealth of literature on the topic, current knowledge lacks a deeper molecular understanding of TM interaction with HPHE signaling, in particular in the kidney. This precludes rationale preventive and therapeutic approaches to TM nephrotoxicity, although recently activators of HPHE signaling have become available for therapy.

Topics: Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Kidney Diseases; Oxygen; Transcription Factors

Cellular hypoxia occurs when the demand for sufficient molecular oxygen needed to produce the levels of ATP required to perform physiological functions exceeds the vascular supply, thereby leading to a state of oxygen depletion with the associated risk of bioenergetic crisis. To protect against the threat of hypoxia, eukaryotic cells have evolved the capacity to elicit oxygen-sensitive adaptive transcriptional responses driven primarily (although not exclusively) by the hypoxia-inducible factor (HIF) pathway. In addition to the canonical regulation of HIF by oxygen-dependent hydroxylases, multiple other input signals, including gasotransmitters, non-coding RNAs, histone modifiers and post-translational modifications, modulate the nature of the HIF response in discreet cell types and contexts. Activation of HIF induces various effector pathways that mitigate the effects of hypoxia, including metabolic reprogramming and the production of erythropoietin. Drugs that target the HIF pathway to induce erythropoietin production are now approved for the treatment of chronic kidney disease-related anaemia. However, HIF-dependent changes in cell metabolism also have profound implications for functional responses in innate and adaptive immune cells, and thereby heavily influence immunity and the inflammatory response. Preclinical studies indicate a potential use of HIF therapeutics to treat inflammatory diseases, such as inflammatory bowel disease. Understanding the links between HIF, cellular metabolism and immunity is key to unlocking the full therapeutic potential of drugs that target the HIF pathway.

Topics: Cell Hypoxia; Erythropoietin; Humans; Hypoxia; Kidney; Oxygen

Renal erythropoietin (Epo)-producing (REP) cells represent a rare and incompletely understood cell type. REP cells are fibroblast-like cells located in close proximity to blood vessels and tubules of the corticomedullary border region. Epo mRNA in REP cells is produced in a pronounced "on-off" mode, showing transient transcriptional bursts upon exposure to hypoxia. In contrast to "ordinary" fibroblasts, REP cells do not proliferate ex vivo, cease to produce Epo, and lose their identity following immortalization and prolonged in vitro culture, consistent with the loss of Epo production following REP cell proliferation during tissue remodelling in chronic kidney disease. Because Epo protein is usually not detectable in kidney tissue, and Epo mRNA is only transiently induced under hypoxic conditions, transgenic mouse models have been developed to permanently label REP cell precursors, active Epo producers, and inactive descendants. Future single-cell analyses of the renal stromal compartment will identify novel characteristic markers of tagged REP cells, which will provide novel insights into the regulation of Epo expression in this unique cell type.

Topics: Animals; Erythropoietin; Hypoxia; Kidney; Mice; Mice, Transgenic; Renal Insufficiency, Chronic; RNA, Messenger

Desidustat (Oxemia™) is an orally bioavailable, small molecule, hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitor developed by Zydus Cadila for the treatment of anaemia associated with chronic kidney disease (CKD), COVID-2019 infections and chemotherapy induced anaemia. Desidustat inhibits prolyl hydroxylase domain enzymes, resulting in the stabilisation of hypoxia-inducible factor which stimulates erythropoietin production and erythropoiesis. In March 2022, desidustat received its first approval in India for the treatment of anaemia in adults with CKD who are either on dialysis or not on dialysis. Desidustat is in clinical development in China for the treatment of anaemia in patients with CKD, in Mexico for the management of COVID-2019 infections and in the USA for the treatment of chemotherapy induced anaemia. This article summarizes the milestones in the development of desidustat leading to this first approval for anaemia associated with CKD.

Topics: Adult; Anemia; Antineoplastic Agents; COVID-19 Drug Treatment; Erythropoietin; Humans; Hypoxia; Prolyl Hydroxylases; Prolyl-Hydroxylase Inhibitors; Quinolones; Renal Insufficiency, Chronic

Anaemia is a common complication of end-stage renal disease (ESRD) that relies on dialysis. Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHI) is a new class of small-molecule oral drugs for the treatment of anaemia in chronic kidney disease. They demonstrate several advantages over traditional exogenous erythropoietin (EPO). We conducted a meta-analysis of studies that compared the efficacy of HIF-PHI in erythropoiesis and iron metabolism, and its safety with EPO in maintenance dialysis patients.. A sensitive search strategy in the PubMed, EMBASE and Cochrane databases identified all citations for randomised controlled trials (RCTs) comparing HIF-PHI agents with EPO/placebo through December 2021.. Fourteen RCTs were identified, which included 2738 patients. No statistical difference was found in haemoglobin increase (p = 0.37) between HIF-PHI treatment and EPO using the random-effects model. HIF-PHI administration upregulated transferrin (MD 36.12, 95% CI 27.04-45.20) and soluble transferrin receptors (sTfR) (MD 1.28, 95% CI 0.44-2.13), but did not statistically reduce hepcidin level (p = 0.37). Total and LDL-cholestrol levels were suppressed by HIF-PHI (MD - 0.99, 95% CI - 1.34 to  - 0.63) (MD - 0.99, 95% CI - 1.34 to - 0.64), while triglyceride (TG) was not different between HIF-PHI and EPO (p = 0.74). The total incident rates of treatment-emergent adverse events (TEAE) (p = 0.20) from HIF-PHI treatment were not different from those of erythropoietin, while the treatment-emergent serious adverse events (TSAE) (p = 0.02) were higher in the HIF-PHI group than those in the EPO controls with the fixed-effect model.. HIF-PHI could effectively upregulate and maintain haemoglobin levels in patients with anaemia receiving maintenance dialysis. Furthermore, HIF-PHI could elevate iron metabolism activity and utility without inducing treatment-associated serious adverse events. Robust data from larger RCTs with longer treatment duration and follow-up are needed.

Topics: Anemia; Erythropoietin; Hepcidins; Humans; Hypoxia; Iron; Prolyl-Hydroxylase Inhibitors; Receptors, Transferrin; Renal Dialysis; Renal Insufficiency, Chronic; Transferrin; Triglycerides

Anemia is a common complication of chronic kidney disease; it is mainly treated with erythropoiesis-stimulating agents (ESAs) and iron. Experimental studies extensively investigated the mechanisms involved in the body's response to hypoxia and led to the discovery of the hypoxia-inducible factor (HIF) pathway and the enzymes regulating its function. HIF-prolyl-hydroxyl domain (PHD) inhibitors are a new class of oral drugs developed to treat anemia in chronic kidney disease. By inhibiting the function of PHD enzymes, they mimic the exposure to moderate hypoxia and stimulate the production of endogenous erythropoietin and very likely increase iron availability. Some data also suggest that their efficacy and, consequently, dose needs are less influenced by inflammation than ESAs. Overall, data from phases 2 and 3 clinical development showed efficacy in anemia correction and maintenance for all of the class molecules compared with placebo (superiority) or erythropoiesis-stimulating agents (noninferiority). Three molecules, roxadustat, vadadustat, and daprodustat, underwent extensive clinical investigation to assess their safety on hard cardiovascular end points, mortality, and special interest events (including cancer and thrombosis). Aside from vadadustat in the nondialysis population, at the prespecified primary analyses, all three molecules met the noninferiority margin for the risk of major cardiovascular events compared with erythropoiesis-stimulating agents or placebo. The reason for this discrepancy is difficult to explain. Other safety signals came from secondary analyses of some of the other randomized clinical trials, including a higher incidence of thrombosis. A more extensive clinical experience with post-marketing data on hard safety issues is needed to define better when and how to use HIF-PHD inhibitors compared with already available ESAs.

Topics: Anemia; Erythropoietin; Hematinics; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Iron; Renal Insufficiency, Chronic

Diabetes is one of the leading causes of chronic kidney disease (CKD), and multiple underlying mechanisms involved in pathogenesis of diabetic nephropathy (DN) have been described. Although various treatments and diagnosis applications are available, DN remains a clinical and economic burden, considering that about 40% of type 2 diabetes patients will develop nephropathy. In the past years, some research found that hypoxia response and hypoxia-inducible factors (HIFs) play critical roles in the pathogenesis of DN. Hypoxia-inducible factors (HIFs) HIF-1, HIF-2, and HIF-3 are the main mediators of metabolic responses to the state of hypoxia, which seems to be the one of the earliest events in the occurrence and progression of diabetic kidney disease (DKD). The abnormal activity of HIFs seems to be of crucial importance in the pathogenesis of diseases, including nephropathies. Studies using transcriptome analysis confirmed by metabolome analysis revealed that HIF stabilizers (HIF-prolyl hydroxylase inhibitors) are novel therapeutic agents used to treat anemia in CKD patients that not only increase endogenous erythropoietin production, but also could act by counteracting the metabolic alterations in incipient diabetic kidney disease and relieve oxidative stress in the renal tissue. In this review, we present the newest data regarding hypoxia response and HIF involvement in the pathogenesis of diabetic nephropathy and new therapeutic insights, starting from improving kidney oxygen homeostasis.

Topics: Basic Helix-Loop-Helix Transcription Factors; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Erythropoietin; Humans; Hypoxia; Oxygen; Prolyl-Hydroxylase Inhibitors; Renal Insufficiency, Chronic

Since fall 2019, SARS-CoV-2 spread world-wide, causing a major pandemic with estimated ~ 220 million subjects affected as of September 2021. Severe COVID-19 is associated with multiple organ failure, particularly of lung and kidney, but also grave neuropsychiatric manifestations. Overall mortality reaches > 2%. Vaccine development has thrived in thus far unreached dimensions and will be one prerequisite to terminate the pandemic. Despite intensive research, however, few treatment options for modifying COVID-19 course/outcome have emerged since the pandemic outbreak. Additionally, the substantial threat of serious downstream sequelae, called 'long COVID' and 'neuroCOVID', becomes increasingly evident. Among candidates that were suggested but did not yet receive appropriate funding for clinical trials is recombinant human erythropoietin. Based on accumulating experimental and clinical evidence, erythropoietin is expected to (1) improve respiration/organ function, (2) counteract overshooting inflammation, (3) act sustainably neuroprotective/neuroregenerative. Recent counterintuitive findings of decreased serum erythropoietin levels in severe COVID-19 not only support a relative deficiency of erythropoietin in this condition, which can be therapeutically addressed, but also made us coin the term 'hypoxia paradox'. As we review here, this paradox is likely due to uncoupling of physiological hypoxia signaling circuits, mediated by detrimental gene products of SARS-CoV-2 or unfavorable host responses, including microRNAs or dysfunctional mitochondria. Substitution of erythropoietin might overcome this 'hypoxia paradox' caused by deranged signaling and improve survival/functional status of COVID-19 patients and their long-term outcome. As supporting hints, embedded in this review, we present 4 male patients with severe COVID-19 and unfavorable prognosis, including predicted high lethality, who all profoundly improved upon treatment which included erythropoietin analogues.. Substitution of EPO may-among other beneficial EPO effects in severe COVID-19-circumvent downstream consequences of the 'hypoxia paradox'. A double-blind, placebo-controlled, randomized clinical trial for proof-of-concept is warranted.

Topics: COVID-19; COVID-19 Drug Treatment; Erythropoietin; Humans; Hypoxia; Lung; Pandemics; Post-Acute COVID-19 Syndrome; Recombinant Proteins; SARS-CoV-2

The 2019 Nobel Prize in Physiology or Medicine was awarded to William G. Kaelin Jr, Sir Peter J. Ratcliffe, and Gregg L. Semenza "for their discoveries of how cells sense and adapt to oxygen availability." The three pioneers discovered the hypoxia-inducible factor (HIF), elucidated the oxygen sensing mechanism of cells, and confirmed the critical role of HIF in hypoxic cellular responses. The broad and profound biological effects of HIF open up the possibilities for clinical translation. HIF stabilizers have been proven effective on anemia of chronic kidney disease in phase III clinical trials. HIF antagonists for cancer treatment are under phase II clinical trials. It is imperative to gain insight into the biology of HIF. In this article, the discovery of HIF and its oxygen-dependent enzymatic regulation will be introduced, based largely on the groundbreaking work of the three Nobel laureates. Next, the biology of HIF in the kidney will be reviewed. Studies on the HIF stabilizers in the context of kidney disease, as well as the manipulation of HIF in different renal cell types will be covered. Lastly, the clinical application of HIF stabilizers and HIF antagonists for the treatment of anemia and cancer, respectively, will be discussed.

Topics: Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Nobel Prize; Oxygen

The cerebral hypoxia-ischemia can induce a wide spectrum of biologic responses that include depolarization, excitotoxicity, oxidative stress, inflammation, and apoptosis, and result in neurodegeneration. Several adaptive and survival endogenous mechanisms can also be activated giving an opportunity for the affected cells to remain alive, waiting for helper signals that avoid apoptosis. These signals appear to help cells, depending on intensity, chronicity, and proximity to the central hypoxic area of the affected tissue. These mechanisms are present not only in a large list of brain pathologies affecting commonly older individuals, but also in other pathologies such as refractory epilepsies, encephalopathies, or brain trauma, where neurodegenerative features such as cognitive and/or motor deficits sequelae can be developed. The hypoxia inducible factor 1α (HIF-1α) is a master transcription factor driving a wide spectrum cellular response. HIF-1α may induce erythropoietin (EPO) receptor overexpression, which provides the therapeutic opportunity to administer pharmacological doses of EPO to rescue and/or repair affected brain tissue. Intranasal administration of EPO combined with other antioxidant and anti-inflammatory compounds could become an effective therapeutic alternative, to avoid and/or slow down neurodegenerative deterioration without producing adverse peripheral effects.

Topics: Animals; Apoptosis; Brain; Erythropoietin; Humans; Hypoxia; Inflammation Mediators; Neurodegenerative Diseases; Oxidative Stress

The origin of polycythemia is often simple to detect. Sometimes it is necessary to look for hereditary forms, the decisive parameters being the dosage of erythropoietin and the measurement of the oxygen dissociation curve (P50). These rare diseases are related to high oxygen-affinity haemoglobins, abnormalities of the erythropoietin receptor or dysfunction of the HIF (hypoxia-inducible factor) pathway.. We report the case of a 56-year-old patient with unexplained polycythemia associated with normal serum erythropoietin and normal P50, in whom the never previously described mutation c.400C>T(p.Gln134*) on exon 1 in the EGLN1 gene (encoding PHD2) was found.. In the face of an unexplained polycythemia a good cooperation between clinicians and biologists is necessary to be able to characterize rare hereditary pathologies.

Topics: Erythropoietin; Family; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Male; Middle Aged; Mutation; Polycythemia

A very recent epidemiological study provides preliminary evidence that living in habitats located at 2500 m above sea level (masl) might protect from the development of severe respiratory symptoms following infection with the novel SARS-CoV-2 virus. This epidemiological finding raises the question of whether physiological mechanisms underlying the acclimatization to high altitude identifies therapeutic targets for the effective treatment of severe acute respiratory syndrome pivotal to the reduction of global mortality during the COVID-19 pandemic. This article compares the symptoms of acute mountain sickness (AMS) with those of SARS-CoV-2 infection and explores overlapping patho-physiological mechanisms of the respiratory system including impaired oxygen transport, pulmonary gas exchange and brainstem circuits controlling respiration. In this context, we also discuss the potential impact of SARS-CoV-2 infection on oxygen sensing in the carotid body. Finally, since erythropoietin (EPO) is an effective prophylactic treatment for AMS, this article reviews the potential benefits of implementing FDA-approved erythropoietin-based (EPO) drug therapies to counteract a variety of acute respiratory and non-respiratory (e.g. excessive inflammation of vascular beds) symptoms of SARS-CoV-2 infection.

Topics: Acclimatization; Altitude Sickness; Coronavirus Infections; COVID-19; Erythropoietin; Humans; Hypoxia; Pandemics; Pneumonia, Viral

Berger, Marc Moritz, Peter H. Hackett, and Peter Bärtsch. No relevant analogy between COVID-19 and acute mountain sickness.

Topics: Acute Disease; Altitude Sickness; Angiotensin-Converting Enzyme 2; COVID-19; COVID-19 Drug Treatment; Erythropoietin; Humans; Hypoxia; Inflammation; SARS-CoV-2; Symptom Assessment

Neonatal hypoxic-ischemic encephalopathy is a disorder with heterogeneous manifestation due to asphyxia during perinatal period. It affects approximately 3-12 children per 1000 live births and cause death of 1 million neonates worldwide per year. Besides, motor disabilities, seizures, impaired muscle tone and epilepsy are few of the consequences of hypoxic-ischemic encephalopathy. Despite an extensive research effort regarding various treatment strategies, therapeutic hypothermia with intensive care unit supportive treatment remains the only approved method for neonates who have suffered from moderate to severe hypoxic-ischemic encephalopathy. However, these protocols are only partially effective given that many infants still suffer from severe brain damage. Thus, further research to systematically test promising neuroprotective treatments in combination with hypothermia is essential. In this review, we discussed the pathophysiology of hypoxic-ischemic encephalopathy and delved into different promising treatment modalities, such as melatonin and erythropoietin. However, preclinical studies and clinical trials are still needed to further elucidate the mechanisms of action of these modalities.

Topics: Central Nervous System Depressants; Erythropoietin; Female; Humans; Hypoxia; Hypoxia-Ischemia, Brain; Infant, Newborn; Melatonin; Pregnancy

The recent bestowal of the Nobel Prize 2019 in Physiology or Medicine to Gregg L. Semenza, Sir Peter J. Ratcliffe, and William G. Kaelin Jr. celebrates a series of remarkable discoveries that span from the physiological research question on how oxygen deficiency (hypoxia) induces the red blood cell forming hormone erythropoietin (Epo) to the first clinical application of a novel family of Epo-inducing drugs to treat patients suffering from renal anemia. This review looks back at the most important findings made by the three Nobel laureates, highlights current research trends, and sheds an eye on future perspectives of hypoxia research, including emerging and potential clinical applications.

Topics: Animals; Erythropoietin; Humans; Hypoxia; Oxygen

Oxidative stress is responsible for microvascular complications (hypertension, nephropathy, retinopathy, peripheral neuropathy) of diabetes, which during pregnancy increase both maternal and fetal complications. Chronic hypoxia and hyperglycemia result in increased oxidative stress and decreased antioxidant enzyme activity. However, oxidative stress induces also anti-oxidative reactions both in pregnant diabetes patients and in their fetuses. Not all type 1 diabetes patients with long-lasting disease develop microvascular complications, which suggests that some of these patients have protective mechanisms against these complications. Fetal erythropoietin (EPO) is the main regulator of red cell production in the mother and in the fetus, but it has also protective effects in various maternal and fetal tissues. This dual effect of EPO is based on EPO receptor (EPO-R) isoforms, which differ structurally and functionally from the hematopoietic EPO-R isoform. The tissue protective effects of EPO are based on its anti-apoptotic, anti-oxidative, anti-inflammatory, cell proliferative and angiogenic properties. Recent experimental and clinical studies have shown that EPO has also positive metabolic effects on hyperglycemia and diabetes, although these have not yet been fully delineated. Whether the tissue protective and metabolic effects of EPO could have clinical benefits, are important topics for future research in diabetic pregnancies.

Topics: Erythropoiesis; Erythropoietin; Female; Fetal Growth Retardation; Fetus; Humans; Hypoxia; Infant, Newborn; Oxidative Stress; Placenta; Pre-Eclampsia; Pregnancy; Pregnancy in Diabetics; Reactive Oxygen Species; Receptors, Erythropoietin

Erythropoietin (EPO) is a glycoprotein produced mainly by the adult kidney in response to hypoxia and is the crucial regulator of red blood cell production. EPO receptors (EPORs), however, are not confined to erythroid cells, but are expressed by many organs including the heart, brain, retina, pancreas, and kidney, where they mediate EPO-induced, erythropoiesis-independent, tissue-protective effects. Some of these tissues also produce and locally release small amounts of EPO in response to organ injury as a mechanism of self-repair. Growing evidence shows that EPO possesses also important immune-modulating effects. Monocytes can produce EPO, and autocrine EPO/EPOR signaling in these cells is crucial in maintaining immunologic self-tolerance. New data in mice and humans also indicate that EPO has a direct inhibitory effect on effector/memory T cells, while it promotes formation of regulatory T cells. This review examines the nonerythropoietic effects of EPO, with a special emphasis on its modulating activity on innate immune cells and T cells and on how it affects transplant outcomes.

Topics: Animals; Apoptosis; Erythrocytes; Erythropoietin; Humans; Hypoxia; Immunity, Innate; Immunologic Memory; Mice; Monocytes; Protein Binding; Receptors, Erythropoietin; Regeneration; Signal Transduction; T-Lymphocytes; T-Lymphocytes, Regulatory; Transplants

Type A lactic acidosis resulted from hypoxic mitochondrial dysfunction is an independent predictor of mortality for critically ill patients. However, current therapeutic agents are still in shortage and can even be harmful. This paper reviewed data regarding lactic acidosis treatment and recommended that pyruvate might be a potential alkalizer to correct type A lactic acidosis in future clinical practice. Pyruvate is a key energy metabolic substrate and a pyruvate dehydrogenase (PDH) activator with several unique beneficial biological properties, including anti-oxidant and anti-inflammatory effects and the ability to activate the hypoxia-inducible factor-1 (HIF-1α) - erythropoietin (EPO) signal pathway. Pyruvate preserves glucose metabolism and cellular energetics better than bicarbonate, lactate, acetate and malate in the efficient correction of hypoxic lactic acidosis and shows few side effects. Therefore, application of pyruvate may be promising and safe as a novel therapeutic strategy in hypoxic lactic acidosis correction accompanied with multi-organ protection in critical care patients.

Topics: Acidosis, Lactic; Antacids; Bicarbonates; Erythropoietin; Fluid Therapy; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Pyruvic Acid; Ringer's Lactate

Congenital Erythrocytosis (CE) represents a rare and heterogeneous clinical entity. It is caused by deregulated erythropoiesis where red blood cell overproduction results in elevated hemoglobin and hematocrit levels. CE may either be primary or secondary to elevated erythropoietin concentrations. The only known form of primary CE is caused by mutations in the EPOR gene. Secondary CE can be a consequence of tissue hypoxia, being caused by congenital defects such as hemoglobin variants with increased oxygen affinity, due to mutations in the α- or β-globin genes (HBB, HBA2, HBA1), or due to mutations in the BPGM gene. Secondary CE can also result from defects in the components of the oxygen-sensing pathway (PHD2, HIF2α and VHL).. The family history and the quantification of serum EPO are mandatory to define the best diagnostic strategy regarding molecular studies. Based upon the serum EPO level, P50 and familial data, it is possible to establish a diagnostic algorithm.. Despite recent important discoveries in the molecular pathogenesis of CE, in about 70% of the patients the genetic causes remain to be identified. Clinical data on patients suffering from CE are sparse. This fact is conditional upon the effective way to predict the disease evolution, the establishment of the best management and the genetic counselling.. The number and phenotypic variability of patients that remain without an identified etiology suggest that other genes have to be implicated. Studies by next generation sequencing methodologies are already being performed and it is expected the identification of other genes involved in the pathophysiology of the CE.

Topics: Erythropoietin; Humans; Hypoxia; Mutation; Polycythemia

Fetal erythropoietin (EPO), in addition to regulating erythropoiesis, has also tissue-protective properties based on its anti-inflammatory, anti-apoptotic, antioxidant, and neurotrophic effects. Notably, EPO concentrations needed for tissue protection are 100-1000 times higher than concentrations needed for regulating erythropoiesis. This dual effect of EPO is based on EPO-receptor (EPO-R) isoforms, which differ structurally and functionally. We hypothesize in this Integrated Mechanism Review that during severe fetal hypoxia the observed, but poorly understood, marked increases of fetal plasma EPO concentrations occur to protect the brain, heart, and other vital fetal organs. We further hypothesize that the concurrent marked increases of EPO in the amniotic fluid during fetal hypoxia, occur to protect newborn infants from necrotizing enterocolitis. This review presents experimental and clinical evidence in support of these hypotheses and points out unknown or poorly understood functions of EPO in the fetus. If these novel hypotheses are correct, the importance of fetal EPO as an antenatal hypoxia biomarker will become apparent. It will also likely point the way to important diagnostic and therapeutic fetal and neonatal interventions.

Topics: Amniotic Fluid; Animals; Biomarkers; Brain; Enterocolitis, Necrotizing; Erythropoietin; Female; Fetal Blood; Fetal Diseases; Hematopoiesis; Humans; Hypoxia; Infant, Newborn; Inflammation; Intestines; Neuroprotection; Pregnancy; Protein Isoforms; Reactive Oxygen Species

Hypoxia increases erythropoiesis mediated by hypoxia-inducible transcription factors (HIF), which regulate erythropoietin transcription. Neocytolysis is a physiological mechanism that corrects polycythemia from chronic sustained hypoxemia by transient, preferential destruction of young RBCs after normoxia is restored. We showed that neocytolysis is caused by excessive mitochondrial-derived reactive oxygen species in reticulocytes mediated by downregulation of HIF-controlled BNIP3L regulated mitophagy and a decrease in RBC antioxidant catalase (CAT) in hypoxia-produced erythrocytes. Decreased CAT results from hypoxia-induced miR-21 that downregulates CAT. This correlates with a transient acute decrease of HIF-1 at normoxic return that is associated with normalization of red cell mass.

Topics: Animals; Catalase; Disease Models, Animal; Erythrocytes; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Membrane Proteins; Mice; Mitophagy; Proto-Oncogene Proteins; Reactive Oxygen Species; Tumor Suppressor Proteins

Humans ascending to high altitude (HA) experience a reduction in arterial oxyhemoglobin saturation and, as a result, arterial O

Topics: Acclimatization; Altitude; Blood Volume; Erythropoietin; Humans; Hypoxia; Oxygen; Oxyhemoglobins

Hypoxia is a common clinical problem that has profound effects on renal homeostasis. Prolyl-4-hydroxylases PHD1, 2 and 3 function as oxygen sensors and control the activity of hypoxia-inducible factor (HIF), an oxygen-sensitive transcription factor that regulates a multitude of hypoxia responses, which help cells and tissues to adapt to low oxygen environments. This review provides an overview of the molecular mechanisms that govern these hypoxia responses and discusses clinical experience with compounds that inhibit prolyl-4-hydroxylases to harness HIF responses for therapy in nephrology.

Topics: Erythropoietin; Homeostasis; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Kidney; Oxygen; Prolyl-Hydroxylase Inhibitors; Renal Insufficiency, Chronic

Hypoxia stimulates a variety of adaptive responses, many mediated via the hypoxia inducible factors (HIF) family of transcriptional complexes. The balance of HIF-1, -2 and -3 controls a variety of genes, directly up-regulating transcription of genes involved in erythropoiesis, angiogenesis, vasomotor tone, metabolic pathways and processes related to cell multiplication and survival, and indirectly reducing the transcription of genes with other effects. HIF transcription factors are heterodimers consisting of an oxygen-regulated alpha chain bound to the constitutive aryl hydrocarbon receptor nuclear translocator. Under circumstances where oxygen is abundant the activity of the alpha chain is blocked by the actions of members of a family of oxygen-, iron- and oxoglutarate-dependent dioxygenase enzymes. Hydroxylation of two critical prolyl residues by the HIF prolyl hydroxylases (PHD1-3) leads to recognition by the von Hippel-Lindau E3 ubiquitin ligase complex, polyubiquitylation of the alpha chain and its consequent destruction by the proteasome. Hydroxylation of an asparaginyl residue by Factor Inhibiting HIF prevents any surviving HIF alpha chains from recruiting p300-CBP proteins, important for maximal transcriptional activation. Under conditions of acute hypoxia enzyme activity is suppressed, the HIF alpha chains are allowed to exist in their active form and target gene transcription is enhanced. In sustained hypoxia, adaptive responses mediated by the HIF pathway reduce oxygen demand and increase oxygen supply and thus ultimately down-regulate the pathway. However, a number of other processes also modulate HIF signalling and the balance between HIF-1 and HIF-2 actions. These include the generation of antisense HIF-1 and micro RNAs, up-regulation of HIF-3 alpha, antagonism of the HIF-p300 interaction by CITED2, increased PHD2 and PHD3 levels and effects on the pool of ankyrins within the cell which compete with HIF for the action of FIH. Additionally, effects on intermediary metabolism, reactive oxygen species, iron availability, nitric oxide levels and redox status within the cell may modulate HIF activity. Together, these effects lead to a reduction in the magnitude of the HIF response even if oxygenation is not restored and are predicted to alter the responsiveness of the system when oxygenation is restored.

Topics: Altitude; Animals; Erythropoietin; Feedback, Physiological; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Up-Regulation

Erythropoietin (Epo) is a heavily glycosylated protein, with its main function being related to erythropoiesis, where it controls red blood cell production via interaction with the Epo receptor (EpoR). It also plays a number of important roles in various hormonal, growth factor, and cytokine pathways. These roles are defined by Epo partners, such as the homodimeric (EpoR)2 receptor, the heterodimeric EpoR/βCR receptor and hypoxia inducing factor (HIF). Although the main structural features of both Epo and EpoR are conserved in vertebrates, the secretion sites of Epo in mammals are different from those in other vertebrates. Both biosynthetic and synthetic analogues of this protein are available on the market. Several side effects, such as pure red cells aplaisa, increase the rate of cancer-related death in patients treated with recombinant Epo. The multifunctionality of Epo and the ability of this protein to serve as a hormone, a cytokine, and a growth factor suggest the presence of functional disorder, which is a typical "structural" feature of moonlighting proteins. The goal of this article is to evaluate the roles of intrinsic disorder in the functions of Epo and its primary interactors, EpoR, βCR, and HIF-1α.

Topics: Animals; Erythropoietin; Evolution, Molecular; Gene Expression Regulation; Genetic Variation; Glycosylation; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Protein Binding; Receptors, Erythropoietin; Signal Transduction; Structure-Activity Relationship

Erythropoietin (Epo) was once considered to be a regulator of erythropoiesis by controlling the apoptosis, proliferation and differentiation of erythroid precursor cells over an extended period of time. However, the expression of Epo and Epo receptor (Epo-R) occurs in the brain and retina in addition to the kidney. These expression behaviors lead to physiological effects in addition to hematocrit elevation. In this review we discuss the protective effect of Epo on retinal cells.

Topics: Animals; Cell Survival; Erythropoietin; Humans; Hyperoxia; Hypoxia; Retinal Degeneration

Renal hypoxia is known to play an important role in the pathophysiology of acute renal injury as well as in chronic kidney diseases. The mediators of hypoxia are the transcription factors HIF (hypoxia-inducible factors), that are highly regulated. Under normoxic conditions constitutively expressed HIF-α subunits are hydroxylated by prolyl hydroxylases (PHD1, PHD2, and PHD3) and subsequently degraded by proteasomes.. This narrative review is based on the material searched for and obtained via PubMed and MEDLINE up to January 2015.. The MAPK organizer 1 (Morg1) has been identified to act as a scaffold protein of PHD3 and suppression of Morg1 leads to the stabilization of HIF-α, which forms in the absence of oxygen a heterodimer with HIF-β, translocates to the nucleus and promotes the transcription of HIF target genes.. This review summarizes the current knowledge regarding the role of hypoxia, HIF signalling, and Morg1 in acute and chronic renal injury.

Topics: Acute Kidney Injury; Adaptor Proteins, Signal Transducing; Aryl Hydrocarbon Receptor Nuclear Translocator; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Failure, Chronic; Signal Transduction

Topics: Adaptation, Physiological; Altitude Sickness; Calcium; Chronic Disease; Endothelium, Vascular; Erythropoietin; Heart Failure; Hemodynamics; Humans; Hypertension, Pulmonary; Hypoxia; Muscle, Smooth; Pulmonary Edema; Vascular Diseases; Vascular Remodeling; Vasoconstriction

Erythrocytes play an essential role in the delivery of oxygen from the lung to every organ; a decrease in erythrocytes (anemia) causes hypoxic stress and tissue damage. To maintain oxygen homeostasis in adult mammals, when the kidney senses hypoxia, it secretes an erythroid growth factor, erythropoietin (Epo), which stimulates erythropoiesis in the bone marrow. Recently, studies using genetically modified mice have shown that the in vivo expression profile of the Epo gene changes dramatically during development. The first Epo-producing cells emerge in the neural crest and neuroepithelium of mid-stage embryos and support primitive erythropoiesis in the yolk sac. Subsequently, Epo from the hepatocytes stimulates erythropoiesis in the fetal liver of later stage embryos in a paracrine manner. In fact, erythroid lineage cells comprise the largest cell population in the fetal liver, and hepatocytes are distributed among the erythroid cell clusters. Adult erythropoiesis in the bone marrow requires Epo that is secreted by renal Epo-producing cells (REP cells). REP cells are widely distributed in the renal cortex and outer medulla. Hypoxia-inducible Epo production both in hepatocytes and REP cells is controlled at the gene transcription level that is mainly mediated by the hypoxia-inducible transcription factor (HIF) pathway. These mouse studies further provide insights into the molecular mechanisms of the cell-type specific, hypoxia-inducible expression of the Epo gene, which involves multiple sets of cis- and trans-regulatory elements.

Topics: Aging; Animals; Erythrocytes; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Hepatocytes; Humans; Hypoxia; Mice; Rats

The review describes the induction of erythropoietin gene expression in liver, reproouctive and hemopoietic systems during hypoxia or a state of proliferation.

Topics: Animals; Erythropoietin; Gene Expression Regulation; Hematopoiesis; Humans; Hypoxia; Liver; Urogenital System

Iron is required for efficient oxygen transport, and hypoxia signaling links erythropoiesis with iron homeostasis. Hypoxia induces a highly conserved signaling pathway in cells under conditions of low levels of O2. One component of this pathway, hypoxia-inducible factor (HIF), is a transcription factor that is highly active in hypoxic cells. The first HIF target gene characterized was EPO, which encodes erythropoietin-a glycoprotein hormone that controls erythropoiesis. In the past decade, there have been fundamental advances in our understanding of how hypoxia regulates iron levels to support erythropoiesis and maintain systemic iron homeostasis. We review the cell type-specific effects of hypoxia and HIFs in adaptive response to changes in oxygen and iron availability as well as potential uses of HIF modulators for patients with iron-related disorders.

Topics: Basic Helix-Loop-Helix Transcription Factors; Erythropoiesis; Erythropoietin; Homeostasis; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Iron; Liver; Oxygen; Signal Transduction

The multipronged drug approach targeting blood pressure and serum levels of glucose, insulin, and lipids fails to fully prevent diabetic nephropathy (DN). Recently, a broad range of anomalies associated with oxygen biology, such as hypoxia, oxidative stress (OS), and dyserythropoiesis, have been implicated in DN. This review delineates the cellular mechanisms of these anomalies to pinpoint novel therapeutic approaches. The PHD-HIF system mitigates hypoxia: HIF activates a broad range of reactions against hypoxia whereas PHD is an intracellular oxygen sensor negatively regulating HIF. The Keap1-Nrf2 system mitigates OS: Nrf2 activates cellular reactions against OS whereas Keap1 negatively regulates Nrf2. Clinical trials of PHD inhibitors to correct anemia in patients with CKD as well as of a Nrf2 activator, bardoxolone methyl, for DN are under way, even if the latter has been recently interrupted. A specific PHD1 inhibitor, a Keap1 inhibitor, and an allosteric effector of hemoglobin may offer alternative, novel therapies. Erythropoietin (EPO) is critical for the development of erythroid progenitors and thus for tissue oxygen supply. Renal EPO-producing (REP) cells, originating from neural crests, but not fibroblasts from injured tubular epithelial cells, transdifferentiate into myofibroblasts and contribute to renal fibrosis. Agents restoring the initial function of REP cells might retard renal fibrosis. These newer approaches targeting oxygen biology may offer new treatments not only for DN but also for several diseases in which hypoxia and/or OS is a final, common pathway.

Topics: Diabetic Nephropathies; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor-Proline Dioxygenases; Intracellular Signaling Peptides and Proteins; Kelch-Like ECH-Associated Protein 1; NF-E2-Related Factor 2; Oxidative Stress; Oxygen; Signal Transduction

Tumoral microenvironments play a key role in the evolution of solid tumors. Tumor hypoxia is actively involved in the promotion of genetic instability, the invasive capacity of tumor cells, metastasis, and a worsening of the clinical evolution. Endogenous hypoxia markers are controlled by hypoxia-related genes, formed by HIF-1, which is related to several target genes that involve the energy metabolism, angiogenesis, and transmembrane carbonic anhydrases (CAs), mainly CA-IX that is one of the tumor-related carbonic anhydrases. The goal of this paper is to establish the role of CA-IX as a hypoxia marker in OSCC, while analyzing its expression in this type of tumors and its relationship with several clinical and pathological parameters and prognosis, evaluating its relationship with angiogenesis, other hypoxia markers, and clarifying its role in chemotherapy and radiotherapy resistance.

Topics: Antigens, Neoplasm; Antineoplastic Agents; Carbonic Anhydrase IX; Carbonic Anhydrases; Carcinoma, Squamous Cell; Drug Resistance, Neoplasm; Erythropoietin; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glucose Transporter Type 1; Head and Neck Neoplasms; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ki-67 Antigen; Mouth Neoplasms; Neovascularization, Pathologic; Radiation Tolerance; Receptors, Erythropoietin

Iron is an important mineral element required for diverse life processes. Its metabolism is almost synonymous to erythrocyte maintenance, erythropoiesis and erythrophagocytosis. Consequently, exercise exertion impacts significantly on red cell haematology. Here, the interactions between exercise and erythropoiesis are explored. Hepcidin, the peptide hormone that regulates systemic iron metabolism, decreases in response to erythropoiesis by facilitating increased iron efflux from ferroportin into circulation. However, during exercise, there is an alarming increase in the expression of hepcidin resulting in a negative iron balance in athletes. In this review, the confounding cause and effect scenarios of exercise, athlete training and haematology and hepcidin interactions are discussed.

Topics: Anemia; Antimicrobial Cationic Peptides; Athletes; Erythropoiesis; Erythropoietin; Exercise; Hemolysis; Hepcidins; Humans; Hypoxia; Iron

A classic physiologic response to systemic hypoxia is the increase in red blood cell production. Hypoxia-inducible factors (HIFs) orchestrate this response by inducing cell-type specific gene expression changes that result in increased erythropoietin (EPO) production in kidney and liver, in enhanced iron uptake and utilization and in adjustments of the bone marrow microenvironment that facilitate erythroid progenitor maturation and proliferation. In particular HIF-2 has emerged as the transcription factor that regulates EPO synthesis in the kidney and liver and plays a critical role in the regulation of intestinal iron uptake. Its key function in the hypoxic regulation of erythropoiesis is underscored by genetic studies in human populations that live at high-altitude and by mutational analysis of patients with familial erythrocytosis. This review provides a perspective on recent insights into HIF-controlled erythropoiesis and iron metabolism, and examines cell types that have EPO-producing capability. Furthermore, the review summarizes clinical syndromes associated with mutations in the O(2)-sensing pathway and the genetic changes that occur in high altitude natives. The therapeutic potential of pharmacologic HIF activation for the treatment of anemia is discussed.

Topics: Basic Helix-Loop-Helix Transcription Factors; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; Erythrocytes; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Iron; Kidney; Liver; Oxygen; Signal Transduction

High altitude training has become a mainstay in endurance sports, with live high-train low as the current protocol of choice. Athletes either live or sleep in artificial or natural hypoxic conditions with the aim to increase serum erythropoietin concentrations, which are thought to improve maximum oxygen uptake and thus exercise performance.. Changes, however, are not very striking and only apparent in so-called responders, who are not a well-defined group and may be as little as 50% of the trained study population. Whereas some studies show minor improvement, others report no change or even worsening. Furthermore, the mechanisms behind the proposed beneficial changes remain obscure and are far from being proven. There is an evident lack of sufficiently powered randomized, double-blinded studies, with training protocols that are identical for all groups and groups that are indeed comparable. Several studies discriminate between responders and non-responders, without clearly assessing the characteristics of the so-called responders. Until this has been done, it remains unclear if such a group really exists and how these subjects are characterized. This, however, would be of immense value, so protocols could be tailored to athletes' needs. Taken together, the current literature on natural or artificial hypoxia somewhat documents improved performance at high but not low altitude.

Topics: Altitude; Athletic Performance; Erythrocyte Volume; Erythropoietin; Exercise; Female; Humans; Hypoxia; Male; Oxygen; Physical Education and Training; Physical Endurance; Randomized Controlled Trials as Topic; Running; Sex Factors; Sports

Photoreceptors and other cells of the retina consume large quantities of energy to efficiently convert light information into a neuronal signal understandable by the brain. The necessary energy is mainly provided by the oxygen-dependent generation of ATP in the numerous mitochondria of retinal cells. To secure the availability of sufficient oxygen for this process, the retina requires constant blood flow through the vasculature of the retina and the choroid. Inefficient supply of oxygen and nutrients, as it may occur in conditions of disturbed hemodynamics or vascular defects, results in tissue ischemia or hypoxia. This has profound consequences on retinal function and cell survival, requiring an adaptational response by cells to cope with the reduced oxygen tension. Central to this response are hypoxia inducible factors, transcription factors that accumulate under hypoxic conditions and drive the expression of a large variety of target genes involved in angiogenesis, cell survival and metabolism. Prominent among these factors are vascular endothelial growth factor and erythropoietin, which may contribute to normal angiogenesis during development, but may also cause neovascularization and vascular leakage under pathologically reduced oxygen levels. Since ischemia and hypoxia may have a role in various retinal diseases such as diabetic retinopathy and retinopathy of prematurity, studying the cellular and molecular response to reduced tissue oxygenation is of high relevance. In addition, the concept of preconditioning with ischemia or hypoxia demonstrates the capacity of the retina to activate endogenous survival mechanisms, which may protect cells against a following noxious insult. Part of these mechanisms is the local production of protective factors such as erythropoietin. Due to its plethora of effects in the retina including neuro- and vaso-protective activities, erythropoietin has gained strong interest as potential therapeutic factor for retinal degenerative diseases.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Erythropoietin; Humans; Hypoxia; Mice; Oxygen; Rats; Retina; Retinal Diseases

Because kidneys consume a large amount of oxygen and are relatively inefficient in oxygen uptake, they are susceptible to hypoxia, especially in patients with advanced chronic kidney disease accompanied by loss of peritubular capillaries. Accumulating evidence suggests that chronic tubulointerstitial hypoxia acts as a final common pathway leading to end-stage renal disease. Some biologically active uremic retention molecules, considered as uremic toxins, accumulate as the renal function declines, and at this moment, more than 90 bioactive uremic toxins have been identified. Uremic toxins per se have been proven to accelerate the progression of renal failure. However, the causal relationship between uremic toxin and tubulointerstitial hypoxia remains unclear. Our studies provided direct evidence that uremic toxin dysregulates oxygen metabolism in the kidney. Indoxyl sulfate (IS), a representative protein-bound uremic toxin, increased oxygen consumption in proximal renal tubules, decreased renal oxygenation, and consequently aggravated hypoxia in the remnant rat kidneys. The increase in tubular oxygen consumption by IS was dependent on sodium-potassium adenosine triphosphatase and oxidative stress. Our work also indicated a possible connection between IS and the desensitization of the oxygen-sensing mechanism in erythropoietin-producing cells, which may partly explain inadequate erythropoietin production in hypoxic kidneys of end-stage renal disease patients. Studies of uremic toxins will open a new avenue in development of novel therapeutic approaches of kidney disease.

Topics: Animals; Cell Proliferation; Erythropoietin; Homeostasis; Humans; Hypoxia; Indican; Kidney; Kidney Failure, Chronic; Kidney Tubules; Oxidative Stress; Oxygen Consumption; Rats; Uremia

Although we have known since the 19th century that oxygen tension affects erythrocyte production, we have only recently begun to understand many subtleties of erythropoietin physiology. The unanticipated increase in mortality associated with erythropoietin use found in recent randomized studies is prompting a reassessment of static hemoglobin targets. Hemoglobin levels in dialysis patients do not correlate with endogenous erythropoietin production and may be related to differences in oxygen delivery resulting from shifts in the oxygen-hemoglobin dissociation curve. The time may have arrived to develop more physiologic targets such as oxygen delivery that would mimic the natural response to hypoxia. There are several equations that already exist that can compensate for the effects of the concentration of inorganic and organic phosphates as well as pH, carbon dioxide, and temperature on the delivery of oxygen. However, since the shape and dispersion of the oxygen-hemoglobin dissociation curve may actually change in different disease states, more work is needed.

Topics: Algorithms; Blood Transfusion; Carbon Dioxide; Epoetin Alfa; Erythropoiesis; Erythropoietin; Feedback, Physiological; Hemoglobins; Humans; Hydrogen-Ion Concentration; Hypoxia; Hypoxia-Inducible Factor 1; Models, Biological; Oxygen; Oxyhemoglobins; Patient Care Planning; Phosphates; Randomized Controlled Trials as Topic; Recombinant Proteins

Original results and literature data on the erythropoietin effects and mechanisms of action are analyzed. The action of this substance on blood oxygen transport is not only manifested by direct change in hemoglobin concentration and, correspondingly, by increased oxygen capacity, but is also mediated by modulators of hemoglobinaffinity to oxygen, in particular, via nitric oxide effect on hemoglobin.

Topics: Aged; Aged, 80 and over; Animals; Erythropoietin; Hemoglobins; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Infant, Newborn; Infant, Premature; Middle Aged; Nitric Oxide; Oxygen; Rats

Although tissues may exist regardless of reduced oxygen pressure, this requires glycolytic ATP generation, which is very expensive from the energetic viewpoint. Hypoxia is defined as the condition in which oxygen pressure is reduced at the level of bodily tissues. There are many clinical situations during which decreased tissue oxygenation may occur. It may be transient or chronic, as well as systemic or local. An emergent need exists for monitoring and diagnosis with respect to numerous possible clinical circumstances leading to hypoxia and its life-threatening consequences. The assessment of global oxygen homeo-stasis relies on blood gas analysis and lactate concentration, but such an approach does not fully reflect the local oxygenation of tissues. Oxygen needle microelectrode measurements reveal great differences in tissue pO2 levels. Local pO2 levels depend on many factors, among which the most important are: the distance to the nearest capillary, the extracellular and intracellular fluid diffusion rates and intracellular measurements of the number and activity levels of mitochondria. Thus, nowadays, it is impossible to establish an accurate normal value ranges for local tissue pO2. Oxygen deficiency is an important gene regulator. A sequence-specific DNA-binding factor, the hypoxia induced factor (HIF), is the fundamental hypoxia response protein. 70 genes identified so far have been found to be HIF-dependent. They are responsible for increased oxygen delivery, i.e. by boosting angiogensis due to vascular endothelial growth factor (VEGF) release and the enhancement of red blood cell production by erythropoietin (EPO). VEGF-induced angiogenesis is one of several key hypoxia adaptations. An enhanced vascular bed in response to hypoxia affects almost every bodily tissue and organ. This was observed particularly in skeletal muscles as well as in the brain. The expression of a few hypoxia markers does not require HIF activation. An especially interesting member of this group is osteopontin (OPN), whose synthesis increases during hypoxia. OPN was originally linked to bone remodeling, but currently it seems to posses an important role in immunity, inflammation and tumor pathogenesis. Quantification of hypoxia is clinically essential both for therapy and prognosis. Taking account of the fact that the concept of oxygen pressure at the tissue level is not quantitative (norms do not exist, results are incomparable), biochemical markers are preferable. Particul

Topics: Adaptation, Physiological; Biomarkers; Cell Hypoxia; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Osteopontin; Oxygen; Vascular Endothelial Growth Factor A

Tissue oxygenation in general and hypoxia in particular are important regulators of retinal physiology and pathophysiology. Reduced oxygen tension and hypoxia-inducible transcription factors along with some of their target genes are critically involved in retinal development, and especially in the generation of a normal retinal vasculature. Well-timed hypoxia is thus vital for the young eye to establish proper retinal function and vision. However, when hypoxia is ill-timed, reduced oxygen tension may be associated with the development of retinal pathologies, including retinopathy of prematurity, diabetic retinopathy, glaucoma, age-related macular degeneration, or high altitude retinopathy. Here, reduced oxygen tension activates a hypoxic response that culminates in an increased expression of vascular endothelial growth factor. This causes pathological neovascularization of the delicate neuronal retina, a process that may ultimately lead to loss of vision. In contrast, preconditioning by well-defined and controlled short-term hypoxia is not devastating for the retina but instead induces a molecular response that provides protection to neuronal cells. Detailed investigation of hypoxic mechanisms during development and adulthood may thus reveal factors, which may be targeted by therapeutic approaches to save and preserve vision in patients.

Topics: Altitude; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Neovascularization, Pathologic; Neovascularization, Physiologic; Oxygen; Partial Pressure; Retina; Retinal Diseases; Retinal Vessels; Vascular Endothelial Growth Factor A

Hypoxia of the placenta is integral to complications of pregnancy, including preeclampsia, intrauterine growth restriction, and small-for-gestational age babies. Hypoxia in the placenta is associated with vascular remodeling, hypertension, metabolic changes, oxidative stress, mitochondrial dysfunction, and endoplasmic reticular stress. Hypoxia induces similar outcomes in other organs such as the lungs, kidney, and gut. Comparing and contrasting the effects of hypoxia on placental functions and functions of lung, kidney, and gut can lead to novel hypotheses and investigations, furthering our understanding of the impact of hypoxia on these diverse yet similar organs. In this review, we compare and contrast hypoxic placental responses to those in the other organ and cell systems.

Topics: Adaptation, Physiological; Altitude; Blood Pressure; Erythropoietin; Female; Gastrointestinal Tract; Humans; Hypertension; Hypoxia; Kidney; Lung; Oxidative Stress; Oxygen; Partial Pressure; Placenta; Pregnancy; Pregnancy Complications

During hypoxic or hypoxemic conditions, tissue oxygenation and arterial O(2) carrying capacity are upregulated by two complementary systems, namely the neural respiratory network (central and peripheral) that leads to increased minute ventilation thereby increasing tissue oxygenation, and erythropoietin (Epo) release by the kidney that activates erythropoiesis in bone marrow to augment arterial blood O(2) carrying capacity. Despite the fact that both neural respiratory control and Epo-mediated elevation of red blood cells are responsible for keeping arterial O(2) content optimal, no interaction between these systems has been described so far. Here we review data obtained in our laboratory demonstrating that ventilatory and erythropoietic systems are tightly connected. We found Epo is the key factor mediating this relationship through modulation of the chemoreflex pathway. Moreover, we showed that this interaction occurs in a sex-dependent manner.

Topics: Animals; Carotid Body; Chemoreceptor Cells; Erythropoietin; Humans; Hypoxia; Reflex; Respiration; Sex Characteristics

The essential biological role of erythropoietin (EPO) in maintaining erythrocyte mass has been well understood for many years. Although EPO is required for the maturation of red cells, it also has strong procoagulant effects on the vascular endothelium and platelets, which limit erythrocyte losses after hemorrhage. Like other members of the type 1 cytokine superfamily, EPO has multiple biological activities. For the past 10 years, multiple investigators have shown that EPO acts as a locally produced antagonist of proinflammatory cytokines that are generated by the innate immune response in response to infection, trauma, or metabolic stress. Specifically, EPO inhibits apoptosis of cells surrounding a locus of injury, reduces the influx of inflammatory cells, and recruits tissue-specific stem cells and endothelial progenitor cells. Available evidence suggests that these multiple, nonerythropoietic effects of EPO are mediated by a tissue protective receptor (TPR) that is distinct from the homodimeric receptor responsible for erythropoiesis. Notably, activation of the TPR requires a higher concentration of EPO than is needed for maximal erythropoiesis. Unfortunately, these higher concentrations of EPO also stimulate hematopoietic and procoagulant pathways, which can cause adverse effects and, therefore, potentially limit the clinical use of EPO for tissue protection. To circumvent these problems, the EPO molecule has been successfully modified in a variety of ways to interact only with the TPR. Early clinical experience has shown that these compounds appear to be safe, and proof of concept trials are ready to begin.

Topics: Animals; Apoptosis; Coagulants; Erythrocytes; Erythropoiesis; Erythropoietin; Hemorrhage; Humans; Hypoxia; Inflammation; Ischemia; Malaria, Cerebral; Neoplasms; Signal Transduction; Stem Cells; Thrombosis

Because of the central role that red blood cells play in the delivery of oxygen to tissues of the body, red blood cell mass must be controlled at precise levels. The glycoprotein hormone erythropoietin (EPO) regulates red blood cell mass. EPO transcription, in turn, is regulated by a distinctive oxygen-sensing mechanism. In this pathway, prolyl hydroxylase domain protein (PHD) site-specifically hydroxylates the α-subunit of the transcription factor hypoxia-inducible factor α (HIF-α), thereby targeting the latter for degradation by the von Hippel-Lindau tumor-suppressor protein (VHL). Under hypoxic conditions, this posttranslational modification of HIF-α is inhibited, which stabilizes it and promotes the transcriptional activation of genes, including that for EPO. Rare patients with erythrocytosis have mutations in the genes encoding for PHD2, HIF-2α, and VHL, which implicates these proteins as critical to the proper control of red blood cell mass in humans.

Topics: Basic Helix-Loop-Helix Transcription Factors; Erythropoietin; Hematopoiesis; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Polycythemia; Procollagen-Proline Dioxygenase; Von Hippel-Lindau Tumor Suppressor Protein

The hormone erythropoietin (Epo) maintains red blood cell mass by promoting the survival, proliferation and differentiation of erythrocytic progenitors. Circulating Epo originates mainly from fibroblasts in the renal cortex. Epo production is controlled at the transcriptional level. Hypoxia attenuates the inhibition of the Epo promoter by GATA-2. More importantly, hypoxia promotes the availability of heterodimeric (α/β) hypoxia-inducible transcription factors (predominantly HIF-2) which stimulate the Epo enhancer. The HIFs are inactivated in normoxia by enzymatic hydroxylation of their α-subunits. Three HIF-α prolyl hydroxylases (PHD-1, -2 and -3) initiate proteasomal degradation of HIF-α, while an asparaginyl hydroxylase ('factor inhibiting HIF-1', FIH-1) inhibits the transactivation potential. The HIF-α hydroxylases contain Fe(2+) and require 2-oxoglutarate as co-factor. The in vivo response is dynamic, i.e. the concentration of circulating Epo increases initially greatly following an anaemic or hypoxaemic stimulus and then declines despite continued hypoxia. Epo and angiotensin II collaborate in the maintenance of the blood volume. Whether extra-renal sites (brain, skin) modulate renal Epo production is a matter of debate. Epo overproduction results in erythrocytosis. Epo deficiency is the primary cause of the anaemia in chronic kidney disease and a contributing factor in the anaemias of chronic inflammation and cancer. Here, recombinant analogues can substitute for the hormone.

Topics: Angiotensin II; Animals; Erythrocytes; Erythropoiesis; Erythropoietin; Feedback, Physiological; Humans; Hypoxia; Kidney

Our understanding of organismal responses to hypoxia has stemmed from studies of erythropoietin regulation by hypoxia that led to the discovery of the master regulator of the hypoxic response, i.e., hypoxia-inducible factor (HIF). This is a transcription factor that is now known to induce the expression of a battery of genes in response to hypoxia. HIF-1 and HIF-2 regulate many genes that are involved in erythropoiesis and iron metabolism, which are essential for tissue oxygen delivery.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Brain; Cell Hypoxia; Cell Lineage; Erythropoietin; Hematopoiesis; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Iron; Kidney; Liver; Oxygen; Pluripotent Stem Cells; Protein Isoforms; Syndrome

Living at high altitude is demanding and thus drives adaptational mechanisms. The Tibetan population has had a longer evolutionary period to adapt to high altitude than other mountain populations such as Andeans. As a result, some Tibetans living at high altitudes do not show markedly elevated red blood cell production as compared to South American high altitude natives such as Quechuas or Aymaras, thereby avoiding high blood viscosity creating cardiovascular risk. Unexpectedly, the responsible mutation(s) reducing red blood cell production do not involve either the gene encoding the blood hormone erythropoietin (Epo), or the corresponding regulatory sequences flanking the Epo gene. Similarly, functional mutations in the hypoxia-inducible transcription factor 1α (HIF-1α) gene that represents the oxygen-dependent subunit of the HIF-1 heterodimer, the latter being the main regulator of over 100 hypoxia-inducible genes, have not been described so far. It was not until very recently that three independent groups showed that the gene encoding HIF-2α, EPAS-1 (Wenger et al. 1997), represents a key gene mutated in Tibetan populations adapted to living at high altitudes (Beall et al. 2010 , Yi et al. 2010 , Simonson et al. 2010). Hypoxia-inducible transcription factors were first identified by the description of HIF-1 (Semenza et al. 1991 , 1992), which was subsequently found to enhance transcription of multiple genes that encode proteins necessary for rescuing from hypoxic exposure, including erythropoietic, angiogenic and glycolytic proteins. Then HIF-2 was identified (Ema et al. 1997 ; Flamme et al. 1997 ; Hogenesch et al. 1997 ; and Tian et al. 1997) and although it is highly similar to HIF-1 and has the potential to bind (Camenisch et al. 2001) and mediate (Mole et al. 2009) many of the same genes as HIF-1, its biological actions in response to hypoxia are distinct from those of HIF-1 (reviewed by Loboda et al. 2010). By now, several of these HIF-2 mediated processes have been implicated in the human response to high altitude exposure including erythropoiesis (Kapitsinou et al. 2010), iron homeostasis (Peyssonnaux et al. 2008), metabolism (Shohet et al. 2007; Tormos et al. 2010; Biswas et al. 2010 ; Rankin et al. 2009) and vascular permeability (Chen et al. 2009; Tanaka et al. 2005), among others. Clearly, mutation of EPAS-1 has the potential to bring far more advantage when adapting to high altitude than solely mutating the Epo gene.

Topics: Adaptation, Biological; Altitude; Asian People; Basic Helix-Loop-Helix Transcription Factors; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Mutation; Tibet

The kidney is a highly sensitive oxygen sensor and plays a central role in mediating the hypoxic induction of red blood cell production. Efforts to understand the molecular basis of oxygen-regulated erythropoiesis have led to the identification of erythropoietin (EPO), which is essential for normal erythropoiesis and to the purification of hypoxia-inducible factor (HIF), the transcription factor that regulates EPO synthesis and mediates cellular adaptation to hypoxia. Recent insights into the molecular mechanisms that control and integrate cellular and systemic erythropoiesis-promoting hypoxia responses and their potential as a therapeutic target for the treatment of renal anemia are discussed in this review.

Topics: Anemia; Animals; Basic Helix-Loop-Helix Transcription Factors; Bone Marrow; Erythrocytes; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Hematinics; Homeostasis; Humans; Hypoxia; Iron; Kidney; Oxygen; Polycythemia; Procollagen-Proline Dioxygenase; Protein Processing, Post-Translational; Von Hippel-Lindau Tumor Suppressor Protein

Sirtuins (silent information regulator 2 [Sir2] proteins) belong to an ancient family of evolutionary conserved nicotinamide adenine dinucleotide (NAD)(+)-dependent enzymes with deacetylase and/or mono-ADP-ribosyltransferase activity. They regulate DNA repair and recombination, chromosomal stability, and gene transcription, and most importantly mediate the health-promoting effects of caloric restriction (CR), which includes the retardation of aging. At least seven Sir2 homologs, sirtuins (SIRT) 1 to 7 have been identified in mammals. Mammalian SIRT1, the most extensively studied family member, couples protein deacetylation with NAD(+) hydrolysis and links cellular energy and redox state to multiple signaling and survival pathways. Cell-type and context-specific activation of sirtuins increases resistance to metabolic, oxidative, and hypoxic stress in different tissues. In particular, SIRT1 plays a central role in mediating the beneficial effects of CR, and its activation associates with longevity and the attenuation of metabolic disorders. SIRT1 in the kidney is cytoprotective and participates in the regulation of BP and sodium balance. Here, we review sirtuin biology and discuss how CR-triggered sirtuin-dependent pathways affect renal physiology and the pathogenesis of kidney diseases and related disorders.

Topics: Animals; Blood Pressure; Caloric Restriction; Erythropoietin; Humans; Hypoxia; Kidney; Kidney Diseases; Sirtuins; Sodium

The erythropoietin is produced by the kidney and other organs. EPO does not only affect erythroid cells, but also other blood cell lines, such as myeloid cells, lymphocytes and megakaryocytes. This hormone can also enhance phagocytes function of the polymorph nuclear cells and reduces the activation of macrophages, thus modulating the inflammatory process. Hematopoietic and endothelial cells probably have the same cellular origin, and the discovery of erythropoietin receptors also on mesangial and myocardial cells and smooth muscle fibro-cells has prompted the study of the pleiotropic actions of this hormone. Through its receptors, spread out over the body, it carries out many actions which range from the erythrogenesis after hypoxic stimuli to the tissue protection of the heart and the brain after ischemia. Erythropoietin also acts in the endothelial proliferation of new vessels involving the tumor genesis, but it opens new frontiers to the employment of rHuEPO in the Regenerative Medicine.

Topics: Anemia; Animals; Brain; Cardiovascular Diseases; Endothelium, Vascular; Erythroid Cells; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Ischemia; Kidney; Mice; Models, Biological; Myeloid Cells; Myocardium; Neovascularization, Pathologic; Neovascularization, Physiologic; Organ Specificity; Oxygen; Receptors, Erythropoietin

High-altitude environments provide ideal testing grounds for investigations of mechanism and process in physiological adaptation. In vertebrates, much of our understanding of the acclimatization response to high-altitude hypoxia derives from studies of animal species that are native to lowland environments. Such studies can indicate whether phenotypic plasticity will generally facilitate or impede adaptation to high altitude. Here, we review general mechanisms of physiological acclimatization and genetic adaptation to high-altitude hypoxia in birds and mammals. We evaluate whether the acclimatization response to environmental hypoxia can be regarded generally as a mechanism of adaptive phenotypic plasticity, or whether it might sometimes represent a misdirected response that acts as a hindrance to genetic adaptation. In cases in which the acclimatization response to hypoxia is maladaptive, selection will favor an attenuation of the induced phenotypic change. This can result in a form of cryptic adaptive evolution in which phenotypic similarity between high- and low-altitude populations is attributable to directional selection on genetically based trait variation that offsets environmentally induced changes. The blunted erythropoietic and pulmonary vasoconstriction responses to hypoxia in Tibetan humans and numerous high-altitude birds and mammals provide possible examples of this phenomenon. When lowland animals colonize high-altitude environments, adaptive phenotypic plasticity can mitigate the costs of selection, thereby enhancing prospects for population establishment and persistence. By contrast, maladaptive plasticity has the opposite effect. Thus, insights into the acclimatization response of lowland animals to high-altitude hypoxia can provide a basis for predicting how altitudinal range limits might shift in response to climate change.

Topics: Adaptation, Biological; Altitude; Animals; Biological Transport; Birds; Climate Change; Erythropoietin; Humans; Hypoxia; Mammals; Oxygen; Phenotype

Topics: Animals; Diabetic Angiopathies; Diabetic Nephropathies; Diabetic Retinopathy; Erythropoietin; Humans; Hypoxia; Kidney Failure, Chronic; Models, Biological; Polymorphism, Single Nucleotide; Promoter Regions, Genetic

Erythropoietin (EPO) is a circulating glycoprotein hormone whose principal function is thought to be red blood cell production. It is a classic example of a hypoxia-inducible gene, and studies of the induction of EPO synthesis by low oxygen led to the discovery of a widespread system of hypoxia-inducible transcription factors. Tissue-specific expression of the EPO gene is tightly controlled, and in the adult organism the kidney produces around 90% of systemic EPO. Before birth, the liver is the main site of EPO production; factors contributing to the liver-to-kidney switch are still elusive, but may provide clues to the tissue-specificity of EPO gene expression. EPO has also been detected in non-erythropoietic tissues such as the brain, where it is suggested to exert local protective effects. Apart from classical ways of regulating renal EPO during hypoxia and anemia, novel pathways have been discovered that demonstrate that other organ systems in the adult might not only be important for the production of EPO but also for modulating the hypoxic EPO response. Knowledge of the molecular bases of these non-renal pathways will eventually help to develop pharmacological strategies to induce endogenous EPO production when the main source, the kidney, is significantly impaired. This review will provide an overview of the molecular aspects of EPO gene regulation by hypoxia-inducible transcription factors and of the tissue-specific regulation of EPO production in adult mammals. Insights into the biology of EPO production in genetically modified animals, with an emphasis on recent advances in the understanding of non-renal EPO regulation, will be discussed.

Topics: Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Organ Specificity; Transcription Factors

Numerous factors involved in general homeostasis are able to modulate ventilation. Classically, this comprises several kind of molecules, including neurotransmitters and steroids that are necessary for fine tuning ventilation under different conditions such as sleep, exercise, and acclimatization to high altitude. Recently, however, we have found that erythropoietin (Epo), the main regulator of red blood cell production, influences both central (brainstem) and peripheral (carotid bodies) respiratory centers when the organism is exposed to hypoxic conditions. Here, we summarize the effect of Epo on the respiratory control in mammals and highlight the potential implication of Epo in the ventilatory acclimatization to high altitude, as well as in the several respiratory sickness and syndromes occurring at low and high altitude.

Topics: Adaptation, Physiological; Animals; Carotid Body; Erythropoietin; Humans; Hypoxia; Models, Biological; Nervous System Physiological Phenomena; Pulmonary Ventilation

Using mice, we demonstrated that when oxygen supply is lowered, erythropoietin (Epo), the main regulator of red blood cell production, modulates the ventilatory response by interacting with central (brainstem) and peripheral (carotid bodies) respiratory centers. We showed that enhanced Epo levels in the brainstem increased the hypoxic ventilatory response, and that intracerebroventricular injection of an Epo antagonist (soluble Epo receptor) abolished the ventilatory acclimatization to hypoxia. More recently, we have found that the impact of Epo on ventilation occurs in a sex-dependent manner. Keeping in mind that women are less susceptible to several respiratory sicknesses and syndromes than men, we suggest that Epo plays a key role in sexually-dimorphic hypoxic ventilation. Accordingly, we foresee that Epo has a potential therapeutic use as treatment for hypoxia-associated ventilatory diseases.

Topics: Animals; Brain Stem; Erythropoietin; Female; Humans; Hypoxia; Male; Mice; Pulmonary Ventilation; Respiratory Center; Sex Factors

Erythrocytosis results when there is an increased red cell mass and thus an increased hemoglobin. The causes can be divided into primary intrinsic defects of the erythroid progenitor cell and secondary defects, where factors external to the erythroid compartment are responsible. Both can then be further divided into congenital and acquired categories. Congenital causes include mutations of the erythropoietin receptor and defects of the oxygen-sensing pathway including VHL, PHD2 and HIF2A mutations. When fully investigated there remain a number of patients in whom no cause can be elucidated who are currently described as having idiopathic erythrocytosis. Investigation should start with a full history and examination. Having eliminated the common entity polycythemia vera, further direction for investigation is guided by the erythropoietin level. Clinical consequences of the various erythrocytoses are not clear, but in some groups thromboembolic events have been described in young patients. Evidence is lacking to define best management, but aspirin and venesection to a target hematocrit should be considered.

Topics: Adult; Aspirin; Blood Viscosity; Diagnosis, Differential; Erythropoiesis; Erythropoietin; Female; Hematocrit; Humans; Hypoxia; Male; Mutation; Phlebotomy; Platelet Aggregation Inhibitors; Polycythemia; Polycythemia Vera; Receptors, Erythropoietin; Signal Transduction; Thrombophilia

Renal parenchymal hypoxia has recently been documented in a host of clinical and experimental conditions characterized by tubulointerstitial changes and progressive chronic kidney disease (CKD). The nature of renal hypoxia under these settings, its causes and modes of detection are outlined in this review. Cellular hypoxia response, mediated in part by hypoxia-inducible factors (HIF), includes protective components, such as erythropoietin and heme-oxygenase-1, as well as the induction of harmful mediators. Prevention of the progression of CKD include strategies that attenuate renal parenchymal hypoxia, perhaps with selective intensification or inhibition of protective and harmful components, respectively of the hypoxia adaptive response.

Topics: Cell Hypoxia; Disease Progression; Erythropoietin; Heme Oxygenase-1; Humans; Hypoxia; Kidney Diseases; Kidney Failure, Chronic; Kidney Tubules

Erythropoietin is a hypoxia-induced cytokine that stimulates erythropoiesis through the promotion of erythroid precursor cell proliferation and differentiation. Recent evidence supports that erythropoietin has a broad spectrum of tissue protecting actions affecting other systems than hemopoietic. Lately, research has focused on the nonhemopoietic effects of erythropoietin against tissue ischemia due to the unexpected observations of erythropoietin receptor expression by various cells, such as endothelial cells, neuronal cells, cardiac myocytes, and vascular smooth muscle cells. It has been shown that erythropoietin exerts its cardioprotective action during cardiac ischemic injury through reducing the infract size and enhancing new vessel formation over a longer time frame. Erythropoietin plays a crucial role in neuroprotection in many types of ischemic injury in the central and the peripheral nervous system. It is also strongly believed that erythropoietin exhibits a critical role in many other disorders that are pathogenetically related to acute tissue ischemia. This article reviews the proposed implications of erythropoietin in tissue ischemia and discusses the possible mechanisms for this action along with its potential therapeutic applications.

Topics: Animals; Apoptosis; Cardiovascular System; Cell Survival; Endothelial Cells; Erythropoietin; Humans; Hypoxia; Ischemia; Models, Biological; Nervous System; Neurons; Oxygen; Reperfusion Injury

Erythropoietin (EPO) has long been recognized as the major hematopoietic cytokine regulating normal erythropoiesis. Moreover, there is a growing interest in the non-erythropoietic, tissue-protective effects of EPO. Because of its potential to correct anemia, EPO has been increasingly prescribed to cancer patients. However, although recombinant human Epo (rHuEPO) significantly reduces the risk for red blood cell transfusions in cancer patients, recent clinical studies have reported decreased survival and disease control following rHuEPO treatment in patients with different cancer types. The issue of EPOR expression in tumor cells is critical in this respect. The expression of EPOR in tumor cells raises the possibility that exogenous rHuEPO may directly influence tumor growth or sensitivity to chemo-radiation therapy. In addition, EPOR expression in endothelial cells suggests what potential effects EPO may have on tumor capillaries, such as the stimulation of angiogenesis. However, as experimental studies reveal, the overall direct effect of EPO-EPOR signaling on cancer progression and therapy is not a straightforward one. The current paper provides an update on the biology of EPO, and discusses its utility in the treatment of cancer patients.

Topics: Erythropoietin; Hematopoietic System; Humans; Hypoxia; Models, Theoretical; Neoplasms; Recombinant Proteins; Signal Transduction; Survival Rate

An absolute erythrocytosis is present when the red cell mass is raised and the haematocrit is elevated above prescribed limits. Causes of an absolute erythrocytosis can be primary where there is an intrinsic problem in the bone marrow and secondary where there an event outside the bone marrow driving erythropoiesis. This can further be divided into congenital and acquired causes. There remain an unexplained group idiopathic erythrocytosis. Investigation commencing with thorough history taking and examination and then investigation depending on initial features is required. Clear simple criteria for polycythaemia vera are now defined. Those who do not fulfil these criteria require further investigation depending on the clinical scenario and initial results. The erythropoietin level provides some guidance as to the direction in which to proceed and the order and extent of investigation necessary in an individual patient. It should thus be possible to make an accurate diagnosis in the majority of patients.

Topics: Erythrocyte Volume; Erythropoietin; Hematocrit; Hemoglobins; Humans; Hypoxia; Mutation; Polycythemia; Polycythemia Vera

Anaemia is the most common haematological abnormality in cancer patients, and unfortunately, it is often under-recognised and undertreated. The aetiopathology of anaemia in cancer patients is complex and is usually multifactorial. There is enough evidence suggesting that tumour hypoxia in anaemic patients has a negative impact on the treatment outcomes in cancer patients. The use of recombinant human erythropoietin is becoming a new standard of care in cancer patients. Various well-controlled studies have shown that the use of erythropoietin (EPO) increases the haemoglobin level, thereby decreasing the need for frequent transfusions and improving the tumour responses, cancer-free survival and quality-of-life parameters in cancer patients. However, a few recent clinical trials failed to replicate the survival benefit. Hence, a free unrestricted use of EPO is to be avoided. The past belief that anaemia does not matter in cancer patients is now considered invalid and is being seriously challenged. This article aims to present some recent findings on the impact of anaemia on outcomes, with discussion on the possible causes and effects. The benefits of the use of EPO analogues in cancer-related anaemia are also presented.

Topics: Anemia; Antineoplastic Agents; Clinical Trials as Topic; Disease-Free Survival; Erythropoietin; Hemoglobins; Humans; Hypoxia; Neoplasms; Quality of Life; Recombinant Proteins; Treatment Outcome

Diabetic nephropathy is traditionally considered to be a primarily glomerular disease, although this contention has recently been challenged. Early tubular injury has been reported in patients with diabetes mellitus whose glomerular function is intact. Chronic hypoxia of the tubulointerstitium has been recognized as a mechanism of progression that is common to many renal diseases. The hypoxic milieu in early-stage diabetic nephropathy is aggravated by manifestations of chronic hyperglycemia-abnormalities of red blood cells, oxidative stress, sympathetic denervation of the kidney due to autonomic neuropathy, and diabetes-mellitus-induced tubular apoptosis; as such, tubulointerstitial hypoxia in diabetes mellitus might be an important early event. Chronic hypoxia could have a dominant pathogenic role in diabetic nephropathy, not only in promoting progression but also during initiation of the condition. Early loss of tubular and peritubular cells reduces production of 1,25-dihydroxyvitamin D3 and erythropoietin, which, together with dysfunction of their receptors caused by the diabetic state, diminishes the local trophic effects of the hormones. This diminution could further compromise the functional and structural integrity of the parenchyma and contribute to the gradual decline of renal function.

Topics: Animals; Calcitriol; Capillaries; Chronic Disease; Diabetic Nephropathies; Disease Progression; Erythropoietin; Filtration; Humans; Hyperglycemia; Hypoxia; Kidney; Kidney Glomerulus; Kidney Tubules; Oxidative Stress; Proteinuria; Receptors, Calcitriol

Erythropoietin (Epo) is synthesized mainly under hypoxic conditions by renal and extrarenal tissues, including liver, spleen, brain, lung, bone marrow, and reproductive organs. Hypoxia abrogates the degradation of hypoxia-inducible factors (HIF)-1 and -2, that can then bind to the hypoxia response element within the Epo gene, activating its transcription. Receptors for Epo are expressed on cells known to synthesize Epo, but also on cardiomyocytes, cardiac fibroblasts, and endothelial, retinal, gastric, prostate and vascular smooth muscle cells. Epo-receptor binding triggers at least three intracellular signalling cascades: (1) janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5); (2) phosphatidylinositol-3 kinase (PI3K)/Akt, and (3) RAS/mitogen-activated protein kinase (MAPK). Epo also enhances nitric oxide (NO) bioavailability through endothelial NO synthase transcription and activation, and exerts antiapoptotic actions through Bcl-2 and Bcl-XL. NO is a powerful vasodilator, insulin-sensitizer, inhibitor of atherothrombosis and apoptosis, and essential for progenitor mobilization. This article is a concise review of recent advances regarding the molecular and cardiovascular effects of Epo.

Topics: Apoptosis; Endothelium, Vascular; Erythropoietin; Female; Heart; Humans; Hypoxia; Male; Nitric Oxide; Nitric Oxide Synthase; Receptors, Erythropoietin; Signal Transduction

In endurance sport the delivery of oxygen to muscles plays a critical role. Indeed, muscle performance declines during prolonged and intense activity as a consequence of the shift from the aerobic to the anaerobic metabolism with an increase of lactate. To enhance the aerobic capacity 2 alternatives may be used: increasing either the transport or the delivery of oxygen. In this setting, blood doping is the practice of illicitly using a drug or blood product to improve athletic performance. Based on this definition, blood doping techniques may include: 1) blood transfusion (autologous or omologous); 2) erythropoiesis-stimulating substances [recombinant human erythropoietin (alpha, beta, omega), darbepoietin-alpha, continuous erythropoiesis receptor activator, hematide]; 3) blood substitutes (hemoglobin-based oxygen carriers, perfluorocarbon emulsions); 4) allosteric modulators of hemoglobin (RSR-13 and RSR-4); 5) gene doping (human erythropoietin gene transfection); 6) gene regulation (hypoxia-inducible transcription factors pathway). In the present overview we will briefly describe the above-mentioned techniques with the aim of underlining potential hematological alternatives to gene doping for increasing aerobic capacity in sport.

Topics: Animals; Biological Transport; Blood Transfusion; Doping in Sports; Drug Delivery Systems; Erythropoietin; Fluorocarbons; Gene Transfer Techniques; Hemoglobins; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Models, Biological; Oxygen; Peptides; Polyethylene Glycols; Recombinant Proteins; Substance Abuse Detection

"Live high-train low" (LH+TL) altitude training allows athletes to "live high" for the purpose of facilitating altitude acclimatization, as characterized by a significant and sustained increase in endogenous erythropoietin and subsequent increase in erythrocyte volume, while simultaneously enabling them to "train low" for the purpose of replicating sea-level training intensity and oxygen flux, thereby inducing beneficial metabolic and neuromuscular adaptations. In addition to natural/terrestrial LH+TL, several simulated LH+TL devices have been developed including nitrogen apartments, hypoxic tents, and hypoxicator devices. One of the key issues regarding the practical application of LH+TL is what the optimal hypoxic dose is that is needed to facilitate altitude acclimatization and produce the expected beneficial physiological responses and sea-level performance effects. The purpose of this review is to examine this issue from a research-based and applied perspective by addressing the following questions: What is the optimal altitude at which to live, how many days are required at altitude, and how many hours per day are required? It appears that for athletes to derive the hematological benefits of LH+TL while using natural/terrestrial altitude, they need to live at an elevation of 2000 to 2500 m for =4 wk for =22 h/d. For athletes using LH+TL in a simulated altitude environment, fewer hours (12-16 h) of hypoxic exposure might be necessary, but a higher elevation (2500 to 3000 m) is required to achieve similar physiological responses.

Topics: Acclimatization; Adaptation, Physiological; Altitude; Erythropoietin; Exercise; Humans; Hypoxia; Oxygen Consumption; Sports; Time Factors

In the light of the enthusiasm regarding the use of recombinant human erythropoietin (Epo) and its analogues for treatment of the anaemias of chronic renal failure and malignancies it is worth remembering that today's success has been based on a century of laborious research. The concept of the humoral regulation of haematopoiesis was first formulated in 1906. The term 'erythropoietin' for the erythropoiesis-stimulating hormone was introduced in 1948. Native human Epo was isolated in 1977 and its gene cloned in 1985. During the last 15 yr, major progress has been made in identifying the molecules controlling Epo gene expression, primarily the hypoxia-inducible transcription factors (HIF) that are regulated by specific O2 and oxoglutarate requiring Fe2+-containing dioxygenases. With respect to the action of Epo, its dimeric receptor (Epo-R) has been characterised and shown to signal through protein kinases, anti-apoptotic proteins and transcription factors. The demonstration of Epo-R in non-haematopoietic tissues indicates that Epo is a pleiotropic viability and growth factor. The neuroprotective and cardioprotective potentials of Epo are reviewed with a focus on clinical research. In addition, studies utilising the Epo derivatives with prolonged half-life, peptidic and non-peptidic Epo mimetics, orally active drugs stimulating endogenous Epo production and Epo gene transfer are reviewed.

Topics: Anemia; Animals; Erythropoiesis; Erythropoietin; History, 16th Century; History, 19th Century; History, 20th Century; History, 21st Century; Hormones; Humans; Hypoxia; Oxygen

The idiopathic erythrocytosis (IE) group of disorders is defined by an absolute increase in red cell mass and hematocrit without elevation of the megakaryocytic or granulocytic lineages. It is associated with a wide range of serum erythropoietin (Epo) levels and broadly falls into groups of raised/inappropriately normal or low/undetectable Epo levels. A spectrum of molecular defects has been described in association with IE, which reflects the heterogeneity of this disorder. To date the most common identified cause of IE has been mutations in the von Hippel Landau (VHL) protein, which results in aberrant oxygen sensing and dysregulated Epo production. Studying the molecular basis of IE will provide insights into the control of Epo synthesis and Epo-induced signaling pathways.

Topics: Dimerization; Erythrocyte Volume; Erythropoiesis; Erythropoietin; Feedback, Physiological; Gene Expression Regulation; Genetic Heterogeneity; Genotype; Humans; Hydroxylation; Hypoxia; Hypoxia-Inducible Factor 1; Kidney; Models, Biological; Mutation; Oxygen; Polycythemia; Procollagen-Proline Dioxygenase; Proteasome Endopeptidase Complex; Protein Interaction Mapping; Protein Kinases; Protein Processing, Post-Translational; Receptors, Erythropoietin; Signal Transduction; Transcription Factors; Ubiquitin; Von Hippel-Lindau Tumor Suppressor Protein

Neurocognitive dysfunction is a common postoperative complication exacerbated by cardiopulmonary bypass triggering a systemic inflammatory response. This clinical column focuses on the up-regulation of endogenous erythropoietin related to neurological inflammation and the use of recombinant erythropoietin as a neuroprotective pharmacotherapeutic agent.

Topics: Cardiac Surgical Procedures; Cognition Disorders; Erythropoietin; Hematinics; Humans; Hypoxia; Inflammation; Recombinant Proteins

Rapid deployment of unacclimatized soldiers to high mountainous environments causes debilitating effects on operational capabilities (physical work performance), and force health (altitude sickness). Most of these altitude-induced debilitations can be prevented or ameliorated by a wide range of physiological responses collectively referred to as altitude acclimatization. Acclimatization to a target altitude can be induced by slow progressive ascents or continuous sojourns at intermediate altitudes. However, this "altitude residency" requirement reduces their utilization in rapid response military missions that exploit the air mobility capability of modern military forces to quickly deploy to an area of operations on short notice. A more recent approach to induce altitude acclimatization is the use of daily intermittent hypoxic exposures (IHE) in lieu of continuous residence at high altitudes. IHE treatments consist of three elements: 1) IHE simulated altitude (inspired oxygen partial pressure: PIO2), 2) IHE session duration, and 3) total number of IHE sessions over the treatment period. This paper reviews and summarizes the results of 25 published IHE studies. This review finds that an IHE altitude>or=4000 m, and daily exposure duration of at least 1.5 h repeated over a week or more are required to have a high probability of developing altitude acclimatization. The efficacy of shorter duration (<1.5 h) hypoxic exposures at >or=4000 m simulated altitudes, and longer exposures (>4 h) at moderate altitudes (2500-3500 m) is not well documented. The predominate IHE-induced altitude acclimatization response appears to be increased arterial oxygen content through ventilatory acclimatization. Thus, IHE is a promising approach to provide the benefits of altitude acclimatization to low-altitude-based soldiers before their deployment to high mountainous regions.

Topics: Acclimatization; Erythrocytes; Erythropoietin; Humans; Hypoxia; Military Medicine; Military Personnel; Mountaineering; Oxygen Consumption; Physical Education and Training; Physical Fitness

The kidney is sensitive to changes in oxygen delivery. This sensitivity has the merit of facilitating the kidneys in their adjustment of erythropoietin (EPO) production to changes in oxygen supply. The main determinant of EPO synthesis is the transcriptional activity of its gene in kidneys, which is related to local oxygen tensions. Regulation of EPO production is mediated by hypoxia-inducible factor (HIF). When local oxygen tension decreases, accumulated HIF binds to the key sequence of the EPO gene, the hypoxia-responsive element (HRE), and activates transcription of EPO. HIF consists of a constitutive beta-subunit and one of alternative oxygen-regulated HIF alpha-subunits (HIF-1alpha, HIF-2alpha, and HIF-3alpha), and HIF-2alpha is responsible for erythropoietin production. However, the high sensitivity to changes in oxygen tension also makes the kidney prone to hypoxic injury. Severe energy depletion and subsequent activation of a number of critical alterations in metabolism occurs under hypoxic conditions. Hypoxia is also a profibrogenic stimulus. In addition to ischemic acute renal failure, hypoxia can also play a crucial role in the development of nephrotoxic acute kidney injury, radiocontrast nephropathy, and acute glomerulonephritis. Furthermore, accumulating evidence suggests that chronic hypoxia is a final common pathway to end-stage kidney failure in chronic kidney disease. Given that renal hypoxia has pivotal roles on the development and progression of both acute and chronic kidney disease, hypoxia can be a valid therapeutic target for chronic kidney disease. Activation of HIF leads to expression of a variety of adaptive genes in a coordinated manner. Studies utilizing HIF-stimulating agents proved efficacy in various kidney disease models, suggesting that HIF activation is an ideal target of future therapeutic approaches.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cobalt; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Iron Chelating Agents; Kidney; Kidney Diseases; Oxygen; Signal Transduction

Hemoglobin, the sole carrier of oxygen to tissues, accounts for most cytoplasmic protein of the erythrocyte, an enucleate cell lacking protein synthesizing machinery and with limited energy metabolism. While a number of genetic mechanisms can result in decreased hemoglobin concentration in the blood, this review concentrates on those that lead to increased hemoglobin mass, i.e. polycythemia or erythrocytosis. Polycythemia may be due to (a) mutations of the enzyme synthesizing 2, 3 BPG, a metabolic intermediate which regulates hemoglobin-oxygen affinity and thus oxygen delivery, (b) mutation of the alpha or beta globin genes that increase hemoglobin-oxygen affinity and thus decrease oxygen delivery, and (c) mutations of the erythropoietin receptor gene or genes regulating erythropoietin production that lead to increased production of erythrocytes. Primary polycythemias are caused by inherited or acquired somatic mutations affecting the hematopoietic progenitors. In contrast, in secondary polycythemia normal progenitors are activated by external factors present in increased concentration, most commonly erythropoietin. Some hypoxia sensing disorders blur the distinction between primary and secondary polycythemias and may deserve their own category. Most polycythemias are acquired, but both primary and secondary polycythemias may be inherited. In this review we will discuss the genetic heterogeneity of individual responses to hypoxia, and the current understanding of inherited primary and secondary polycythemias.

Topics: Erythropoietin; Genetic Variation; Hemoglobins; Humans; Hypoxia; Mutation; Polycythemia

Topics: Endothelin A Receptor Antagonists; Erythropoietin; Humans; Hypoxia; Neovascularization, Physiologic; Neuroprotective Agents; Wounds and Injuries

The aims of the present review are to summarize and to discuss the role of hypoxia-inducible factor-1 (HIF-1) and the expression and functions of vasoactive substances in chronic hypoxemia with specific focus in the liver and the carotid body. Vascular remodelling and vasoactive substances play important functional roles in the adaptive response to chronic hypoxemia for the maintenance of oxygen homeostasis in all systems in man. HIF-1 regulates the gene expression of vasoactive substances such as vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) and enzymes for producing nitric oxide (NO). Recent studies have shown the effect of chronic hypoxia on the expression of HIF-1alpha and HIF-1-target genes in multiple organ systems including the liver and the carotid body. Results are consistent with increases in the hematocrit levels, pulmonary arterial pressure and right heart mass developed during chronic hypoxia. In addition, the carotid body is also hyperplastic and increases in organ mass with increased levels of HIF-1alpha and the vasoactive substances. These molecules increase the mitotic activity and modulate the excitability of the chemoreceptor. Intriguingly, the liver morphology, serum alanine aminotransferase and 8-isoprostane levels are within normal range in chronic hypoxia, suggesting the absence of significant oxidative stress. Yet, the HIF-1alpha is upregulated and the mRNA and protein levels of VEGF, ET-1, inducible and constitutive NO synthases are elevated in the liver during chronic hypoxia. In conclusion, the adaptive response to long-term hypoxemia involves compensatory mechanisms mediated by expressing significant levels of HIF-1alpha and vasoactive substances regulated by HIF-1.

Topics: Animals; Carotid Arteries; Chronic Disease; Endothelin-1; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Liver; Nitric Oxide; Oxygen; Transcription Factors; Vascular Endothelial Growth Factor A

Erythropoietin (EPO) is a hypoxia-inducible hormone required for erythroid differentiation. Expression of the EPO receptor is not restricted to hematopoietic cells and exhibits a multi-tissue distribution that includes neural cells, vascular endothelium and muscle progenitor cells. The ability for EPO to stimulate progenitor cell proliferation and prevent apoptosis is critical for maintenance of the erythroid lineage, but is also observed in neural and muscle progenitor cells. Mice lacking the EPO receptor die in utero due to severe anemia. However, even prior to lack of erythroid cell production in the embryo proper, these mice exhibit increased apoptosis in the brain as early as E10.5 and a reduction in the number of neural progenitor cells. Corresponding cultures of primary neural cells exhibit decreased neuron generation and increased sensitivity to reduced oxygen tension, and neurons do not survive after 24 h at low oxygen tension. In contrast, hypoxia induces EPO and EPO receptor in wild-type neuronal cells, and EPO enhances neuron survival at low oxygen tension. In vivo EPO is neuroprotective in adult animal models for brain ischemia. Induction of EPO and its receptor by hypoxia likely contributes to its neuroprotective activity and selective cell survival in the brain during hypoxic stress.

Topics: Animals; Brain; Erythropoietin; Hypoxia; Receptors, Erythropoietin

The mechanisms controlling the expression of the gene encoding for the hormone erythropoietin (EPO) are exemplary for oxygen-regulated gene expression. In humans and other mammals, hypoxia modulates EPO levels by increasing expression of the EPO gene. An association between polycythaemia and people living at high altitudes was first reported more than 100 years ago. Since the identification of EPO as the humoral regulator of red blood cell production and the cloning of the EPO gene, considerable progress has been made in understanding the regulation of EPO gene expression. This has finally led to the identification of a widespread cellular oxygen-sensing mechanism. Central to this mechanism is the transcription factor complex hypoxia-inducible factor (HIF)-1. The abundance and activity of HIF-1, a heterodimer of an alpha- and beta-subunit, is predominantly regulated by oxygen-dependent post-translational hydroxylation of the alpha-subunit. Non-heme ferrous iron containing hydroxylases use dioxygen and 2-oxoglutarate to specifically target proline and an asparagine residue in HIF-1alpha. As such, the three prolyl hydroxylases (prolyl hydroxylase domain-containing protein (PHD) 1, PHD2 and PHD3) and the asparagyl hydroxylase (factor inhibiting HIF (FIH)-1) act as cellular oxygen sensors. In addition to erythropoiesis, HIF-1 regulates a broad range of physiologically relevant genes involved in angiogenesis, apoptosis, vasomotor control and energy metabolism. Therefore, the HIF system is implicated in the pathophysiology of many human diseases. In addition to the tight regulation by oxygen tension, temporal and tissue-specific signals limit expression of the EPO gene primarily to the fetal liver and the adult kidney.

Topics: Animals; Dioxygenases; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Kidney; Models, Biological; Oxygen; Protein Denaturation; Protein Processing, Post-Translational; Protein Subunits; Transcriptional Activation

Erythropoietin (EPO), already known as the stimulating hormone for erythropoiesis, has shown different and interesting pleiotropic actions. It does not only affect erythroid cells, but also myeloid cells, lymphocytes and megakaryocytes. This hormone can also enhance phagocytic function of the polymorphonuclear cells and reduce the activation of macrophages, thus modulating the inflammatory process.Moreover, hematopoietic and endothelial cells probably have the same cellular origin, and the discovery of erythropoietin receptors (EPO-R) also on mesangial and myocardial cells, smooth muscle fibrocells and neurons has prompted the study of the non-erythropoietic functions of this hormone.The interaction between EPO and VEGF may be of particular importance in neovascularization and wound healing. Different studies have demonstrated that EPO has an important direct hemodynamic and vasoactive action, which does not depend exclusively on any increase in hematocrit and viscosity. Moreover EPO showed protective effects on myocardial cells against apoptosis induced by ischemia/repefusion injury, but it could negatively affect pulmonary hypertension in patient with chronic cor pulmonale.This review aims to stress the importance of the increasing interest in EPO applications and the necessity of further studies to gain a deeper knowledge of this hormone and its pleiotropic and complex actions.

Topics: Anemia; Angiogenesis Modulating Agents; Clinical Trials as Topic; Erythropoietin; Humans; Hypoxia; Shock; Vascular Endothelial Growth Factor A

Reduced tissue oxygenation stabilizes the alpha-subunit of the transcription factor hypoxia-inducible factor-1 (HIF-1). This leads to the induction of a number of hypoxia responsive genes. One of the best known HIF-1 targets is erythropoietin that exerts neuroprotective effects on ischemia-related injury in the brain. Thus, pre-exposure to low environmental oxygen concentrations might be exploited as a preconditioning procedure to protect tissues against a variety of harmful conditions. We present recent work on neuroprotection of retinal photoreceptors induced by hypoxic preconditioning or by systemically elevated levels of Epo in mouse plasma.

Topics: Animals; Brain; Erythropoietin; Hypoxia; Ischemia; Ischemic Preconditioning; Mice; Mice, Transgenic; Neurons; Oxygen; Retina; Retinal Degeneration; Retinal Ganglion Cells; Time Factors; Up-Regulation

Altitude training continues to be a key adjunctive aid for the training of competitive athletes throughout the world. Over the past decade, evidence has accumulated from many groups of investigators that the "living high--training low" approach to altitude training provides the most robust and reliable performance enhancements. The success of this strategy depends on two key features: 1) living high enough, for enough hours per day, for a long enough period of time, to initiate and sustain an erythropoietic effect of high altitude; and 2) training low enough to allow maximal quality of high intensity workouts, requiring high rates of sustained oxidative flux. Because of the relatively limited access to environments where such a strategy can be practically applied, numerous devices have been developed to "bring the mountain to the athlete," which has raised the key issue of the appropriate "dose" of altitude required to stimulate an acclimatization response and performance enhancement. These include devices using molecular sieve technology to provide a normobaric hypoxic living or sleeping environment, approaches using very high altitudes (5,500m) for shorter periods of time during the day, and "intermittent hypoxic training" involving breathing very hypoxic gas mixtures for alternating 5 minutes periods over the course of 60-90 minutes. Unfortunately, objective testing of the strategies employing short term (less than 4 hours) normobaric or hypobaric hypoxia has failed to demonstrate an advantage of these techniques. Moreover individual variability of the response to even the best of living high--training low strategies has been great, and the mechanisms behind this variability remain obscure. Future research efforts will need to focus on defining the optimal dosing strategy for these devices, and determining the underlying mechanisms of the individual variability so as to enable the individualized "prescription" of altitude exposure to optimize the performance of each athlete.

Topics: Acclimatization; Altitude; Altitude Sickness; Erythropoietin; Hemoglobins; Humans; Hypoxia; Nitrogen; Oxygen Consumption; Physical Education and Training; Physical Endurance; Physical Fitness; Time Factors

Erythropoietin (Epo) is a pleiotropic agent, that is to say, it can act on several cell types in different ways. An independent system Epo/Epo receptor (EpoR) was detected in brain, leading to the hypothesis that this hormone could be involved in cerebral functions. Epo/EpoR expression changes during ontogenesis, thus indicating the importance of this system in neurodevelopment. Moreover, the hypoxia-induced production of Epo in the adult brain suggests that it could exert a neurotrophic and neuroprotective effect in case of brain injury. Epo could also influence neurotransmission, inducing neurotransmitters (NT) release. Epo therapy in anemic cancer patients is still a controversial issue, because of its possible action as a growth and an angiogenic factor. In our speculative hypothesis Epo could be involved in a "two steps process" that, after a neovascularization phase, leads to its down regulation. Moreover, Epo-activated signaling pathways could be modulated as possible targets to interfere in neoplastic cells cycle. In conclusion, treatment with rHuEpo could change therapeutical perspectives in different pathological conditions, such as central nervous system (CNS) diseases, but further studies are needed to clarify its physiopathological activities in different clinical fields.

Topics: Animals; Brain; Erythropoietin; Humans; Hypoxia; Models, Biological; Neoplasms; Neovascularization, Pathologic; Neurotransmitter Agents; Receptors, Erythropoietin

This review will focus on the molecular basis of certain polycythemic disorders. Primary polycythemias are characterized by acquired somatic or inherited germ-line mutations expressed within hematopoietic progenitors that cause increased accumulation of red blood cells. Polycythemia vera (PV), an acquired condition, is the most common primary polycythemia; although some progress has been made in the understanding of PV, its molecular basis remains unknown. In contrast, recent advances in delineating the molecular defects of some inherited polycythemias have greatly furthered our knowledge of the regulation of erythropoiesis and hypoxia sensing; however, more work needs to be done.

Topics: Adult; Animals; Child; Cohort Studies; Disease Models, Animal; DNA-Binding Proteins; Erythropoiesis; Erythropoietin; Ethnicity; Female; Founder Effect; Germ-Line Mutation; GPI-Linked Proteins; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Isoantigens; Janus Kinase 2; Male; Membrane Glycoproteins; Mice; Nuclear Proteins; Polycythemia; Polycythemia Vera; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Cell Surface; Receptors, Erythropoietin; Russia; Transcription Factors; Tumor Suppressor Proteins; Ubiquitin; Ubiquitin-Protein Ligases; Von Hippel-Lindau Tumor Suppressor Protein

Cancer has a negative systemic impact on its host in addition to its local or metastatic effects, and no cancer complication is more ubiquitous than anaemia, a condition for which there is now a specific remedy, the recombinant growth factor erythropoietin. This is not a trivial therapeutic consideration, because cancer-associated anaemia has an adverse influence on survival regardless of tumour type. However, the pharmacological correction of anaemia with recombinant erythropoietin could promote tumour growth, whereas the use of tumour-necrosis factor-alpha (TNFalpha) and TNF-related apoptosis-inducing ligand as antitumour agents could exacerbate anaemia, thereby perpetuating tissue hypoxia and tumour progression.

Topics: Anemia; Carcinogens; Cell Death; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Neoplasms; Recombinant Proteins; Signal Transduction

To review the impact of anemia/tumor hypoxemia on the quality of life and survival in cancer patients, and to assess the problems associated with the correction of this difficulty.. MEDLINE searches were performed to find relevant literature regarding anemia and/or tumor hypoxia in cancer patients. Articles were evaluated in order to assess the epidemiology, adverse patient effects, anemia correction guidelines, and mechanisms of hypoxia-induced cancer cell growth and/or therapeutic resistance. Past and current clinical studies of radiosensitization via tumor oxygenation/hypoxic cell sensitization were reviewed. All clinical studies using multi-variate analysis were analyzed to show whether or not anemia and/or tumor hypoxemia affected tumor control and patient survival. Articles dealing with the correction of anemia via transfusion and/or erythropoietin were reviewed in order to show the impact of the rectification on the quality of life and survival of cancer patients.. Approximately 40-64% of patients presenting for cancer therapy are anemic. The rate of anemia rises with the use of chemotherapy, radiotherapy, and hormonal therapy for prostate cancer. Anemia is associated with reductions both in quality of life and survival. Tumor hypoxemia has been hypothesized to lead to tumor growth and resistance to therapy because it leads to angiogenesis, genetic mutations, resistance to apoptosis, and a resistance to free radicals from chemotherapy and radiotherapy. Nineteen clinical studies of anemia and eight clinical studies of tumor hypoxemia were found that used multi-variate analysis to determine the effect of these conditions on the local control and/or survival of cancer patients. Despite differing definitions of anemia and hypoxemia, all studies have shown a correlation between low hemoglobin levels and/or higher amounts of tumor hypoxia with poorer prognosis. Radiosensitization through improvements in tumor oxygenation/hypoxic cell sensitization has met with limited success via the use of hyperbaric oxygen, electron-affinic radiosensitizers, and mitomycin. Improvements in tumor oxygenation via the use of carbogen and nicotinamide, RSR13, and tirapazamine have shown promising clinical results and are all currently being tested in Phase III trials. The National Comprehensive Cancer Network (NCCN) guidelines recommend transfusion or erythropoietin for symptomatic patients with a hemoglobin of 10-11 g/dl and state that erythropoietin should strongly be considered if hemoglobin falls to less than 10 g/dl. These recommendations were based on studies that revealed an improvement in the quality of life of cancer patients, but not patient survival with anemia correction. Phase III studies evaluating the correction of anemia via erythropoietin have shown mixed results with some studies reporting a decrease in patient survival despite an improvement in hemoglobin levels. Diverse functions of erythropoietin are reviewed, including its potential to inhibit apoptosis via the JAK2/STAT5/BCL-X pathway. Correction of anemia by the use of blood transfusions has also shown a decrement in patient survival, possibly through inflammatory and/or immunosuppressive pathways.. Anemia is a prevalent condition associated with cancer and its therapies. Proper Phase III trials are necessary to find the best way to correct anemia for specific patients. Future studies of erythropoietin must evaluate the possible anti-apoptotic effects by directly assessing the tumor for erythropoietin receptors or the presence of the JAK2/STAT5/BCL-X pathway. Due to the ability of transfusions to cause immunosuppression, most probably through inflammatory pathways, it may be best to study the effects of transfusion with the prolonged use of anti-inflammatory medications.

Topics: Anemia; Blood Transfusion; Erythropoietin; Humans; Hyperbaric Oxygenation; Hypoxia; Neoplasms; Practice Guidelines as Topic; Quality of Life; Radiation Tolerance; Radiation-Sensitizing Agents; Recombinant Proteins

Hypoxia and/or anaemia have an adverse prognostic impact in locally-advanced cancers of uterine cervix. Moreover, these parameters are independent of other well-known prognostic factors. However, the mechanisms by which treatment efficacy and survival are compromised by anaemia are not fully understood. Although it is clear that erythropoietin can reduce the need for transfusions for cancer patients with anaemia, there is no proof that the use of erythropoietin is in any way superior to transfusions with respect to the impact on clinical outcome, especially for patients receiving radiation therapy. Whether haemoglobin levels at the start of therapy, during therapy, or at the end of therapy are of prognostic value for better disease-free and overall survival, are matters for further studies as is the question of the best option for increasing the level of the patient's haemoglobin.

Topics: Anemia; Erythropoietin; Female; Humans; Hypoxia; Uterine Neoplasms

The dramatic increase in knowledge during the last half century about the hormone erythropoietin is reviewed. The description of these events has been separated into two parts. The first part describes how the rapid changes in response to tissue oxygenation in the kidneys changes the rate of erythropoietin production. The second part describes how changes in erythropoietin concentrations act on erythroid progenitor cells, resulting in prompt changes in rates of erythrocyte production. Together these two aspects of erythropoietin biology provide an explanation for the tight physiological regulation of the numbers of circulating erythrocytes and, in a more general manner, provide a model for the control of the numbers of other specific blood cells by their respective hematopoietic growth factors.

Topics: Erythroid Precursor Cells; Erythropoietin; Hematopoietic Cell Growth Factors; Homeostasis; Humans; Hypoxia; Oxygen

The glycoprotein hormone erythropoietin (EPO) is an essential growth and survival factor for erythroid progenitor cells, and the rate of red blood cell production is normally determined by the serum EPO concentration. EPO production is inversely related to oxygen availability, so that an effective feedback loop is established, which controls erythropoiesis. Since recombinant EPO became available as an effective therapeutic agent, significant progress has also been made in understanding the basis of this feedback control. The main determinant of EPO synthesis is the transcriptional activity of its gene in liver and kidneys, which is related to local oxygen tensions. This control is achieved by hypoxia-inducible transcription factors (HIF), consisting of a constitutive beta-subunit and one of two alternative oxygen-regulated HIFalpha subunits (HIF-1alpha and HIF-2alpha). In the presence of oxygen (normoxia) the HIFalpha subunits are hydroxylated, which targets them for proteasomal degradation. Under hypoxia, because of the lack of molecular oxygen, HIF cannot be hydroxylated and is thereby stabilized. Although HIF-1alpha was the first transcription factor identified through its ability to bind to an enhancer sequence of the EPO gene, more recent evidence suggests that HIF-2alpha is responsible for the regulation of EPO. Although EPO is a prime example for an oxygen- regulated gene, the role of the HIF system goes far beyond the regulation of EPO, because it operates widely in almost all cells and controls a broad transcriptional response to hypoxia, including genes involved in cell metabolism, angiogenesis and vascular tone. Further evidence suggests that apart from its effect as an erythropoietic hormone EPO acts as a paracrine, tissue-protective protein in the brain and possibly also in other organs.

Topics: Erythropoiesis; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Kidney; Kidney Diseases; Oxygen; Transcription Factors

Death from malaria occurs from the complications of the infection: cerebral manifestations leading to coma and a severe and refractory anemia leading to hypoxia and cardiac decompensation. Several mechanisms have been identified to play a role in the pathogenesis of malarial anemia, such as erythrocyte lysis and phagocytosis, and sequestration of parasitized red blood cells, but recent data indicate that these mechanisms (singly or in combination) do not adequately explain the severity of this anemia. By contrast, hematologic studies have shown that bone marrow suppression and ineffective erythropoiesis contribute importantly to the severe anemia of malaria infection. The host mechanisms responsible for suppression of erythropoiesis may involve an excessive or sustained innate immune response or a pathologic skewing of the T-cell differentiation response with the attendant production of certain proinflammatory cytokines. Experimental data also indicate that severe malarial anemia is associated with the immunologic expression of a circulating inhibitor of erythropoiesis that functionally antagonizes the action of erythropoietin. We review the clinical and experimental basis for these concepts and discuss ongoing experimental and genetic studies aimed at unraveling the molecular basis of this malaria-induced bone marrow suppression.

Topics: Adult; Anemia; Animals; Aotus trivirgatus; Bone Marrow; Child; Child, Preschool; Cytokines; Erythropoiesis; Erythropoietin; Genetic Predisposition to Disease; Heart Failure; Humans; Hypoxia; Infant; Inflammation Mediators; Malaria; Malaria Vaccines; T-Lymphocyte Subsets

Topics: 2,3-Diphosphoglycerate; Bisphosphoglycerate Mutase; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Hemoglobinopathies; Homeostasis; Humans; Hypoxia; Kidney Diseases; Liver Diseases; Neoplasms; Oxygen; Polycythemia; Polycythemia Vera

Topics: Animals; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Kidney Diseases

Oxygen, carried mainly by erythrocytes, plays a crucial role in human organisms--as the terminal electron acceptor enables mitochondria functioning and energy production. Oxygen excess may be a cause of the damage of basic structural organism components--proteins, lipids and nucleinic acids. Thus, from the biological point of view, regulation of oxygen supply with its detection mechanisms is a critical process. Erythrocyte content optima-lization, in means of "benefit-loss" is a compromise between the necessity of anaerobic metabolism transit (in case of too low erythrocyte count), excessive increase in blood viscosity and non-productive, increased red blood cell turn-over with increased erythrocyte level. It is in fact a process of adaptation to changeable environmental conditions, life style and possible pathological processes. The study is a review of knowledge on the oxygen sensor and its connection to erythropoiesis regulation.

Topics: Adenosine Triphosphate; Apoptosis; DNA-Binding Proteins; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Mitochondria; Nuclear Proteins; Oxygen; Transcription Factors

Topics: Anemia; Animals; Erythropoietin; Gene Expression Regulation; Genetic Therapy; Hypoxia; Mice

Included in the acute response of lowlanders exposed to reduced oxygen availability is an elevated red blood cell count due to increased erythropoietin (Epo) synthesis. According to current thinking, hypoxia is "sensed" by hydroxylases that permit Hypoxia Inducible Factor 1alpha (HIF-1alpha) to complex with HIF-1beta to form a transcriptional activator (HIF-1) that drives expression of hypoxia-sensitive genes (such as EPO) under hypoxic conditions. In altitude-adapted Andean natives, the Epo hypoxic response may be blunted; however, our data indicate that the DNA sequences of the genes encoding Epo (including the 3' regulatory region) and HIF-1alpha appear to be conserved. Hence, adaptive changes in the Andean hypoxic response are not a consequence of changes in the primary sequence of these proteins or of known transcriptional regulatory domains of EPO. These results suggest that the altered erthropoietic response in Andean natives reflects adaptations in hypoxia sensing, rather than hypoxia response, mechanisms.

Topics: Altitude; Atmospheric Pressure; DNA-Binding Proteins; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Nuclear Proteins; Oxygen; Peru; Transcription Factors

Multiple myeloma (MM) is commonly associated with anemia. Several causes have been implicated, but anemia of chronic disease with inadequate erythropoietin (EPO) production related to the inflammatory cytokines appears to be of utmost importance. Interleukin-1 and tumor necrosis factor are capable of suppressing erythropoiesis. Anemia has broad implications. First, the low hemoglobin and hematocrit are associated with poor quality of life and performance and affect daily activity. Second, anemia has an impact on the cardiovascular system. Considering that most MM patients are elderly, this may be even more important. Anemia has been shown to induce or aggravate hypoxia and ischemic complications. Third, anemia has been shown to be a poor prognostic factor in MM. Traditionally, patients with symptomatic anemia were treated with red blood cell transfusions as needed. The introduction of epoetin alfa and epoetin beta into clinical practice opened new avenues to these patients. The administration of epoetins to patients with MM and anemia have been shown to be very useful. Several studies in more than 1000 patients have demonstrated a high response rate (range, 25%-85%; mean, 60%). This response is characterized by a significant increase of hemoglobin, hematocrit, and the number of red blood cells together with a reduction in the blood transfusion requirements. This is also associated with an improved quality of life. Although there is no complete agreement about the role of pretreatment serum EPO levels, many investigators believe that relatively low levels may help in predicting response, thereby limiting the number of potential candidates to receive this expensive therapy. The epoetins are safe and well tolerated with minimal toxicity; however, some concern has been recently raised regarding several dozen patients who developed pure red cell aplasia while on epoetin therapy. However, this adverse effect appears to be extremely rare. Recent data suggest that EPO has additional biologic effects, such as longer-than-expected survival in patients with MM. This observation is further supported by animal studies, demonstrating an antimyeloma effect of EPO in mice models. This effect has been shown to be immune mediated. If these exciting data are confirmed in future clinical trials, this may have significant implications on the treatment of MM.

Topics: Anemia; Animals; Cytokines; Epoetin Alfa; Erythrocyte Transfusion; Erythropoietin; Hemoglobins; Humans; Hypoxia; Mice; Mice, Inbred BALB C; Multiple Myeloma; Prognosis; Quality of Life; Recombinant Proteins; Time Factors

Topics: Animals; Cell Division; DNA-Binding Proteins; Endothelins; Erythropoietin; Humans; Hypertension, Pulmonary; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nuclear Proteins; Transcription Factors; Vascular Endothelial Growth Factor A

Increased erythropoietin plasma levels and the consequent augmented production of red blood cells is the best known systemic adaptation to reduced oxygen partial pressure (pO2). Intensive research during the last years revealed that the molecular mechanism behind the regulation of erythropoietin is ubiquitous and has far more implications than first thought. Erythropoietin regulation results from the activation of the hypoxia-inducible factor-1 (HIF-1) pathway under hypoxic conditions. HIF-1 is a heterodimer consisting of an oxygen sensitive--HIF-1--and an oxygen-independent subunit--HIF-1beta (also known as the aryl hydrocarbon receptor nuclear translocator--ARNT). In addition to erythropoietin, more than 30 genes are now known to be up-regulated by HIF-1. Recently, the critical involvement of HIF-1alpha post-translational modifications in the cellular oxygen sensing mechanism was discovered. In this review we will focus on the regulation of the HIF-1 pathway and the cellular oxygen sensor and discuss their implications in high altitude hypoxia.

Topics: Adaptation, Physiological; Altitude; Altitude Sickness; Animals; Aryl Hydrocarbon Receptor Nuclear Translocator; DNA-Binding Proteins; Erythropoietin; Hematocrit; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Models, Biological; Nuclear Proteins; Oxygen; Phosphorylation; Receptors, Aryl Hydrocarbon; Transcription Factors

Polycythemia is one of the key factors involved in the chronic mountain sickness syndrome, a condition frequent in Andean natives but whose causes still remain unclear. In theory, polycythemia may be secondary to abnormalities in ventilation, occurring during day or night (e.g. due to sleep abnormalities) stimulating excessive erythropoietin (Epo) production, or else it may result from either autogenous production, or from co-factors like cobalt. To assess the importance of these points, we studied subjects with or without polycythemia, born and living in Cerro de Pasco (Peru, 4330m asl, CP) and evaluated the relationship between Epo and respiratory variables both in CP and sea level. We also assessed the relationship between sleep abnormalities and the circadian rhythm of Epo. Polycythemic subjects showed higher Epo in all conditions, lower SaO2 and hypoxic ventilatory response, higher physiological dead space and higher CO2, suggesting ventilatory inefficiency. Epo levels could be highly modified by the level of oxygenation, and were related to similar directional changes in SaO2. Cobalt levels were normal in all subjects and correlated poorly with hematologic variables. The diurnal variations in Epo were grossly abnormal in polycythemic subjects, with complete loss of the circadian rhythm. These abnormalities correlated with the levels of hypoxemia during the night, but not with sleep abnormalities, which were only minor even in polycythemic subjects. The increased Epo production is mainly related to a greater ventilatory inefficiency, and not to altered sensitivity to hypoxia, cobalt or sleep abnormalities. Improving oxygenation can represent a possible therapeutic option for this syndrome.

Topics: Altitude Sickness; Autonomic Nervous System; Chronic Disease; Circadian Rhythm; Erythropoietin; Humans; Hypoxia; Indians, South American; Peru; Polycythemia; Respiratory Physiological Phenomena; Sleep

This article summarizes recent advances in understanding the production and action of the hormone erythropoietin (Epo) with respect to high altitude physiology and sports medicine. Hypoxia is the main stimulus for Epo gene expression. An O2-labile protein (hypoxia-inducible factor 1, HIF-1) has been identified that is hydroxylated and degraded under normoxic conditions but active in hypoxia, where it enhances Epo gene transcription resulting in elevated hemoglobin levels and O2 capacity of the blood. The stimulation of Epo production at lowered arterial O2 tension can be maladaptive, if erythrocytosis develops such as seen in high altitude habitants. Within physiological limits the aerobic power increases in parallel with blood O2 capacity. Therefore, some elite athletes have misused recombinant human Epo (rhEpo), which is a beneficial anti-anemic drug in clinical practice. Indirect and direct methods to detect rhEpo doping have been recently developed.

Topics: Altitude; Doping in Sports; Erythropoietin; Exercise; Hemoglobins; Humans; Hypoxia; Oxygen; Polycythemia; Sports Medicine

The regulation of blood red cell production by the hormone erythropoietin (Epo) provides a paradigm for control of gene expression by oxygen. Analysis of this pathway has revealed a widespread system of gene regulation based on a transcriptional complex termed hypoxia-inducible factor (HIF). Hydroxylation of specific prolyl and asparinyl residues in the alpha subunit of HIF by a series of non-haem iron-dependent dioxygenases has been defined as a novel mechanism of protein modification that transduces the oxygen-sensitive signal.

Topics: Animals; DNA-Binding Proteins; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Ligases; Mixed Function Oxygenases; Nuclear Proteins; Oxygen; Procollagen-Proline Dioxygenase; Repressor Proteins; Signal Transduction; Transcription Factors; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Von Hippel-Lindau Tumor Suppressor Protein

Topics: Animals; Apoptosis; DNA-Binding Proteins; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Light; Mice; Nuclear Proteins; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinal Ganglion Cells; Transcription Factors; Up-Regulation

Adaptation to hypoxia is a topic of considerable clinical relevance, as it influences the pathophysiology of anaemia, polycythaemia, tissue ischaemia and cancer. A growing number of physiologically relevant genes are regulated in response to changes in intracellular oxygen tension. These include genes encoding erythropoietin, vascular endothelial growth factor and tyrosine hydroxylase. Studies on the regulation of the erythropoietin gene have provided insights into the common mechanism of oxygen sensing and signal transduction, leading to activation of the hypoxia-inducible transcription factor 1 (HIF-1). Activation of HIF-1 by hypoxia depends on rescue of its alpha-subunit from oxygen-dependent degradation in the proteasome, allowing it to form a heterodimer with HIF-1 beta. This then translocates to the nucleus. There, HIF-1 assembles with a highly conserved orphan nuclear receptor, HNF-4, and a critical transcriptional adaptor, p300. This complex binds to a 3' enhancer on the erythropoietin gene, enabling transcription of erythropoietin. HIF-1 also activates other genes, the cis-acting elements of which contain cognate hypoxia response elements. There is growing evidence that the oxygen sensor is a flavohaem protein and that the signal transduction pathway involves changes in the level of intracellular reactive oxygen intermediates. We have recently cloned a novel fusion protein called cytochrome b5/b5 reductase, which is a cyanide-insensitive NADPH oxidase and, therefore, a candidate to be the oxygen sensor. This flavohaem protein is widely expressed in cell lines and tissues, with localization in the perinuclear space. In the presence of oxygen and iron, it may induce oxidative modifications that target HIF-1 alpha for ubiquitination and degradation.

Topics: Adaptation, Physiological; DNA-Binding Proteins; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Models, Biological; Nuclear Proteins; Oxygen; Structure-Activity Relationship; Transcription Factors

Topics: Anemia; Apoptosis; Erythropoietin; Fibrosis; Humans; Hypoxia; Kidney Failure, Chronic; Kidney Tubules; Models, Biological; Oxidative Stress; Recombinant Proteins

Topics: Animals; DNA-Binding Proteins; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Nuclear Proteins; Signal Transduction; Transcription Factors

Anaemia is a common occurrence in patients with cancer and contributes to the clinical symptomatology and reduced quality of life (QOL) seen in cancer patients. Many aspects of reduced QOL, including fatigue, are known to be associated with suboptimally low levels of haemoglobin. Even mild-to-moderate anaemia adversely affects patient-reported QOL parameters. Red blood cell transfusions are associated with many real and perceived risks, inconveniences, costs, and only temporary benefits. Recombinant human erythropoietin (rHuEPO) is an effective therapy to increase haemoglobin values in over half of anaemic cancer patients receiving concurrent chemotherapy. These increased haemoglobin values are closely correlated with improvements in QOL. Despite these objectively defined benefits, less than 50% of anaemic patients undergoing cytotoxic chemotherapy receive rHuEPO, in contrast to patients with chronic renal failure on dialysis, where anaemia is universally and aggressively treated to more optimal haemoglobin values. However, there are several barriers that may limit more widespread use of rHuEPO. These include inconvenience associated with frequent dosing; failure of a large proportion (40 to 50%) of patients to respond; relatively slow time to response; absence of reliable early indicators of response; and current lack of rigorous pharmacoeconomic data demonstrating cost-effectiveness. Darbepoetin alfa is a novel erythropoiesis stimulating protein (NESP) that is biochemically distinct from rHuEPO, and which has been proven to stimulate red blood cell production. The molecule has a 3-fold longer half-life and increased biological activity that will allow less frequent dosing, facilitating improved management of the anaemia of cancer. With this new option for therapy, further avenues of investigation should lead to renewed interest in the clinical benefits of optimal haemoglobin levels for patients with cancer.

Topics: Anemia; Antineoplastic Agents; Case Management; Clinical Trials as Topic; Darbepoetin alfa; Erythrocyte Transfusion; Erythropoietin; Fatigue; Forecasting; Health Care Costs; Humans; Hypoxia; Incidence; Kidney Failure, Chronic; Multicenter Studies as Topic; Neoplasms; Quality of Life; Radiation Tolerance; Recombinant Proteins; Treatment Outcome

Recently, endurance athletes have used several novel approaches and modalities for altitude training including: (i) normobaric hypoxia via nitrogen dilution (hypoxic apartment); (ii) supplemental oxygen; (iii) hypoxic sleeping devices; and (iv) intermittent hypoxic exposure (IHE). A normobaric hypoxic apartment simulates an altitude environment equivalent to approximately 2000 to 3000m (6560 to 9840ft). Athletes who use a hypoxic apartment typically 'live and sleep high' in the hypoxic apartment for 8 to 18 hours a day, but complete their training at sea level, or approximate sea level conditions. Several studies suggest that using a hypoxic apartment in this manner produces beneficial changes in serum erythropoietin (EPO) levels, reticulocyte count and red blood cell (RBC) mass, which in turn may lead to improvements in postaltitude endurance performance. However, other studies failed to demonstrate significant changes in haematological indices as a result of using a hypoxic apartment. These discrepancies may be caused by differences in methodology, the hypoxic stimulus that athletes were exposed to and/or the training status of the athletes. Supplemental oxygen is used to simulate either normoxic (sea level) or hyperoxic conditions during high-intensity workouts at altitude. This method is a modification of the 'high-low' strategy, since athletes live in a natural terrestrial altitude environment but train at 'sea level' with the aid of supplemental oxygen. Limited data regarding the efficacy of hyperoxic training suggests that high-intensity workouts at moderate altitude (1860m/6100ft) and endurance perfor- mance at sea level may be enhanced when supplemental oxygen training is utilised at altitude over a duration of several weeks. Hypoxic sleeping devices include the Colorado Altitude Training (CAT) Hatch (hypobaric chamber) and Hypoxico Tent System (normobaric hypoxic system), both of which are designed to allow athletes to sleep high and train low. These devices simulate altitudes up to approximately 4575 m/15006 ft and 4270 m/14005 ft, respectively. Currently, no studies have been published on the efficacy of these devices on RBC production, maximal oxygen uptake and/or performance in elite athletes. IHE is based on the assumption that brief exposures to hypoxia (1.5 to 2.0 hours) are sufficient to stimulate the release of EPO, and ultimately bring about an increase in RBC concentration. Athletes typically use IHE while at rest, or in conjunction w

Topics: Acclimatization; Altitude; Erythrocytes; Erythropoietin; Female; Humans; Hypoxia; Male; Nitrogen; Oxygen; Oxygen Consumption; Physical Fitness; Reticulocyte Count; Sports

By definition, idiopathic erythrocytosis (IE) applies to a group of patients characterised by having a measured RCM above their predicted normal range (an absolute erythrocytosis) and following investigation do not have a form of primary or secondary erythrocytosis. Patients with IE are heterogenous. The possibilities include physiological variation, 'early' polycythaemia vera (10-15% develop clear features of PV over a few years), unrecognized congenital erythrocytosis, unrecognized or unrecognizable secondary acquired erythrocytosis or a currently undescribed form of primary or secondary erythrocytosis. Patients are more commonly male with a median age at presentation of 55-60 years. Approximately half of the patients present with vascular occlusive complications. Retrospective evidence indicates that vascular occlusion occurs less frequently when the PCV is controlled at normal levels. Venesection is the treatment of choice to lower the PCV. As a general approach to management, all patients with a PCV above 0.54 should be venesected to a PCV less than 0.45. This target PCV should also apply to patients with lesser degrees of raised PCV who have additional other risk factors for vascular occlusion.

Topics: Aged; Arterial Occlusive Diseases; Bone Marrow; Chlorambucil; Diagnosis, Differential; Endocrine System Diseases; Erythrocyte Volume; Erythroid Precursor Cells; Erythropoietin; Genetic Predisposition to Disease; Humans; Hypoxia; Kidney Diseases; Leukemia; Leukemia, Radiation-Induced; Middle Aged; Phosphorus Radioisotopes; Polycythemia; Polycythemia Vera; Receptors, Erythropoietin; Sequence Deletion; Smoking; Stroke

Acute exposure to moderate altitude is likely to enhance cycling performance on flat terrain because the benefit of reduced aerodynamic drag outweighs the decrease in maximum aerobic power [maximal oxygen uptake (VO2max)]. In contrast, when the course is mountainous, cycling performance will be reduced at moderate altitude. Living and training at altitude, or living in an hypoxic environment (approximately 2500 m) but training near sea level, are popular practices among elite cyclists seeking enhanced performance at sea level. In an attempt to confirm or refute the efficacy of these practices, we reviewed studies conducted on highly-trained athletes and, where possible, on elite cyclists. To ensure relevance of the information to the conditions likely to be encountered by cyclists, we concentrated our literature survey on studies that have used 2- to 4-week exposures to moderate altitude (1500 to 3000 m). With acclimatisation there is strong evidence of decreased production or increased clearance of lactate in the muscle, moderate evidence of enhanced muscle buffering capacity (beta m) and tenuous evidence of improved mechanical efficiency (ME) of cycling. Our analysis of the relevant literature indicates that, in contrast to the existing paradigm, adaptation to natural or simulated moderate altitude does not stimulate red cell production sufficiently to increase red cell volume (RCV) and haemoglobin mass (Hb(mass)). Hypoxia does increase serum erthyropoietin levels but the next step in the erythropoietic cascade is not clearly established; there is only weak evidence of an increase in young red blood cells (reticulocytes). Moreover, the collective evidence from studies of highly-trained athletes indicates that adaptation to hypoxia is unlikely to enhance sea level VO2max. Such enhancement would be expected if RCV and Hb(mass) were elevated. The accumulated results of 5 different research groups that have used controlled study designs indicate that continuous living and training at moderate altitude does not improve sea level performance of high level athletes. However, recent studies from 3 independent laboratories have consistently shown small improvements after living in hypoxia and training near sea level. While other research groups have attributed the improved performance to increased RCV and VO2max, we cite evidence that changes at the muscle level (beta m and ME) could be the fundamental mechanism. While living at altitude but training near sea le

Topics: 2,3-Diphosphoglycerate; Altitude; Bicycling; Cardiac Output; Epoetin Alfa; Erythrocyte Volume; Erythropoietin; Glycolysis; Hemoglobinometry; Humans; Hypoxia; Muscle, Skeletal; Receptors, Transferrin; Recombinant Proteins; Reticulocytes; Task Performance and Analysis

Normal cell homeostasis relies on the ordered flow of nutrients and substrates through metabolic pathways. Any perturbation of this flow eventually leads to dysfunction, impairment of defense mechanisms, loss of viability and death. High altitude and pathological hypoxia represent a serious and frequent cause for the loss of cell viability. Although organisms customarily respond by triggering adaptive or maladaptive mechanisms, all forms of life eventually succumb to hypoxia if it is severe enough, irrespectively of the primary cause. This paper reviews one of the mechanisms by which organisms respond to hypoxia: erythropoiesis. Although such response is not always beneficial, the discovery of the biochemical mechanisms underlying erythropoiesis has triggered an active field of research that is actually applying lessons learned in the mountains to a more clinical environment.

Topics: Adaptation, Physiological; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia

Reduced oxygenation activates specific expression of erythropoietin (EPO) and other hypoxia-regulated genes. The cellular response to hypoxia is mediated by the hypoxia-inducible factor-1 (HIF-1). Unregulated overexpression of EPO results in a hematocrit value of about 80%; transgenic mice were shown to adapt to this polyglobulia.

Topics: Animals; DNA-Binding Proteins; Erythropoietin; Gene Expression; Gene Expression Regulation; Hematocrit; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Transgenic; Nuclear Proteins; Oxygen; Transcription Factors

For training at altitude to be effective, it must provide some advantage above and beyond similar training at sea level. This advantage could be provided by: 1) acclimatization to altitude which improves oxygen transport and/or utilization; 2) hypoxic exercise which "intensifies" the training stimulus; or 3) some combination of both. Controlled studies of "typical" altitude training, involving both altitude acclimatization and hypoxic exercise have never been shown to improve sea level performance. This failure has been attributed to reduced training loads at altitude. One approach developed by Levine and Stray-Gundersen, called "living high-training low" has been shown to improve sea level performance over events lasting 8-20 minutes. This strategy combines altitude acclimatization (2,500 m) with low altitude training to get the optimal effect. The opposite strategy, "living low-training high" is proposed by Dr. Hoppeler in this debate. In defense of the primacy of the altitude acclimatization effect, data will be presented to support the following: 1). Living high-training low clearly improves performance in athletes of all abilities; 2). The mechanism of this improvement is primarily an increase in erythropoietin leading to increased red cell mass, VO2max, and running performance; 3). Rather than intensifying the training stimulus, training at altitude leads to the opposite effect--reduced speeds, reduced power output, reduced oxygen flux--and, following the principal of symmorphosis, is not likely to provide any advantage for a well trained athlete; 4). At the moderate altitudes used by most athletes, resting oxygen delivery to skeletal muscle is well preserved, arguing against any detrimental effect on "protein synthesis"; 5). It is possible however, that at significantly higher altitudes, acclimatization leads to appetite suppression, inhibition of protein synthesis, muscle wasting, excessive ventilatory work, and metabolic compensation that is NOT advantageous for a competitive athlete.

Topics: Acclimatization; Altitude; Animals; Blood Gas Analysis; Erythropoietin; Exercise; Hemoglobins; Humans; Hypoxia; Myoglobin; Oxygen; Physical Fitness

Because molecular oxygen is essential for generating cellular energy in aerobic organisms, and because survival depends on this fundamental requirement for oxygen, all higher organisms have evolved numerous diversely regulated mechanisms to detect and respond to potentially life-threatening occurrences of decreased oxygen availability (hypoxia). While the oxygen-dependent regulation of gene expression involves both transcriptional- and post-transcriptional mechanisms, investigations have focused mainly on mechanisms working at the transcriptional level. In this review, the focus is on a growing body of work that looks at post-transcriptional mechanisms acting at a level of mRNA stability.

Topics: Animals; Endothelial Growth Factors; Erythropoietin; Humans; Hypoxia; Lymphokines; RNA Stability; RNA, Messenger; Tyrosine 3-Monooxygenase; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

During the past few years, the investigation of altitude hypoxia and its effect on metabolic functions in humans has increasingly attracted the attention of endocrinologists. Most of the studies have been performed as field studies at moderate or high altitude, but with conflicting results. One of the possible reasons certainly is the fact that standardisation of field studies is almost impossible. Furthermore, many factors such as wind, temperature, radiation and others, may affect certain endocrine parameters, but they cannot be individually quantified. Hence, their inclusion into statistical analyses of the obtained data is not useful. Nevertheless, several endocrine parameters were shown to be affected by altitude hypoxia. Among them, there is erythropoietin, a hormone which is well known to stimulate erythropoiesis. This hormone shows a rapid increase after ascent to moderate or high altitude. There is also evidence that urinary and serum noradrenalin levels increase significantly, whereas adrenalin seems to be less affected. Another "stress-hormone", cortisol, also shows a significant increase. Furthermore, the biologically active fraction of the thyroidal hormones thyroxine and triiodothyronine increases significantly. And last but not least, one of the most important proinflammatory cytokines, interleukin-6, shows a manyfold increase compared to the basal level. However, the clinical significance of most of these studies is not yet clear. Hence, from an endocrinological point of view, no specific recommendations may be given to people staying at moderate or high altitude.

Topics: Adaptation, Physiological; Altitude; Catecholamines; Cytokines; Erythropoietin; Humans; Hydrocortisone; Hypoxia; Interleukin-6; Thyroid Hormones

NADPH oxidase is classically regarded as a key enzyme of neutrophils, where it is involved in the pathogenic production of reactive oxygen species. However, NADPH oxidase-like enzymes have recently been identified in non-neutrophil cells, supporting a separate role for NADPH-oxidase derived oxygen species in oxygen sensitive processes. This article reviews the current literature surrounding the potential role of NADPH oxidase in the oxygen sensing processes which underlie hypoxic pulmonary vasoconstriction, systemic vascular smooth muscle proliferation, carotid and airways chemoreceptor activation, erythropoietin gene expression, and oxytropic responses of plant cells.

Topics: Animals; Carotid Body; Erythropoietin; Humans; Hypoxia; NADPH Oxidases; Neutrophils; Oxygen; Pulmonary Circulation; Reactive Oxygen Species; Vasodilation

A growing number of physiologically relevant genes are regulated in response to changes in intracellular oxygen tension. It is likely that cells from a wide variety of tissues share a common mechanism of oxygen sensing and signal transduction leading to the activation of the transcription factor hypoxia-inducible factor 1 (HIF-1). Besides hypoxia, transition metals (Co2+, Ni2+ and Mn2+) and iron chelation also promote activation of HIF-1. Induction of HIF-1 by hypoxia is blocked by the heme ligands carbon monoxide and nitric oxide. There is growing, albeit indirect, evidence that the oxygen sensor is a flavoheme protein and that the signal transduction pathway involves changes in the level of intracellular reactive oxygen intermediates. The activation of HIF-1 by hypoxia depends upon signaling-dependent rescue of its alpha-subunit from oxygen-dependent degradation in the proteasome, allowing it to form a heterodimer with HIF-1beta (ARNT), which then translocates to the nucleus and impacts on the transcription of genes whose cis-acting elements contain cognate hypoxia response elements.

Topics: Animals; Carotid Body; DNA-Binding Proteins; Erythropoietin; Gene Expression Regulation; Hemeproteins; Humans; Hydrogen Peroxide; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; In Vitro Techniques; Models, Biological; NADH, NADPH Oxidoreductases; Nuclear Proteins; Oxygen; Signal Transduction; Transcription Factors

Topics: Adenosine Triphosphate; Animals; Apoptosis; DNA-Binding Proteins; Endothelial Growth Factors; Erythropoietin; Genes, Tumor Suppressor; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphokines; Nuclear Proteins; Signal Transduction; Stress, Physiological; Transcription Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Amino Acid Sequence; Animals; DNA-Binding Proteins; Endothelial Growth Factors; Erythropoietin; Gene Expression Regulation; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphokines; Molecular Sequence Data; Nuclear Proteins; Signal Transduction; Transcription Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Anemia; Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; DNA-Binding Proteins; Erythropoietin; Event-Related Potentials, P300; Gene Expression Regulation; Genetic Therapy; Hepatocyte Nuclear Factor 4; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Failure, Chronic; Nuclear Proteins; omega-N-Methylarginine; Phosphoproteins; Transcription Factors; Transcription, Genetic

The optimal delivery of oxygen to tissues is essential both to ensure adequate energy provision and to avoid the toxic effects of higher oxygen concentrations. For this to occur, organisms must be able to sense oxygen and respond to changes in oxygen tension by altering gene expression. The analysis of the regulation of erythropoiesis has provided important insights into the mechanisms of oxygen-regulated gene expression. These mechanisms have a role in the regulation of many genes, in many cell types and appear to be of relevance to many common pathologies in which disturbances of oxygen supply are central.

Topics: Animals; DNA-Binding Proteins; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Neovascularization, Physiologic; Nuclear Proteins; Oxygen; Transcription Factors; Transcription, Genetic

Erythropoietin, a hormone produced in the adult kidney, controls erythrocyte production. In response to ischemic or hypoxic hypoxia, the level of expression of the gene increases markedly. We have a limited understanding of the factors governing the tissue specificity of erythropoietin gene expression but considerable progress has been made toward understanding the mechanisms that increase erythropoietin gene expression in response to hypoxia. An increase in transcription occurs, mediated (at least in part) by formation of a heterodimeric DNA binding complex termed hypoxia inducible factor 1. Transcriptional regulation mediated by this complex has now been shown to modulate the expression of a number of other genes in a wide range of cell types.

Topics: Animals; Embryonic and Fetal Development; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation; Gene Expression Regulation, Developmental; Humans; Hypoxia; Organ Specificity; Oxygen

Erythropoietin (Epo) controls the proliferation, differentiation and survival of the erythroid progenitors. This cytokine was cloned in 1985 and rapidly became used for treatment of anemia of renal failure, opening the way to the first clinical trials of a hematopoietic growth factor. The clonage of one chain of the Epo receptor followed in 1989, thereby opening the research on intracellular signal transduction induced by Epo. Epo is synthesized mainly by the kidney and the liver and sequences required for tissue-specific expression have been localized in the Epo gene. A 3'enhancer is responsible for hypoxia-inducible Epo gene expression. HIF-1 alpha and beta proteins bind to this enhancer. Gene regulation by hypoxia is widespread in many cells and involves numerous genes in addition to the Epo gene. The Epo receptor belongs to the cytokine receptor family and includes a p66 chain which is dimerized upon Epo activation; two accessory proteins defined by cross-linking remain to be characterized. Epo binding induces the stimulation of Jak2 tyrosine kinase. Jak2 activation leads to the tyrosine phosphorylation of several proteins including the Epo receptor itself. As a result, different intracellular pathways are activated: Ras/MAP kinase, phosphatidylinositol 3-kinase and STAT transcription factors. However, the exact mechanisms by which the proliferation and/or the differentiation of erythroid cells are regulated after Epo stimulation are not known. Furthermore, target disruption of both Epo and Epo receptor showed that Epo was not involved in the commitment of the erythroid lineage and seemed to act mainly as a survival factor.

Topics: Animals; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Phosphorylation; Protein Processing, Post-Translational; Receptors, Erythropoietin; Signal Transduction; Structure-Activity Relationship

An erythrocytosis describes an increased peripheral blood packed cell volume (PCV) and is deemed to be absolute or apparent depending on whether or not the measured red cell mass (RCM) is above the reference range. This reference range must be related to the individual's height and weight to avoid erroneous interpretations using ml/kg total body weight expressions in obesity. Absolute erythrocytoses are divided into primary, where the erythropoietic compartment is intrinsically abnormal, secondary, where the erythropoietic compartment is normal but is responding to external pathological events leading to an increased erythropoietin drive, and idiopathic, where neither a primary nor a secondary erythrocytosis can be established. Both primary and secondary erythrocytoses have congenital and acquired forms. The only form of primary acquired erythrocytosis that has been defined is the clonal myeloproliferative disorder, polycythaemia vera (PV). Modified diagnostic markers for PV are proposed. Thrombocytoses can be classified into primary, where megakaryopoiesis is intrinsically abnormal, secondary, where megakaryopoiesis is normal but increased platelet production is a reaction to some other unrelated pathology, and finally idiopathic. This latter new group would be used for patients not satisfying the criteria for primary or secondary thrombocytoses, if these were more precise and rigidly used than currently is the case. While theoretically congenital and acquired forms of primary and secondary thrombocytoses might exist, only one cause of secondary congenital thrombocytosis has been established, and primary congenital thrombocytosis has not yet been precisely defined. Primary (essential) thrombocythaemia (PT) is one of the forms of primary acquired thrombocytoses. The diagnostic criteria of PT traditionally involve the exclusion of secondary thrombocytoses and other myeloproliferative disorders but marrow histology could hold a key positive diagnostic role if objective histological features of PT were agreed.

Topics: Anthropometry; Bone Marrow; Bone Marrow Diseases; Cells, Cultured; Diagnostic Imaging; Diagnostic Tests, Routine; Erythrocyte Volume; Erythroid Precursor Cells; Erythropoietin; Female; Hematocrit; Hemoglobinopathies; Humans; Hypoxia; Kidney Diseases; Liver Diseases; Male; Neoplasms; Polycythemia; Receptors, Erythropoietin; Thrombocytosis

Oxygen sensing is a determinant function of mammals, especially humans, to maintain their activity under acute or chronic exposure to hypoxia. True O2 sensors (chemoreceptors, erythropoietin secreting cells) are involved in regulation loops, which aim to restore O2 availability to the cells. Pseudo O2 sensors are cells activated by the lack of oxygen but not clearly involved in regulation processes. Potassium channels in the carotid bodies have been suspected to be O2 sensitive and could mediate the chemosensitivity to hypoxia. Na,K-ATPase related ion transport in alveolar pneumocytes could be sensitive to O2 availability and regulate the flux of water and sodium in the alveolar space. Signal transduction in G-protein-dependent receptor systems is modified in hypoxia, such as in cardiac beta-receptors and adenosinergic and muscarinic receptors. Recent studies have provided some evidence to the possible role of hypoxia inducible factors (HIF-1) in the regulation of protein synthesis at the transcriptional level. Similarities between O2-sensing mechanisms in erythropoiesis and in the synthesis of vascular endothelial growth factor were recently evidenced. Both genes are upregulated in hypoxia. However, the precise structure (heme-linked enzyme?) of all these O2-sensitive sites is not known, either in the erythropoietic system or in the chemoreceptor function. An adequate balance between hypoxia-induced upregulation and downregulation processes is necessary for optimal survival in a hypoxic environment.

Topics: Altitude; Animals; Calcium; Down-Regulation; Erythropoiesis; Erythropoietin; Hypoxia; Mammals; Oxygen; Parathyroid Hormone; Pulmonary Alveoli; Up-Regulation

Topics: Animals; Bacteria; Biosensing Techniques; Energy Metabolism; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Nitrogen; Oxygen; Promoter Regions, Genetic; Signal Transduction; Transcription, Genetic

Erythropoietin gene (EPO) expression is activated by tissue hypoxia in renal peritubular interstitial fibroblasts and, to a lesser extent, in hepatocytes and ito cells of the liver. A hypoxia-inducible enhancer spanning approximately 50 bp within the 3'-flanking region of the EPO gene is required for transcriptional activation in hypoxic cells. Hypoxia-inducible factor 1 is a basic helix-loop-helix protein that binds at the 5' end of the enhancer. The binding of hypoxia-inducible factor 1 is absolutely required for enhancer function. Hepatocyte nuclear factor 4 is an orphan receptor that binds at the 3' end of the enhancer. The binding of hepatocyte nuclear factor 4 augments hypoxia-inducible transcription mediated by the enhancer but is not absolutely required for enhancer function. Factors binding to the enhancer may interact synergistically with factors binding to the EPO promoter to activate transcription in hypoxic cells. Indirect evidence suggests that oxygen tension may be sensed by a hemoprotein. In one model, the putative hemoprotein adopts different conformational states depending on whether O2 is bound. Another model proposes that the hemoprotein converts O2 to H2O2. The protein tyrosine kinase c-Src, GTP-binding protein Ras, and MAP kinase signal pathways have been implicated in hypoxia signal transduction, but no direct evidence links these pathways to EPO transcriptional activation.

Topics: Animals; DNA-Binding Proteins; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Molecular Biology; Nuclear Proteins; Signal Transduction; Tissue Distribution; Transcription Factors

All forms of oxygen deprivation act as a stimulus for the production of the hormone erythropoietin. The main sites of production are specialized renal fibroblasts in adult mammals and hepatic cells in mammalian fetuses and neonates. The hormone's name is a succint description of its main effect: erythropoietin stimulates red cell production from bone marrow precursors and hence controls the O2-carrying capacity of the blood. The peripheral red cell count is kept constant by a closely controlled feedback mechanism involving O2 supply, erythropoietin secretion and erythropoiesis; the system may become unbalanced in conditions such as chronic renal disease, chronic inflammation and prematurity. Recombinant human erythropoietin is used as hormonal replacement therapy to correct various types of anemia and replenish the red cell count following hemorrhage or blood donation for autologous transfusion.

Topics: Anemia; Animals; Erythropoietin; Humans; Hypoxia; Recombinant Proteins

Topics: Amino Acid Sequence; Animals; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Kidney; Liver; Mice; Models, Biological; Models, Molecular; Protein Structure, Tertiary; Receptors, Erythropoietin; Recombinant Fusion Proteins; Second Messenger Systems; Signal Transduction

Topics: Adenosine Triphosphate; Animals; Carotid Body; Chemoreceptor Cells; Epithelium; Erythropoietin; Humans; Hypoxia; Lung; Models, Biological; NADH, NADPH Oxidoreductases; Oxygen; Oxygen Consumption; Potassium Channels

Low oxygen tension is a feature of many physiologic and pathologic conditions, including wound healing, fibrosis, and neoplasia. Increasing evidence suggests that low oxygen tension induces the transcription of a number of genes, and that this process depends on the cellular context. The proteins synthesized from these genes enable cells to adapt to the hypoxic environment and/or to fulfill their functional roles. The regulatory regions responsible for the induction of erythropoietin gene transcription and synthesis in response to hypoxia/anemia appear to be cis-acting deoxyribonucleic acid sequences located within the 5' and 3' flanking regions of the erythropoietin gene. Other proteins induced by hypoxia include cytokines (platelet-derived growth factor-beta chain, endothelin-1, transforming growth factor-beta), enzymes (tyrosine hydroxylase, glycolytic enzymes), and stress proteins. The molecular mechanisms of the hypoxia-induced expression of these genes are poorly understood. A heme protein may act as the oxygen tension sensor, or the redox state of certain nuclear transcription factors may function as second messengers.

Topics: Animals; Cell Hypoxia; Cytokines; Erythropoietin; Gene Expression; Gene Expression Regulation; Humans; Hypoxia; Transcription, Genetic

Topics: Biomarkers; Erythroid Precursor Cells; Erythropoietin; Fetal Hemoglobin; Humans; Hypoxia; Infant, Newborn

Hypoxia is the fundamental stimulus for erythropoietin (EP) production. It is clear that hypoxia increases erythropoietin messenger RNA in a renal cell, which leads to the production of increased amounts of erythropoietin in the kidney. Hypoxia also increases external messenger substances that amplify the effects of hypoxia and increases erythropoietin messenger RNA to further accelerate erythropoietin production. Some of these messenger substances are adenosine, eicosanoids, oxygen-derived metabolites, and beta-2 adrenergic agonists that are postulated to act through the activation of cell membrane receptors and are coupled to an increase in a G stimulatory protein which activates adenylate cyclase. This leads to increased production of cyclic adenosine monophosphate (AMP) for the production of key phosphoproteins that are involved in the biosynthesis/secretion of erythropoietin. This paper considers the physicochemical properties of human erythropoietin, pharmacologic agents that increase and decrease erythropoietin production/secretion, serum erythropoietin levels in normal human subjects and in patients with several types of anemia, and a model for the role of adenosine and other external messenger substances in erythropoietin biosynthesis/secretion.

Topics: Adenosine; Animals; Erythropoietin; Humans; Hypoxia; Kidney

Topics: Anemia; Animals; Blotting, Northern; Cobalt; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Mice; Rats; Restriction Mapping; RNA; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The kidney is the main site of erythropoietin (EPO) formation. Oxygen sensing in the kidney itself plays a major role in the control of EPO synthesis. By in situ hybridization it has been established that the EPO-producing cells are situated in the interstitium of the cortical labyrinth, but they have not been precisely identified. Morphological findings provide new insights into the location and mechanism of oxygen sensing in the kidney. In addition to causing an increase in the number of cells containing EPO messenger RNA, anemia provokes structural changes exclusively in the cortical labyrinth. Specifically, the fibroblasts become enlarged and show increased activity of 5'-nucleotidase, and the S1 segment of the proximal tubule shows similar alterations as in various models of hypoxia. Thus, structures that are situated in the close vicinity of the EPO-producing cells appear to be sensitive to decreased oxygen delivery.

Topics: Anemia; Animals; Endothelium, Vascular; Erythropoietin; Humans; Hypoxia; Kidney; Kidney Tubules, Proximal; Oxygen

Topics: Animals; Blotting, Northern; Erythropoietin; Humans; Hypoxia; Kidney; Kidney Diseases; Mice; RNA, Messenger

Topics: Animals; Erythropoietin; Hemoglobins; Hormones; Hydrogen-Ion Concentration; Hypoxia; Kidney; Kinetics; Oxygen; Rats

Topics: Animals; Erythropoietin; Humans; Hypoxia; Kidney; Kidney Failure, Chronic; Parathyroidectomy; Polycystic Kidney Diseases; Polycythemia

The glycoprotein hormone erythropoietin (EPO) counteracts tissue hypoxia by increasing the systemic oxygen-carrying capacity. It induces augmentation of red blood cell mass by stimulating the formation and differentiation of erythroid precursor cells in the bone marrow. EPO production is increased under various forms of diminished oxygen supply such as anemic or hypoxic hypoxia. In the adult organism, the kidneys are the major source of EPO. The precise nature of the cells responsible for renal EPO production, however, has not yet been elucidated. Most likely, peritubular cortical cells, e.g. interstitial or endothelial cells, are involved in the elaboration of the hormone. From the observation that isolated perfused rat kidneys produce EPO in an oxygen-dependent fashion we conclude that the 'oxygen sensor' that controls hypoxia-induced EPO synthesis is located in the kidney itself. Within the kidneys, the local venous oxygen tension which reflects the ratio of oxygen supply to oxygen consumption is measured and transformed into a signal that regulates the formation of EPO. However, the mechanism by which a decrease of oxygen delivery to the kidneys is linked to an enhanced EPO gene expression is not yet known. Two possible mechanisms of regulation are discussed: First, renal hypoxia could lead to enhanced formation of metabolic mediators, for example prostaglandins or adenosine, which might stimulate EPO gene transcription by increasing cellular levels of second messenger molecules. Second, some kind of molecular 'oxygen receptor' such as a heme protein, that controls EPO formation by an oxygen-dependent conformational change, could mediate signal transduction.

Topics: Animals; Erythropoietin; Humans; Hypoxia; Kidney; Signal Transduction

Topics: Anemia; Anemia, Refractory; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Kidney Failure, Chronic; Polycythemia; Recombinant Proteins

Topics: Angiotensin II; Animals; Atmospheric Pressure; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Indomethacin; Liver; Nephrectomy; Prostaglandins; Rabbits; Rats; Secretory Rate

Topics: Adenosine Triphosphate; Animals; Biological Availability; Biomechanical Phenomena; Chemoreceptor Cells; Erythropoietin; Humans; Hypoxia; Kidney; Kidney Cortex; Oxygen; Prostaglandins; Signal Transduction

Topics: Animals; Diffusion; Erythropoietin; Hypoxia; Kidney; Oxygen; Rats

Topics: Animals; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Kidney

The discovery and present state of knowledge of the specific hormonal mechanism controlling erythropoiesis are briefly reviewed. 80 years ago, Carnot and Deflandre postulated the existence of a humoral erythropoietic factor ('hémopoïétine') produced in response to anemia. The general approval of this concept required almost 50 years, when the factor was recognized to be a glycoprotein hormone and termed 'erythropoietin'. Very recently, human erythropoietin has been purified and its amino acid sequence been identified. The hormone is mainly of renal origin. Its production is stimulated by tissue hypoxia of different causes. The mechanism of O2 sensing and the exact site of erythropoietin synthesis in the kidney still remain to be clarified.

Topics: Animals; Biomechanical Phenomena; Dogs; Erythropoietin; Guinea Pigs; Humans; Hypoxia; Kidney; Methods; Mice; Physiology; Rabbits

The reversible period of hypoxia and ischemia is a consequence of the function of numerous regulatory mechanisms which convert cells to a quiescent state. Thus, early changes in metabolism reflect regulatory events rather than pathological events. O2-dependent enzymes (oxidases and oxygenases) are the primary sensors for physiological responses to hypoxia, and failure of their functions are ultimately responsible for hypoxic and ischemic cell injury. At least 30 of these enzymes are known to occur in kidney, but only cytochrome oxidase has been extensively studied with regard to the above processes. Heterogeneity of subcellular oxygenation occurs as a result of the existence of clusters of mitochondria in the basolateral regions of proximal and distal tubule cells. This creates regions with very high O2 consumption rates, and results in diffusion limitations in O2 supply. Finally, dramatic progress has been made in protecting against ischemic injury through use of nonpermeant solutes to reduce cell swelling, addition of ATP-MgCl2 to stimulate recovery of cellular adenylates upon reoxygenation, use of a Ca2+ uptake blocker to prevent cellular loading of Ca2+, and addition of compounds to inhibit superoxide and H2O2 production or scavenge reactive O2 species. While the mechanistic details and complete description of metabolic effects are not yet available, the ability to alter cellular metabolism and delay or prevent irreversible injury marks a very important advance in renal physiology.

Topics: Adenosine Triphosphate; Animals; Calcium; Cell Membrane Permeability; Energy Metabolism; Erythropoietin; Free Radicals; Humans; Hypoxia; Ischemia; Kidney; Kidney Tubules; Mice; Mitochondria; Oxidoreductases; Oxygen; Oxygenases; Rats

Topics: Animals; Cobalt; Erythropoiesis; Erythropoietin; Hormones; Humans; Hypoxia; Kidney; Oxygen

Topics: Altitude; Circadian Rhythm; Erythrocyte Volume; Erythropoietin; Hematocrit; Hemoglobins; Humans; Hypoxia; Lung Diseases, Obstructive; Polycythemia; Smoking; Testosterone

Aerobic living demands a constant supply of oxygen at the cellular mitochondrial level. Numerous interlocking feedback mechanisms insure the constancy of this supply by adapting the supply lines to changes in the intra and extracellular milieu. Among these mechanisms is the feedback circuit which, triggered by an intracellular decrease in oxygen supply, initiates the production of a polypeptide hormone, erythropoietin, which in turn augments the production of an oxygen carrier, the hemoglobin-containing red cell. The biogenesis of this hormone is an important biologic process which is being studied intensively and slowly unravelled.

Topics: Animals; Erythrocytes; Erythropoietin; Feedback; Female; Hypoxia; Kidney; Liver; Male; Oxygen Consumption; Rabbits; Rats; Rats, Inbred Strains

Topics: Animals; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Kidney; Renal Artery

A regulatory role for adenosine 3',5'-monophosphate (cyclic AMP) in the production of the renal hormone rythropoietin following erythropoietic stimulation with cobaltous chloride hexahydrate is proposed. Studies in rates reveal a temporal relationship between renal cyclic AMP levels and plasma titers of erythropoietin. In addition, cobalt increases the activity of an erythropoietin-generating enzyme (renal erythropoietic factor) with maximal enzyme activity occurring after the rise in cyclic AMP levels but before the increase in erythropoietin titers. This increase in renal cyclic AMP is localized to the renal cortex. Cobalt stimulates renal cortical adenylate cyclase but has no effect on renal cyclic nucleotide phosphodiesterase. The addition of cyclic AMP (3 time 10-6 M) and a partially purified cyclic AMP-dependent protein kinase from rat kidney to an inactive preparation of renal erythropoietic factor increases the ability of renal erythropoietic factor to generate erythropoietin. Data from the polycythemic mouse assay, a bioassay used to quantitate erythropoietic activity of test substances, indicate that dibutyryl cyclic AMP is erythropoietically active with respect to its ability to increase radioactive-labelled iron (59Fe) incorporation into heme of newly formed red blood cells. Theophylline, which by itself is erythropoietically inactive, potentiated the erythropoietic effect of cobalt in polycythemic mice. These results suggest that cyclic AMP plays a significant role in the renal production of erythropoietin following cobalt administration. It is postulated that cobalt stimulates renal cortical adenyoate cyclase, thus increasing renal cyclic AMP levels. Cyclic AMP then activates a protein kinase which subsequently stimulates renal erythropoietic factor to generate erythropoietin. A similar cyclic AMP mechanism may be operative after erythropoietic stimulation by exposure to hypoxia or prostaglandin treatment.

Topics: Adenine Nucleotides; Adenylyl Cyclases; Animals; Bucladesine; Cobalt; Cyclic AMP; Cyclic GMP; Enzyme Activation; Erythropoietin; Humans; Hypoxia; Kidney; Kidney Cortex; Mice; Phosphoric Diester Hydrolases; Prostaglandins; Protein Kinases; Rats; Stimulation, Chemical; Theophylline

The widespread and ever expanding use of dialysis in the maintenance of patients with chronic renal disease has added an urgency to the study of the biogenesis of erythropoietin. It seems almost certain that erythropoietin could ameliorate, if not eliminate, the anemia of uremia, but unfortunately, erythropoietin is still not available in therapeutic quantities. Initially, erythropietin was though to be produced by the kidneys" but then the attention became directed at the liver. It was proposed that erythropoietin was produced there as an inactive precursor and that the kidney only acted as an oxygen sensor and as a producer of an erythropoietin-activating enzyme. Recent studies summarized here show that an isolated perfused kidney in the absence of any extrarenal substrate or precursor can synthesize erythropoietin. Consequently, it appears almost certain that the kidney is the endocrine organ of origin of erythropoietin. Further studies suggest that erythropoietin formation involves a phase of oxygen sensing and programming and a phase of synthesis. These phases probably occur in the same cell, and the renal cortex appears to be the most likely location for such cells. The current inability to extract erythropoietin from kidney homogenates is discussed but, unfortunately, not adequately explained.

Topics: Animals; Erythropoietin; Humans; Hypoxia; Kidney; Kidney Cortex; Oxygen Consumption; Perfusion; Rabbits; Time Factors; Uremia

Topics: Altitude; Biological Transport; Chronic Disease; Erythropoietin; Heart Diseases; Hemoglobins, Abnormal; Humans; Hypoxia; Lung Diseases; Lung Diseases, Obstructive; Oxygen; Oxygen Consumption; Polycythemia

Topics: Cell Differentiation; DNA; Erythropoiesis; Erythropoietin; Ganglia, Autonomic; Hematopoietic Stem Cells; Humans; Hypoxia; Iron Radioisotopes; Kidney; Liver; Mitosis; RNA; Splanchnic Nerves; Spleen; Vagus Nerve

Topics: Erythropoietin; Hormones, Ectopic; Humans; Hypoxia; Kidney; Kidney Diseases, Cystic; Kidney Neoplasms; Neoplasms; Oxygen; Polycythemia; Prostaglandins; Regional Blood Flow

Topics: Adaptation, Physiological; Blood Preservation; Diphosphoglyceric Acids; Erythrocytes; Erythropoietin; Glycolysis; Heme; Hemoglobins; Humans; Hypoxia; Iron; Oxygen; Oxyhemoglobins; Time Factors

Topics: Alkylating Agents; Blood Cell Count; Blood Volume; Erythrocyte Count; Erythropoiesis; Erythropoietin; Hematocrit; Hemoglobinopathies; Hemoglobins; Humans; Hypoxia; Oxygen Consumption; Polycythemia; Polycythemia Vera; Protein Binding

Topics: Aminolevulinic Acid; Anemia; Animals; Bone Marrow; Bone Marrow Cells; Depression, Chemical; Erythrocytes; Erythropoiesis; Erythropoietin; Estrogens; Feedback; Female; Heme; Hemoglobins; Humans; Hypoxia; Immunoglobulin G; In Vitro Techniques; Infant, Newborn; Iron; Male; Mice; Models, Biological; Nephritis; Polycythemia; Polycythemia Vera; Rats

Topics: Acclimatization; Altitude; Animals; Animals, Domestic; Blood Coagulation Factors; Blood Pressure; Blood Viscosity; Blood Volume; Carbon Dioxide; Cardiac Output; Cardiomegaly; Cats; Cattle; Colorado; Coronary Circulation; Dogs; Erythrocyte Count; Erythropoietin; Guinea Pigs; Heart Rate; Humans; Hypertension, Pulmonary; Hypoxia; Indians, South American; Myocardium; Organ Size; Oxygen; Oxygen Consumption; Partial Pressure; Peru; Physical Exertion; Polycythemia; Pulmonary Circulation; Pulmonary Diffusing Capacity; Rabbits; Rats; Vascular Resistance

Topics: Anemia, Hypochromic; Animals; Bile; Chelating Agents; Chemical Phenomena; Chemistry; Diet; Erythropoietin; Gastric Mucosa; Heme; Hemoglobins; Humans; Hydrogen-Ion Concentration; Hypoxia; Intestinal Absorption; Intestinal Mucosa; Iron; Iron Isotopes; Kinetics; Models, Biological; Pancreas; Transferrin

Topics: Bone Marrow; Erythrocyte Count; Erythropoiesis; Erythropoietin; Female; Fetus; Gestational Age; Glycerophosphates; Heart Defects, Congenital; Hematocrit; Hemoglobinometry; Humans; Hypoxia; Infant; Infant, Newborn; Infant, Premature; Kidney; Liver; Oxygen; Oxygen Consumption; Placenta; Pregnancy; Vitelline Membrane

Topics: Acute Disease; Animals; Arteries; Chronic Disease; Erythropoietin; Hematopoietic System; Hemoglobins; Humans; Hypoxia; Methods; Mice; Oxygen; Partial Pressure; Time Factors

Topics: Anemia; Animals; Chemical Phenomena; Chemistry; Erythrocyte Aging; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Kidney Failure, Chronic; Neoplasms; Oxygen Consumption; Polycythemia Vera

Topics: Animals; Chemical Phenomena; Chemistry, Physical; Dogs; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Kidney; Mice; Neurons, Efferent; Neurosecretory Systems; Neurotransmitter Agents; Polycythemia; Polycythemia Vera; Rabbits; Rats; Reticulocytes

Topics: Animals; Central Nervous System Diseases; Erythropoiesis; Erythropoietin; Female; Genes, Regulator; Genital Diseases, Female; Hemodynamics; Humans; Hypertension; Hypoxia; Kidney; Kidney Diseases; Liver Diseases; Male; Polycythemia; Polycythemia Vera; Rats

Topics: Anemia; Animals; Bone Marrow; Dogs; Erythrocytes; Erythropoiesis; Erythropoietin; Hematopoiesis; Hemorrhage; Humans; Hypoxia; Iron Isotopes; Kidney; Kidney Diseases; Mice; Placenta; Polycythemia Vera; Rabbits; Rats; Stimulation, Chemical

Athletes incorporate altitude training programs into their conventional training to improve their performance. The purpose of this study was to determine the effects of an 8-week altitude training program that was supplemented with intermittent hypoxic training (IHE) on the blood biomarkers, sports performance, and safety profiles of elite athletes. In a single-blind randomized clinical trial that followed the CONSORT recommendations, 24 male athletes were randomized to an IHE group (HA, n = 12) or an intermittent normoxia group (NA, n = 12). The IHE consisted of 5-min cycles of hypoxia−normoxia with an FIO2 of between 10−13% for 90 min every day for 8 weeks. Hematological (red blood cells, hemoglobin, hematocrit, hematocrit, reticulated hemoglobin, reticulocytes, and erythropoietin), immunological (leukocytes, monocytes, and lymphocytes), and renal (urea, creatinine, glomerular filtrate, and total protein) biomarkers were assessed at the baseline (T1), day 28 (T2), and day 56 (T3). Sports performance was evaluated at T1 and T3 by measuring quadriceps strength and using three-time trials over the distances of 60, 400, and 1000 m on an athletics track. Statistically significant increases (p < 0.05) in erythropoietin, reticulocytes, hemoglobin, and reticulocyte hemoglobin were observed in the HA group at T3 with respect to T1 and the NA group. In addition, statistically significant improvements (p < 0.05) were achieved in all performance tests. No variations were observed in the immunological or renal biomarkers. The athletes who were living and training at 1065 m and were supplemented with IHE produced significant improvements in their hematological behavior and sports performance with optimal safety profiles.

Topics: Altitude; Athletes; Athletic Performance; Biomarkers; Erythropoietin; Exercise; Hemoglobins; Humans; Hypoxia; Male; Oxygen Consumption; Physical Fitness; Single-Blind Method

Erythropoietin (EPO) and vascular endothelial growth factor (VEGF) are important factors regulating erythropoiesis and angiogenesis. Altitude/hypoxic training may induce elevated VEGF-A and EPO levels. However, it appears that the range of adaptive changes depends largely on the training method used. Therefore, we investigated the changes in EPO and VEGF-A levels in athletes using three different altitude/hypoxic training concepts.. Thirty-four male cyclists were randomly divided into four groups: LH-TL group ("live high-train low" protocol), HiHiLo ("live high - base train high - interval train low" procedure), IHT ("intermittent hypoxic training") and control group (CN, normoxic training). The same 4-week training program was used in all groups. Blood samples were taken before and after each training week in order to evaluate serum EPO and VEGF-A levels.. In the LH-TL and HiHiLo groups, EPO increased (P<0.001) after 1st week and remained elevated until 3rd week of altitude training. In the IHT and CN groups, EPO did not change significantly. VEGF-A was higher (P<0.001) after 2nd and 3rd week of training in the IHT group. In the HiHiLo group, VEGF-A changed (P<0.05) only after 3rd week. No significant changes of VEGF-A were noted in the LH-TL and CN groups.. Altitude/hypoxic training is effective in increasing VEGF-A and EPO levels. However, a training method plays a key role in the pattern of adaptations. EPO level increase only when an adequate hypoxic dose is provided, whereas VEGF-A increases when the hypoxic exposure is combined with exercise, particularly at high intensity.

Topics: Acclimatization; Adult; Altitude; Erythropoietin; Exercise; Humans; Hypoxia; Male; Vascular Endothelial Growth Factor A; Young Adult

Roxadustat (FG-4592), an oral hypoxia-inducible factor prolyl hydroxylase inhibitor that stimulates erythropoiesis, was evaluated in a phase 1b study in patients with end-stage renal disease with anemia on hemodialysis. Seventeen patients, on epoetin-alfa maintenance therapy with stable hemoglobin levels ≥10 g/dL, had epoetin-alfa discontinued on day 3 and were enrolled in this double-blind placebo-controlled study. Two cohorts were randomized 3:1 (roxadustat: placebo). Patients received single doses of roxadustat (1 or 2 mg/kg) or placebo 1 hour after hemodialysis on day 1 and 2 hours before dialysis on day 8. Maximum plasma concentration and area under the plasma concentration-time curve for patients receiving roxadustat were slightly more than dose proportional and elimination half-life ranged from 14.7 to 19.4 hours. Roxadustat was highly protein bound (99%) in plasma, and dialysis contributed a small fraction of the total clearance: only 4.56% and 3.04% of roxadustat recovered from the 1 and 2 mg/kg dose groups, respectively. Roxadustat induced transient elevations of endogenous erythropoietin that peaked between 7 and 14 hours after dosing and returned to baseline by 48 hours after dosing. Peak median endogenous erythropoietin levels were 96 mIU/mL and 268 mIU/mL for the 1- and 2-mg/kg doses, respectively, within physiologic range of endogenous erythropoietin responses to hypoxia at high altitude or after blood loss. No serious adverse events were reported, and there were no treatment- or dose-related trends in adverse event incidence.

Topics: Administration, Oral; Adult; Aged; Anemia; Area Under Curve; Dose-Response Relationship, Drug; Double-Blind Method; Erythropoiesis; Erythropoietin; Female; Glycine; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Isoquinolines; Kidney Failure, Chronic; Male; Middle Aged; Prolyl-Hydroxylase Inhibitors; Renal Dialysis; Treatment Outcome

Intermittent hypoxia, defined as alternating bouts of breathing hypoxic and normoxic air, has the potential to improve oxygen-carrying capacity through an erythropoietin-mediated increase in hemoglobin mass. The purpose of this study was to determine the effect of a single session of intermittent hypoxia on erythropoietin levels and hemoglobin mass in young healthy individuals. Nineteen participants were randomly assigned to an intermittent hypoxia group (Hyp,

Topics: Adult; Erythrocytes; Erythropoietin; Female; Hemoglobins; Humans; Hypoxia; Male; Oxygen; Oxygen Consumption

The production of erythropoietin (Epo) is modulated by renal tissue oxygen tension, which in principle depends on both arterial oxygen content (CaO2) and arterial oxygen tension (PaO2). Uncontrolled observational studies suggest that alterations in CaO2 fundamentally regulate Epo synthesis. We sought to establish whether reduced CaO2 enhances plasma Epo concentration independently of PaO

Topics: Adult; Arteries; Blood Gas Analysis; Carbon Monoxide; Cross-Over Studies; Erythropoietin; Healthy Volunteers; Humans; Hypoxia; Kidney; Male; Oxygen; Oxygen Consumption; Young Adult

Tissue hypoxia stimulates the production of erythropoietin (EPO), the main effect of which is, in turn, to stimulate erythropoiesis. Sleep apnea-hypopnea syndrome (SAHS) is an entity characterized by repeated episodes of hypoxemia during sleep.. To analyze whether hypoxemia stimulated increased urinary excretion of EPO, and if so, to evaluate if treatment with continuous positive airway pressure (CPAP) can inhibit this phenomenon.. We studied 25 subjects with suspected SAHS who underwent a polysomnography study (PSG). EPO levels in first morning urine (uEPO) and blood creatinine and hemoglobin were determined in all patients. Patients with severe SAHS repeated the same determinations after CPAP treatment.. Twelve subjects were diagnosed with severe SAHS (mean ± SD, AHI 53.1 ± 22.7). Creatinine and hemoglobin levels were normal in all subjects. uEPO was 4 times higher in the SAHS group than in the control group (1.32 ± 0.83 vs. 0.32 ± 0.35 UI/l, p <.002). CPAP treatment reduced uEPO to 0.61 ± 0.9 UI/l (p <.02), levels close to those observed in healthy subjects. No dose-response relationship was observed between severity of PSG changes and uEPO values.. Patients with severe SAHS show increased uEPO excretion, but this normalizes after treatment with CPAP.

Topics: Adult; Aged; Cell Hypoxia; Continuous Positive Airway Pressure; Creatinine; Erythropoietin; Female; Hemoglobins; Humans; Hypoxia; Male; Middle Aged; Polysomnography; Sleep Apnea Syndromes

This study exanimated the effects of intermittent hypoxic training (IHT) conducted at a high level of hypoxia with recovery at ambient air on aerobic/anaerobic capacities at sea level and hematological variations. According to a double-blind randomized design, fifteen highly endurance-trained runners completed a 6-weeks regimented training with 3 sessions per week consisting of intermittent runs (6x work-rest ratio of 5':5') on a treadmill at 80-85% of maximal aerobic speed ([Formula: see text]). Nine athletes (hypoxic group, HG) performed the exercise bouts at FI0

Topics: Adaptation, Physiological; Altitude; Cross-Over Studies; Double-Blind Method; Erythropoietin; Hematocrit; Humans; Hypoxia; Male; Physical Conditioning, Human; Physical Endurance; Running; Time Factors

Wonnabussapawich, P, Hamlin, MJ, Lizamore, CA, Manimmanakorn, N, Leelayuwat, N, Tunkamnerdthai, O, Thuwakum, W, and Manimmanakorn, A. Living and training at 825 m for 8 weeks supplemented with intermittent hypoxic training at 3,000 m improves blood parameters and running performance. J Strength Cond Res 31(12): 3287-3294, 2017-We aimed to investigate the effect of an 8-week low-altitude training block supplemented with intermittent hypoxic training, on blood and performance parameters in soccer players. Forty university-level male soccer players were separated into altitude (n = 20, 825 m) or sea-level (n = 20, 125 m) groups. Before (1-2 days ago) and after (1 and 14 days later) training, players were asked to give a resting venous blood sample and complete a series of performance tests. Compared with sea level, the altitude group increased erythropoietin, red blood cell (RBC) count, and hematocrit 1 day after training (42.6 ± 24.0%, 1.8 ± 1.3%, 1.4 ± 1.1%, mean ± 95% confidence limits (CL), respectively). By 14 days after training, only RBC count and hemoglobin were substantially higher in the altitude compared with the sea-level group (3.2 ± 1.8%, 2.9 ± 2.1% respectively). Compared with sea level, the altitude group 1-2 days after training improved their 50-m (-2.9 ± 1.4%) and 2,800-m (-2.9 ± 4.4%) run times and demonstrated a higher maximal aerobic speed (4.7 ± 7.4%). These performance changes remained at 14 days after training with the addition of a likely higher estimated V[Combining Dot Above]O2max in the altitude compared with the sea-level group (3.2 ± 3.0%). Eight weeks of low-altitude training, supplemented with regular bouts of intermittent hypoxic training at higher altitude, produced beneficial performance improvements in team-sport athletes, which may increase the viability of such training to coaches and players that cannot access more traditional high altitude venues.

Topics: Adult; Altitude; Athletes; Blood; Dietary Supplements; Erythrocyte Count; Erythropoietin; Hematocrit; Hemoglobins; Humans; Hypoxia; Male; Physical Conditioning, Human; Running; Soccer

Topics: Adult; Altitude; Erythropoietin; Humans; Hypoxia; Interleukin-6; Male; Single-Blind Method; Time Factors; Tumor Necrosis Factor-alpha; Young Adult

Both acute hypoxia and physical exercise are known to increase oxidative stress. This randomized prospective trial investigated whether the addition of moderate exercise can alter oxidative stress induced by continuous hypoxic exposure.. Fourteen male participants were confined to 10-d continuous normobaric hypoxia (FIO2 = 0.139 ± 0.003, PIO2 = 88.2 ± 0.6 mm Hg, ∼4000-m simulated altitude) either with (HCE, n = 8, two training sessions per day at 50% of hypoxic maximal aerobic power) or without exercise (HCS, n = 6). Plasma levels of oxidative stress markers (advanced oxidation protein products [AOPP], nitrotyrosine, and malondialdehyde), antioxidant markers (ferric-reducing antioxidant power, superoxide dismutase, glutathione peroxidase, and catalase), nitric oxide end-products, and erythropoietin were measured before the exposure (Pre), after the first 24 h of exposure (D1), after the exposure (Post) and after the 24-h reoxygenation (Post + 1). In addition, graded exercise test in hypoxia was performed before and after the protocol.. Maximal aerobic power increased after the protocol in HCE only (+6.8%, P < 0.05). Compared with baseline, AOPP was higher at Post + 1 (+28%, P < 0.05) and nitrotyrosine at Post (+81%, P < 0.05) in HCS only. Superoxide dismutase (+30%, P < 0.05) and catalase (+53%, P < 0.05) increased at Post in HCE only. Higher levels of ferric-reducing antioxidant power (+41%, P < 0.05) at Post and lower levels of AOPP (-47%, P < 0.01) at Post + 1 were measured in HCE versus HCS. Glutathione peroxidase (+31%, P < 0.01) increased in both groups at Post + 1. Similar erythropoietin kinetics was noted in both groups with an increase at D1 (+143%, P < 0.01), a return to baseline at Post, and a decrease at Post + 1 (-56%, P < 0.05).. These data provide evidence that 2 h of moderate daily exercise training can attenuate the oxidative stress induced by continuous hypoxic exposure.

Topics: Adult; Advanced Oxidation Protein Products; Atmospheric Pressure; Catalase; Erythropoietin; Exercise; Exercise Test; Glutathione Peroxidase; Heart Rate; Humans; Hypoxia; Male; Malondialdehyde; Oxidative Stress; Oxygen; Physical Exertion; Superoxide Dismutase; Tyrosine; Young Adult

Recent findings suggest that besides renal tissue hypoxia, relative decrements in tissue oxygenation, using a transition of the breathing mixture from hyperoxic to normoxic, can also stimulate erythropoietin (EPO) production. To further clarify the importance of the relative change in tissue oxygenation on plasma EPO concentration [EPO], we investigated the effect of a consecutive hyperoxic and hypoxic breathing intervention. Eighteen healthy male subjects were assigned to either IHH (N = 10) or CON (N = 8) group. The IHH group breathed pure oxygen (F(i)O(2) ~ 1.0) for 1 h, followed by a 1-h period of breathing a hypoxic gas mixture (F(i)O(2) ~ 0.15). The CON group breathed a normoxic gas mixture (F(i)O(2) ~ 0.21) for the same duration (2 h). Blood samples were taken just before, after 60 min, and immediately after the 2-h exposure period. Thereafter, samples were taken at 3, 5, 8, 24, 32, and 48 h after the exposure. During the breathing interventions, subjects remained in supine position. There were significant increases in absolute [EPO] within groups at 8 and 32 h in the CON and at 32 h only in the IHH group. No significant differences in absolute [EPO] were observed between groups following the intervention. Relative (∆[EPO]) levels were significantly lower in the IHH than in the CON group, 5 and 8 h following exposure. The tested protocol of consecutive hyperoxic-hypoxic gas mixture breathing did not induce [EPO] synthesis stimulation. Moreover, the transient attenuation in ∆[EPO] in the IHH group was most likely due to a hyperoxic suppression. Hence, our findings provide further evidence against the "normobaric O(2) paradox" theory.

Topics: Acute Disease; Adult; Erythropoietin; Humans; Hyperoxia; Hypoxia; Male; Models, Theoretical; Nitrogen; Oxygen; Oxygen Consumption; Respiration; Time Factors; Young Adult

Circulating hematopoietic progenitor cells (CPCs) may be triggered by physical exercise and/or normobaric hypoxia from the bone marrow. The aim of the study was to investigate the influence of physical exercise and normobaric hypoxia on CPC number and functionality in the peripheral blood as well as the involvement of oxidative stress parameters as possibly active agents. Ten healthy male subjects (25.3±4.4 years) underwent a standardized cycle incremental exercise test protocol (40 W+20 W/min) under either normoxic (FiO2 ∼0.21) or hypoxic conditions (FiO2<0.15, equals 3,500 m, 3 h xposure) within a time span of at least 1 week. Blood was drawn from the cubital vein before and 10, 30, 60, and 120 min after exercise. The number of CPCs in the peripheral blood was analyzed by flow cytometry (CD34/CD45-positive cells). The functionality of cells present was addressed by secondary colony-forming unit-granulocyte macrophage (CFU-GM) assays. To determine a possible correlation between the mobilization of CPCs and reactive oxygen species, parameters for oxidative stress such as malondialdehyde (MDA) and myeloperoxidase (MPO) were obtained. Data showed a significant increase of CPC release under normoxic as well as hypoxic conditions after 10 min of recovery (P<0.01). Most interestingly, although CD34+/CD45dim cells increased in number, the proliferative capacity of CPCs decreased significantly 10 min after cessation of exercise (P<0.05). A positive correlation between CPCs and MDA/MPO levels turned out to be significant for both normoxic and hypoxic conditions (P<0.05/P<0.01). Hypoxia did not provoke an additional effect. Although the CPC frequency increased, the functionality of CPCs decreased significantly after exercise, possibly due to the influence of increased oxidative stress levels.

Topics: Adult; Antigens, CD34; Blood Cell Count; Cell Movement; Cell Proliferation; Colony-Forming Units Assay; Erythropoietin; Exercise; Flow Cytometry; Hematopoiesis; Hematopoietic Stem Cells; Humans; Hypoxia; Kinetics; Leukocyte Common Antigens; Male; Malondialdehyde; Oxidative Stress; Peroxidase

There is an expectation that repeated daily exposures to normobaric hypoxia (NH) will induce ventilatory acclimatization and lessen acute mountain sickness (AMS) and the exercise performance decrement during subsequent hypobaric hypoxia (HH) exposure. However, this notion has not been tested objectively. Healthy, unacclimatized sea-level (SL) residents slept for 7.5 h each night for 7 consecutive nights in hypoxia rooms under NH [n = 14, 24 ± 5 (SD) yr] or "sham" (n = 9, 25 ± 6 yr) conditions. The ambient percent O(2) for the NH group was progressively reduced by 0.3% [150 m equivalent (equiv)] each night from 16.2% (2,200 m equiv) on night 1 to 14.4% (3,100 m equiv) on night 7, while that for the ventilatory- and exercise-matched sham group remained at 20.9%. Beginning at 25 h after sham or NH treatment, all subjects ascended and lived for 5 days at HH (4,300 m). End-tidal Pco(2), O(2) saturation (Sa(O(2))), AMS, and heart rate were measured repeatedly during daytime rest, sleep, or exercise (11.3-km treadmill time trial). From pre- to posttreatment at SL, resting end-tidal Pco(2) decreased (P < 0.01) for the NH (from 39 ± 3 to 35 ± 3 mmHg), but not for the sham (from 39 ± 2 to 38 ± 3 mmHg), group. Throughout HH, only sleep Sa(O(2)) was higher (80 ± 1 vs. 76 ± 1%, P < 0.05) and only AMS upon awakening was lower (0.34 ± 0.12 vs. 0.83 ± 0.14, P < 0.02) in the NH than the sham group; no other between-group rest, sleep, or exercise differences were observed at HH. These results indicate that the ventilatory acclimatization induced by NH sleep was primarily expressed during HH sleep. Under HH conditions, the higher sleep Sa(O(2)) may have contributed to a lessening of AMS upon awakening but had no impact on AMS or exercise performance for the remainder of each day.

Topics: Acclimatization; Adult; Altitude; Altitude Sickness; Atmospheric Pressure; Carbon Dioxide; Erythropoietin; Exercise; Female; Heart Rate; Hematocrit; Hemoglobins; Humans; Hydrocortisone; Hypoxia; Male; Norepinephrine; Oxygen; Oxygen Consumption; Partial Pressure; Physical Exertion; Pulmonary Gas Exchange; Pulmonary Ventilation; Sleep; Young Adult

Recombinant human erythropoietin (EPO) increases exercise capacity by stimulating erythropoiesis and subsequently enhancing oxygen delivery to the working muscles. In a large dose, EPO crosses the BBB and may reduce central fatigue and improve cognition. In turn, this would augment exercise capacity independent of erythropoiesis. To test this hypothesis, 15 healthy young men (18-34 years old, 74 + or - 7 kg) received either 3 days of high-dose (30,000 IU/day; n = 7) double-blinded placebo controlled or 3 mo of low-dose (5,000 IU/wk; n = 8) counter-balanced open but controlled administration of EPO. We recorded exercise capacity, transcranial ultrasonography-derived middle cerebral artery blood velocity, and arterial-internal jugular venous concentration differences of glucose and lactate. In addition, cognitive function, ratings of perceived exertion, ventilation, and voluntary activation by transcranial magnetic stimulation-induced twitch force were evaluated. Although EPO in a high dose increased cerebrospinal fluid EPO concentration approximately 20-fold and affected ventilation and cerebral glucose and lactate metabolism (P < 0.05), 3 days of high-dose EPO administration had no effect on cognition, voluntary activation, or exercise capacity, but ratings of perceived exertion increased (P < 0.05). We confirmed that 3 mo of administration of EPO increases exercise capacity, but the improvement could not be accounted for by other mechanisms than enhanced oxygen delivery. In conclusion, EPO does not attenuate central fatigue or change cognitive performance strategy, suggesting that EPO enhances exercise capacity exclusively by increased oxygen delivery to the working muscles.

Topics: Adolescent; Adult; Biomarkers; Blood Flow Velocity; Blood Glucose; Blood-Brain Barrier; Brain; Cerebrovascular Circulation; Cognition; Cross-Over Studies; Double-Blind Method; Drug Administration Schedule; Erythropoietin; Exercise Tolerance; Hematinics; Humans; Hypoxia; Lactic Acid; Male; Middle Cerebral Artery; Muscle, Skeletal; Oxygen; Oxygen Consumption; Perception; Placebo Effect; Pulmonary Ventilation; Recombinant Proteins; Time Factors; Transcranial Magnetic Stimulation; Ultrasonography, Doppler, Transcranial; Young Adult

Hypoxia is a major cause of pulmonary hypertension. Gene expression activated by the transcription factor hypoxia-inducible factor (HIF) is central to this process. The oxygen-sensing iron-dependent dioxygenase enzymes that regulate HIF are highly sensitive to varying iron availability. It is unknown whether iron similarly influences the pulmonary vasculature. This human physiology study aimed to determine whether varying iron availability affects pulmonary arterial pressure and the pulmonary vascular response to hypoxia, as predicted biochemically by the role of HIF. In a controlled crossover study, 16 healthy iron-replete volunteers undertook two separate protocols. The 'Iron Protocol' studied the effects of an intravenous infusion of iron on the pulmonary vascular response to 8 h of sustained hypoxia. The 'Desferrioxamine Protocol' examined the effects of an 8 h intravenous infusion of the iron chelator desferrioxamine on the pulmonary circulation. Primary outcome measures were pulmonary artery systolic pressure (PASP) and the PASP response to acute hypoxia (DeltaPASP), assessed by Doppler echocardiography. In the Iron Protocol, infusion of iron abolished or greatly reduced both the elevation in baseline PASP (P < 0.001) and the enhanced sensitivity of the pulmonary vasculature to acute hypoxia (P = 0.002) that are induced by exposure to sustained hypoxia. In the Desferrioxamine Protocol, desferrioxamine significantly elevated both PASP (P < 0.001) and DeltaPASP (P = 0.01). We conclude that iron availability modifies pulmonary arterial pressure and pulmonary vascular responses to hypoxia. Further research should investigate the potential for therapeutic manipulation of iron status in the management of hypoxic pulmonary hypertensive disease.

Topics: Adult; Blood Pressure; Cross-Over Studies; Deferoxamine; Erythropoietin; Female; Ferritins; Humans; Hypoxia; Infusions, Intravenous; Iron; Male; Oxygen; Partial Pressure; Pulmonary Artery; Pulmonary Circulation; Siderophores; Young Adult

Investigations studying the secretion of EPO (erythropoietin) in response to acute hypoxia have produced mixed results. Further, the errors associated with the various methods used to determine EPO are not well documented. The purpose of the current study was to determine the EPO response of 17 trained male subjects to either an acute bout of normobaric hypoxia (Hy; n = 10) or normoxia (Con; n = 7). A secondary aim was to determine the error associated with the measurement of EPO. After baseline tests, the treatment group (Hy) underwent a single bout of hypoxic exposure (F(I(O(2))) approximately 0.148; 3100 m) consisting of a 90-min rest period followed by a 30-min exercise phase (50% V(O)(2max)). Venous blood samples were drawn pre (0 min) and post (120 min) each test to assess changes in plasma EPO (DeltaEPO). The control (Con) group was subjected to the same general experimental design, but placed in a normoxic environment (F(I(O(2))) approximately 0.2093). The Hy group demonstrated a mean increase in EPO [19.3 (4.4) vs. 24.1 (5.1) mU/mL], p < 0.04, post 120 min of normobaric hypoxia. The calculated technical error of measurement for EPO was 2.1 mU/mL (9.8%). It was concluded that an acute bout of hypoxia, has the capacity to elevate plasma EPO. This study also demonstrates that the increase in EPO accumulation was 2 times greater than the calculated measurement of error.

Topics: Acclimatization; Adult; Altitude Sickness; Erythropoietin; Exercise; Hemoglobins; Humans; Hypoxia; Male; Physical Education and Training; Physical Endurance

The aim of this study was to assess the effect of intermittent hypoxia exposure on direct and indirect methods used to evaluate recombinant human erythropoietin (rhEPO) misuse. Sixteen male triathletes were randomly assigned to either the intermittent hypoxia exposure group (experimental group) or the control normoxic group (control group). The members of the experimental group were exposed to simulated altitude (from 4000 to 5500 m) in a hypobaric chamber for 3 h per day, 5 days a week, for 4 weeks. Blood and urine samples were collected before and after the first and the final exposures, and again 2 weeks after the final exposure. While serum EPO significantly increased after the first [from a mean 8.3 IU x l(-1) (s = 3.2) to 16.6 IU x l(-1) (s = 4.7)] and final exposures [from 4.6 IU x l(-1) (s = 1.4) to 24.8 IU x l(-1) (s = 9.3)], haemoglobin, percentage of reticulocytes, and soluble transferrin receptor were not elevated. Second-generation ON/OFF models (indirect rhEPO misuse detection) were insensitive to intermittent hypoxia exposure. The distribution of the urinary EPO isoelectric profiles (direct rhEPO misuse detection) was altered after intermittent hypoxia exposure with a slight shift towards more basic isoforms. However, those shifts never resulted in misinterpretation of results. The intermittent hypoxia exposure protocol studied did not produce any false-positive result for indirect or direct detection of rhEPO misuse in spite of the changes in EPO serum concentrations and urinary EPO isoelectric profiles, respectively.

Topics: Adult; Altitude; Doping in Sports; Erythropoietin; Humans; Hypoxia; Isoelectric Focusing; Male; Spain

Live high-train low (LH+TL) altitude training was developed in the early 1990s in response to potential training limitations imposed on endurance athletes by traditional live high-train high (LH+TH) altitude training. The essence of LH+TL is that it allows athletes to "live high" for the purpose of facilitating altitude acclimatization, as manifest by a profound and sustained increase in endogenous erythropoietin (EPO) and ultimately an augmented erythrocyte volume, while simultaneously allowing athletes to "train low" for the purpose of replicating sea-level training intensity and oxygen flux, thereby inducing beneficial metabolic and neuromuscular adaptations. In addition to "natural/terrestrial" LH+TL, several simulated LH+TL devices have been developed to conveniently bring the mountain to the athlete, including nitrogen apartments, hypoxic tents, and hypoxicator devices. One of the key questions regarding the practical application of LH+TL is, what is the optimal hypoxic dose needed to facilitate altitude acclimatization and produce the expected beneficial physiological responses and sea-level performance effects? The purpose of this paper is to objectively answer that question, on the basis of an extensive body of research by our group in LH+TL altitude training. We will address three key questions: 1) What is the optimal altitude at which to live? 2) How many days are required at altitude? and 3) How many hours per day are required? On the basis of consistent findings from our research group, we recommend that for athletes to derive the physiological benefits of LH+TL, they need to live at a natural elevation of 2000-2500 m for >or=4 wk for >or=22 h.d(-1).

Topics: Acclimatization; Adult; Altitude; Erythropoiesis; Erythropoietin; Female; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Nitrogen; Oxygen; Oxygen Consumption; Physical Fitness; Running; Sports; Task Performance and Analysis

Long-term intermittent hypoxia, characterized by several days or weeks at altitude with periodic stays at sea level, is a frequently occurring pattern of life in mountainous countries demanding a good state of physical performance. The aim of the study was to determine the effects of a typical South American type of long-term intermittent hypoxia on VO2max at altitude and at sea level. We therefore compared an intermittently exposed group of soldiers (IH) who regularly (6 months) performed hypoxic-normoxic cycles of 11 days at 3550 m and 3 days at sea level with a group of soldiers from sea level (SL, control group) at 0 m and in acute hypoxia at 3550 m. VO2max was determined in both groups 1 day after arrival at altitude and at sea level. At altitude, the decrease in VO2max was less pronounced in IH (10.6 +/- 4.2%) than in SL (14.1 +/- 4.7%). However, no significant differences in VO2max were found between the groups either at sea level or at altitude, although arterial oxygen content (Ca(O(2) )) at maximum exercise was elevated (p < 0.001) in IH compared to SL by 11.7% at sea level and by 8.9% at altitude. This higher Ca(O(2) ) mainly resulted from augmented hemoglobin mass (IH: 836 +/- 103 g, SL: 751 +/- 72 g, p < 0.05) and at altitude also from increased arterial O(2)-saturation. In conclusion, acclimatization to long-term intermittent hypoxia substantially increases Ca(O(2) ), but has no beneficial effects on physical performance either at altitude or at sea level.

Topics: Adaptation, Physiological; Adult; Altitude; Altitude Sickness; Analysis of Variance; Blood Volume; Chile; Erythrocyte Count; Erythrocyte Volume; Erythropoietin; Hemoglobins; Humans; Hypoxia; Male; Military Personnel; Oxygen Consumption

This study aimed to determine the impact of short-term normobaric hypoxia on physiology and performance in highly trained athletes. Twelve (7 male and 5 female) athletes were randomly assigned into two groups and spent 8 h per night for two consecutive nights a week over 3 weeks under either short-term normobaric hypoxia (simulating 3636 m altitude, inspired O2=13%) or in normobaric normoxia in a single-blind study. Following a 3 week washout period, athletes were then exposed to the other condition. Athletes were tested for maximal oxygen consumption and time to exhaustion on an electromagnetically braked cycle ergometer before and after each treatment in addition to being tested for anaerobic performance (Wingate test) on a modified Monark cycle ergometer. Blood samples were taken throughout the experiment and vastus lateralis muscle biopsies were taken before and after each treatment. Increases in red blood cell count, haematocrit, haemoglobin, platelet number and erythropoietin concentration were observed following short-term normobaric hypoxia. Except for a modest decrease in phosphofructokinase activity following short-term normobaric hypoxia, no changes were observed in muscle enzyme activities, buffer capacity, capillary density or morphology. No performance measures were changed following short-term normobaric hypoxia or normobaric normoxia. Although short-term normobaric hypoxia exposure increased levels of a number of haematological parameters, this was not associated with improved aerobic or anaerobic performance in highly trained athletes.

Topics: Altitude; Atmospheric Pressure; Cross-Over Studies; Erythrocyte Count; Erythropoietin; Exercise Test; Female; Hematocrit; Humans; Hypoxia; Male; Muscle, Skeletal; Oxygen Consumption; Phosphofructokinase-1, Muscle Type; Physical Exertion; Platelet Count; Single-Blind Method; Sports

This study was performed to examine the effect of diurnal normobaric hypoxia on hematological parameters. Eleven healthy male volunteers were randomly selected to be in either the hypoxic group (n=6) or the control group (n=5). The hypoxic group was exposed to 8 h of normobaric hypoxia in hypoxic tent systems that elicited a target peripheral O(2) saturation of 81+/-2% on three consecutive days. The control group spent three consecutive 8-h days in modified tent systems that delivered normoxic air into the tent. Venous blood samples were collected before the exposure (days -5, 0), after each day of the exposure (days 1, 2, 3), and for 3 weeks after the exposure (days 7, 10, 13, 17, 24). Serum erythropoietin concentration significantly increased from 9.1+/-3.3 U.L(-1) to 30.7+/-8.6 U.L(-1) in the hypoxic group. Although there were significant increases in hematocrit (4%), hemoglobin concentration (5%), red blood cell count (4%) on day 7 in the hypoxic group, these observations were likely due to dehydration or biological variation over time. There was no significant change in early erythropoietic markers (reticulocyte counts or serum ferritin concentration), which provided inconclusive evidence of accelerated erythroid differentiation and proliferation. The results suggest that the degree of hypoxia was sufficient to stimulate increased erythropoietin production and release. However, the duration of hypoxic exposure was insufficient to propagate the erythropoietic cascade.

Topics: Adult; Circadian Rhythm; Erythrocyte Count; Erythropoiesis; Erythropoietin; Ferritins; Hematocrit; Hemoglobins; Humans; Hypoxia; Male; Oxygen; Reticulocytes

The effect of live high-train low on hemoglobin mass (Hbmass) and red cell volume (RCV) in elite endurance athletes is still controversial. We expected that Hb(mass) and RCV would increase, when using a presumably adequate hypoxic dose. An altitude group (AG) of 10 Swiss national team orienteers (5 men and 5 women) lived at 2,500 m (18 h per day) and trained at 1,800 and 1,000 m above sea level for 24 days. Before and after altitude, Hbmass, RCV (carbon monoxide rebreathing method), blood, iron, and performance parameters were determined. Seven Swiss national team cross-country skiers (3 men and 4 women) served as "sea level" (500-1,600 m) control group (CG) for the changes in Hbmass and RCV. The AG increased Hbmass (805+/-209 vs. 848+/-225 g; P<0.01) and RCV (2,353+/-611 vs. 2,470+/-653 ml; P<0.01), whereas there was no change for the CG (Hbmass: 849+/-197 vs. 858+/-205 g; RCV: 2,373+/-536 vs. 2,387+/-551 ml). Serum erythropoietin (P<0.001), reticulocytes (P<0.001), transferrin (P<0.001), soluble transferrin receptor (P<0.05), and hematocrit (P<0.01) increased, whereas ferritin (P<0.05) decreased in the AG. These changes were associated with an increased maximal oxygen uptake (3,515+/-837 vs. 3,660+/-770 ml/min; P<0.05) and improved 5,000-m running times (1,098+/-104 vs. 1,080+/-98 s; P<0.01) from pre- to postaltitude. Living at 2,500 m and training at lower altitudes for 24 days increases Hbmass and RCV. These changes may contribute to enhance performance of elite endurance athletes.

Topics: Adult; Altitude; Anthropometry; Erythrocyte Indices; Erythrocyte Volume; Erythropoietin; Exercise; Female; Hematocrit; Hemoglobins; Humans; Hypoxia; Male; Oxygen Consumption; Physical Endurance; Receptors, Transferrin; Reticulocyte Count; Transferrin

This study tested the hypothesis that athletes exposed to 4 wk of intermittent hypobaric hypoxia exposure (3 h/day, 5 days/wk at 4,000-5,500 m) or double-blind placebo increase their red blood cell volume (RCV) and hemoglobin mass (Hbmass) secondary to an increase in erythropoietin (EPO). Twenty-three collegiate level athletes were measured before (Pre) and after (Post) the intervention for RCV via Evans blue (EB) dye and in duplicate for Hbmass using CO rebreathing. Hematological indexes including EPO, soluble transferrin receptor, and reticulocyte parameters were measured on 8-10 occasions spanning the intervention. The subjects were randomly divided among hypobaric hypoxia (Hypo, n = 11) and normoxic (Norm, n = 12) groups. Apart from doubling EPO concentration 3 h after hypoxia there was no increase in any of the measures for either Hypo or Norm groups. The mean change in RCV from Pre to Post for the Hypo group was 2.3% (95% confidence limits = -4.8 to 9.5%) and for the Norm group was -0.2% (-5.7 to 5.3%). The corresponding changes in Hbmass were 1.0% (-1.3 to 3.3%) for Hypo and -0.3% (-2.6 to 3.1%) for Norm. There was good agreement between blood volume (BV) from EB and CO: EB BV = 1.03 x CO BV + 142, r2 = 0.85, P < 0.0001. Overall, evidence from four independent techniques (RCV, Hbmass, reticulocyte parameters, and soluble transferrin receptor) suggests that intermittent hypobaric hypoxia exposure did not accelerate erythropoiesis despite the increase in serum EPO.

Topics: Adaptation, Physiological; Altitude; Double-Blind Method; Erythrocyte Volume; Erythropoiesis; Erythropoietin; Female; Hematologic Tests; Humans; Hypoxia; Male; Running; Swimming

Despite the severe derangement of gas exchange in the advanced stages of idiopathic pulmonary fibrosis (IPF), secondary erythrocytosis is either absent or much lower than is seen in chronic obstructive pulmonary disease (COPD) with comparable hypoxemia. This study investigates the differences in erythropoiesis between IPF and COPD, searching for the possible underlying mechanisms.. The study included 32 patients with COPD, 18 patients with IPF, all with overt hypoxemia (PO(2) <65 mmHg), and 34 healthy controls. Erythrocytic parameters and serum erythropoietin (EPO) levels were assessed for all subjects. In a number of patients from both groups, the development of erythroid colonies grown from peripheral blood mononuclear cells was assayed in semisolid methylcellulose cultures and compared to cultures of control cells, in the presence of patient or control serum.. Hb and serum EPO levels were significantly higher in the COPD group than in IPF patients and controls. However, the number of BFU-E colonies obtained from mononuclear cells of IPF patients was clearly higher than in COPD patients when the same culture medium was used. Unlike COPD sera, IPF sera induced a significant growth inhibition of erythroid bursts arising from mononuclear cells of both patients and controls.. Our findings suggest a kind of ineffective erythropoiesis in IPF. Defective EPO production and inhibitory effect on erythropoiesis exerted by pro-inflammatory cytokines released from alveolar macrophages may be implicated in the suboptimal erythropoietic response. However, the possible involvement of other factors affecting erythropoiesis in IPF requires further investigation.

Topics: Aged; Blood Gas Analysis; Erythrocyte Count; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Female; Hemoglobins; Humans; Hypoxia; Male; Middle Aged; Pulmonary Disease, Chronic Obstructive; Pulmonary Fibrosis

This study was designed to test the hypothesis that intermittent normobaric hypoxia at rest is a sufficient stimulus to elicit changes in physiological measures associated with improved performance in highly trained distance runners. Fourteen national-class distance runners completed a 4-wk regimen (5:5-min hypoxia-to-normoxia ratio for 70 min, 5 times/wk) of intermittent normobaric hypoxia (Hyp) or placebo control (Norm) at rest. The experimental group was exposed to a graded decline in fraction of inspired O2: 0.12 (week 1), 0.11 (week 2), and 0.10 (weeks 3 and 4). The placebo control group was exposed to the same temporal regimen but breathed fraction of inspired O2 of 0.209 for the entire 4 wk. Subjects were matched for training history, gender, and baseline measures of maximal O2 uptake and 3,000-m time-trial performance in a randomized, balanced, double-blind design. These parameters, along with submaximal treadmill performance (economy, heart rate, lactate, and ventilation), were measured in duplicate before, as well as 1 and 3 wk after, the intervention. Hematologic indexes, including serum concentrations of erythropoietin and soluble transferrin receptor and reticulocyte parameters (flow cytometry), were measured twice before the intervention, on days 1, 5, 10, and 19 of the intervention, and 10 and 25 days after the intervention. There were no significant differences in maximal O2 uptake, 3,000-m time-trial performance, erythropoietin, soluble transferrin receptor, or reticulocyte parameters between groups at any time. Four weeks of a 5:5-min normobaric hypoxia exposure at rest for 70 min, 5 days/wk, is not a sufficient stimulus to elicit improved performance or change the normal level of erythropoiesis in highly trained runners.

Topics: Adult; Altitude; Biomarkers; Double-Blind Method; Erythropoiesis; Erythropoietin; Female; Humans; Hypoxia; Male; Physical Education and Training; Receptors, Transferrin; Reticulocyte Count; Running; Solubility; Task Performance and Analysis

Recent research has demonstrated that reactive oxygen species (ROS) participate in intracellular signaling processes initiated during hypoxia. We investigated the role of ROS in the response of plasma erythropoietin (Epo) to short-term normobaric hypoxia in humans. Twelve male subjects were exposed twice to 4 h of normobaric hypoxia (H; inspired oxygen fraction 12.5%) with a period of 6 wk between both experiments (H1 and H2). With the use of a randomized placebo-controlled crossover design, the subjects received orally a combination of the antioxidants all-rac-alpha-tocopherol (800 mg/day for 3 wk) and alpha-lipoic acid (600 mg/day for 2 wk) or placebo before H1 and H2, respectively. Three weeks before H1, the subjects underwent one control experiment in normoxia (N; inspired oxygen fraction 20.9%) without any treatment. Serum alpha-tocopherol was significantly higher after treatment with antioxidants compared with placebo. Capillary Po(2) declined during H without significant differences between antioxidants and placebo. Plasma peroxide levels were lower under antioxidant treatment but not affected by hypoxia. The response of Epo to H did not show significant differences between antioxidant [maximum increase (means, 95% confidence interval): +121%, +66 to +176%] and placebo conditions (+108%, +68 to +149%). Similarly, hypoxia-induced increase of Epo corrected for diurnal variations, as revealed during N, did not differ between antioxidants and placebo. Individual variability of Epo in response to H was not related to the individual degree of hypoxemia during H. Our results do not support the assumption that ROS play a major modulating role in the response of Epo to short-term normobaric hypoxia in humans.

Topics: Antioxidants; Confidence Intervals; Cross-Over Studies; Double-Blind Method; Erythropoietin; Humans; Hypoxia; Linear Models; Male; Reactive Oxygen Species

While it is well established that highlanders have optimized their oxygen transport system, little is known about the acclimatization of those who move between different altitudes. The purpose of this study was to establish whether the acclimatization to long-term intermittent hypoxic exposure in members of the Chilean Army who frequently move from sea level to 3,550 m altitude is correlated with acute acclimatization or chronic adaptation to hypoxia. A group of officers was exposed intermittently to hypoxia for about 22 years (OI, officers at intermittent hypoxia) and a group of soldiers for 6 months (SI, soldiers at intermittent hypoxia). Both groups were compared to residents at altitude (RA) and to soldiers at sea level (SL). When compared to SL, we observed an 11% increase in total hemoglobin mass (tHb) as well as a corresponding increase in red cell volume (RCV), hemoglobin concentration and hematocrit in all three groups at altitude. Plasma volume (PV) and blood volume (BV) decreased at altitude but increased when OI and SI returned to sea level. Moreover, intermittent hypoxic exposure of OI and SI resulted in increased plasma erythropoietin (Epo) levels, which peaked on day 2 at high altitude followed by decreasing levels during the successive days, and reaching pre-altitude values in SI even when staying at altitude. In conclusion, with regard to tHb and RCV, the acclimatization to long-term intermittent hypoxia resembles the adaptation to chronic hypoxia, while PV and BV regulation mimicked acclimatization to acute hypoxia. Remarkably, finely controlled regulation of Epo expression still occurs after up to 22 years of weekly exposure to altitude.

Topics: Adaptation, Physiological; Adolescent; Adult; Altitude; Altitude Sickness; Blood Volume; Chile; Erythrocyte Count; Erythrocyte Volume; Erythropoietin; Hemoglobins; Humans; Hypoxia; Male; Military Personnel; Plasma Volume

Oxygen-sensing chemoreceptors contribute significantly to the regulation of the respiratory drive and arterial PO(2) levels. The hypoxic ventilatory response (HVR) decreases strongly with age and is modulated by prolonged hypoxia and physical exercise. Several earlier studies indicated that the regulation of the ventilatory response and erythropoietin (EPO) production by the respective oxygen sensors involves redox-sensitive signaling pathways, which are triggered by the O(2)-dependent production of reactive oxygen species. The hypothesis that HVR and EPO production are modulated by thiol compounds or changes in the plasma thiol-disulfide redox state (REDST) was investigated. It was demonstrated that both responses are enhanced by oral treatment with N-acetyl-cysteine (NAC) and that HVR is correlated with plasma thiol level and REDST. Results suggest the possibility that age-related changes in plasma REDST may account for the age-related changes in HVR.

Topics: Acetylcysteine; Adult; Erythropoietin; Humans; Hypoxia; Male; Oxidation-Reduction; Oxygen; Pulmonary Ventilation; Respiration; Sulfhydryl Compounds; Time Factors

Serum erythropoietin levels (s-[epo]), haemoglobin concentration ([Hb]), haematocrit (hct), and ferritin concentration ([fer]) were measured in seven healthy male volunteers (20-23 years) exposed continuously to hypoxia (PO(2) 14 kPa) for 10 days. Serum erythropoietin concentration increased significantly from 9.5 +/- 3.51 to 33.6 +/- 11.64 U L(-1) (P < 0.05) after 2 days of hypoxia. Thereafter, s-[epo] decreased. However, after 10 days s-[epo] was 18.7 +/- 5.83 U L(-1) which was still increased above the pre-hypoxia level (P < 0.05). Serum haemoglobin concentration and hct increased over the 10 days of hypoxia, [Hb] from 152 +/- 8.9 to 168 +/- 9.2 gL(-1) (P < 0.001), and hct from 43 +/- 2.4 to 49 +/- 2.6% (P < 0.001). Ferritin concentration decreased significantly during the hypoxic exposure from 82 +/- 46.9 to 44 +/- 31.7 mmol L(-1) after 10 days (P < 0.01). In conclusion, the initial increase of s-[epo] under controlled normobaric hypoxia was marked, 353%, and levelled off after 5-10 days at 62-97% above normoxia level. There was also a significant increase in [Hb] and hct and a decrease in [fer] after 10 days of exposure to normobaric hypoxia.

Topics: Adaptation, Physiological; Adult; Carbon Dioxide; Erythropoietin; Exercise; Humans; Hypoxia; Male; Oxygen; Respiratory Function Tests; Time Factors

The present study was carried out to evaluate a model of a hypoxic stimulus of erythropoietin (EPO) production in humans and to investigate the role of free oxygen radicals in human EPO production. The study was conducted as an open, randomized, parallel, placebo-controlled trial. Thirty-six healthy male volunteers received a hypoxic treatment (13% O(2)) with a respiration mask for 6 h. During the period of hypoxia, the volunteers received as a short-term treatment either 1200 mg thioctic acid, or N-acetylcysteine 600 mg or placebo (0.9% sodium chloride). The EPO concentration in plasma increased up to 290% of the baseline level in all three groups. No statistically significant differences of AUC(EPO(0-48 h)) could be demonstrated between the groups. The malondialdehyde (MDA) concentration in plasma increased significantly (P < 0.001) 2 h after termination of hypoxia (mean 129.8 +/- 6.8% of the baseline) in all three groups.. Taken together, our in-vivo results do not support a gross modulatory effect of a short-term treatment with radical scavenging agents on EPO-production during or after hypoxia in humans, as derived from the detected changes of MDA-concentrations in peripheral plasma.

Topics: Acetylcysteine; Adult; Antioxidants; Area Under Curve; Erythropoietin; Free Radical Scavengers; Humans; Hypoxia; Male; Malondialdehyde; Thioctic Acid

The purpose of this study was to investigate whether the modest increases in serum erythropoietin (sEpo) experienced after brief sojourns at simulated altitude are sufficient to stimulate reticulocyte production. Six well-trained middle-distance runners (HIGH, mean maximum oxygen uptake, VO2max = 70.2 ml x kg(-1) x min(-1) spent 8-11 h per night for 5 nights in a nitrogen house that simulated an altitude of 2650 m. Five squad members (CONTROL, mean VO2max= 68.9 ml x kg(-1) x min(-1) undertook the same training, which was conducted under near-sea-level conditions (600 m altitude), and slept in dormitory-style accommodation also at 600 m altitude. For both groups, this 5-night protocol was undertaken on three occasions, with a 3-night interim between successive exposures. Venous blood samples were measured for sEpo after 1 and 5 nights of hypoxia on each occasion. The percentage of reticulocytes was measured, along with a range of reticulocyte parameters that are sensitive to changes in erythropoiesis. Mean serum erythropoietin levels increased significantly (P < 0.01) above baseline values [mean (SD) 7.9 (2.4) mU x ml(-1)] in the HIGH group after the 1st night [11.8 (1.9) mU x ml(-1), 57%], and were also higher on the 5th night [10.7 (2.2) mU x ml(-1), 42%] compared with the CONTROL group, whose erythropoietin levels did not change. After athletes spent 3 nights at near sea level, the change in sEpo during subsequent hypoxic exposures was markedly attenuated (13% and -4% change during the second exposure; 26% and 14% change during the third exposure; 1st and 5th nights of each block, respectively). The increase in sEpo was insufficient to stimulate reticulocyte production at any time point. We conclude that when daily training loads are controlled, the modest increases in sEpo known to occur following brief exposure to a simulated altitude of 2650 m are insufficient to stimulate reticulocyte production.

Topics: Adult; Altitude; Erythropoietin; Heart Rate; Humans; Hypoxia; Male; Oxygen; Oxygen Consumption; Physical Fitness; Reticulocytes; Running

Erythropoietic response in 10 healthy nonsmoking volunteers exposed to normobaric hypoxia continuously or intermittently 12 h daily for 7 d was evaluated in a randomized cross-over study.. An oxygen content of 15.4% corresponding to an altitude of 2500 m was created by adding nitrogen into room air in a flat. Venous blood samples for hemoglobin (Hb), hematocrit (Hct), reticulocytes, serum erythropoietin (S-EPO), red cell 2,3-diphosphoglycerate (2,3-DPG), serum ferritin (S-Ferrit), and serum soluble transferrin receptor (S-TransfR) were drawn at 8:00 a.m.. S-EPO was increased from baseline values of 22.9+/-9.6 and 20.5+/-10.1 U x L(-1) to 40.7+/-12.9 (P < 0.05) and 35+/-14.3 U x L(-1) (P < 0.05) after the first night in continuous and intermittent hypoxia, respectively, and remained elevated throughout both exposures. Hb and Hct values did not show any significant changes. Red cell 2,3-DPG rose from baseline a value of 5.0+/-0.8 to 5.9+/-0.7 mmol x L(-1) (P < 0.05) after the first day in continuous hypoxia and from 5.2+/-0.7 mmol x L(-1) to 6.1+/-0.5 mmol x L(-1) on day 3 (P < 0.05) during intermittent hypoxia. The reticulocyte count rose significantly (P < 0.05) after 5 d in both experiments. S-transferrin receptor level rose significantly from 2.2+/-0.4 and 2.1+/-0.5 mg x L(-1) to 2.6+/-0.5 mg x L(-1) and 2.3+/-0.6 mg x L(-1) on day 5 (P < 0.05), to 2.7+/-0.5 mg x L(-1) and 2.5+/-0.6 mg x L(-1) on day 7 (P < 0.05) under continuous and intermittent hypoxia, respectively.. We suggest that intermittent exposure to moderate normobaric hypoxia 12 h daily for 1 wk induces a similar stimulation of erythropoiesis as continuous exposure.

Topics: Adult; Cross-Over Studies; Erythrocytes; Erythropoietin; Female; Humans; Hypoxia; Male; Random Allocation; Receptors, Transferrin

In cell culture, hypoxia stabilizes a transcriptional complex called hypoxia-inducible factor-1 (HIF-1) that increases erythropoietin (Epo) formation. One hallmark of HIF-1 responses is that they can be induced by iron chelation. The first aim of this study was to examine whether an infusion of desferrioxamine (DFO) increased serum Epo in humans. If so, this might provide a paradigm for identifying other HIF-1 responses in humans. Consequently a second aim was to determine whether an infusion of DFO would mimic prolonged hypoxia and increase the acute hypoxic ventilatory response (AHVR). Sixteen volunteers undertook two protocols: 1) continuous infusion of DFO over 8 h and 2) control. Epo and AHVR were measured at fixed times during and after the protocols. The results show that 1) compared with control, Epo increased in most subjects at 8 h [52.8 +/- 57.7 vs. 6.9 +/- 2.5 (SD) mIU/ml, for DFO = 4 g/70 kg body wt, P < 0.05] and 12 h (63.7 +/- 76.3 vs. 7.3 +/- 2.5 mIU/ml, P < 0.001) after the start of DFO administration and 2) DFO had no significant effect on AHVR. We conclude that, whereas infusions of DFO mimic hypoxia by increasing Epo, they do not mimic prolonged hypoxia by augmenting AHVR.

Topics: Acute Disease; Adolescent; Adult; Carbon Dioxide; Carotid Body; Deferoxamine; Erythropoietin; Female; Humans; Hypoxia; Infusions, Intravenous; Male; Middle Aged; Respiratory Mechanics

Topics: Adult; Aged; Anemia; Biocompatible Materials; Biological Factors; Cardiovascular Diseases; Erythroid Precursor Cells; Erythropoietin; Female; Humans; Hypoxia; Informed Consent; Male; Membranes, Artificial; Middle Aged; Permeability; Polymethyl Methacrylate; Recombinant Proteins; Renal Dialysis; Uremia

Effects on erythropoiesis and blood pressure as well as physical performance and mental effects were studied in 15 healthy subjects during intermittent exposure to normobaric hypoxia corresponding to either 2000 m (6 persons) or 2700 m (9 persons) above sea level; another group (5 persons) also served as controls at normoxia. The concept "live high-train low" was used for 10 d consecutively and the exposure to hypoxia was 12 h/d. Blood pO2 and oxygen saturation were significantly decreased during the 10 d at hypoxia. [Hb] and Hct decreased significantly after 2 d in hypoxia and then returned to pre-study levels. Erythropoietin was significantly elevated in both hypoxia groups during the initial 3-5 d. Reticulocytes were significantly increased during 7 d of hypoxia. Submaximal and maximal oxygen uptake, blood pressure at rest and during exercise and the profile of mood states (POMS test) did not change during the study. In conclusion, intermittent normobaric hypoxia for 10 d resulted in a significant stimulation of erythropoiesis. Staying at normobaric hypoxia may serve as a complement to an ordinary altitude level sojourn.

Topics: Adult; Blood Pressure; Erythropoiesis; Erythropoietin; Exercise; Female; Humans; Hypoxia; Male; Mental Processes; Oxygen Consumption; Time Factors

Serum erythropoietin (EPO) and its relationship to hemoglobin (Hb), iron status markers and iron supplementation during normal pregnancy was assessed in a longitudinal, placebo-controlled study on 118 women, 61 took daily tablets containing 66 mg ferrous iron from the second trimester until delivery and 57 took placebo. Blood samples were obtained at 4-week intervals until delivery as well as post-partum. In the placebo-treated women, median serum EPO rose from 22.5 U/l at inclusion to 35.0 U/l at delivery (P = 0.0001). In the iron-treated women, median serum EPO rose from 23.9 to 29.9 U/l (P = 0.0001). Serum EPO showed a steeper increase in the placebo-treated women than in the iron-treated women (P < 0.05). After delivery, serum EPO became normal in both groups (P = 0.0001). Median Hb was lower in placebo-treated (iron depleted) than in iron-treated (iron repleted) women (P < 0.05). In the placebo-treated women there was a negative correlation and in the iron-treated women a positive correlation between serum EPO and Hb. In the placebo-treated women, inverse correlations existed between serum EPO and serum transferrin saturation and serum ferritin, reflecting the consequences of iron deficiency, whereas the iron-treated women displayed no correlation. A physiological, nonhypoxia-induced increase in EPO production accounts for the basic expansion of the red cell mass during pregnancy. In placebo-treated women, iron deficient erythropoiesis constitutes an additional hypoxic stimulus, which induces a further increase in serum EPO.

Topics: Anemia, Hypochromic; Double-Blind Method; Erythropoiesis; Erythropoietin; Female; Ferritins; Ferrous Compounds; Hemoglobins; Humans; Hypoxia; Iron; Iron Deficiencies; Longitudinal Studies; Placental Lactogen; Postpartum Period; Pregnancy; Pregnancy Complications, Hematologic; Transferrin

Objective of this study was to investigate whether adenosine modulates renal erythropoietin production.. In the present study erythropoietin production was stimulated by hypobaric hypoxia by subjecting healthy volunteers to a simulated altitude of 4000 m in a low pressure chamber for 5.5 h. During exposure to hypoxia the subjects received i.v. in a randomized, single-blind, cross-over fashion the non-specific adenosine antagonist theophylline, the adenosine reuptake inhibitor dipyridamole and placebo.. Contrary to the working hypothesis, theophylline did not decrease and dipyridamole did not further boost erythropoietin concentrations.. The results are in agreement with our earlier study using haemorrhage as a controlled physiological stimulus of erythropoietin production, and would question a major role for adenosine as a mediator of renal erythropoietin production.

Topics: Adenosine; Adult; Area Under Curve; Atmosphere Exposure Chambers; Dipyridamole; Erythropoietin; Humans; Hypoxia; Kidney; Male; Placebos; Reference Values; Single-Blind Method; Theophylline

This study tested the hypothesis that the diurnal variations of serum-erythropoietin concentration (serum-EPO) observed in normoxia also exist in hypoxia. The study also attempted to investigate the regulation of EPO production during sustained hypoxia. Nine subjects were investigated at sea level and during 4 days at an altitude of 4350 m. Median sea level serum-EPO concentration was 6 (range 6-13) U.l-1. Serum-EPO concentration increased after 18 and 42 h at altitude, [58 (range 39-240) and 54 (range 36-340) U.l-1, respectively], and then decreased after 64 and 88 h at altitude [34 (range 18-290) and 31 (range 17-104) U.l-1, respectively]. These changes of serum-EPO concentration were correlated to the changes in arterial blood oxygen saturation (r = -0.60, P = 0.0009), pH (r = 0.67, P = 0.003), and in-vivo venous blood oxygen half saturation tension (r = -0.68, P = 0.004) but not to the changes in 2, 3 diphosphoglycerate. After 64 h at altitude, six of the nine subjects had down-regulated their serum-EPO concentrations so that median values were three times above those at sea level. These six subjects had significant diurnal variations of serum-EPO concentration at sea level; the nadir occurred between 0800-1600 hours [6 (range 4-13) U.l-1], and peak concentrations occurred at 0400 hours [9 (range 8-14) U.l-1, P = 0.02]. After 64 h at altitude, the subjects had significant diurnal variations of serum-EPO concentration; the nadir occurred at 1600 hours [20 (range 16-26) U.l-1], and peak concentrations occurred at 0400 hours [31 (range 20-38) U.l-1, P = 0.02]. This study demonstrated diurnal variations of serum-EPO concentration in normoxia and hypoxia, with comparable time courses of median values. The results also suggested that EPO production at altitude is influenced by changes in pH and haemoglobin oxygen affinity.

Topics: Adult; Altitude; Blood Pressure; Blood Volume; Circadian Rhythm; Erythropoietin; Glucosephosphates; Heart Rate; Humans; Hypoxia; Male; Oxygen

The effect of nasal continuous positive pressure (CPAP) treatment on erythropoietin (EPO) was examined by measuring diurnal serum EPO levels before and twice (over the 3rd day and over 1 day on recall after > or = 1 mo of therapy) after initiation of treatment in 12 obstructive sleep apnea syndrome patients with normal hemoglobin, hematocrit, creatinine, blood urea nitrogen, and albumin levels. Over each study day, oxygen saturation was measured by an ambulatory pulse oximetry system. Patients spent 27 +/- 9% (SE) of time below oxygen saturation of 88% vs. 2.1 +/- 0.6% after initiation of nasal CPAP treatment (P < 0.01). The number of desaturation events per hour of sleep before nasal CPAP treatment was 62 +/- 6 vs. 9 +/- 2 with nasal CPAP (P < 0.01). EPO levels measured by radioimmunoassay were drawn every hour before and at 3 days (n = 9) and before and at recall (n = 0) after initiation of CPAP therapy. The mean serum EPO level was higher before treatment (61 +/- 14 mU/ml) than that at 3 days (38 +/- 10 mU/ml, P < 0.01) or at recall (32 +/- 7 mU/ml, P < 0.01). We conclude that nasal CPAP treatment of sleep-disordered breathing will reduce diurnal levels of EPO.

Topics: Adult; Aged; Blood Gas Monitoring, Transcutaneous; Circadian Rhythm; Erythropoietin; Female; Humans; Hypoxia; Male; Middle Aged; Oxygen; Positive-Pressure Respiration; Sleep Apnea Syndromes

Chronic hypoxemia is associated with development of secondary polycythemia. To evaluate effects of transient hypoxemia on serum EPO activity in patients with chronic lung disease, we studied six oxygen-dependent patients who underwent either a 4-h oxygen withdrawal or their routine therapy, in a randomized, blinded fashion, on two separate days. Serum EPO did not differ at baseline between study days. Erythropoietin levels did not change significantly over time during normoxic conditions. Under hypoxic conditions, serum EPO levels rose over 4 h with the change from baseline first becoming significant at 2 h. The log of serum EPO response showed an inverse correlation with the level of arterial oxygen saturation. We conclude that patients with chronic lung disease are able to produce EPO in response to acute hypoxemic stress. Transient episodes of hypoxemia, such as occur during sleep or exercise, may result in increased red blood cell production stimulated by this EPO response.

Topics: Acute Disease; Animals; Biological Assay; Erythropoietin; Female; Humans; Hypoxia; Lung Diseases, Obstructive; Male; Mice; Oxygen; Oxygen Inhalation Therapy; Time Factors

A blinded cooperative assay of several androstane and pregnane steroid metabolites has been carried out in order to determine whether 5beta-H derivatives are as active as testosterone in stimulating in vivo erythropoiesis. The steroids tested were: testosterone, 5alpha-dihydrotestosterone, 5beta-dihydrotestosterone, 5beta-pregnane-3,20-dione, 3alpha-dihydroxy-5beta-pregnane-11,20-dione and 3beta-hydroxy-5beta-pregnan-20-one. The incorporation of radioactive iron into newly formed red cells in exhypoxic polycythemic mice was used to compare the effects of the steroids. Testosterone and 5alpha-dihydrotestosterone both produced significant increases in 59Fe incorporation. 5beta-dihydrotestosterone, 5beta-pregnane-3,20-dione, 3alpha-hydroxy-5beta-pregnane-11,20-dione and 3beta-hydroxy-5beta-pregnan-20-one were all devoid of significant erythropoietic activity in polycythemic mice in almost all instances. Thus, under the conditions chosen, this study failed to demonstrate that 5beta-steroids increase radioactive iron incorporation in red cells of exhypoxic polycythemic mice.

Topics: Androstanes; Animals; Clinical Trials as Topic; Dihydrotestosterone; Double-Blind Method; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Hydroxysteroids; Hypoxia; Iron; Mice; Polycythemia; Pregnanediones; Pregnanes; Stereoisomerism; Testosterone

Topics: Anemia; Chronic Disease; Erythropoietin; Humans; Hypoxia; Renal Insufficiency, Chronic

Little is known about the impact of multiple trauma (MT)-related systemic hypoxia on osseous protein concentration of the hypoxia transcriptome. To shed light on this issue, we investigated erythropoietin (Epo), erythropoietin receptor (EpoR), and Y-box binding protein 1 (YB-1) concentrations in the fracture zone in a porcine MT + traumatic hemorrhage (TH) model. Sixteen male domestic pigs were randomized into two groups: an MT + TH group and a sham group. A tibia fracture, lung contusion, and TH were induced in the MT + TH group. The total observation period was 72 h. YB-1 concentrations in bone marrow (BM) were significantly lower in the fracture zone of the MT + TH animals than in the sham animals. Significant downregulation of BM-localized EpoR concentration in both unfractured and fractured bones was observed in the MT + TH animals relative to the sham animals. In BM, Epo concentrations were higher in the fracture zone of the MT + TH animals compared with that in the sham animals. Significantly higher Epo concentrations were detected in the BM of fractured bone compared to that in cortical bone. Our results provide the first evidence that MT + TH alters hypoxia-related protein concentrations. The impacts of both the fracture and concomitant injuries on protein concentrations need to be studied in more detail to shed light on the hypoxia transcriptome in fractured and healthy bones after MT + TH.

Topics: Animals; Erythropoietin; Fractures, Bone; Hypoxia; Male; Multiple Trauma; Receptors, Erythropoietin; Swine

Erythropoietin (EPO), hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), and vascular endothelial growth factor (VEGF) are key factors in the regulation of hypoxia, and can transcriptionally activate multiple genes under hypoxic conditions, thereby initiating large hypoxic stress in the network. The liver and kidneys are important metabolic organs of the body. We assessed the expression of EPO, HIF-1α, HIF-2α, and VEGF in liver and kidney tissues of plain and Tibetan sheep using hematoxylin and eosin staining, immunohistochemistry, and RT-qPCR. The results showed that EPO, HIF-1α, HIF-2α, and VEGF were expressed in tubular epithelial cells, collecting duct epithelial cells, mural epithelial cells, and the glomerular cytoplasm of Tibetan sheep, and their expression was significantly higher in Tibetan sheep than in plain sheep (P<0.05). EPO, HIF-1α, HIF-2α, and VEGF are expressed in hepatocytes, interlobular venous endothelial cells, and interlobular bile duct epithelial cells. In plain sheep, positive signals for EPO, HIF-1α, HIF-2α, and VEGF were localized mainly in interlobular venous endothelial cells, whereas VEGF and HIF-2α were negatively expressed in interlobular bile duct epithelial cells and positively expressed in EPO and HIF-1α. The differences in EPO, HIF-1α, and HIF-2α in Tibetan sheep were significantly higher than those in plain sheep (P<0.001). In the liver and kidney tissues of Tibetan sheep, EPO was associated with HIF-1α, HIF-2α, and VEGF (P<0.05). RT-qPCR results showed that EPO was not expressed, and HIF-1α, HIF-2α, and VEGF were expressed (P<0.05). The results showed that the expression of EPO, HIF-1α, HIF-2α, and VEGF in the kidney and liver of Tibetan sheep was higher than that in of plain sheep. Therefore, EPO, HIF-1α, HIF-2α, and VEGF may be involved in the adaptive response of plateau animals, which provides theoretical clarity to further explore the adaptive mechanism of plateau hypoxia in Tibetan sheep.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Endothelial Cells; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Liver; Sheep; Tibet; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Erythropoietin (EPO) is a hypoxia-responsive cytokine that induces neuroprotective effect in hypoxic-ischaemic, traumatic, excitotoxic and inflammatory injuries. Recently, utilizing a clinically relevant murine model of TBI and delayed hypoxemia, we have found that ongoing recombinant human EPO (rhEPO) administration influenced neurogenesis, neuroprotection, synaptic density and, behavioral outcomes early after TBI, and the impact on long-lasting outcomes 6 months after injury. We also demonstrated that the 1-month behavioral improvement was associated with mitogen-activated protein kinase (MAPK)/cAMP response element-binding protein (CREB) signaling activation and increased of excitatory synaptic density in the amygdala. However, we did not uncover which type of cells were involved in fear memory response enhancement after rhEPO treatment in the setting of TBI with delayed hypoxemia. In this report, using chemogenetic tools in our controlled cortical impact (CCI) model, we were able to inactivate excitatory neurons and eliminate rhEPO-induced fear memory recall enhancement. In summary, these data demonstrate that rhEPO treatment initiated after TBI enhances contextual fear memory in the injured brain via activation of excitatory neurons in the amygdala.

Topics: Animals; Brain Injuries; Erythropoietin; Fear; Humans; Hypoxia; Mice; Neurons

The erythroid growth factor erythropoietin (EPO) is mainly produced by the kidneys in adult mammals and induces expansion of erythroid cells and iron use for hemoglobin synthesis. The liver also produces EPO at a lower level than the kidneys. Renal and hepatic EPO production is fundamentally regulated by hypoxia-inducible transcription factors (HIFs) in a hypoxia/anemia-inducible manner. Recently, small compounds that activate HIFs and EPO production in the kidneys by inhibiting HIF-prolyl hydroxylases (HIF-PHIs) have been launched to treat EPO-deficiency anemia in patients with kidney disease. However, the roles of the liver in the HIF-PHI-mediated induction of erythropoiesis and iron mobilization remain controversial. Here, to elucidate the liver contributions to the therapeutic effects of HIF-PHIs, genetically modified mouse lines lacking renal EPO-production ability were analyzed. In the mutant mice, HIF-PHI administration marginally increased plasma EPO concentrations and peripheral erythrocytes by inducing hepatic EPO production. The effects of HIF-PHIs on the mobilization of stored iron and on the suppression of hepatic hepcidin, an inhibitory molecule for iron release from iron-storage cells, were not observed in the mutant mice. These findings demonstrate that adequate induction of EPO mainly in the kidney is essential for achieving the full therapeutic effects of HIF-PHIs, which include hepcidin suppression. The data also show that HIF-PHIs directly induce the expression of duodenal genes related to dietary iron intake. Furthermore, hepatic EPO induction is considered to partially contribute to the erythropoietic effects of HIF-PHIs but to be insufficient to compensate for the abundant EPO induction by the kidneys.

Topics: Anemia; Animals; Erythropoiesis; Erythropoietin; Hepcidins; Hypoxia; Iron; Kidney; Mammals; Mice; Pharmaceutical Preparations

Flavonoids are the most common phytochemicals in vegetables and herbal products. The beneficial functions of flavonoids in the brain and erythropoietic system have been proposed. Erythropoietin (EPO) is a potent protective agent in the brain; but which has difficulty to cross the blood brain barrier (BBB). Here, about 60 flavonoids were screened for their potential activation on the transcription of EPO mRNA in the neuronal embryonic stem cell lines, NT2/D1 and PC12. Amongst the screened flavonoids, formononetin, calycosin, ononin, chrysin, baicalein and apigenin showed robust up regulation of EPO production via enhancement of hypoxia response element (HRE) activity in cultured embryonic stem cells. In addition, the flavonoids showed activation of HRE activity by having increased accumulation of HIF-1α, but not on level of HIF-1β, in the cultures. The accumulation of HIF-1α was attributed to up regulation of HIF-1α mRNA and blockade of HIF-1α degradation upon treatment of the flavonoids. These results suggested a promising trend of developing commercial products of flavonoids as food supplements tailored for brain health.

Topics: Cell Line; Erythropoietin; Flavonoids; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; RNA, Messenger

Von Hippel-Lindau protein (VHL) is essential to hypoxic regulation of cellular processes. VHL promotes proteolytic clearance of hypoxia-inducible transcription factors (HIFs) that have been modified by oxygen-dependent HIF-prolyl hydroxylases. A homozygous loss-of-function VHL

Topics: Erythropoietin; Ferritins; Humans; Hypoxia; Iron Deficiencies; Polycythemia; Thrombosis; Transferrin; Von Hippel-Lindau Tumor Suppressor Protein

Traumatic brain injury (TBI) can result in axonal loss and demyelination, leading to persistent damage in the white matter. Demyelinated axons are vulnerable to pathologies related to an abnormal myelin structure that expose neurons to further damage. Oligodendrocyte progenitor cells (OPCs) mediate remyelination after recruitment to the injury site. Often this process is inefficient due to inadequate OPC proliferation. To date, no effective treatments are currently available to stimulate OPC proliferation in TBI. Recombinant human erythropoietin (rhEPO) is a pleiotropic neuroprotective cytokine, and its receptor is present in all stages of oligodendroglial lineage cell differentiation. Therefore, we hypothesized that rhEPO administration would enhance remyelination after TBI through the modulation of OPC response. Utilizing a murine model of controlled cortical impact and a primary OPC culture in vitro model, we characterized the impact of rhEPO on remyelination and proliferation of oligodendrocyte lineage cells. Myelin black gold II staining of the peri-contusional corpus callosum revealed an increase in myelinated area in association with an increase in BrdU-positive oligodendrocytes in injured mice treated with rhEPO. Furthermore, morphological analysis of OPCs showed a decrease in process length in rhEPO-treated animals. RhEPO treatment increased OPC proliferation after in vitro CSPG exposure. Erythropoietin receptor (EPOr) gene knockdown using siRNA prevented rhEPO-induced OPC proliferation, demonstrating that the rhEPO effect on OPC response is EPOr activation dependent. Together, our findings demonstrate that rhEPO administration may promote myelination by increasing oligodendrocyte lineage cell proliferation after TBI.

Topics: Animals; Brain Injuries, Traumatic; Cell Differentiation; Cell Proliferation; Erythropoietin; Humans; Hypoxia; Mice; Myelin Sheath; Oligodendrocyte Precursor Cells; Oligodendroglia; Recombinant Proteins

The occurrence and development of chronic obstructive pulmonary disease (COPD) are regulated by environmental and genetic factors. In hypoxia, Erythropoietin (. We conducted a case-control study enrolled 1095 COPD patients and 1144 healthy controls in Guangdong Province to evaluate the association between. In conclusion, this study found that

Topics: Case-Control Studies; Erythropoietin; Female; Genetic Predisposition to Disease; Humans; Hypoxia; Male; Polymorphism, Single Nucleotide; Protective Factors; Pulmonary Disease, Chronic Obstructive

Gliflozins provide a breakthrough in the management of type-2 diabetes. In addition to facilitating normoglycemia, these sodium-glucose cotransporter type 2 (SGLT2) inhibitors attenuate obesity, hypertension, dyslipidemia, and fluid retention, reduce cardiovascular morbidity, retard the progression of renal dysfunction, and improve survival. The administration of gliflozins also triggers erythropoietin (EPO) production, with the consequent induction of reticulocytosis and erythrocytosis. The mechanism(s) by which gliflozins induce erythropoiesis is a matter of debate. Whereas the canonical pathway of triggering EPO synthesis is through renal tissue hypoxia, it has been suggested that improved renal oxygenation may facilitate EPO synthesis via noncanonical trails. The latter proposes that recovery of peritubular interstitial fibroblasts producing erythropoietin (EPO) is responsible for enhanced erythropoiesis. According to this hypothesis, enhanced glucose/sodium reuptake by proximal tubules in uncontrolled diabetes generates cortical hypoxia, with injury to these cells. Once transport workload declines with the use of SGLT2i, they recover and regain their capacity to produce EPO. In this short communication, we argue that this hypothesis is incorrect. First, there is no evidence for interstitial cell injury related to hypoxia in the diabetic kidney. Tubular, rather than interstitial cells are prone to hypoxic injury in the diabetic kidney. Moreover, hypoxia, not normoxia, stimulates EPO synthesis by hypoxia-inducible factors (HIFs). Hypoxia regulates EPO synthesis as it blocks HIF prolyl hydroxylases (that initiate HIF alpha degradation), hence stabilizing HIF signals, inducing HIF-dependent genes, including EPO located in the deep cortex, and its production is initiated by the apocrinic formation of HIF-2, colocalized in these same cells.

Topics: Diabetic Nephropathies; Erythropoietin; Glucose; Humans; Hypoxia; Kidney; Polycythemia; Reticulocytosis; Sodium; Sodium-Glucose Transporter 2 Inhibitors

Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHI) are a new drug class for the treatment of renal anemia. HIF-PHI increase the expression of genes such as erythropoietin and genes involved in iron homeostasis. HIF-PHI were found to be superior to placebo in increasing hemoglobin levels and non-inferior to erythropoiesis stimulating agents (ESA). Furthermore, HIF-PHI appeared to positively influence iron parameters and also appeared to be effective in patients with elevated inflammatory values. The cardiovascular safety of HIF-PHI was found to be similar to ESA in most studies. However, a stronger risk of deep vein thrombosis and thrombosis of the shunt was found with treatment of HIF-PHI compared to ESA. HIF-PHI can be considered as an alternative to ESA, with the positive effect on iron homeostasis, the oral administration and the potential possibility to treat patients with ESA hyporesponsiveness as additional benefits, although effectiveness in this subgroup has yet to be demonstrated.

Topics: Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Iron; Prolyl Hydroxylases; Prolyl-Hydroxylase Inhibitors; Renal Insufficiency, Chronic

To investigate the neuroprotection of recombinant human erythropoietin (rhEPO) against hypoxic/ischemic (HI) insult in three-day-old rats. Postnatal day 3 (PD3) rats were randomly divided into three groups: Sham group, HI group and HI+rhEPO group. Ligation of the right common carotid artery and hypoxia to induce HI brain injury. After HI insult, the rats received intraperitoneal injection of rhEPO (5000 IU/Kg, qod) in HI+rhEPO group or equal saline in other groups. On PD10, damage of brain tissue was examined by hematoxylin-eosin (HE) staining, observation of neuronal apoptosis in the hippocampus and cortex using immunofluorescence assay (marker: TUNEL). Immunohistochemical staining or western blotting was performed to detect the expression of cyclooxygenase-2 (COX-2), Caspase-3 and phosphorylated Akt (p-Akt) protein. On PD28, cognitive ability of rats was assessed by Morris water maze test. HI injury causes brain pathological morphology and cognitive function damage in PD3 rats, which can be alleviated by rhEPO intervention. Compared with the HI group, the HI+rhEPO group showed an increase in platform discovery rate and cross platform frequency, while the search platform time was shortened (P < 0.05). The proportion of TUNEL positive neurons and the expression of COX-2 and Caspase-3 proteins in brain tissue in the hippocampus and cortex was decreased, while the expression of p-Akt protein was upregulated (P < 0.05). RhEPO could protect against the pathological and cognitive impairment of immature brain induced by HI insult. This neuroprotective activity may involve in inhibiting inflammatory and apoptosis by activation of PI3K/Akt signaling pathway.

Topics: Animals; Animals, Newborn; Brain; Caspase 3; Cyclooxygenase 2; Erythropoietin; Humans; Hypoxia; Hypoxia-Ischemia, Brain; Ischemia; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley

Topics: COVID-19; Erythropoietin; Humans; Hypoxia; Polycythemia; SARS-CoV-2

Aquatic hypoxia is both a naturally-occurring and anthropogenically-generated event. Fish species have evolved different adaptations to cope with hypoxic environments, including gill modifications and air breathing. However, little is known about the molecular mechanisms involved in the respiration of embryonic and larval fishes during critical windows of development. We assessed expression of the genes hif-1α, fih-1, nhe1, epo, gr and il8 using the developing tropical gar as a piscine model during three developmental periods (fertilization to hatch, 1 to 6 days post hatch (dph) and 7 to 12 dph) when exposed to normoxia (~7.43 mg/L DO), hypoxia (~2.5 mg/L DO) or hyperoxia (~9.15 mg/L DO). All genes had higher expression when fish were exposed to either hypoxia or hyperoxia during the first two developmental periods. However, fish continuously exposed to hypoxia had increased expression of the six genes by hatching and 6 dph, and by 12 dph only hif-1α still had increased expression. The middle developmental period was the most hypoxia-sensitive, coinciding with several changes in physiology and morphology. The oldest larvae were the most resilient to gene expression change, with little variation in expression of the six genes compared. This study is the first to relate the molecular response of an air-breathing fish to oxygen availability to developmental critical windows and contributes to our understanding of some molecular responses of developing fish to changes in oxygen availability.

Topics: Animals; Aquaculture; Erythropoietin; Female; Fish Diseases; Fish Proteins; Fishes; Gene Expression Regulation, Developmental; Hyperoxia; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-8; Male; Receptors, Glucocorticoid; Respiratory Physiological Phenomena; Sodium-Hydrogen Exchanger 1

Fibroblast-like renal erythropoietin (Epo) producing (REP) cells of the corticomedullary border region "sense" a decrease in blood oxygen content following anaemia or hypoxaemia. Burst-like transcription of Epo during tissue hypoxia is transient and is lost during fibrotic tissue remodelling, as observed in chronic kidney disease. The reason for this loss of Epo expression is under debate. Therefore, we tested the hypothesis that REP cell migration, loss and/or differentiation may cause Epo inhibition.. Using a reporter mouse that allows permanent labelling of active REP cells at any given time point, we analysed the spatiotemporal fate of REP cells following their initial hypoxic recruitment in models of hypoxaemia and renal tissue remodelling.. In long-term tracing experiments, tagged REP reporter cells neither died, proliferated, migrated nor transdifferentiated into myofibroblasts. Approximately 60% of tagged cells re-expressed Epo upon a second hypoxic stimulus. In an unilateral model of tissue remodelling, tagged cells proliferated and ceased to produce Epo before a detectable increase in myofibroblast markers. Treatment with a hypoxia-inducible factor (HIF) stabilizing agent (FG-4592/roxadustat) re-induced Epo expression in the previously active REP cells of the damaged kidney to a similar extent as in the contralateral healthy kidney.. Rather than cell death or differentiation, these results suggest cell-intrinsic transient inhibition of Epo transcription: following long-term dormancy, REP cells can repeatedly be recruited by tissue hypoxia, and during myofibrotic tissue remodelling, dormant REP cells are efficiently rescued by a pharmaceutic HIF stabilizer, demonstrating persistent REP cell functionality even during phases of Epo suppression.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoietin; Hypoxia; Kidney; Mice; Renal Insufficiency, Chronic

Topics: Animals; Erythropoietin; Hypoxia; Kidney; Mice

Erythropoietin (EPO), a central protein of erythropoiesis, plays an important role during hypoxia adaptation and is regulated by hypoxia-inducible factor (HIF). However, there is no report on EPO-producing cells and their regulatory mechanisms in yak (Bos grunniens). To understand EPO production and regulation of yak, kidneys from different age of yak were collected and expression of EPO, hypoxia-inducible factor 1 alpha (HIF-1α), and hypoxia-inducible factor 2 alpha (HIF-2α) were detected. Then renal tubule epithelial cells (RTECs) and peritubular interstitial fibroblast-like (RIFs) cells were isolated and cultured to determine their EPO production abilities. Subsequently, the cells were treated with dimethyloxalylglycine (DMOG) and Geldanamycin (GA), which are inhibitors of prolyl-4-hydroxylase domain (PHD) and heat shock protein 90 (HSP90) respectively, and siRNAs of HIF-1α and HIF-2α to explore their effect on EPO production and regulation. The results showed that expressions of EPO, HIF-1α, and HIF-2α were different in the different age groups of yak. High DMOG concentration caused a corresponding increase in the levels of HIF-1α and HIF-2α in RIFs and RTECs, however, EPO levels increased in RIFs only and was not detected at any concentration in RTECs; suggesting that EPO was produced in RIFs. Upon treating RIFs with siRNAs of HIF-1α and HIF-2α, we found that EPO was regulated by PHD through HIF-2α. In addition, increasing GA concentration caused a decrease in expression of HSP90, HIF-1α, HIF-2α, and EPO in RIFs. In conclusion, these findings support our proposition that PHD regulates EPO via HIF-2α in yak RIFs, while HSP90 impelled EPO expression.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cattle; Erythropoietin; Fibroblasts; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Prolyl Hydroxylases

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.

Topics: Animals; Erythropoietin; Female; Glycine; Hypoxia; Isoquinolines; Kidney; Male; Prolyl-Hydroxylase Inhibitors; Protein Biosynthesis; Rats; Rats, Sprague-Dawley; Up-Regulation

Inhibition of the prolyl-4-hydroxylase domain (PHD) enzymes, leading to the stabilization of hypoxia-inducible factor (HIF) α as well as to the stimulation of erythropoietin (Epo) synthesis, is the functional mechanism of the new anti-anemia drug roxadustat. Little is known about the effects of roxadustat on the Epo-producing cell pool. To gain further insights into the function of PHD inhibitors, we characterized the abundance of mesenchymal stem cell (MSC)-like cells after roxadustat treatment of mice. The number of Sca-1

Topics: Anemia; Animals; Erythropoietin; Glycine; Hypoxia; Isoquinolines; Kidney; Mice; Procollagen-Proline Dioxygenase; Prolyl Hydroxylases

The protective effects of recombinant human lactoferrin rhLF (branded "CAPRABEL™") on the cognitive functions of rat offspring subjected to prenatal hypoxia (7% O

Topics: Animals; Cognition; Erythropoietin; Female; Hypoxia; Lactoferrin; Pregnancy; Rats; Recombinant Proteins; Vitamins

Hypoxia is associated with increased erythropoietin (EPO) release to drive erythropoiesis. At high altitude, EPO levels first increase and then decrease, although erythropoiesis remains elevated at a stable level. The roles of hypoxia and related EPO adjustments are not fully understood, which has contributed to the formulation of the theory of neocytolysis. We aimed to evaluate the role of oxygen exclusively on erythropoiesis, comparing in vitro erythroid differentiation performed at atmospheric oxygen, a lower oxygen concentration (three percent oxygen) and with cultures of erythroid precursors isolated from peripheral blood after a 19-day sojourn at high altitude (3450 m). Results highlight an accelerated erythroid maturation at low oxygen and more concave morphology of reticulocytes. No differences in deformability were observed in the formed reticulocytes in the tested conditions. Moreover, hematopoietic stem and progenitor cells isolated from blood affected by hypoxia at high altitude did not result in different erythroid development, suggesting no retention of a high-altitude signature but rather an immediate adaptation to oxygen concentration. This adaptation was observed during in vitro erythropoiesis at three percent oxygen by a significantly increased glycolytic metabolic profile. These hypoxia-induced effects on in vitro erythropoiesis fail to provide an intrinsic explanation of the concept of neocytolysis.

Topics: Acclimatization; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Oxygen

Erythropoietin (EPO) is a glycoprotein hormone involved in proerythropoiesis, antioxidation, and antiapoptosis. It also contributes to cellular immune function in high-altitude species, such as the schizothoracine fish Gymnocypris dobula. Six mutation sites previously identified in EPO from G. dobula (GD-EPO) were injected into zebrafish embryos, and their effects were compared with EPO from the low-altitude schizothoracine Schizothorax prenanti. The key mutation site in GD-EPO was identified as H131S. Under hypoxic conditions, the levels of superoxide dismutase and malondialdehyde were decreased, whereas that of nitric oxide was increased in zebrafish injected with GD-EPO compared with those injected with S. prenanti-EPO (SP-EPO). The results suggest that EPO in high-altitude schizothoracine species is both antioxidative and antiapoptotic, driven by the H131S mutation site. Thus, this enhanced the ability of this species to adapt to the high-altitude hypoxic environment. These results provide a basis for investigating further the hypoxia adaptation mechanisms of teleosts.

Topics: Animals; Cyprinidae; Erythropoietin; Hypoxia; Mutation; Zebrafish

Experimental in vivo data have recently shown complementary neuroprotective actions of rhEPO and growth hormone (rhGH) in a neonatal murine model of hypoxic brain injury. Here, we hypothesized that rhGH and rhEPO mediate stabilization of the blood−brain barrier (BBB) and regenerative vascular effects in hypoxic injury to the developing brain. Using an established model of neonatal hypoxia, neonatal mice (P7) were treated i.p. with rhGH (4000 µg/kg) or rhEPO (5000 IU/kg) 0/12/24 h after hypoxic exposure. After a regeneration period of 48 h or 7 d, cerebral mRNA expression of Vegf-A, its receptors and co-receptors, and selected tight junction proteins were determined using qRT-PCR and ELISA. Vessel structures were assessed by Pecam-1 and occludin (Ocln) IHC. While Vegf-A expression increased significantly with rhGH treatment (p < 0.01), expression of the Vegfr and TEK receptor tyrosine kinase (Tie-2) system remained unchanged. RhEPO increased Vegf-A (p < 0.05) and Angpt-2 (p < 0.05) expression. While hypoxia reduced the mean vessel area in the parietal cortex compared to controls (p < 0.05), rhGH and rhEPO prevented this reduction after 48 h of regeneration. Hypoxia significantly reduced the Ocln+ fraction of cortical vascular endothelial cells. Ocln signal intensity increased in the cortex in response to rhGH (p < 0.05) and in the cortex and hippocampus in response to rhEPO (p < 0.05). Our data indicate that rhGH and rhEPO have protective effects on hypoxia-induced BBB disruption and regenerative vascular effects during the post-hypoxic period in the developing brain.

Topics: Animals; Animals, Newborn; Brain Injuries; Endothelial Cells; Erythropoietin; Growth Hormone; Humans; Hypoxia; Mice; Neuroprotective Agents; Occludin; Recombinant Proteins; Vascular Endothelial Growth Factor A

In the present work, we studied the effect of short-term acute hypoxia on the cellular composition of the blood and the head kidney of the black scorpionfish. Dissolved oxygen concentration was decreased from 8.5-8.7 mg O

Topics: Animals; Erythropoietin; Head Kidney; Hypoxia; Methemoglobin; Oxygen; Perciformes; Water

Therapeutic interventions targeting secondary insults, such as delayed hypoxemia, provide a unique opportunity for treatment in severe traumatic brain injury (TBI). Erythropoietin (EPO) is a hypoxia-responsive cytokine with important roles in neurodevelopment, neuroprotection and neuromodulation. We hypothesized that recombinant human erythropoietin (rhEPO) administration would mitigate injury in a combined injury model of TBI and delayed hypoxemia. Utilizing a clinically relevant murine model of TBI and delayed hypoxemia, we characterized how ongoing rhEPO administration influenced neurogenesis, neuroprotection, synaptic density and, behavioral outcomes early after TBI, and the impact on long-lasting outcomes 6 months after injury. We employed novel object recognition (NOR) and fear conditioning to assess long-term memory. At 1-month post-injury, we observed a significant increase in cued-fear memory response in the rhEPO-injured mice compared with vehicle-injured mice. This was associated with neuroprotection and neurogenesis in the hippocampus and mitogen-activated protein kinase (MAPK)/cAMP response element-binding protein (CREB) signaling activation and increased of excitatory synaptic density in the amygdala. Early rhEPO treatment after injury reduced neurodegeneration and increased excitatory synaptic density in the hippocampus and amygdala at 6 months post-injury. However at 6 months post-injury (4 months after discontinuation of rhEPO), we did not observe changes in behavioral assessments nor MAPK/CREB pathway activation. In summary, these data demonstrate that ongoing rhEPO treatment initiated at a clinically feasible time point improves neurological, cognitive, and histological outcomes after TBI in the setting of secondary hypoxemic insults.

Topics: Animals; Brain Injuries, Traumatic; Cyclic AMP Response Element-Binding Protein; Erythropoietin; Fear; Humans; Hypoxia; Mice; Mitogen-Activated Protein Kinases; Neuroprotection; Recombinant Proteins

Anemia is common in heart failure (HF) patients with chronic kidney disease (CKD) and is associated with worse outcomes. Iron supplementation improves symptoms and is associated with reduced risk of hospitalization for HF in iron-deficiency HF patients. However, iron deficiency is present in <30% of anemic HF patients. Erythropoiesis stimulating agents (ESAs) improve symptoms but are associated with increased risk of thromboembolic events in anemic HF patients with CKD. Hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitors are a new class of agents for the treatment of anemia. These agents work by stabilizing the HIF complex, thereby stimulating endogenous erythropoietin production. We hypothesized that HIF-PH inhibitors may be associated with reduced risk of cardiovascular outcomes compared with ESAs in anemic HF patients with CKD. Accordingly, we aim to perform the meta-analysis of studies on the efficacy and safety of HIF-PH inhibitors compared with ESAs in anemic HF patients with CKD.. This meta-analysis will include prospective cohort studies and randomized controlled trials on the effect of HIF-PH inhibitors compared with ESAs in anemic HF patients with CKD. Information of studies will be collected from PubMed, Web of Science, Cochrane Library, and ClinicalTrials.gov. The primary outcome will be cardiovascular death. The secondary outcomes will be all-cause death, hospitalization for HF, HF symptoms, exercise capacity, health-related quality of life, and hemoglobin levels.. This meta-analysis will evaluate the effect of HIF-PH inhibitors in anemic HF patients with CKD, providing evidence regarding the use of HIF-PH inhibitors in these patients.. INPLASY202230103.

Topics: Anemia; Erythropoietin; Heart Failure; Hematinics; Hemoglobins; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Iron; Meta-Analysis as Topic; Prolyl-Hydroxylase Inhibitors; Prospective Studies; Quality of Life; Renal Insufficiency, Chronic; Systematic Reviews as Topic

Topics: Erythropoietin; Humans; Hypoxia

Coronavirus Disease (COVID-19) can be considered as a human pathological model of inflammation combined with hypoxia. In this setting, both erythropoiesis and iron metabolism appear to be profoundly affected by inflammatory and hypoxic stimuli, which act in the opposite direction on hepcidin regulation. The impact of low blood oxygen levels on erythropoiesis and iron metabolism in the context of human hypoxic disease (e.g., pneumonia) has not been fully elucidated. This multicentric observational study was aimed at investigating the prevalence of anemia, the alterations of iron homeostasis, and the relationship between inflammation, hypoxia, and erythropoietic parameters in a cohort of 481 COVID-19 patients admitted both to medical wards and intensive care units (ICU). Data were collected on admission and after 7 days of hospitalization. On admission, nearly half of the patients were anemic, displaying mild-to-moderate anemia. We found that hepcidin levels were increased during the whole period of observation. The patients with a higher burden of disease (i.e., those who needed intensive care treatment or had a more severe degree of hypoxia) showed lower hepcidin levels, despite having a more marked inflammatory pattern. Erythropoietin (EPO) levels were also lower in the ICU group on admission. After 7 days, EPO levels rose in the ICU group while they remained stable in the non-ICU group, reflecting that the initial hypoxic stimulus was stronger in the first group. These findings strengthen the hypothesis that, at least in the early phases, hypoxia-driven stimuli prevail over inflammation in the regulation of hepcidin and, finally, of erythropoiesis.

Topics: Anemia; COVID-19; Erythropoiesis; Erythropoietin; Hepcidins; Humans; Hypoxia; Inflammation; Iron

Cytokine receptor-like factor 3 (CRLF3) is a conserved but largely uncharacterized orphan cytokine receptor of eumetazoan animals. CRLF3-mediated neuroprotection in insects can be stimulated with human erythropoietin. To identify mechanisms of CRLF3-mediated neuroprotection we studied the expression and proapoptotic function of acetylcholinesterase in insect neurons. We exposed primary brain neurons from Tribolium castaneum to apoptogenic stimuli and dsRNA to interfere with acetylcholinesterase gene expression and compared survival and acetylcholinesterase expression in the presence or absence of the CRLF3 ligand erythropoietin. Hypoxia increased apoptotic cell death and expression of both acetylcholinesterase-coding genes ace-1 and ace-2. Both ace genes give rise to single transcripts in normal and apoptogenic conditions. Pharmacological inhibition of acetylcholinesterases and RNAi-mediated knockdown of either ace-1 or ace-2 expression prevented hypoxia-induced apoptosis. Activation of CRLF3 with protective concentrations of erythropoietin prevented the increased expression of acetylcholinesterase with larger impact on ace-1 than on ace-2. In contrast, high concentrations of erythropoietin that cause neuronal death induced ace-1 expression and hence promoted apoptosis. Our study confirms the general proapoptotic function of AChE, assigns a role of both ace-1 and ace-2 in the regulation of apoptotic death and identifies the erythropoietin/CRLF3-mediated prevention of enhanced acetylcholinesterase expression under apoptogenic conditions as neuroprotective mechanism.

Topics: Acetylcholinesterase; Animals; Erythropoietin; Humans; Hypoxia; Insecta; Neurons; Receptors, Cytokine; Receptors, Erythropoietin

Liu, Na, Yanbo Zhang, Pu Zhang, Kerui Gong, Chunyang Zhang, Kai Sun, and Guo Shao. Vascular endothelial growth factor and erythropoietin show different expression patterns in the early and late hypoxia preconditioning phases and may correlate with DNA methylation status in the mouse hippocampus.

Topics: Animals; DNA Methylation; Erythropoietin; Hippocampus; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Vascular Endothelial Growth Factor A

The hematological module of the Athlete Biological Passport (ABP) is used for indirect detection of blood manipulations; however, the use of this method to detect doping, such as with microdoses of recombinant human erythropoietin (rhEPO), is problematic. For this reason, the sensitivity of ABP must be enhanced by implementing novel biomarkers. Here, we show that 5'-aminolevulinate synthase 2 (ALAS2) mRNAs are useful transcriptomic biomarkers to improve the indirect detection of rhEPO microdosing. Moreover, the sensitivity was sufficient to distinguish rhEPO administration from exposure to hypoxic conditions. Levels of mRNAs encoding carbonate anhydrase 1 (CA1) and solute carrier family 4 member 1 (SLC4A1) RNA, as well as the linear (L) and linear + circular (LC) forms of ALAS2 mRNA, were monitored for 16 days after rhEPO microdosing and during exposure to hypoxic conditions. ALAS2 mRNAs increased by 300% compared with the baseline values after rhEPO microdosing. Moreover, ALAS2 mRNAs were not significantly increased under hypoxic conditions. By contrast, CA1 mRNA was increased after both rhEPO microdosing and hypoxia, whereas SLC4A1 mRNA did not significantly increase under either condition. Furthermore, the analyses described here were performed using dried blood spots (DBSs), which provide advantages in terms of the sample collection, transport, and storage logistics. This study demonstrates that ALAS2 mRNA levels are sensitive and specific transcriptomic biomarkers for the detection of rhEPO microdosing using the hematological module of the ABP, and this method is compatible with the use of DBSs for anti-doping analyses.

Topics: 5-Aminolevulinate Synthetase; Biomarkers; Doping in Sports; Erythropoietin; Humans; Hypoxia; Recombinant Proteins; RNA; RNA, Messenger

Oxygen biology is currently a focus of intensive scientific research. Three scientists received the Nobel prize in physiology or medicine for their outstanding scientific efforts in revealing the mechanisms of oxygen sensing and defense against hypoxia. Hypoxia is a final common pathway to end-stage kidney disease and plays a crucial role in the pathogenesis of cardiovascular complications. Hypoxia-inducible factors (HIFs) are master regulators of defensive mechanisms against hypoxia. Erythropoietin is one of the main targets of HIFs that enhances oxygen delivery by increasing the production of red blood cells. HIF levels are regulated by HIF-prolyl hydroxylase (HIF-PH) inhibitors, which are now available as a new therapeutic modality against anemia in chronic kidney disease. HIF-PH inhibitors raise some theoretical concerns, but should be noted for their potential organ-protective effects.

Topics: Anemia; Erythropoietin; Humans; Hypoxia; Prolyl-Hydroxylase Inhibitors; Renal Insufficiency, Chronic

Altitude training increases haemoglobin, erythropoietin values among athletes, but may have negative physiological consequences. An alternative, although less explored, that has the potential to positively influence performance while avoiding some of the negative physiological consequences of hypoxia is sand training. Ten endurance-trained athletes (age: 20.8 ± 1.4, body mass: 57.7 ± 8.2 kg, stature: 176 ± 6 cm; 5000 m 14:55.00 ± 0:30 min) performed three 21-day training camps at different locations: at a high altitude (HIGH), at the sea-level (CTRL), at the sea-level on the sand (SAND). Differences in erythropoietin (EPO) and haemoglobin (Hb) concentration, body weight, VO

Topics: Adult; Altitude; Erythropoietin; Hemoglobins; Humans; Hypoxia; Oxygen Consumption; Running; Young Adult

Topics: Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Sirtuins

In the Preterm Erythropoietin (Epo) NeUroproTection (PENUT) Trial, potential biomarkers of neurological injury were measured to determine their association with outcomes at two years of age and whether Epo treatment decreased markers of inflammation in extremely preterm (<28 weeks' gestation) infants.. Plasma Epo was measured (n=391 Epo, n=384 placebo) within 24h after birth (baseline), 30min after study drug administration (day 7), 30min before study drug (day 9), and on day 14. A subset of infants (n=113 Epo, n=107 placebo) had interferon-gamma (IFN-γ), Interleukin (IL)-6, IL-8, IL-10, Tau, and tumour necrosis factor-α (TNF-α) levels evaluated at baseline, day 7 and 14. Infants were then evaluated at 2 years using the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III).. Elevated baseline Epo was associated with increased risk of death or severe disability (BSID-III Motor and Cognitive subscales <70 or severe cerebral palsy). No difference in other biomarkers were seen between treatment groups at any time, though Epo appeared to mitigate the association between elevated baseline IL-6 and lower BSID-III scores in survivors. Elevated baseline, day 7 and 14 Tau concentrations were associated with worse BSID-III Cognitive, Motor, and Language skills at two years.. Elevated Epo at baseline and elevated Tau in the first two weeks after birth predict poor outcomes in infants born extremely preterm. However, no clear prognostic cut-off values are apparent, and further work is required before these biomarkers can be widely implemented in clinical practice.. PENUT was funded by the National Institute of Neurological Disorders and Stroke (U01NS077955 and U01NS077953).

Topics: Adult; Biomarkers; Cerebral Palsy; Cognition; Erythropoietin; Female; Gestational Age; Humans; Hypoxia; Infant, Newborn; Infant, Premature; Inflammation; Interleukin-6; Male; Neuroprotection

Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A). The mutation reduces activation of erythropoietin receptor homodimers that induce erythropoiesis and thrombosis, but maintains the tissue-protective signaling. The binding of the nanobody element to glycophorin A rescues homodimeric erythropoietin receptor activation on red blood cell precursors. In a cell proliferation assay, the fusion protein is active at 10-14 M, allowing an estimate of the number of receptor-ligand complexes needed for signaling. This fusion protein stimulates erythroid cell proliferation in vitro and in mice, and shows neuroprotective activity in vitro. Our erythropoietin fusion protein presents a novel molecule for treating hypoxia.

Topics: Animals; Erythropoiesis; Erythropoietin; Hypoxia; Mice; Protein Binding; Receptors, Erythropoietin

Erythropoietin (Epo) controls a variety of signal transduction pathways during oxidative stress. The main function of Epo and its receptor (EpoR) is the stimulation of erythropoiesis.. The role of Epo and EpoR on non-hematopoietic normal and cancerous tissues is still poorly understood. This is the first report in which we aimed to investigate the role of Epo and EpoR systems at oxidative condition in human basal cell carcinoma (BCC), which is the most common tumour in the world.Materials and methods: Fresh normal and cancerous skin paired tissue was obtained from 63 patients who underwent curative BCC resection in Kahramanmaras, Turkey. Preliminary diagnosis of BCC was made in the dermatology clinic by excision and then the diagnosis was confirmed as histopathologic findings. Oxidative stress biomarkers such as superoxide dismutase (SOD) and catalase (CAT) activities, and malondialdehyde (MDA) levels in biopsy samples were measured spectrophotometrically, and also the levels of Epo and EpoR were measured by ELISA.. While the levels of MDA in cancerous tissue of patients with skin BCC were significantly higher than normal neighbouring skin tissue (p<0.05), SOD and CAT activities decreased (p<0.05). Furthermore, a remarkable increase was found in the Epo level ofpatients with skin BCC in comparison with the normal neighbouring skin tissue (p<0.05). However, we found that EpoR levels decreased (p<0.05).. Results indicate that there is an active oxidative process in BCC biopsies. The levels of increased Epo and decreased EpoR in oxidative condition due to hypoxia may aggravate tumour growth by its angiogenic activity.

Topics: Carcinoma; Erythropoietin; Humans; Hypoxia; Oxidative Stress; Receptors, Erythropoietin; Signal Transduction; Superoxide Dismutase

Although erythropoiesis-stimulating agents (ESAs) exert renoprotective effects in renal disease models, it has not been revealed whether the prolonged duration of action of ESAs contributes to their renoprotective effects.. We examined whether the prolonged duration of ESAs' action contributes to their renoprotective effects by comparing a divided administration of a short-acting ESA, epoetin beta (EPO), or a single administration of a long-acting ESA, epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.), to a single administration of EPO in chronic glomerulonephritis (GN) rats.. Chronic GN was induced by intravenous injection of anti-Thy 1.1 antibody (0.6 mg/kg) into uninephrectomized rats (day 0). Chronic GN rats were intravenously injected once with vehicle (disease control; DC), EPO 5,000 IU/kg (single EPO), or C.E.R.A. 25 μg/kg (single C.E.R.A.) on day 1; or 3 times during the first week with EPO 1,667 IU/kg from day 1 (divided EPO; total 5,000 IU/kg). Hemoglobin (Hb) level and urinary total protein (U-TP) level which are the indexes of hematopoiesis and renoprotective effects, respectively, were measured several times over 8 weeks.. Divided EPO and single C.E.R.A. increased Hb levels more greatly than did single EPO. In all chronic GN rats, elevated U-TP levels decreased transiently 2 weeks after chronic GN induction and then flared again. Single EPO significantly suppressed this exacerbation of U-TP levels compared to DC. Divided EPO and single C.E.R.A. each significantly suppressed the exacerbation of U-TP levels compared to single EPO.. Prolonged duration of ESAs' action contributed significantly to their renoprotective effects.

Topics: Anemia; Animals; Disease Models, Animal; Disease Progression; Drug Administration Schedule; Erythropoiesis; Erythropoietin; Glomerulonephritis; Hematinics; Hemoglobins; Hypoxia; Injections, Intravenous; Iron; Isoantibodies; Kidney; Male; Polyethylene Glycols; Protective Agents; Proteinuria; Rats, Inbred F344; Recombinant Proteins

This study evaluated whether recombinant human erythropoietin (rhEpo) treatment combined with chronic hypoxia provided an additive erythropoietic response and whether the athlete biological passport (ABP) sensitivity improved with hypoxia. Two interventions were completed, each containing 4 weeks baseline, 4 weeks exposure at sea level or 2,320 m of altitude, and 4 weeks follow-up. Participants were randomly assigned to 20 IU·kg bw

Topics: Adult; Altitude; Athletes; Erythropoiesis; Erythropoietin; Female; Humans; Hypoxia; Male; Recombinant Proteins; Young Adult

Hypoxia-inducible factors (HIFs) play important roles in the pathogenesis of erythrocytosis in chronic mountain sickness (CMS). von Hippel-Lindau (VHL) is a key regulator of hypoxia that can direct the poly-ubiquitylation and degradation of HIFs. Epigenetic mechanisms are believed to contribute toward adaption to chronic hypoxia. Here, we investigated the contribution and mechanism of VHL methylation in rats with erythrocytosis in CMS.. The methylation status of VHL was measured via bisulfite sequencing PCR, while VHL, DNMT1, DNMT3α, and DNMT3β expression were assessed using real-time reverse transcription PCR and western blotting. HIF-2α and EPO expression levels in bone marrow were determined via immunohistochemical staining, and erythroid hyperplasia in bone marrow sections were observed with hematoxylin and eosin staining.. We found that chronic hypoxia triggered erythroid hyperplasia in the bone marrow and increased the quantity of peripheral red blood cells in CMS rats. Chronic hypoxia significantly induced methylation at the CpG site in the VHL promoter, decreased VHL expression, and increased HIF-2α and EPO expression. Chronic hypoxia increased DNMT3α and DNMT3β expression, consistent with the decrease in VHL expression. The DNA methyltransferase inhibitor 5-azacytidine reduced chronic hypoxia-induced erythroid proliferation in the bone marrow of rats with CMS by suppressing VHL methylation and DNMTs expression.. Our study suggests that VHL methylation contributes toward excessive erythrocytosis in CMS by upregulating the HIF-2α/EPO pathway in the bone marrow of rats. We demonstrated that the DNMT inhibitor 5-azacytidine can attenuate erythroid hyperplasia in the bone marrow by demethylating the VHL promoter.

Topics: Altitude Sickness; Animals; Basic Helix-Loop-Helix Transcription Factors; Chronic Disease; Disease Models, Animal; DNA Methylation; Erythropoietin; Gene Expression Regulation; Hypoxia; Male; Polycythemia; Rats; Rats, Sprague-Dawley; Von Hippel-Lindau Tumor Suppressor Protein

Erythrocyte mass contributes to maintaining systemic oxygen delivery and blood viscosity, with the latter being one of the determinants of blood pressure. However, the physiological response to blood pressure changes under anaemic conditions remain unknown.. We show that anaemia decreases blood pressure in human patients and mouse models. Analyses of pathways related to blood pressure regulation demonstrate that anaemia enhances the expression of the gene encoding the vasopressor substance renin in kidneys. Although kidney juxtaglomerular cells are known to continuously produce renin, renal interstitial fibroblasts are identified in the present study as a novel site of renin induction under anaemic hypotensive conditions in mice and rats. Notably, some renal interstitial fibroblasts are found to simultaneously express renin and the erythroid growth factor erythropoietin in the anaemic mouse kidney. Antihypertensive agents but not hypoxic stimuli induced interstitial renin expression, suggesting that blood pressure reduction triggers interstitial renin induction in anaemic mice. The interstitial renin expression was also detected in injured fibrotic kidneys of the mouse and human, and the renin-expressing interstitial cells in murine fibrotic kidneys were identified as myofibroblasts originating from renal interstitial fibroblasts. Since the elevated expression levels of renin in fibrotic kidneys along with progression of renal fibrosis were well correlated to the systemic blood pressure increase, the renal interstitial renin production seemed to affect systemic blood pressure.. Renal interstitial fibroblasts function as central controllers of systemic oxygen delivery by producing both renin and erythropoietin.. Grants-in-Aid from Japan Society for the Promotion of Science (JSPS) KAKENHI (17K19680, 15H04691, and 26111002) and the Takeda Science Foundation.

Topics: Aged; Anemia; Animals; Biomarkers; Blood Pressure; Chronic Disease; Disease Models, Animal; Erythropoietin; Female; Fibroblasts; Fibrosis; Gene Expression; Humans; Hypotension; Hypoxia; Kidney; Kidney Diseases; Male; Mice; Mice, Knockout; Mice, Transgenic; Middle Aged; Renin; Signal Transduction

Olfactory receptor (Olfr) 78 is expressed in the carotid bodies (CB) and participates in CB responses to acute hypoxia. Olfr78 is also expressed in the kidney, which is a major site of erythropoietin (Epo) production by hypoxia. The present study examined the role of Olfr78 in cardiorespiratory and renal

Topics: Animals; Carotid Body; Erythropoietin; Hypoxia; Male; Mice; Receptors, Odorant; Respiration

Chronic intermittent hypoxia (CIH), one of the main features of obstructive sleep apnea (OSA), enhances carotid body-mediated chemoreflex and induces hypertension and breathing disorders. The carbamylated form of erythropoietin (cEpo) may have beneficial effects as it retains its antioxidant/anti-inflammatory and neuroprotective profile without increasing red blood cells number. However, no studies have evaluated the potential therapeutic effect of cEpo on CIH-related cardiorespiratory disorders. We aimed to determine whether cEpo normalized the CIH-enhanced carotid body ventilatory chemoreflex, the hypertension and ventilatory disorders in rats.. Male Sprague-Dawley rats (250 g) were exposed to CIH (5% O2, 12/h, 8 h/day) for 28 days. cEPO (20 μg/kg, i.p) was administrated from day 21 every other day for one more week. Cardiovascular and respiratory function were assessed in freely moving animals.. Twenty-one days of CIH increased carotid body-mediated chemoreflex responses as evidenced by a significant increase in the hypoxic ventilatory response (FiO2 10%) and triggered irregular eupneic breathing, active expiration, and produced hypertension. cEpo treatment significantly reduced the carotid body--chemoreflex responses, normalizes breathing patterns and the hypertension in CIH. In addition, cEpo treatment effectively normalized carotid body chemosensory responses evoked by acute hypoxic stimulation in CIH rats.. Present results strongly support beneficial cardiorespiratory therapeutic effects of cEpo during CIH exposure.

Topics: Animals; Erythropoietin; Humans; Hypoxia; Male; Rats; Rats, Sprague-Dawley; Respiration; Sleep Apnea Syndromes

Prolyl hydroxylase domain enzyme (PHD) inhibitors are effective in the treatment of chronic kidney disease (CKD)-associated anemia by stabilizing hypoxia inducible factor (HIF), thereby increasing erythropoietin and consequently erythropoiesis. However, concern for CKD progression needs to be addressed in clinical trials. Although pre-clinical studies showed an anti-inflammatory effect in kidney disease models, the effect of PHD inhibitors on kidney fibrosis was inconsistent probably because the effects of HIF are cell type and context dependent. The major kidney erythropoietin-producing cells are pericytes that produce erythropoietin through HIF-2α-dependent gene transcription. The concern for the impact of HIF in pericytes on kidney fibrosis arises from the fact that pericytes are the major precursor cells of myofibroblasts in CKD. Since cells expressing Gli1 fulfill the morphologic and anatomic criteria for pericytes, we induced Gli1

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Erythropoiesis; Erythropoietin; Fibrosis; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Kidney; Mice; Pericytes

Exposure to intermittent hypoxia has been demonstrated to be an efficient tool for hypoxic preconditioning, preventing damage to cells and demonstrating therapeutic benefits. We aimed to evaluate the effects of respiratory intermittent hypobaric hypoxia (IHH) to avoid brain injury caused by exposure to acute severe hypoxia (ASH).. biomarkers of oxidative damage, mitochondrial apoptosis, and transcriptional factors in response to hypoxia were assessed by Western blot and immunohistochemistry in brain tissue. Four groups of rats were used: (1) normoxic (NOR), (2) exposed to ASH (FiO. ASH animals underwent increased oxidative-stress-related parameters, an upregulation in apoptotic proteins and had astrocytes with phenotype forms compatible with severe diffuse reactive astrogliosis. These effects were attenuated and even prevented when the animals were preconditioned with IHH. These changes paralleled the inhibition of NF-κB expression and the increase of erythropoietin (EPO) levels in the brain.. IHH exerted neuroprotection against ASH-induced oxidative injury by preventing oxidative stress and inhibiting the apoptotic cascade, which was associated with NF-κB downregulation and EPO upregulation.

Topics: Animals; Antioxidants; Apoptosis; Astrocytes; Brain; Erythropoietin; Gliosis; Hypoxia; Male; Mitochondria; Neurons; Neuroprotection; NF-kappa B; Oxidative Stress; Rats; Rats, Sprague-Dawley

Anemia is a significant complication of chronic kidney disease (CKD), caused by erythropoietin deficiency and reduced iron availability. Erythropoiesis-stimulating agents have been used with iron supplementation to treat anemia; however, they are associated with some problems. Hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) is a promising new class of oral therapy for the treatment of anemia associated with CKD. HIF-PHI inhibits HIF-prolyl hydroxylase enzymes and results in the HIF-α accumulation, which leads to increased expression of HIF-responsive genes, including erythropoietin and vascular endothelial growth factor (VEGF). HIF stimulates endogenous erythropoietin production and also reduces circulating hepcidin concentrations, resulting in improved anemia. Many clinical trials demonstrate that HIF-PHI improves anemia in patients with CKD and on dialysis. In addition to treating anemia, HIF-PHI may have multiple potential effects. Several animal experiments show that HIF-PHI protects against ischemic kidney damage that progresses to CKD and also improves metabolic disorders and ameliorates cardiovascular complications. In contrast, malignant tumor and retinopathy should be carefully evaluated due to theoretical concerns that HIF stabilization may result in increased VEGF protein expression. Some adverse events such as shunt occlusion reported in large clinical trials also need attention and warrant further investigations.

Topics: Anemia; Animals; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Prolyl-Hydroxylase Inhibitors; Renal Insufficiency, Chronic; Vascular Endothelial Growth Factor A

COVID-19 virus has caused the world's deadliest pandemic. Early April 2020, the Delhi Government made it compulsory for people to wear face masks while going outdoors to curb disease spread. Prolonged use of surgical masks during the pandemic has been reported to cause many adverse effects. Intermittent hypoxia has been shown to activate erythropoietin (EPO leading to increased hemoglobin mass.. To analyze whether face mask induced intermittent hypoxia has any effect on the hemoglobin levels of healthy blood donors.. We retrospectively analyzed donor data from 1st July 2019-31st December 2020 for hemoglobin distribution across hemoglobin ranges and donor deferral on basis of hemoglobin. Study population was divided into two cohorts Group 1- (1st July 2019-31 st March 2020): before implementation of mandatory face masks Group 2- (1st April 2020-31 st December 2020): after implementation of mandatory face masks RESULTS: Mean Hb of blood donors in Group 2 (15.01 ± 1.1 g/dl) was higher than Group1 (14.49 ± 1.15 g/dl), (p < 0.0001). 47.1 % group2 donors had Hb of 16.1-18 g/dl compared to group1 (38.4 %). 52.9 % group 2 donors had Hb between 12.5-15 g/dl compared to 61.6 % Group 1 (p < 0.05). Deferral due to anemia was lesser in group 2 compared to group 1 (p < 0.00001). Group 2 had significantly higher deferral due to high Hb (>18 gm/dl) was than Group 1 (p = 0.0039).. This study including 19504 blood donors spanning over one and a half year shows that prolonged use of face mask by blood donors may lead to intermittent hypoxia and consequent increase in hemoglobin mass.

Topics: Adolescent; Adult; Aged; Blood Donors; COVID-19; Cross-Sectional Studies; Donor Selection; Erythropoietin; Female; Hemoglobins; Humans; Hypoxia; Male; Masks; Middle Aged; Pandemics; Retrospective Studies; SARS-CoV-2; Young Adult

Topics: Animals; Cell Culture Techniques; Disease Models, Animal; Erythropoietin; Fetal Growth Retardation; Fetal Weight; Fetus; Hep G2 Cells; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; In Vitro Techniques; Insulin-Like Growth Factor Binding Protein 1; Mechanistic Target of Rapamycin Complex 1; Microscopy, Fluorescence; Organ Size; Papio; Phosphorylation; Protein Kinase C-alpha; Receptors, Erythropoietin; Transcription Factors; Vascular Endothelial Growth Factor A

Excessive erythrocytosis (EE) is the main sign of chronic mountain sickness (CMS), a maladaptive clinical syndrome prevalent in Andean and other high-altitude populations worldwide. The pathophysiological mechanism of EE is still controversial, as physiological variability of systemic respiratory, cardiovascular, and hormonal responses to chronic hypoxemia complicates the identification of underlying causes. Induced pluripotent stem cells derived from CMS highlanders showed increased expression of genes relevant to the regulation of erythropoiesis, angiogenesis, cardiovascular, and steroid-hormone function that appear to explain the exaggerated erythropoietic response. However, the cellular response to hypoxia in native CMS cells is yet unknown. This study had three related aims: to determine the hypoxic proliferation of native erythroid progenitor burst-forming unit-erythroid (BFU-E) cells derived from CMS and non-CMS peripheral blood mononuclear cells; to examine their sentrin-specific protease 1 (SENP1), GATA-binding factor 1 (GATA1), erythropoietin (EPO), and EPO receptor (EPOR) expression; and to investigate the functional upstream role of SENP1 in native progenitor differentiation into erythroid precursors. Native CMS BFU-E colonies showed increased proliferation under hypoxic conditions compared with non-CMS cells, together with an upregulated expression of SENP1, GATA1, EPOR; and no difference in EPO expression. Knock-down of the SENP1 gene abolished the augmented proliferative response. Thus, we demonstrate that native CMS progenitor cells produce a larger proportion of erythroid precursors under hypoxia and that SENP1 is essential for proliferation. Our findings suggest a significant intrinsic component for developing EE in CMS highlanders at the cellular and gene expression level that could be further enhanced by systemic factors such as alterations in respiratory control, or differential hormonal patterns.

Topics: Altitude; Altitude Sickness; Chronic Disease; Erythroid Precursor Cells; Erythropoietin; Gene Expression Regulation; Genetic Predisposition to Disease; Homeostasis; Humans; Hypoxia; Iron; Leukocytes, Mononuclear; Oxygen; Transcriptome

We suggested previously that hippocampal slices were protected from hypoxic depolarization and swelling by preincubating them at room temperature (Kreisman et al., 2000). We postulated that hypothermic preconditioning induced tolerance in our slices, which protected against hypoxic depolarization and swelling. Control hippocampal slices were incubated at 34-35 °C for two hours and the response to 10 min of severe hypoxia was compared to slices which were preconditioned for two hours at room temperature (22-23 °C) prior to warming to 34-35 °C. Recordings of the extracellular DC potential provided an index of tissue depolarization and changes in tissue light transmittance provided an index of swelling. Hypothermic preconditioning significantly reduced hypoxia-induced swelling, particularly in CA3 and the dentate inner blade. Since erythropoietin (EPO) had been shown to mediate hypoxic preconditioning, we tested whether EPO also mediated hypothermic preconditioning in our slices. Recombinant rat EPO (1-10 micromolar) mitigated hypoxia-induced swelling and depolarization in dentate inner blade of unconditioned slices in a dose-dependent manner. We also blocked the protective effects of hypothermic preconditioning on hypoxic depolarization and swelling in the inner blade of the dentate gyrus by administering soluble EPO receptor in the bath and treating slices with wortmannin to block phosphorylation of PI3 kinase, a critical step in the activation of the downstream neuroprotectant, Akt. These results suggest that EPO mediates tolerance to hypoxic depolarization and swelling induced by hypothermic preconditioning. They also emphasize that various preincubation protocols used in experiments with hippocampal slices may differentially affect basal electrophysiological and metabolic properties of those slices.

Topics: Animals; Erythropoietin; Hippocampus; Hypothermia; Hypoxia; Ischemic Preconditioning; Male; Neuroprotective Agents; Rats, Sprague-Dawley

Besides their many other functions, hair shafts (HS) also are a repository for potentially noxious compounds. These are neutralized by their deposition within terminally differentiated, avital epithelial cells (trichocytes) that also facilitate the interaction of potential toxins with melanin, a toxin-adsorbent biopolymer. Trichocytes are completely extruded via HS shedding during exogen, an actively controlled process. This underappreciated functional property of the human hair follicle (HF) makes it a bona fide excretory (mini-) organ. Here, we ask whether the ca. 2 million HFs of the human integument operate in part as primitive, spatially dispersed kidney-like excretory organs. Despite the many obvious differences between kidneys and HFs, this provocative hypothesis is also supported by other underappreciated renal-follicular similarities such as anatomical parallels between Bowman's capsule and the anagen hair bulb, renal podocytes and HF winged cells ["Fuegelzellen"], and hypoxia-dependent production of erythropoietin and extensive prostaglandin synthesis by human scalp HFs-just as in the kidney. The proposed kidney-like excretory function of HFs may have constituted a major selection advantage of mammals during evolution and could be clinically relevant. We explain how the many open questions (eg, how are molecules destined to be excreted by hair shaft entrapment recognized, taken up and deposited into hair matrix cells?) can be tested experimentally. Finally, we explore how the therapeutic targeting of kidney-like excretory HF functions may usefully complement classical nephrological therapy (dialysis) and ask whether stimulation of intrafollicular erythropoietin synthesis might become exploitable for the benefit of patients with renal anaemia.

Topics: Anemia; Animals; Apoptosis; Bowman Capsule; Cell Differentiation; Erythropoietin; Hair; Hair Follicle; Humans; Hypoxia; Keratinocytes; Kidney; Melanins; Mice; Models, Biological; Organ Culture Techniques; Oxygen; Podocytes; Polymers; Scalp

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Topics: Animals; Brazil; Disease Models, Animal; Erythropoietin; Euterpe; Hematinics; Hematopoiesis; Hypoxia; Kidney; Mice; Plant Extracts; Up-Regulation

Erythropoietin (EPO), named after its role in hematopoiesis, is also expressed in mammalian brain. In clinical settings, recombinant EPO treatment has revealed a remarkable improvement of cognition, but underlying mechanisms have remained obscure. Here, we show with a novel line of reporter mice that cognitive challenge induces local/endogenous hypoxia in hippocampal pyramidal neurons, hence enhancing expression of EPO and EPO receptor (EPOR). High-dose EPO administration, amplifying auto/paracrine EPO/EPOR signaling, prompts the emergence of new CA1 neurons and enhanced dendritic spine densities. Single-cell sequencing reveals rapid increase in newly differentiating neurons. Importantly, improved performance on complex running wheels after EPO is imitated by exposure to mild exogenous/inspiratory hypoxia. All these effects depend on neuronal expression of the Epor gene. This suggests a model of neuroplasticity in form of a fundamental regulatory circle, in which neuronal networks-challenged by cognitive tasks-drift into transient hypoxia, thereby triggering neuronal EPO/EPOR expression.

Topics: Animals; Brain; Cell Differentiation; Cognition; Dendritic Spines; Erythropoietin; Female; Gene Deletion; Humans; Hypoxia; Male; Mice, Inbred C57BL; Models, Neurological; Motor Activity; Neurogenesis; Neuronal Plasticity; Physical Conditioning, Animal; Physical Endurance; Proto-Oncogene Proteins c-fos; Pyramidal Cells; Receptors, Erythropoietin; Transcriptome

This study aimed to evaluate the changes in the erythropoietin level and hematological variables in wrestlers after intermittent hypoxic exposure (IHE). Twelve wrestlers were assigned into two groups: hypoxia (sports training combined with IHE, n=6) and control (sports training, n=6). An IHE was performed for 10 days, with one day off after 6 days, once a day for about an hour. The concentrations of hydrogen peroxide ( H(2)O(2) ), nitric oxide (NO), vascular endothelial growth factor (VEGF) and erythropoietin (EPO), as well as total creatine kinase activity (CK) were measured. Also, the hematological markers (Hb - hemoglobin, Ht - hematocrit, RBC - red blood cell, WBC - white blood cell, Ret - reticulocytes) were analyzed. The 6-day IHE caused an increase in the levels of H(2)O(2), NO and VEGF. Similarly, the EPO level and WBC count reached the highest value after 6 days of IHE. The total Ret number increase constantly during 10 days of IHE. The hypoxia group showed a higher CK activity compared to the control. In conclusion, 10-day IHE in combination with wrestling training elevates levels of H(2)O(2), NO and VEGF, and improves the oxygen transport capacity by the release of EPO and Ret in circulation.

Topics: Adult; Athletes; Blood Cell Count; Erythropoiesis; Erythropoietin; Exercise; Humans; Hypoxia; Male; Oxygen Consumption; Random Allocation; Wrestling; Young Adult

Hepcidin is a novel factor for iron deficiency in athletes, which is suggested to be regulated by interleukin-6 (IL-6) or erythropoietin (EPO).. The purpose of the present study was to compare endurance exercise-induced hepcidin elevation among "normoxia", "hypoxia" and "combined heat and hypoxia".. Twelve males (21.5 ± 0.3 years, 168.1 ± 1.2 cm, 63.6 ± 2.0 kg) participated in the present study. They performed 60 min of cycling at 60% of [Formula: see text] in either "heat and hypoxia" (HHYP; F. Plasma IL-6 level significantly increased immediately after exercise (P < 0.05), with no significant difference among the trials. A significant elevation in serum EPO was observed 3 h after exercise in hypoxic trials (HHYP and HYP, P < 0.05), with no significant difference between HHYP and HYP. Serum hepcidin level increased 3 h after exercise in all trials (NOR, before 18.3 ± 3.9 and post180 31.2 ± 6.3 ng/mL; HYP, before 13.5 ± 2.5 and post180 23.3 ± 3.6 ng/mL, HHYP; before 15.8 ± 3.3 and post180 31.4 ± 5.3 ng/mL, P < 0.05). However, there was no significant difference among the trials during post-exercise.. Endurance exercise in "combined heat and hypoxia" did not exacerbate exercise-induced hepcidin elevation compared with the same exercise in "hypoxia" or "normoxia".

Topics: Bicycling; Erythropoietin; Exercise; Heat-Shock Response; Hepcidins; Humans; Hypoxia; Interleukin-6; Male; Physical Endurance; Young Adult

Genetic induction of hypoxia signaling by deletion of the von Hippel-Lindau (Vhl) protein in mesenchymal PDGFR-β

Topics: Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Receptor, Platelet-Derived Growth Factor beta; Signal Transduction

Osteonecrosis of the femoral head (ONFH) is a disease in which the blood supply of the femoral head is interrupted or damaged, resulting in joint dysfunction. Hypoxic environments increase the expression of EPO, VEGF, and HIF causes vascular proliferation and increases the blood supply. It also causes the organism to be in a state of hypercoagulability and increases thrombosis. Therefore, the purpose of this study was to explore the occurrence of ONFH after the use of glucocorticoids (GCs) under conditions of hypoxia tolerance for a long time.. Sprague-Dawley rats were fed in a hypobaric hypoxic chamber at an altitude of 4000 m, the whole blood viscosity, and plasma viscosity were determined to analyze the blood flow and hemagglutination. Western blotting, polymerase chain reaction, and immunohistochemistry were used to detect EPO, VEGF, CD31, and osteogenesis related proteins. Femoral head angiography was used to examine the local blood supply and micro-CT scanning was used to detect the structure of the bone trabecula.. Under hypoxic environments, the expression of EPO and VEGF increased, which increased the local blood supply of the femoral head, but due to more severe thrombosis, the local blood supply of the femoral head decreased.. Hypoxic environments can aggravate ONFH in SD rats; this aggravation may be related to the hypercoagulable state of the blood. We suggest that long-term hypoxia should be regarded as one of the risk factors of ONFH and we need to conduct a more extensive epidemiological investigation on the occurrence of ONFH in hypoxic populations.

Topics: Alkaline Phosphatase; Animals; Blood Coagulation; Cell Differentiation; Cell Proliferation; Erythropoietin; Femur Head Necrosis; Glucocorticoids; Hypoxia; Male; Osteogenesis; Oxygen; Partial Pressure; Rats, Sprague-Dawley; Vascular Endothelial Growth Factor A; X-Ray Microtomography

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin β pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O

Topics: Anemia; Animals; Blotting, Western; Disease Models, Animal; Erythropoietin; Glycosylation; Humans; Hypoxia; Kidney; Liver; Male; Rats; Rats, Sprague-Dawley

The level of erythropoietin (EPO) and vascular endothelial growth factor (VEGF-A) was investigated in blood serum and brain of Wistar rats by the enzyme immunoassay with specific rat antibodies. These growth factors are actively studied as biomarkers of ischemia or cytoprotection, as well as targets for agents initiating preconditioning (PreC). Pharmacological (amtizol administration), hypoxic (hypobaric hypoxia), and combined PreC (amtizol+hypobaric hypoxia) were used as neuroprotective approaches in this experimental work. In normoxia groups blood and brain tissue were collected 1 h (early period) or 48 h (delayed period) after the PreC. In addition we studied groups of animals with cerebral ischemia (induced by bilateral ligation of the common carotid arteries) 1 h and 48 h after the combined PreC: the levels of EPO and VEGF-A in the blood serum and the brain supernatant were determined in one day after the ligation. Experiments have shown that amtizol (3,5-diamino-1,2,4-thiadiazole) in normoxia increased the EPO level in the brain, and did not change EPO in blood serum and VEGF-A levels in both serum and the brain. A three-day (60 min exposure with 48 h intervals) hypobaric hypoxia (410 mm Hg) increased EPO and VEGF-A in the blood serum and brain tissues, but in most experimental groups differences did not reach the level of statistical significance versus intact control. The combined PreC was accompanied by a significant increase of EPO and VEGF-A in normoxia conditions both in early and delayed period of PreC. In cerebral ischemia the EPO level in the blood serum and brain tissues was higher than in intact control. The serum level of VEGF-A of the ischemia control group tended to increase while the brain level of VEGF-A remained basically unchanged versus the intact control group. In combined PreC before ischemia, the EPO level was lower in serum as compared with the ischemia control in the delayed PreC period, but did not differ significantly from the ischemia control in serum in early period and in brain tissues in both PreC periods. The VEGF-A level in the groups of combined PreC was significantly lower in serum as compared with the ischemia control in both the early and delayed PreC; in brain tissues it did not differ from the level of both the intact and ishemia control in early PreC period and was higher than in both control groups in the delayed PreC period.. Soderzhanie éritropoétina (EPO) i faktora rosta éndoteliia sosudov (VEGF-A) v syvorotke krovi i golovnom mozge (GM) krys linii Vistar izuchali s pomoshch'iu immunofermentnogo analiza s ispol'zovaniem spetsificheskikh krysinykh antitel. Dannye rostovye faktory aktivno issleduiutsia v kachestve biomarkerov kletochnogo povrezhdeniia ili, naprotiv, tsitoprotektsii, a takzhe misheneĭ dlia sredstv, sposobnykh initsiirovat' prekonditsionirovanie (PreK). V kachestve izuchaemykh neĭroprotektivnykh podkhodov ispol'zovali farmakologicheskoe (vvedenie amtizola), gipoksicheskoe (gipobaricheskaia gipoksiia) i kombinirovannoe (amtizol+gipobaricheskaia gipoksiia) PreK. Cherez 1 ch (ranniĭ period) i 48 ch (pozdniĭ period) posle okonchaniia PreK proizvodili zabor materiala (seriia opytov v usloviiakh normoksii). V drugoĭ serii opytov zhivotnym cherez 1 ch i 48 ch posle okonchaniia PreK modelirovali ishemiiu GM putem pereviazki obshchikh sonnykh arteriĭ i cherez sutki posle operatsii opredeliali soderzhanie EPO i VEGF-A. Pri sravnenii éffektivnosti farmakologicheskogo i gipoksicheskogo PreK v vozmozhnoĭ induktsii EPO i VEGF-A pri normoksii vyiavleno, chto amtizol (3,5-diamino-1,2,4-tiadiazol) povyshaet uroven' EPO v GM, ne izmeniaia soderzhanie EPO v syvorotke krovi i VEGF-A v oboikh substratakh. Trekhkratnaia (60 min ékspozitsii s intervalom 48 ch) gipobaricheskaia gipoksiia (410 mm rt.st.) povyshaet soderzhanie EPO i VEGF-A v syvorotke krovi i GM, no v bol'shinstve opytnykh grupp razlichiia s intaktnym kontrolem okazyvaiutsia statisticheski neznachimymi. Kombinirovannoe PreK privodit k znachimomu povysheniiu soderzhaniia EPO i VEGF-A pri normoksii kak v rannem, tak i pozdnem periode PreK. Pri ishemii GM v kontrol'noĭ gruppe soderzhanie EPO kak v syvorotke krovi, tak i v tkaniakh GM bylo vyshe znacheniĭ intaktnogo kontrolia. Pri otsenke soderzhaniia VEGF-A v kontrol'noĭ gruppe s ishemieĭ obnaruzhena tendentsiia k povysheniiu ego v syvorotke krovi i otsutstvie znachimykh razlichiĭ v tkaniakh GM. Pri ispol'zovanii do operatsii ishemii kombinirovannogo PreK soderzhanie EPO bylo nizhe v syvorotke krovi v sravnenii s kontrolem s ishemieĭ v pozdniĭ period PreK, ne otlichalos' znachimo ot znacheniĭ kontrolia s ishemiĭ v syvorotke krovi v ranniĭ period i v tkaniakh GM v oba perioda PreK. Soderzhanie VEGF-A pri ispol'zovanii kombinirovannogo PreK bylo znachimo nizhe v syvorotke krovi v sravnenii s kontrolem s ishemieĭ kak v ranniĭ, tak i v pozdniĭ periody PreK, v tkaniakh GM krys n

Topics: Animals; Brain Ischemia; Erythropoietin; Hypoxia; Rats; Rats, Wistar; Vascular Endothelial Growth Factor A

Topics: Erythropoietin; Gene Expression; Humans; Hypoxia; Response Elements

While it is well-established that distal hypoxia response elements (HREs) regulate hypoxia-inducible factor (HIF) target genes such as erythropoietin (Epo), an interplay between multiple distal and proximal (promoter) HREs has not been described so far. Hepatic Epo expression is regulated by a HRE located downstream of the EPO gene, but this 3' HRE is dispensable for renal EPO gene expression. We previously identified a 5' HRE and could show that both HREs direct exogenous reporter gene expression. Here, we show that whereas in hepatic cells the 3' but not the 5' HRE is required, in neuronal cells both the 5' and 3' HREs contribute to endogenous Epo induction. Moreover, two novel putative HREs were identified in the EPO promoter. In hepatoma cells HIF interacted mainly with the distal 3' HRE, but in neuronal cells HIF most strongly bound the promoter, to a lesser extent the 3' HRE, and not at all the 5' HRE. Interestingly, mutation of either of the two distal HREs abrogated HIF binding to the 3' and promoter HREs. These results suggest that a canonical functional HRE can recruit multiple, not necessarily HIF, transcription factors to mediate HIF binding to different distant HREs in an organ-specific manner.

Topics: Cell Hypoxia; Erythropoietin; Gene Expression; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Response Elements

Topics: Erythropoietin; Hematocrit; Humans; Hypoxia; Polycythemia; Thrombosis

Hypoxia associated with the high metabolic demand of rods has been implicated in the pathology of age-related macular degeneration (AMD), the most common cause of adult blindness in the developed world. The majority of AMD-associated severe vision loss cases are due to exudative AMD, characterized by neovascularization. To further investigate the causes and histopathology of exudative AMD, we conditionally induced hypoxia in a novel preclinical AMD model (Pde6gcreERT2/+;Vhl-/-) by targeting Vhl and used multimodal imaging and immunohistochemistry to track the development of hypoxia-induced neovascularization. In addition to developing a preclinical model that phenocopies exudative AMD, our studies revealed that the photoreceptor hypoxic response initiates and drives type 3 neovascularization, mainly in the outer retina. Activation of the VHL-HIF1a-VEGF-EPO pathway in the adult retina led to long-term neovascularization, retinal hemorrhages and compromised retinal layers. Our novel preclinical model would accelerate the testing of therapies that use metabolomic approaches to ameliorate AMD.

Topics: Animals; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Macular Degeneration; Mice; Neovascularization, Pathologic; Vascular Endothelial Growth Factor A

Topics: Anemia; Biomedical Research; Erythrocytes; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Neoplasms; Nobel Prize; Oxygen; Transcription, Genetic; United Kingdom; United States

Dyslipidemia is commonly observed in various kidney diseases, renal specific secreted erythropoietin (EPO) may participate in this process. However, how this process is regulated remains elusive.. Dyslipidemia was evaluated in chronic kidney disease and ischemia kidney injury animal model. Primary cultured adipocytes were harvested to investigate the lipid metabolic effect of EPO. Lipidemia was evaluated in EPO treated animals. Blood samples from cardiac surgery-induced kidney injury patient were collected to assess correlationship between EPO and lipidemia.. We found a decrease in secreted EPO and hypertriglyceridemia in chronic kidney disease (CKD) mice. In contrast, in renal ischemia animal model, increased EPO triggered by hypoxia signaling activation, was accompanied by decreased triglyceride (TG) in serum. Mechanistically, circulating EPO modulated JAK2-STAT5 signaling, which in turn enhanced lipid catabolism in peripheral adipose tissue and contributed to dysregulated lipidemia. Delivering of recombinant EPO into both wild type and CKD mice suppressed TG in serum by accelerating lipid catabolism in adipose tissue. In a cohort of patients diagnosed with acute kidney injury after cardiopulmonary bypass surgery, the decreased TG and cholesterol negatively correlated with increased EPO in serum.. This study depicted a new mechanism by which renal secreted EPO controlled lipidemia in kidney diseases including chronic kidney disease. Circulating EPO stimulated lipid catabolism by targeting JAK2-STATA5 signaling in peripheral adipose tissue, providing new therapeutic target for dyslipidemia treatment.. This work was supported by grants from the National Natural Science Foundation of China (Nos. 81700640 and 81970608).

Topics: Adipose Tissue; Animals; Disease Models, Animal; Erythropoietin; Hyperlipidemias; Hypoxia; Janus Kinase 2; Kidney; Kidney Diseases; Lipid Metabolism; Lipids; Male; Mice; Signal Transduction; STAT5 Transcription Factor

Erythropoietin is released from the kidney in response to tissue hypoxia. Montero and Lundby found that increases in plasma erythropoietin induced by reducing arterial oxygen content in healthy humans were independent of arterial oxygen tension. Their observations accord with the established physiology of kidney oxygenation and can be predicted by a computational model of renal oxygen transport. However, model simulations indicate that the interpretation implicit in the title of their paper may be an oversimplification.

Topics: Blood Gas Analysis; Cross-Over Studies; Erythropoietin; Humans; Hypoxia; Kidney; Oxygen

Erythropoietin (EPO) is a secreted hormone that stimulates the production of red blood cells, and the level of EPO is increased under hypoxia. The expression of EPO is regulated not only by the hypoxia-inducible factor (HIF) but also partly through epigenetic modifications, including histone acetylation and methylation. In this study, we report that histone H3K9 demethylase JMJD1 A is regulated by HIF-2α in HepG2 cells under hypoxia. Knockdown or over-expression of JMJD1 A can decrease or increase EPO expression, respectively. JMJD1 A can interact with HIF-2α to form a co-activator complex, which binds to the hypoxia response elements of EPO and increases EPO expression by catalyzing demethylation of H3K9me2, a transcription suppression marker. The results demonstrate that JMJD1 A is a co-activator of EPO expression.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Biocatalysis; Erythropoietin; Gene Expression Regulation; Hep G2 Cells; Histones; Humans; Hypoxia; Jumonji Domain-Containing Histone Demethylases; Lysine; Male; Methylation; Mice; Mice, Inbred C57BL; Transcription, Genetic

Intermittent hypobaric hypoxia can produce a protective effect on both the nervous system and non-nervous system tissues. Intermittent hypobaric hypoxia preconditioning has been shown to protect rats from cardiac ischemia-reperfusion injury by decreasing cardiac iron levels and reactive oxygen species (ROS) production, thereby decreasing oxidative stress to achieve protection. However, the specific mechanism underlying the protective effect of hypobaric hypoxia is unclear. To shed light on this phenomenon, we subjected Sprague-Dawley rats to hypobaric hypoxic preconditioning (8 hours/day). The treatment was continued for 4 weeks. We then measured the iron content in the heart, liver, spleen, and kidney. The iron levels in all of the assessed tissues decreased significantly after hypobaric hypoxia treatment, corroborating previous results that hypobaric hypoxia may produce its protective effect by decreasing ROS production by limiting the levels of catalytic iron in the tissue. We next assessed the expression levels of several proteins involved in iron metabolism (transferrin receptor, L-ferritin, and ferroportin1 [FPN1]). The increased transferrin receptor and decreased L-ferritin levels after hypobaric hypoxia were indicative of a low-iron phenotype, while FPN1 levels remained unchanged. We also examined hepcidin, transmembrane serine proteases 6 (TMPRSS6), erythroferrone (ERFE), and erythropoietin (EPO) levels, all of which play a role in the regulation of systemic iron metabolism. The expression of hepcidin decreased significantly after hypobaric hypoxia treatment, whereas the expression of TMPRSS6 and ERFE and EPO increased sharply. Finally, we measured serum iron and total iron binding capacity in the serum, as well as red blood cell count, mean corpuscular volume, hematocrit, red blood cell distribution width SD, and red blood cell distribution width CV. As expected, all of these values increased after the hypobaric hypoxia treatment. Taken together, our results show that hypobaric hypoxia can stimulate erythropoiesis, which systemically draws iron away from nonhematopoietic tissue through decreased hepcidin levels.

Topics: Animals; Apoferritins; Cation Transport Proteins; Erythrocyte Indices; Erythrocytes; Erythropoietin; Hematocrit; Hepcidins; Hypoxia; Iron; Male; Membrane Proteins; Organ Specificity; Rats, Sprague-Dawley; Receptors, Transferrin; RNA, Messenger; Serine Endopeptidases

HIF prolyl hydroxylase 2 (PHD2) inhibitors represent a novel approach for treating HIF-related diseases. This study reports the first application of photoremovable protecting group to the photoactivatable inhibitor (

Topics: Cell Line, Tumor; Enzyme Stability; Erythropoietin; Gene Expression Regulation; HEK293 Cells; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Light; Models, Chemical; Molecular Structure; Prolyl-Hydroxylase Inhibitors

The hypoxic response is a stress response triggered by low oxygen tension. Hypoxia-inducible factors (HIFs) play a prominent role in the pathobiology of hypoxia-associated conditions, including pulmonary hypertension (PH) and polycythemia. The c-Jun N-terminal protein kinase (JNK), a stress-activated protein kinase that consists of two ubiquitously expressed isoforms, JNK1 and JNK2, and a tissue-specific isoform, JNK3, has been shown to be activated by hypoxia. However, the physiological role of JNK1 and JNK2 in the hypoxic response remains elusive. Here, using genetic knockout cells and/or mice, we show that JNK2, but not JNK1, up-regulates the expression of HIF-1α and HIF-2α and contributes to hypoxia-induced PH and polycythemia. Knockout or silencing of JNK2, but not JNK1, prevented the accumulation of HIF-1α in hypoxia-treated cells. Loss of JNK2 resulted in a decrease in HIF-1α and HIF-2α mRNA levels under resting conditions and in response to hypoxia. Consequently, hypoxia-treated

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Hypoxia; Erythropoiesis; Erythropoietin; Hypertension, Pulmonary; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinase 9; Polycythemia; RNA, Messenger; Transcriptional Activation; Up-Regulation

Various hypoxia-related proteins are differentially expressed in the retina and secreted to the vitreous and/or aqueous humor of patients affected by dry or neovascular age-related macular degeneration (nAMD). To determine whether these conditions alter concentrations of cytokines also in the systemic circulation, we measured plasma levels of six hypoxia-related proteins.. Plasma was prepared from EDTA blood that was collected from patients affected by dry AMD (n = 5), nAMD (n = 11), proliferative diabetic retinopathy (PDR; n = 9), and patients with an epiretinal membrane (ERM; n = 11). ERM samples served as negative controls, PDR samples as positive controls. Protein concentrations of vascular endothelial growth factor (VEGF), erythropoietin (EPO), angiopoietin-like 4 (ANGPTL4), placental growth factor (PlGF), tumor necrosis factor alpha (TNF-α), and pigment epithelium-derived factor (PEDF) were determined by enzyme-linked immunosorbent assay (ELISA).. The concentration of PlGF was significantly increased in plasma of patients affected by nAMD. Although no statistically significant differences were found for EPO, ANGPTL4, PlGF, TNF-α, and PEDF, the mean concentration of VEGF was lowest in the nAMD group. Plasma concentrations of the six factors did not correlate with gender or age of patients.. nAMD may increase plasma concentrations of PlGF, making it a candidate as a biomarker for the neovascular form of AMD. Other factors, however, were not differentially regulated, suggesting that their systemic concentrations are not generally increased in hypoxia-related retinal diseases.

Topics: Aged; Aged, 80 and over; Angiopoietin-Like Protein 4; Biomarkers; Cytokines; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Eye Proteins; Female; Humans; Hypoxia; Macula Lutea; Male; Membrane Proteins; Middle Aged; Nerve Growth Factors; Serpins; Tomography, Optical Coherence; Vascular Endothelial Growth Factor A; Wet Macular Degeneration

The content of regulatory proteins involved in adaptation to hypoxia and ischemia was studied in brain rat homogenate under conditions of normoxia and after bilateral ligation of the common carotid arteries. Preconditioning with amtizole in combination with moderate hypoxia increased the levels of HIF-1α, erythropoietin, vascular endothelial growth factor under conditions of normoxia. During experimental ischemia, combined preconditioning led to stabilization of the content of these regulatory proteins at the level of intact control and to a decrease in glycogen synthase-3β kinase activity. This pattern of changes in regulatory proteins was noted during the early and late periods of preconditioning.

Topics: Animals; Brain; Brain Chemistry; Carotid Artery, Common; Erythropoietin; Gene Expression Regulation; Glycogen Synthase Kinase 3 beta; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning; Ligation; Protective Agents; Rats; Rats, Wistar; Signal Transduction; Thiadiazoles; Vascular Endothelial Growth Factor A

Topics: Adult; Anemia, Sickle Cell; Animals; Benzaldehydes; Cerebrovascular Circulation; Child; Clinical Trials as Topic; Double-Blind Method; Drug Evaluation, Preclinical; Erythropoietin; Gene Knock-In Techniques; Hematologic Agents; Hemoglobin, Sickle; Humans; Hypoxia; Mice; Mice, Transgenic; Oxygen; Polymerization; Pyrazines; Pyrazoles; Reticulocytes

Recent studies indicate that erythropoietin (EPO) is present in many areas of the brain and is active in the restoration of impaired neurons. In this study, we examined the presence of EPO and its role in bulbospinal neurons in the rostral ventrolateral medulla (RVLM). Hypoxia is often accompanied by a high blood pressure (BP). We hypothesized that EPO is produced in response to hypoxia in RVLM neurons and then activates them. To investigate whether RVLM neurons are sensitive to EPO, we examined the changes in the membrane potentials (MPs) of bulbospinal RVLM neurons using the whole cell patch-clamp technique during superfusion with EPO. A brainstem-spinal cord preparation was used for the experiments. EPO depolarized the RVLM neurons, and soluble erythropoietin receptor (SEPOR), an antagonist of EPO, hyperpolarized them. Furthermore, hypoxia-depolarized RVLM neurons were significantly hyperpolarized by SEPOR. In histological examinations, the EPO-depolarized RVLM neurons showed the presence of EPO receptor (EPOR). The RVLM neurons that possessed EPORs showed the presence of EPO and hypoxia-inducible factor (HIF)-2α. We also examined the levels of HIF-2α and EPO messenger RNA (mRNA) in the ventral sites of the medullas (containing RVLM areas) in response to hypoxia. The levels of HIF-2α and EPO mRNA in the hypoxia group were significantly greater than those in the control group. These results suggest that EPO is produced in response to hypoxia in RVLM neurons and causes a high BP via the stimulation of those neurons. EPO may be one of the neurotransmitters produced by RVLM neurons during hypoxia.

Topics: Action Potentials; Animals; Animals, Newborn; Basic Helix-Loop-Helix Transcription Factors; Blood Pressure; Cell Hypoxia; Erythropoietin; Hypertension; Hypoxia; In Vitro Techniques; Medulla Oblongata; Neurons; Rats, Wistar; Receptors, Erythropoietin; Up-Regulation

Iron is integral for erythropoietic adaptation to hypoxia, yet the importance of supplementary iron compared with existing stores is poorly understood. The aim of the present study was to compare the magnitude of the hemoglobin mass (Hbmass) in response to altitude in athletes with intravenous (IV), oral, or placebo iron supplementation.. Thirty-four, nonanemic, endurance-trained athletes completed 3 wk of simulated altitude (3000 m, 14 h·d), receiving two to three bolus iron injections (ferric carboxymaltose), daily oral iron supplementation (ferrous sulfate), or a placebo, commencing 2 wk before and throughout altitude exposure. Hbmass and markers of iron regulation were assessed at baseline (day -14), immediately before (day 0), weekly during (days 8 and 15), and immediately, 1, 3, and 6 wk after (days 22, 28, 42, and 63) the completion of altitude exposure.. Hbmass significantly increased after altitude exposure in athletes with IV (mean % [90% confidence interval (CI)], 3.7% [2.8-4.7]) and oral (3.2% [2.2-4.2]) supplementation and remained elevated at 7 d postaltitude in oral (2.9% [1.5-4.3]) and 21 d after in IV (3.0% [1.5-4.6]) supplementation. Hbmass was not significantly higher than baseline at any time point in placebo.. Iron supplementation appears necessary for optimal erythropoietic adaptation to altitude exposure. IV iron supplementation during 3 wk of simulated live high-train low altitude training offered no additional benefit in terms of the magnitude of the erythropoietic response for nonanemic endurance athletes compared with oral supplementation.

Topics: Adaptation, Physiological; Administration, Intravenous; Administration, Oral; Adult; Altitude; Dietary Supplements; Erythropoietin; Female; Ferric Compounds; Ferrous Compounds; Hemoglobins; Humans; Hypoxia; Male; Maltose; Physical Conditioning, Human; Physical Endurance; Young Adult

Impairment of cardiac function causes renal damage. Renal failure after heart failure is attributed to hemodynamic derangement including reduced renal perfusion and increased venous pressure. One mechanism involves apoptosis and is defined as cardiorenal syndrome type 1. Erythropoietin (EPO) is a cytokine that induces erythropoiesis under hypoxic conditions. Hypoxia inducible factor 1 alpha (HIF-1α) plays a regulatory role in cellular response to hypoxia. Protective effects of EPO on heart, kidney and nervous system are unrelated to red blood cell production. We investigated early changes in and effects of EPO on renal tissues of rats with myocardial infarction by morphology and immunohistochemistry. Coronary artery ligation was used to induce myocardial infarction in Wistar rats. Group 1 comprised sham operated rats; groups 2, 3 and 4 included rats after coronary artery ligation that were sacrificed 6 h after ligation and that were treated with saline, 5,000 U/kg EPO or 10,000 U/kg EPO, respectively; group 5 included rats sacrificed 1 h after ligation. Group 2 showed increased renal tubule damage. Significantly less tubule damage was observed in EPO treated groups. EPO and EPO receptor (EPO-R) immunostaining intensities increased slightly for group 5 and became more intense for group 2. EPO and EPO-R immunostaining was observed in the interstitial area, glomerular cells and tubule epithelial cells of EPO treated groups. HIF-1α immunostaining was observed in collecting tubules in the medulla only in group 2. Caspase-3 immunostaining is an indicator of apoptosis. Caspase-3 staining intensity decreased in renal medulla of EPO treated groups. EPO treatment may exert a protective effect on the renal tissues of patients with cardiorenal syndrome.

Topics: Animals; Apoptosis; Erythropoietin; Heart Failure; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Kidney; Male; Rats, Wistar

Hemodilutional anemia is associated with acute kidney injury (AKI) and mortality in patients undergoing cardiac surgery by mechanisms that may include tissue hypoxia. Our hypothesis was to assess if changes in the potential hypoxic biomarkers, including methemoglobin and erythropoietin, correlated with a decrease in hemoglobin (Hb) concentration following hemodilution on cardiopulmonary bypass (CPB).. Arterial blood samples were taken from patients (n = 64) undergoing heart surgery and CPB at baseline, during CPB, following CPB, and in the intensive care unit (ICU). Potential hypoxic biomarkers were measured, including methemoglobin, plasma Hb, and erythropoietin. Data were analyzed by repeated measures one-way analysis of variance on ranks and linear regression.. Hemoglobin levels decreased following CPB and methemoglobin increased in the ICU (P < 0.001 for both). No correlation was observed between the change in Hb and methemoglobin (P = 0.23). By contrast, reduced Hb on CPB correlated with increased lactate, reduced pH, and increased erythropoietin levels following CPB (P ≤ 0.004 for all). Increased plasma Hb (P < 0.001) also correlated with plasma erythropoietin levels (P < 0.001).. These data support the hypothesis that erythropoietin rather than methemoglobin is a potential biomarker of anemia-induced tissue hypoxia. The observed relationships between decreased Hb during CPB and the increase in lactate, reduced pH, and increase in erythropoietin levels suggest that early changes in plasma erythropoietin may be a pragmatic early biomarker of anemia-induced renal hypoxia. Further study is required to determine if anemia-induced increases in erythropoietin may predict AKI in patients undergoing cardiac surgery.. www.clinicaltrials.gov (NCT01883713). Registered 21 June 2013.

Topics: Acute Kidney Injury; Aged; Anemia; Biomarkers; Cardiac Surgical Procedures; Cardiopulmonary Bypass; Erythropoietin; Female; Hemodilution; Hemoglobins; Humans; Hypoxia; Male; Methemoglobin; Middle Aged; Prospective Studies

Based on the fact that erythropoietin (Epo) administration in rodents protects against spatial learning and cognitive deficits induced by chronic intermittent hypoxia (CIH)-mediated oxidative damage, here we tested the hypothesis that Epo in the brain protects against cardiorespiratory disorders and oxidative stress induced by CIH in adult mice.. Adult control and transgenic mice overexpressing Epo in the brain only (Tg21) were exposed to CIH (21%-10% O2-10 cycles/hour-8 hours/day-7 days) or room air. After CIH exposure, we used the tail cuff method to measure arterial pressure, and whole-body plethysmography to assess the frequency of apneic episodes at rest, minute ventilation, and ventilatory responses to hypoxia and hypercapnia. Finally, the activity of pro-oxidant (XO-xanthine oxidase, and NADPH) and antioxidant (super oxide dismutase) enzymes was evaluated in the cerebral cortex and brainstem.. Exposure of control mice to CIH significantly increased the heart rate and arterial pressure, the number of apneic events, and the ventilatory response to hypoxia and hypercapnia. Furthermore, CIH increased the ratio of pro-oxidant to antioxidant enzymes in cortex and brainstem tissues. Both physiological and molecular changes induced by CIH were prevented in transgenic Tg21 mice.. We conclude that the neuroprotective effect of Epo prevents oxidative damage in the brain and cardiorespiratory disorders induced by CIH. Considering that Epo is used in clinics to treat chronic kidney disease and stroke, our data show convincing evidence suggesting that Epo may be a promising alternative drug to treat sleep-disorder breathing.

Topics: Animals; Apnea; Arterial Pressure; Brain; Brain Stem; Cerebral Cortex; Erythropoietin; Heart Rate; Hypercapnia; Hypoxia; Male; Mice; Mice, Transgenic; NADP; Oxidative Stress; Plethysmography, Whole Body; Pulmonary Ventilation; Reactive Oxygen Species; Rest; Sleep Apnea Syndromes; Superoxide Dismutase; Xanthine Oxidase

Suzuki et al. analyzed a mouse model of erythropoietin-deficient anemia to show that during iron overload renal interstitial fibroblasts accumulate iron, and this impairs the hypoxia-driven transcription of the erythropoietin gene. The authors show that excess iron decreases levels of hypoxia-inducible transcription factor 2α (HIF2α), the main driver of erythropoietin production in hypoxia and anemia. The work advances our understanding of the effect of iron on hypoxia signaling, but its implications for anemia treatment are less clear.

Topics: Anemia; Animals; Basic Helix-Loop-Helix Transcription Factors; Erythropoietin; Fibroblasts; Hypoxia; Iron; Mice

We studied the expression of Hif-1α, Nf-κb, and Vegf genes in the liver and serum levels of HIF-1α, erythropoietin, VEGF, TGF-β, 8-isoprostane, and corticosterone in Wistar rats with different resistance to hypoxia in 5 and 90 min after acute exposure to hypobaric hypoxia. In 5 min after hypoxic exposure, Hif-1α expression in the liver and serum levels of erythropoietin, VEGF, and TGF-β in high-resistant rats were higher than in low-resistant animals. In highresistant rats, the increment in expression of Nf-κb gene responsible for the control over the inflammatory processes was more pronounced than in low-resistant animals. In 90 min after hypoxic exposure, the serum levels of HIF-1α, erythropoietin, VEGF, and TGF-β returned to normal in high-resistant rats, while in low-resistant animals, an increase in 8-isoprostane and TGF-β concentrations was observed. The rats with different resistance to hypoxia were characterized by different changes in biomolecular parameters determining predilection to inflammatory diseases.

Topics: Animals; Corticosterone; Dinoprost; Erythropoietin; Gene Expression Profiling; Gene Expression Regulation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Liver; Male; NF-kappa B; Rats; Rats, Wistar; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Topics: Brain; Erythropoietin; Fetus; Humans; Hypoxia; Infant, Newborn

BackgroundWe determined whether maternal nutrient restriction (MNR) in guinea pigs leading to fetal growth restriction (FGR) impacts markers for tissue hypoxia, implicating a mechanistic role for chronic hypoxia.MethodsGuinea pigs were fed ad libitum (Control) or 70% of the control diet before pregnancy, switching to 90% at mid-pregnancy (MNR). Near term, hypoxyprobe-1 (HP-1), a marker of tissue hypoxia, was injected into pregnant sows. Fetuses were then necropsied and liver, kidney, and placental tissues were processed for erythropoietin (EPO), EPO-receptor (EPOR), and vascular endothelial growth factor (VEGF) protein levels, and for HP-1 immunoreactivity (IR).ResultsFGR-MNR fetuses were 36% smaller with asymmetrical growth restriction compared to controls. EPO and VEGF protein levels were increased in the female FGR-MNR fetuses, providing support for hypoxic stimulus and linkage to increased erythropoiesis, but not in the male FGR-MNR fetuses, possibly reflecting a weaker link between oxygenation and erythropoiesis. HP-1 IR was increased in the liver and kidneys of both male and female FGR-MNR fetuses as an index of local tissue hypoxia, but with no changes in the placenta.ConclusionChronic hypoxia is likely to be an important signaling mechanism for the decreased fetal growth seen with maternal undernutrition and appears to be post-placental in nature.

Topics: Animals; Cohort Studies; Erythropoietin; Female; Fetal Development; Fetal Growth Retardation; Guinea Pigs; Hypoxia; Immunohistochemistry; Male; Maternal Nutritional Physiological Phenomena; Maternal-Fetal Exchange; Nitroimidazoles; Placenta; Pregnancy; Signal Transduction; Vascular Endothelial Growth Factor A

What is the central question of this study? Do the pulmonary vascular responses to hypoxia change during progressive exposure to high altitude and can alterations in these responses be related to changes in concentrations of circulating biomarkers that affect the pulmonary circulation? What is the main finding and its importance? In our field study with healthy volunteers, we demonstrate changes in pulmonary artery pressure suggestive of remodelling in the pulmonary circulation, but find no changes in the acute responsiveness of the pulmonary circulation to changes in oxygenation during 2 weeks of exposure to progressive hypoxia. Pulmonary artery pressure changes were associated with changes in erythropoietin, 8-isoprostane, nitrite and guanosine 3',5'-cyclic monophosphate. We sought to determine whether changes in pulmonary artery pressure responses to hypoxia suggestive of vascular remodelling occur during progressive exposure to high altitude and whether such alterations are related to changes in concentrations of circulating biomarkers with known or suspected actions on the pulmonary vasculature during ascent. We measured tricuspid valve transvalvular pressure gradients (TVPG) in healthy volunteers breathing air at sea level (London, UK) and in hypoxic conditions simulating the inspired O

Topics: Altitude; Altitude Sickness; Blood Gas Analysis; Blood Pressure; Erythropoietin; Female; Humans; Hypoxia; Interleukin-18; Lung; Male; Middle Aged; Nitric Oxide; Oxidative Stress; Oxygen; Pulmonary Artery; Pulmonary Circulation; Pulmonary Gas Exchange

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are rare, neuroendocrine tumors derived from adrenal or extra-adrenal chromaffin cells, respectively. Metastases are discovered in 3-36% of patients at the time of diagnosis. Currently, only suboptimal treatment options exist. Therefore, new therapeutic compounds targeting metastatic PHEOs/PGLs are urgently needed. Here, we investigated if anthracyclines were able to suppress the progression of metastatic PHEO. We explored their effects on experimental mouse PHEO tumor cells using in vitro and in vivo models, and demonstrated that anthracyclines, particularly idarubicin (IDA), suppressed hypoxia signaling by preventing the binding of hypoxia-inducible factor 1 and 2 (HIF-1 and HIF-2) to the hypoxia response element (HRE) sites on DNA. This resulted in reduced transcriptional activation of HIF target genes, including erythropoietin (EPO), phosphoglycerate kinase 1 (PGK1), endothelin 1 (EDN1), glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and vascular endothelial growth factor (VEGFA), which consequently inhibited the growth of metastatic PHEO. Additionally, IDA downregulated hypoxia signaling by interfering with the transcriptional activation of HIF1A and HIF2A. Furthermore, our animal model demonstrated the dose-dependent suppressive effect of IDA on metastatic PHEO growth in vivo. Our results indicate that anthracyclines are prospective candidates for inclusion in metastatic PHEO/PGL therapy, especially in patients with gene mutations involved in the hypoxia signaling pathway.

Topics: Adrenal Gland Neoplasms; Animals; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Cell Growth Processes; Cell Line, Tumor; Endothelin-1; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Idarubicin; Mice; Mice, Nude; Neoplasm Metastasis; Pheochromocytoma; Phosphoglycerate Kinase; Protein Binding; Signal Transduction; Xenograft Model Antitumor Assays

After hypoxia, reoxygenation with air is the consensus treatment for full-term neonates; however, the effect of hyperoxic reoxygenation of adults is unknown. The present study was designed to investigate the effects of reoxygenation with 100% oxygen after hypoxia on inflammation and apoptosis in mice. Eight-week-old mice were either subjected to hypoxia in 8% oxygen for 30 min or air served as controls. Following hypoxia, mice underwent reoxygenation for 30 min with 21% or 100% oxygen. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), caspase-3 and brain derived neurotrophic factor (BDNF) mRNA study and histopathological study were performed. Reoxygenation with 100% oxygen significantly increased TNF-α (2.5 h after hypoxia), IL-1β (5 h after hypoxia), caspase-3 (8 h after hypoxia) mRNA levels in the whole brain compared with 21% oxygen, and significantly decreased erythropoietin mRNA expression compared with 21% oxygen 9 h after reoxygenation. However, reoxygenation with 100% oxygen and 21% oxygen significantly decreased BDNF mRNA levels compared with control air group. There were no clear abnormal findings showing neuronal death among the three groups. Reoxygenation with 100% oxygen after hypoxia induced inflammation and apoptosis in adult mice. Therefore, these results suggest that the reoxygenation with 100% oxygen after hypoxia has harmful effects on adult brain as well as on neonatal brain.

Topics: Animals; Apoptosis; Brain-Derived Neurotrophic Factor; Caspase 3; Erythropoietin; Hypoxia; Interleukin-1; Male; Mice; Mice, Inbred C57BL; Oxygen; RNA, Messenger; Tumor Necrosis Factor-alpha

Generation of multiple mRNAs by alternative splicing is well known in the group of cytokines and has recently been reported for the human erythropoietin (EPO) gene. Here, we focus on the alternatively spliced EPO transcript characterized by deletion of exon 3 (hEPOΔ3). We show co-regulation of EPO and hEPOΔ3 in human diseased tissue. The expression of hEPOΔ3 in various human samples was low under normal conditions, and distinctly increased in pathological states. Concomitant up-regulation of hEPOΔ3 and EPO in response to hypoxic conditions was also observed in HepG2 cell cultures. Using LC-ESI-MS/MS, we provide first evidence for the existence of hEPOΔ3 derived protein EV-3 in human serum from healthy donors. Contrary to EPO, recombinant EV-3 did not promote early erythroid progenitors in cultures of human CD34+ haematopoietic stem cells. Repeated intraperitoneal administration of EV-3 in mice did not affect the haematocrit. Similar to EPO, EV-3 acted anti-apoptotic in rat hippocampal neurons exposed to oxygen-glucose deprivation. Employing the touch-screen paradigm of long-term visual discrimination learning, we obtained first in vivo evidence of beneficial effects of EV-3 on cognition. This is the first report on the presence of a naturally occurring EPO protein isoform in human serum sharing non-erythropoietic functions with EPO.

Topics: Amino Acid Sequence; Cell Line, Tumor; Colony-Forming Units Assay; Erythropoietin; Gene Expression Regulation; Hematopoietic Stem Cells; Humans; Hypoxia; Immunoprecipitation; Male; Models, Molecular; Protein Conformation; Protein Isoforms; Pyramidal Cells; Recombinant Proteins; RNA Splicing; Structure-Activity Relationship

Previous studies suggest that chronic erythropoietin (Epo) deficiency in male mice does not alter normoxic/hypoxic ventilation. As effects of Epo are sex specific and as progesterone could be a respiratory stimulant, we evaluated the impact of Epo deficiency and its possible interaction with progesterone in ventilatory control in female mice during estrous cycle phases. Compared to wild type (WT) animals, Epo-TAg

Topics: Animals; Erythropoietin; Estrous Cycle; Female; Hormone Antagonists; Hyperventilation; Hypoxia; Mice, Inbred CBA; Mice, Transgenic; Mifepristone; Plethysmography, Whole Body; Progesterone; Receptors, Progesterone; Respiration

The pathogenesis of post-transplantation erythrocytosis (PTE) is not well understood and appears to be multifactorial. Our hypothesis in this study was that several factors, including toxicity of calcineurin inhibitor, immunologic factors, and chronic allograft nephropathy, can trigger local tissue hypoxia in peritubular interstitium, which is where production of erythropoietin (EPO) takes place. This local interstitial tissue hypoxia can cause an increase in renal EPO production, which induces the development of PTE.. This cross-sectional study included 15 renal transplant recipients, in whom polycythemia developed after kidney transplantation, with elevated hematocrit level to >51%. Forty-eight age- and gender-matched renal transplant recipients with normal hematocrit level were included as the renal transplant control group. In addition, 13 age- and gender-matched healthy subjects were also included as the healthy control group. We used urine hypoxia-inducible factor-2 alpha (HIF-2α) levels to evaluate whether there is local tissue hypoxia in renal allograft. HIF-2α levels were measured by double antibody sandwich enzyme-linked immunosorbent assay (ELISA). Serum EPO and insulin-like growth factor-1 (IGF-1) levels were also measured.. HIF-2α levels were significantly lower in the polycythemia group than the other two groups, but there was no significant difference between the healthy control group and the renal transplant control group with regard to HIF-2α levels. There was no significant difference among the 3 study groups in terms of levels of serum EPO and IGF-1.. Local tissue hypoxia in renal allograft does not seem to play an important role in the development of PTE.

Topics: Adult; Basic Helix-Loop-Helix Transcription Factors; Cross-Sectional Studies; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Hematocrit; Humans; Hypoxia; Insulin-Like Growth Factor I; Kidney Transplantation; Kidney Tubules; Male; Middle Aged; Polycythemia

The study was carried out at Concordia Station (Antarctic Plateau). The aim was to investigate the respiratory and haematological responses to hypoxia in healthy subjects living at constant altitude. Thirteen men and women (34.1 ± 3.1 years) were exposed for 10 months to hypobaric hypoxia (oxygen level equivalent to 3800 m asl). These unique conditions enable a greater accuracy of monitoring human responses to chronic hypoxia than can be achieved elsewhere. Blood haemoglobin and erythropoietin concentrations were determined at sea level (Pre), and after 3, 7, 20, 90 and 300 days at altitude. Blood gas analysis, base excess and arterial oxygen saturation were measured at Pre, and after 150 and 300 days at altitude. Erythropoietin returned quickly to baseline level after a transient increase in the first days. Blood haemoglobin concentration started increasing at day 7 and remained markedly higher for the entire duration of the mission. At day 150 the blood carbon dioxide partial pressure was markedly reduced, and consequently blood pH remained higher at negative base excess until day 300. The arterial oxygen saturation remained lower than Pre throughout. In conclusion, humans display little capacity of hypoxia acclimatization even after ten months of constant exposure to low oxygen partial pressure.

Topics: Acclimatization; Adult; Altitude; Antarctic Regions; Erythropoietin; Female; Hemoglobins; Humans; Hypoxia; Male

Erythropoietin (EPO) plays an essential role in the regulation of erythropoiesis. Its production is under the control of the Hypoxia Inducible Factor (HIF) protein whose stability varies according to the oxygen level. During chronic renal failure, EPO deficiency is the main cause of anemia, but other factors such as iron deficiency and inflammatory syndrome are also involved. More recently, it is hypothesized that other factors such an excess of GDF-11 production may be also involved. Thus, beside Epo treatment HIF and GDF-11 are potentially new therapeutic targets in anemia of chronic kidney disease.

Topics: Anemia; Animals; beta-Thalassemia; Bone Morphogenetic Proteins; Disease Management; Disease Models, Animal; Erythropoiesis; Erythropoietin; Forecasting; Growth Differentiation Factors; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Immunoglobulin A; Intestinal Absorption; Iron Deficiencies; Iron, Dietary; Kidney Failure, Chronic; Mice; Models, Biological; Receptors, Transferrin

Linear alkylbenzene sulfonate (LAS) is an anionic surfactant commonly used in cleaning agents such as laundry detergents. Trace amounts of LAS are released into environmental waters after processing in wastewater treatment plants after the use of this chemical. Acute toxicity of LAS has been well-studied using various organisms, and its effects are particularly well known in fish. LAS damages fish gill morphology and induces mucous excretion from these organs. LAS also causes hematological changes. These observations suggest that LAS might induce hypoxic conditions in fish. However, the connections between hypoxia and hematological changes at the cellular and molecular levels remain unknown. Common carp were exposed to LAS at concentrations of 625, 1250, and 2500 μg/L for 96 h. A total of 9-10 fish were sampled at the end of the exposure period for each concentration. For hematological analysis, carp blood was sampled from the caudal vein. Gill tissue was used for real-time PCR analysis to evaluate transcriptional changes of hypoxia-induced genes. The number of normal red blood cells and the number of immature red blood cells were significantly decreased and increased, respectively, in fish exposed to 2500 µg/L LAS. The hypoxic marker genes hypoxia inducible factor 1α, myoglobin 1, and erythropoietin 2 were upregulated in these fish. Our results suggest that LAS decreases erythrocyte numbers and induces hypoxic conditions. In addition, LAS-exposed fish increase production of immature erythrocytes and upregulate myoglobin expression in gills to improve oxygen transport and absorption. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 122-130, 2017.

Topics: Alkanesulfonic Acids; Animals; Blood Cell Count; Body Weight; Carps; Dose-Response Relationship, Drug; Erythrocyte Count; Erythropoietin; Gene Expression; Gills; Growth; Hypoxia; Hypoxia-Inducible Factor 1; Myoglobin; Surface-Active Agents

Erythropoietin seems to play an important role in the regulation of fetal hypoxemia. The present prospective study was designed to determine if changes in erythropoietin levels can be found in fetuses with severe early-onset growth restriction and hemodynamic compromise.. Erythropoietin, hemoglobin, hematocrit, platelet counts, normoblasts, lacate, arterial and venous blood gasses in the umbilical cord were determined in 42 fetuses with fetal growth restriction (IUGR) with absent (zero-flow) and 26 IUGR fetuses with retrograde end-diastolic flow (reverse-flow) in the umbilical artery. Color Doppler measurements were performed on the middle cerebral artery (PI) and ductus venosus [(S-a)/D and (S-a)/Vmean]. Erythropoietin concentrations were significantly lower in the zero-flow group (median: 128.0 mU/mL; range: 60.3-213 mU/mL) compared with the reverse-flow group (median: 202.5 mU/mL; range: 166-1182 mU/mL). Significant differences in median lactate concentrations were observed between the zero-flow group: 3.28 mmol/L (range; 2.3-4.7 mmol/L), and reverse-flow group: 5.6 mmol/L (range: 3.8-7.5 mmol/L). Fetuses with reverse-flow had significantly lower median platelet counts than fetuses with zero-flow (74 vs. 155/μL) and significantly lower normoblast counts (63 vs. 342/100 WBC).. Fetuses with severe IUGR due to chronic placental insufficiency and absent or reversed flow in the umbilical artery show increased erythropoietin levels.

Topics: Blood Flow Velocity; Erythropoietin; Female; Fetal Growth Retardation; Humans; Hypercapnia; Hypoxia; Platelet Count; Pregnancy; Prospective Studies; Thrombocytopenia; Ultrasonography, Doppler, Color; Ultrasonography, Prenatal; Umbilical Arteries

Carbamylated erythropoietin (CEPO), an EPO derivative, is attracting widespread interest due to neuroprotective effects without erythropoiesis. However, little is known about molecular mechanisms behind CEPO-mediated neuroprotection. In primary neurons with oxygen-glucose deprivation (OGD) and mice with hypoxia-reoxygenation, the neuroprotection and possible molecular mechanism of CEPO were performed by immunohistochemistry and immunocytochemistry, Western blot, RT-PCR, and ELISA. The comparisons were analyzed by ANOVA followed by unpaired two-tailed Student's t test. Both CEPO and EPO showed the neuroprotective effects in OGD model and hypoxic brain. CEPO did not trigger JAK-2 but activated AKT through glial cell line-derived neurotrophic factor (GDNF). It has been shown that CEPO acts upon a heteroreceptor complex comprising both the EPO receptor and the common β receptor subunit (βcR, also known as CD131). The blockage of CD131 reduced CEPO-mediated GDNF production, while GFR receptor blockage and GDNF neutralization inhibited CEPO-induced neurogenesis. Addition of GDNF to cultured neurons increased phosphorylation of AKT. CEPO protects neurons possible through the CD131/GDNF/AKT pathway.

Topics: Animals; Cytokine Receptor Common beta Subunit; Erythropoietin; Female; Glial Cell Line-Derived Neurotrophic Factor; Hypoxia; Mice, Inbred C57BL; Neurons; Neuroprotection; Neuroprotective Agents; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction

An imbalance of proangiogenic and antiangiogenic factors is thought to induce the widespread vascular dysfunction characteristic of preeclampsia (PreE). Erythropoietin (Epo), a pleiotropic cytokine, has important angiogenic and vasoactive properties; however, its contribution to maternal vascular dysfunction in PreE is unknown.. Because high altitude (HA) raises the incidence of PreE, we asked whether HA increased maternal Epo and soluble Epo receptor (sEpoR) levels and whether such effects differed between PreE and normotensive controls at HA.. Longitudinal studies were conducted in pregnant Andean residents at HA (n = 28; 3600 m) or sea level (SL; n = 16; 300 m). Cross-sectional studies included 34 gestational age‒matched Andean PreE cases (n = 17) and controls (n = 17) in La Paz-El Alto, Bolivia (3600 to 4100 m).. HA augmented the pregnancy-associated rise in Epo relative to SL (P = 0.002), despite similar reductions in hemoglobin (Hb) across pregnancy at each altitude (7% to 9%, P < 0.001 for both). HA PreE cases had circulating Epo levels equivalent to those of controls but greater sEpoR (P < 0.05) and reduced Hb (P = 0.06, trend).. Our findings suggest that an augmented pregnancy-associated rise in Epo may be important for successful vascular adaptation to pregnancy at HA. We further speculate that the elevated sEpoR observed in PreE vs controls at HA impedes the effect of Epo to maintain endothelial function and may, in turn, be of pathological relevance for PreE at HA.

Topics: Adaptation, Physiological; Adult; Altitude; Biomarkers; Bolivia; California; Case-Control Studies; Cross-Sectional Studies; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Follow-Up Studies; Humans; Hypoxia; Incidence; Longitudinal Studies; Male; Pregnancy; Pregnancy Complications; Prognosis; Receptors, Erythropoietin; Vascular Diseases

This study compared the hematopoietic capacities of erythropoietin (Epo) and antioxidant drug U-74389G, based on 2 preliminary studies. The provided results on hematocrit levels augmentation were co-evaluated in a hypoxia reoxygenation protocol of an animal model.. Hematocrit levels were evaluated at the 60th reoxygenation min (for groups A, C and E) and at the 120th reoxygenation min (for groups B, D and F) in 60 rats. Groups A and B received no drugs, rats from groups C and D were administered with Epo; whereas rats from groups E and F were administered with U-74389G.. The first preliminary study of Epo non-significantly increased the hematocrit levels by 0.24%+1.38% (p-value=0.8586). The second preliminary study of U-74389G significantly raised the hematocrit levels by 3.16%+1.33% (p-value=0.0196). These 2 studies were co-evaluated since they came from the same experimental setting. The outcome of the co-evaluation was that U-74389G has approximately 12.66-fold higher hematopoietic potency than Epo (p-value=0.0000).. The anti-oxidant capacities of U-74389G provide satisfactory acute hematopoietic properties; presenting approximately 12.66-fold hematocrit level rise than epo (p-value=0.0000).

Topics: Animals; Antioxidants; Disease Models, Animal; Erythropoietin; Female; Hematocrit; Hematopoiesis; Humans; Hypoxia; Male; Pregnatrienes; Rats, Wistar; Reperfusion Injury

Physical inactivity and a low maximal aerobic capacity (VO

Topics: Blood Volume; Cardiac Output; Cardiovascular Diseases; Diabetes Mellitus, Type 2; Erythrocytes; Erythropoietin; Exercise; Hematocrit; Hemoglobins; Humans; Hypoxia; Muscle, Skeletal; Oxygen; Oxygen Consumption

The aim of this study was to determine the effect of cyclooxygenase-2 (COX-2) gene deletion on the adaptive responses during prolonged moderate hypobaric hypoxia. Wild-type (WT) and COX-2 knockout (KO) mice of both genders (3 months old) were exposed to hypobaric hypoxia (~0.4 ATM) or normoxia for 21 days and brain capillary densities were determined. Hematocrit was measured at different time intervals; brain hypoxia-inducible factor -1

Topics: Acclimatization; Angiopoietin-2; Animals; Brain; Cyclooxygenase 2; Erythropoietin; Female; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Male; Mice; Mice, Knockout; Sex Characteristics; Sex Factors; Vascular Endothelial Growth Factor A

Anemia of end stage renal disease affects 90% of patients on hemodialysis and it is a tremendous concern of patients and health care providers. Renal disease creates a state of renal hypoxia, which may contribute to a lack of erythropoietin production from the kidney when low oxygen levels are sensed. This necessitates the use of exogenous erythropoietin preparations.. Recent evidence suggests that endogenously derived hydrogen sulfide may mediate oxygen sensing in tissues. Given the known involvement of other small molecules such as nitric oxide in erythropoietin production and the observation of decreased urinary H2S levels in patients with renal failure, we postulated that H2S may be the primary mediator of erythropoietin production during hypoxia. PK1, 786-O and Hep3B cells were incubated in hypoxia (1% O2) for 24 hours. Hypoxic cells were treated with the H2S donor GYY 4137 and the H2S inhibitor hydroxylamine. Following hypoxia erythropoietin, HIF-1α, HIF-2α and CBS expression was measured by quantitative real-time polymerase chain reaction and Western blot.. Hydroxylamine administration led to a significant decrease in erythropoietin, HIF-1α, HIF-2α and CBS protein levels during hypoxia. This was rescued by administration of GYY 4137 for erythropoietin, CBS and HIF-2α. Additionally, CSE -/- mice placed in hypoxia for 72 hours showed decreased renal erythropoietin production compared to wild-type mice.. These data suggest previously undocumented interplay of the production and action of H2S during hypoxia with subsequent erythropoietin production. The use of novel hydrogen sulfide donors could represent an alternative to standard therapies of anemia of renal failure.

Topics: Anemia; Animals; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Blotting, Western; Cell Line; Cystathionine beta-Synthase; Cystathionine gamma-Lyase; Erythropoietin; Humans; Hydrogen Sulfide; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Failure, Chronic; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Real-Time Polymerase Chain Reaction; Sus scrofa

Erythropoietin is responsible for regulating the growth and development of red blood cells. Reports conflict on whether skeletal muscle is able to produce erythropoietin and release it into circulation and if exercise affects this. We set out to determine how erythropoietin is regulated in skeletal muscle and to determine whether skeletal muscle-derived erythropoietin can stimulate erythropoiesis.. Using an in vitro approach, we exposed proliferating and differentiated skeletal muscle cells to various forms of exercise-induced physiological stimuli and measured erythropoietin gene expression. To understand if skeletal muscle cells were able to stimulate erythropoiesis, independent of other cell types found in skeletal muscle, we used myoblast-conditioned media to treat bone marrow and to measure erythropoiesis through flow cytometry. We also measured erythropoietin expression and hypoxia in mice subjected to an exercise protocol designed to induce skeletal muscle oxygen stress.. Hypoxia increased erythropoietin expression in C2C12 myoblasts, myotubes, and primary myoblasts in vitro by 50% to 130%. Bone marrow treated with media conditioned with hypoxic myoblasts for 24 h increased the number of Ter-119-positive cells by 32%. An erythropoietin-neutralizing antibody prevented this increase. Compared with unexercised controls, exhaustive exercise increased skeletal muscle HIF1α levels by 50% and HIF2α levels by 20%. Moreover, exercised skeletal muscle erythropoietin expression was 70% higher.. These results demonstrate that skeletal muscle produces erythropoietin in a hypoxia and HIF-dependent manner and that hypoxia-treated muscle is capable of stimulating erythropoiesis in vitro.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Hypoxia; Cell Line; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Inbred C57BL; Muscle Fibers, Skeletal; Muscle, Skeletal; Physical Conditioning, Animal

The study examined the distinct and synergistic effects of hypoxia and bed rest on the erythropoietin (EPO) concentration and relative changes in plasma volume (PV). Eleven healthy male lowlanders underwent three 21-day confinement periods, in a counterbalanced order: (1) normoxic bed rest (NBR; PIO2: 133.1 ± 0.3 mmHg); (2) hypoxic bed rest (HBR; PIO2: 90.0 ± 0.4 mmHg, ambient simulated altitude of ~4000 m); and (3) hypoxic ambulation (HAMB; PIO2: 90.0 ± 0.4 mmHg). Blood samples were collected before, during (days 2, 5, 14, and 21) and 2 days after each confinement to determineEPOconcentration. Qualitative differences inPVchanges were also estimated by changes in hematocrit and hemoglobin concentration along with concomitant changes in plasma renin concentration.NBRcaused an initial reduction inEPOby ~39% (P = 0.04). By contrast,HBRenhancedEPO(P = 0.001), but the increase was less than that induced byHAMB(P < 0.01). All three confinements caused a significant reduction inPV(P < 0.05), with a substantially greater drop inHBRthan in the other conditions (P < 0.001). Thus, present results suggest that hypoxia prevents theEPOsuppression, whereas it seems to exaggerate thePVreduction induced by bed rest.

Topics: Activities of Daily Living; Adult; Bed Rest; Biomarkers; Blood Pressure; Down-Regulation; Erythropoietin; Exercise; Healthy Volunteers; Heart Rate; Humans; Hypovolemia; Hypoxia; Male; Motor Activity; Oxyhemoglobins; Plasma Volume; Renin; Time Factors; Young Adult

Renal peritubular interstitial fibroblast-like cells are critical for adult erythropoiesis, as they are the main source of erythropoietin (EPO). Hypoxia-inducible factor 2 (HIF-2) controls EPO synthesis in the kidney and liver and is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3, which function as cellular oxygen sensors. Renal interstitial cells with EPO-producing capacity are poorly characterized, and the role of the PHD/HIF-2 axis in renal EPO-producing cell (REPC) plasticity is unclear. Here we targeted the PHD/HIF-2/EPO axis in FOXD1 stroma-derived renal interstitial cells and examined the role of individual PHDs in REPC pool size regulation and renal EPO output. Renal interstitial cells with EPO-producing capacity were entirely derived from FOXD1-expressing stroma, and Phd2 inactivation alone induced renal Epo in a limited number of renal interstitial cells. EPO induction was submaximal, as hypoxia or pharmacologic PHD inhibition further increased the REPC fraction among Phd2-/- renal interstitial cells. Moreover, Phd1 and Phd3 were differentially expressed in renal interstitium, and heterozygous deficiency for Phd1 and Phd3 increased REPC numbers in Phd2-/- mice. We propose that FOXD1 lineage renal interstitial cells consist of distinct subpopulations that differ in their responsiveness to Phd2 inactivation and thus regulation of HIF-2 activity and EPO production under hypoxia or conditions of pharmacologic or genetic PHD inactivation.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Erythropoietin; Forkhead Transcription Factors; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Kidney; Mice; Mice, Knockout; Procollagen-Proline Dioxygenase; Stromal Cells

Iron bioavailability has been identified as a factor that influences cellular hypoxia sensing, putatively via an action on the hypoxia-inducible factor (HIF) pathway. We therefore hypothesized that clinical iron deficiency would disturb integrated human responses to hypoxia.. We performed a prospective, controlled, observational study of the effects of iron status on hypoxic pulmonary hypertension. Individuals with absolute iron deficiency (ID) and an iron-replete (IR) control group were exposed to two 6-hour periods of isocapnic hypoxia. The second hypoxic exposure was preceded by i.v. infusion of iron. Pulmonary artery systolic pressure (PASP) was serially assessed with Doppler echocardiography.. Thirteen ID individuals completed the study and were age- and sex-matched with controls. PASP did not differ by group or study day before each hypoxic exposure. During the first 6-hour hypoxic exposure, the rise in PASP was 6.2 mmHg greater in the ID group (absolute rises 16.1 and 10.7 mmHg, respectively; 95% CI for difference, 2.7-9.7 mmHg, P = 0.001). Intravenous iron attenuated the PASP rise in both groups; however, the effect was greater in ID participants than in controls (absolute reductions 11.1 and 6.8 mmHg, respectively; 95% CI for difference in change, -8.3 to -0.3 mmHg, P = 0.035). Serum erythropoietin responses to hypoxia also differed between groups.. Clinical iron deficiency disturbs normal responses to hypoxia, as evidenced by exaggerated hypoxic pulmonary hypertension that is reversed by subsequent iron administration. Disturbed hypoxia sensing and signaling provides a mechanism through which iron deficiency may be detrimental to human health.. ClinicalTrials.gov (NCT01847352).. M.C. Frise is the recipient of a British Heart Foundation Clinical Research Training Fellowship (FS/14/48/30828). K.L. Dorrington is supported by the Dunhill Medical Trust (R178/1110). D.J. Roberts was supported by R&D funding from National Health Service (NHS) Blood and Transplant and a National Institute for Health Research (NIHR) Programme grant (RP-PG-0310-1004). This research was funded by the NIHR Oxford Biomedical Research Centre Programme.

Topics: Adult; Arterial Pressure; Cardiac Output; Case-Control Studies; Echocardiography, Doppler; Erythropoietin; Female; Hepcidins; Humans; Hypertension, Pulmonary; Hypoxia; Interleukin-6; Iron; Iron Deficiencies; Male; Middle Aged; Oxyhemoglobins; Prospective Studies; Pulmonary Artery; Respiration; Signal Transduction

Acute kidney injury (AKI) is common in hospitalized patients and has a poor prognosis, the severity of AKI being linked to progression to chronic kidney disease. This stresses the need to search for protective mechanisms during the acute phase. We investigated kidney repair after hypoxic injury using a rat model of renal artery branch ligation, which led to an oxygen gradient vertical to the corticomedullary axis. Three distinct zones were observed: tubular necrosis, infarction border zone and preserved normal tissue. EphA2 is a receptor tyrosine kinase with pivotal roles in cell architecture, migration and survival, upon juxtacrine contact with its membrane-bound ligand EphrinA1. Following hypoxia, EphA2 was up-regulated in cortical and medullary tubular cells, while EphrinA1 was up-regulated in interstitial cells adjacent to peritubular capillaries. Moreover, erythropoietin (EPO) messenger RNA (mRNA) was strongly expressed in the border zone of infarcted kidney within the first 6 h. To gain more insight into the biological impact of EphA2 and EphrinA1 up-regulation, we activated the signalling pathways in vitro using recombinant EphrinA1/Fc or EphA2/Fc proteins. Stimulation of EphA2 forward signalling in the proximal tubular cell line HK2 increased cell attachment and laminin secretion at the baso-lateral side. Conversely, activation of reverse signalling through EphrinA1 expressed by Hep3B cells promoted EPO production at both the transcriptional and protein level. Strikingly, in co-culture experiments, juxtacrine contact between EphA2 expressing MDCK and EphrinA1 expressing Hep3B was sufficient to induce a significant up-regulation of EPO mRNA production in the latter cells, even in the absence of hypoxic conditions. The synergistic effects of EphA2 and hypoxia led to a 15-20-fold increase of EPO expression. Collectively, our results suggest an important role of EphA2/EphrinA1 signalling in kidney repair after hypoxic injury through stimulation of (i) tubular cell attachment, (ii) secretion of basal membrane proteins and (iii) EPO production. These findings could thus pave the way to new therapeutic approaches.

Topics: Acute Kidney Injury; Animals; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Ephrin-A1; Erythropoietin; Humans; Hypoxia; Laminin; Male; Rats; Rats, Sprague-Dawley; Receptor, EphA2; RNA, Messenger; Signal Transduction; Up-Regulation

Erythropoietin (EPO) has beneficial tissue-protective effects in several diseases but erythrocytosis may cause deleterious effects in EPO-treated patients. Thus carbamylated-EPO (C-EPO) and other derivatives retaining tissue-protective but lacking bone marrow-stimulating actions have been developed. Although EPO modulates ventilatory responses, the effects of C-EPO on ventilation have not been investigated. Here, basal breathing and respiratory chemoreflexes were measured by plethysmography after acute and chronic treatments with recombinant human C-EPO (rhC-EPO; 15,000 IU/kg during 5days) or saline (control group). Hematocrit, plasma and brainstem rhC-EPO levels were also quantified. Chronic rhC-EPO significantly elevated tissue rhC-EPO levels but not hematocrit. None of the drug regimen altered basal ventilation (normoxia). Chronic but not acute rhC-EPO enhanced hyperoxic ventilatory depression, and sustained the hypoxic ventilatory response mainly via a reduction of the roll-off phase. By contrast, rhC-EPO did not blunt the ventilatory response to hypercapnia. Thus, chronic C-EPO may be a promising therapy to improve breathing during hypoxia while minimizing adverse effects on cardiovascular function.

Topics: Analysis of Variance; Animals; Brain Stem; Carbon Dioxide; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Hematocrit; Hypoxia; Male; Mice; Mice, Inbred C57BL; Oxygen; Plethysmography; Pulmonary Ventilation; Respiration; Time Factors

To reduce side effects due to non-specific expression, the heme oxygenase-1 (HO-1) gene under control of a hypoxia-inducible erythropoietin (Epo) enhancer (pEpo-SV-HO-1) was developed for site-specific gene therapy of ischemic stroke.. pEpo-SV-HO-1 was constructed by insertion of the Epo enhancer into pSV-HO-1. Dexamethasone-conjugated polyamidoamine (PAMAM-Dexa) was used as a gene carrier. In vitro transfection assays were performed in the Neuro2A cells. In vivo efficacy of pEpo-SV-HO-1 was evaluated in the transient middle cerebral artery occlusion (MCAO) model.. In vitro transfection assay with the PAMAM-Dexa/pEpo-SV-HO-1 complex showed that pEpo-SV-HO-1 had higher HO-1 gene expression than pSV-HO-1 under hypoxia. In addition, pEpo-SV-HO-1 reduced the level of apoptosis more efficiently than pSV-HO-1 in Neuro2A cells under hypoxia. For in vivo evaluation, the PAMAM-Dexa/pEpo-SV-HO-1 complex was injected into the ischemic brain of the transient MCAO model. pEpo-SV-HO-1 increased HO-1 expression and reduced the number of apoptotic cells in the ischemic brain, compared with the pSV-HO-1 injection group. As a result, the infarct volume was more efficiently decreased by pEpo-SV-HO-1 than by pSV-HO-1.. pEpo-SV-HO-1 induced HO-1 gene expression and therapeutic effect in the ischemic brain. Therefore, pEpo-SV-HO-1 may be useful for site-specific gene therapy of ischemic stroke.

Topics: Animals; Brain; Cell Line, Tumor; Dexamethasone; Disease Models, Animal; Erythropoietin; Gene Transfer Techniques; Genetic Therapy; Heme Oxygenase-1; Hypoxia; Infarction, Middle Cerebral Artery; Male; Polyamines; Rats; Rats, Sprague-Dawley; Stroke; Transfection

Hypoxia induces angiogenesis while hyperoxia promotes vasoregression in the retina. We investigated herein the effect of prolonged hyperoxia on retinal angiogenesis and the underlying mechanism in an oxygen-induced retinopathy (OIR) model.. Vascular morphology was quantified in whole-mount retina from the mice subjected to the conventional OIR model (c-OIR) or the OIR model with prolonged hyperoxia (p-OIR). Expressions of genes related to angiogenesis were determined by real-time PCR.. p-OIR retinas showed few intraretinal neovascular tufts at the border of avascular zones, lacking preretinal neovascularization, whereas c-OIR retinas had numerous preretinal neovascularizations. p-OIR retinas demonstrated outgrowth of capillaries in the deep layers despite persistent hyperoxia and possess a larger avascular zone compared with the c-OIR retinas. The capillaries in the p-OIR retinas were well-formed in contrast to those in the c-OIR retinas. p-OIR retinas expressed significantly higher TNFα (∼4 fold) than c-OIR retinas. The expression of vascular endothelial growth factor, Erythropoietin, Angiopoietin 1 and 2 remained unchanged.. Our data demonstrate that TNFα transcription is increased in hyperoxia-promoted retinal angiogenesis, implicating it, in association with low VEGF levels, as a possible proponent in retinal angiogenesis under hyperoxia.

Topics: Angiopoietin-1; Angiopoietin-2; Animals; Erythropoietin; Fluorescent Antibody Technique; Gene Expression Regulation; Hyperoxia; Hypoxia; Mice, Inbred C57BL; Retinal Neovascularization; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Tumor Necrosis Factor-alpha; Up-Regulation; Vascular Endothelial Growth Factor A

Inflammation resolution is an active process, the failure of which causes uncontrolled inflammation which underlies many chronic diseases. Therefore, endogenous pathways that regulate inflammation resolution are fundamental and of wide interest. Here, we demonstrate that phagocyte respiratory burst-induced hypoxia activates macrophage erythropoietin signalling to promote acute inflammation resolution. This signalling is activated following acute but not chronic inflammation. Pharmacological or genetical inhibition of the respiratory burst suppresses hypoxia and macrophage erythropoietin signalling. Macrophage-specific erythropoietin receptor-deficient mice and chronic granulomatous disease (CGD) mice, which lack the capacity for respiratory burst, display impaired inflammation resolution, and exogenous erythropoietin enhances this resolution in WT and CGD mice. Mechanistically, erythropoietin increases macrophage engulfment of apoptotic neutrophils via PPARγ, promotes macrophage removal of debris and enhances macrophage migration to draining lymph nodes. Together, our results provide evidences of an endogenous pathway that regulates inflammation resolution, with important implications for treating inflammatory conditions.

Topics: Animals; Cell Movement; Erythropoietin; Hypoxia; Inflammation; Lymph Nodes; Macrophages; Male; Mice, Inbred C57BL; Mice, Knockout; Phagocytosis; PPAR gamma; Receptors, Erythropoietin; Respiratory Burst; Signal Transduction

In Caucasians and Native Americans living at altitude, hemoglobin mass is increased in spite of erythropoietin concentrations ([Epo]) not markedly differing from sea level values. We hypothesized that a nocturnal decrease of arterial oxygen saturation (SaO2) causes a temporary rise of [Epo] not detected by morning measurements. SaO2 (continuous, finger oximeter) and [Epo] (ELISA, every 4 h) were determined in young highlanders (altitude 2600 m) during 24 h of usual daily activity. In Series I (six male, nine female students), SaO2 fell during the night with the nadir occurring between 01:00 and 03:00; daily means (range 92.4-95.2%) were higher in females (+1.7%, P < 0.01). [Epo] showed opposite changes with zenith occurring at 04:00 without a sex difference. Mean daily values (22.9 ± 10.7SD U/L) were higher than values obtained at 08:00 (17.2 ± 9.5 U/L, P < 0.05). In Series II (seven females), only SaO2 was measured. During follicular and luteal phases, SaO2 variation was similar to Series I, but the rhythm was disturbed during menstruation. While daily [Epo] variations at sea level are not homogeneous, there is a diurnal variation at altitude following changes in SaO2 Larger hypoventilation-dependent decreases of alveolar PO2 decreases during the night probably cause a stronger reduction of SaO2 in highlanders compared to lowlanders. This variation might be enlarged by a diurnal fluctuation of Hb concentration. In spite of a lower [Hb], the higher SaO2 in women compared to men led to a similar arterial oxygen content, likely explaining the absence of differences in [Epo] between sexes.

Topics: Adolescent; Altitude; Circadian Rhythm; Erythropoietin; Female; Hemoglobins; Hormones; Humans; Hypoxia; Male; Oximetry; Oxygen; Sex Factors; Young Adult

Erythropoietin (EPO) is important for angiogenesis after hypoxia/ischemia. In this study, we investigated whether recombinant human erythropoietin (rhEPO) can enhance angiogenesis, and promote cognitive function through vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway in a rat model of hypoxic-ischemic encephalopathy (HIE). RhEPO, selective VEGFR2 inhibitor (SU5416) or vehicle was administrated by intraperitoneal injection. The assessment for cognitive function begins on day 60 after anoxia. Vascular density in hippocampus and white matter damage within corpus callosum were examined on day 28 after anoxia. The expression of erythropoietin receptor (EPOR), VEGF, rapidly accelerated fibrosarcoma 1 (Raf1), and extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in hippocampus were evaluated on day 7 after anoxia. RhEPO-treated anoxia rats had better cognitive recovery, higher vascular density, and less white matter damage than in the vehicle anoxia rats. These protective effects associated with increased expression of EPOR, VEGF; and increased phosphorylation of Raf1 and ERK1/2. While this up-regulation, and changes in the histopathologic and functional outcomes were abolished by SU5416. Our data indicate that rhEPO can enhance angiogenesis, reduce white matter damage, and promote cognitive recovery through VEGF/VEGFR2 signaling pathway in anoxia rats.

Topics: Angiogenesis Inducing Agents; Animals; Animals, Newborn; Cognition; Disease Models, Animal; Erythropoietin; Hippocampus; Hypoxia; Hypoxia-Ischemia, Brain; Indoles; Male; Pyrroles; Random Allocation; Rats, Sprague-Dawley; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; White Matter

Topics: Adaptation, Physiological; Awards and Prizes; Biomedical Research; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Models, Biological; Oxygen; Signal Transduction; Von Hippel-Lindau Tumor Suppressor Protein

Hypoxia induces innumerable changes in humans and other animals, including an increase in peripheral red blood cells (polycythemia) caused by the activation of erythropoiesis mediated by increased erythropoietin (EPO) production. However, the elevation of EPO is limited and levels return to normal ranges under normoxia within 5-7 days of exposure to hypoxia, whereas polycythemia continues for as long as hypoxia persists. We investigated erythropoiesis in bone marrow and spleens from mouse models of long-term normobaric hypoxia (10 % O2) to clarify the mechanism of prolonged polycythemia in chronic hypoxia. The numbers of erythroid colony-forming units (CFU-E) in the spleen remarkably increased along with elevated serum EPO levels indicating the activation of erythropoiesis during the first 7 days of hypoxia. After 14 days of hypoxia, the numbers of CFU-E returned to normoxic levels, whereas polycythemia persisted for >140 days. Flow cytometry revealed a prolonged increase in the numbers of TER119-positive cells (erythroid cells derived from pro-erythroblasts through mature erythrocyte stages), especially the TER119 (high) CD71 (high) population, in bone marrow. The numbers of annexin-V-positive cells among the TER119-positive cells particularly declined under chronic hypoxia, suggesting that the numbers of apoptotic cells decrease during erythroid cell maturation. Furthermore, RT-PCR analysis showed that the RNA expression of BMP-4 and stem cell factor that reduces apoptotic changes during erythroid cell proliferation and maturation was increased in bone marrow under hypoxia. These findings indicated that decreased apoptosis of erythroid cells during erythropoiesis contributes to polycythemia in mice during chronic exposure to long-term hypoxia.

Topics: Animals; Apoptosis; Bone Marrow Cells; Cell Survival; Erythropoiesis; Erythropoietin; Female; Flow Cytometry; Hypoxia; Mice, Inbred C57BL; Models, Animal; Polycythemia; Spleen; Time Factors

Free diving is associated with extreme hypoxia. This study evaluated the combined effect of maximal static breath holding and underwater swimming on plasma biomarkers of tissue hypoxemia: erythropoietin, neuron-specific enolase and S100B, C-reactive protein, pro-atrial natriuretic peptide, and troponin T. Venous blood samples were obtained from 17 competing free divers before and 3 h after sessions of static apnea and underwater swimming. The heart was evaluated by echocardiography. Static apnea for 293 ± 78 s (mean ± SD) and subsequent 88 ± 21 m underwater swimming increased plasma erythropoietin from 10.6 ± 3.4 to 12.4 ± 4.1 mIU/L (P = 0.013) and neuron-specific enolase from 14.5 ± 5.3 to 24.6 ± 6.4 ng/mL (P = 0.017); C-reactive protein decreased from 0.84 ± 1.0 to 0.71 ± 0.67 mmol/L (P = 0.013). In contrast, plasma concentrations of S100B (P = 0.394), pro-atrial natriuretic peptide (P = 0.549), and troponin T (P = 0.125) remained unchanged and, as assessed by echocardiography, the heart was not affected. In competitive free divers, bouts of static and dynamic apnea increase plasma erythropoietin and neuron-specific enolase, suggesting that renal and neural tissue, rather than the heart, is affected by the hypoxia developed during apnea and underwater swimming.

Topics: Adaptation, Physiological; Adult; Athletes; Atrial Natriuretic Factor; Breath Holding; C-Reactive Protein; Diving; Echocardiography; Erythropoietin; Female; Heart; Humans; Hypoxia; Male; Phosphopyruvate Hydratase; S100 Calcium Binding Protein beta Subunit; Swimming; Troponin T

Hypoxia, or a low concentration of O2, is encountered in humans undertaking activities such as mountain climbing and scuba diving and is important pathophysiologically as a limiting factor in tumor growth. Although data on the interplay between hypoxia and gastrins are limited, gastrin expression is upregulated by hypoxia in gastrointestinal cancer cell lines, and gastrins counterbalance hypoxia by stimulating angiogenesis in vitro and in vivo. The aim of this study was to determine if higher concentrations of the gastrin precursor progastrin are protective against hypoxia in vivo. hGAS mice, which overexpress progastrin in the liver, and mice of the corresponding wild-type FVB/N strain were exposed to normoxia or hypoxia. Iron status was assessed by measurement of serum iron parameters, real-time PCR for mRNAs encoding critical iron regulatory proteins, and Perls' stain and atomic absorption spectrometry for tissue iron concentrations. FVB/N mice lost weight at a faster rate and had higher sickness scores than hGAS mice exposed to hypoxia. Serum iron levels were lower in hGAS than FVB/N mice and decreased further when the animals were exposed to hypoxia. The concentration of iron in the liver was strikingly lower in hGAS than FVB/N mice. We conclude that increased circulating concentrations of progastrin provide a physiological advantage against systemic hypoxia in mice, possibly by increasing the availability of iron stores. This is the first report of an association between progastrin overexpression, hypoxia, and iron homeostasis.

Topics: Animals; Colon; Erythropoietin; Female; Gastrins; Gene Expression; Hypoxia; Iron; Liver; Male; Mice; Real-Time Polymerase Chain Reaction; RNA, Messenger

In mammals, hypoxia-triggered erythropoietin release increases red blood cell mass to meet tissue oxygen demands. Using male Wistar rats, we unmask a previously unrecognized regulatory pathway of erythropoiesis involving suppressor control by the NO metabolite and ubiquitous dietary component nitrate. We find that circulating hemoglobin levels are modulated by nitrate at concentrations achievable by dietary intervention under normoxic and hypoxic conditions; a moderate dose of nitrate administered via the drinking water (7 mg NaNO3/kg body weight/d) lowered hemoglobin concentration and hematocrit after 6 d compared with nonsupplemented/NaCl-supplemented controls. The underlying mechanism is suppression of hepatic erythropoietin expression associated with the downregulation of tissue hypoxia markers, suggesting increased pO2. At higher nitrate doses, however, a partial reversal of this effect occurred; this was accompanied by increased renal erythropoietin expression and stabilization of hypoxia-inducible factors, likely brought about by the relative anemia. Thus, hepatic and renal hypoxia-sensing pathways act in concert to modulate hemoglobin in response to nitrate, converging at an optimal minimal hemoglobin concentration appropriate to the environmental/physiologic situation. Suppression of hepatic erythropoietin expression by nitrate may thus act to decrease blood viscosity while matching oxygen supply to demand, whereas renal oxygen sensing could act as a brake, averting a potentially detrimental fall in hematocrit.

Topics: Animals; Dietary Supplements; Epoetin Alfa; Erythropoiesis; Erythropoietin; Hemoglobins; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoenzyme Techniques; Kidney; Liver; Male; Nitrates; Oxygen; Rats; Rats, Wistar; Recombinant Proteins

Erythropoietin stimulation modulates the central respiratory command in newborn mice. Specifically, the central respiratory depression induced by hypoxia is attenuated by acute (1h) or abolished by chronic erythropoietin stimulation. However, the underlying mechanisms remain unknown. As MEK and PI3K pathways are commonly involved in Epo-mediated effects of neuroprotection and erythropoiesis, we investigated here the implication of PI3K and MEK1/2 in the Epo-mediated regulation of the central respiratory command. To this end, in vitro brainstem-spinal cord preparations from 3 days old transgenic (Tg21; constitutively overexpressing erythropoietin in the brain specifically) and control mice were used. Our results show that blockade of PI3K or MEK1/2 stimulates normoxic bursts frequency in Tg21 preparations and abolish hypoxia-induced frequency depression in control preparations. These results show that MEK1/2 and PI3K pathways are involved in the Epo-mediated regulation of the central respiratory command. Moreover, this is the first demonstration that MEK1/2 and PI3K are involved in the brainstem central respiratory command.

Topics: Animals; Animals, Newborn; Butadienes; Central Nervous System; Chromones; Dose-Response Relationship, Drug; Enzyme Inhibitors; Erythropoietin; Hypoxia; In Vitro Techniques; Long-Term Synaptic Depression; MAP Kinase Kinase 1; Mice; Mice, Inbred C57BL; Mice, Transgenic; Morpholines; Motor Neurons; Nitriles; Phosphatidylinositol 3-Kinases; Respiration; Signal Transduction

Tissue hypoxia induces a systemic response designed to increase oxygen delivery to tissues. One component of this response is increased erythropoiesis. Steady-state erythropoiesis is primarily homeostatic, producing new erythrocytes to replace old erythrocytes removed from circulation by the spleen. In response to anemia, the situation is different. New erythrocytes must be rapidly made to increase hemoglobin levels. At these times, stress erythropoiesis predominates. Stress erythropoiesis is best characterized in the mouse, where it is extramedullary and utilizes progenitors and signals that are distinct from steady-state erythropoiesis. In this report, we use an in vitro culture system that recapitulates the in vivo development of stress erythroid progenitors. We identify cell-surface markers that delineate a series of stress erythroid progenitors with increasing maturity. In addition, we use this in vitro culture system to expand human stress erythroid progenitor cells that express analogous cell-surface markers. Consistent with previous suggestions that human stress erythropoiesis is similar to fetal erythropoiesis, we demonstrate that human stress erythroid progenitors express fetal hemoglobin upon differentiation. These data demonstrate that similar to murine bone marrow, human bone marrow contains cells that can generate BMP4-dependent stress erythroid burst-forming units when cultured under stress erythropoiesis conditions.

Topics: AC133 Antigen; Anemia; Animals; Antigens, CD; Antigens, CD34; Bone Marrow Transplantation; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; gamma-Globins; Glycoproteins; Hemoglobins; Humans; Hypoxia; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oxygen; Peptides; Stress, Physiological

Painschab, Matthew S., Gary E. Malpartida, Victor G. Davila-Roman, Robert H. Gilman, Todd M. Kolb, Fabiola Leon-Velarde, J. Jaime Miranda, and William Checkley. Association between serum concentrations of hypoxia inducible factor responsive proteins and excessive erythrocytosis in high altitude Peru. High Alt Med Biol 16:26-33, 2015.-Long-term residence at high altitude is associated with the development of chronic mountain sickness (CMS), which is characterized by excessive erythrocytosis (EE). EE occurs under chronic hypoxia, and a strongly selected mutation in hypoxia-inducible factor 2α (HIF2A) has been found in native Tibetans that correlates with having a normal hemoglobin at high altitude. We sought to evaluate differences in plasma levels of four HIF-responsive proteins in 20 participants with EE (hemoglobin >21 g/dL in men and >19 in women) and in 20 healthy, age- and sex-matched participants without EE living at high altitude in Puno, Peru. We performed ELISA to measure plasma levels of the four HIF-responsive proteins: vascular endothelial growth factor (VEGF), soluble VEGF receptor 1 (sVEGF-R1), endothelin-1, and erythropoietin. As a secondary aim, we evaluated the association between HIF-responsive proteins and echocardiography-estimated pulmonary artery systolic pressure (PASP) in a subset of 26 participants. sVEGF-R1 was higher in participants with vs. without EE (mean 107 pg/mL vs. 90 pg/mL; p=0.007). Although plasma concentrations of endothelin-1, VEGF, and erythropoietin were higher in participants with vs. without EE, they did not achieve statistical significance (all p>0.25). Both sVEGF-R1 (p=0.04) and erythropoietin (p=0.04) were positively associated with PASP after adjustment for age, sex, and BMI. HIF-responsive proteins may play a pathophysiological role in altitude-related, chronic diseases but our results did not show consistent changes in all measured HIF-responsive proteins. Larger studies are needed to evaluate for additional genetic and environmental risk factors.

Topics: Adult; Aged; Altitude; Altitude Sickness; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Blood Pressure; Case-Control Studies; Endothelin-1; Erythropoietin; Female; Humans; Hypoxia; Male; Middle Aged; Peru; Polycythemia; Pulmonary Artery; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors

We tested the hypothesis that endogenous brain Epo is a respiratory stimulant. Adult (3 mo) and newborn (10 days) male and female mice received an intracisternal (cisterna magna) injection of soluble Epo receptor (sEpoR; competes with EpoR to bind Epo; 50 μg/ml) or vehicle (0.1% BSA in PBS). Twenty-four hours after injection, we used whole body plethysmography to record minute ventilation (V̇e) tidal volume (VT), respiratory frequency (fR), O2 consumption (V̇o2), and CO2 production (V̇co2) under normoxia and progressive exposure to hypoxia (12-10-6% O2; 10 min each). In adult male and female mice sEpoR decreased normoxic V̇e (-25%), due to a decrease of VT in males and fR in females. Moreover, sEpoR injection decreased the ventilatory response to 12% O2, assessed as V̇e/V̇o2 or V̇e/V̇co2, in male but not in female mice. In newborn male and female mice sEpoR decreased V̇e (-37% in males, -59% in females) and VT (-38% in males, -47% in females) in normoxia and fR in females. During hypoxia, sEpoR decreased V̇e/V̇o2 and V̇e/V̇co2 in mice of both sexes. Upon extreme hypoxia (6% O2), the newborn mice treated with sEpoR showed respiratory depression, signs of asphyxia (gasping) and a high mortality rate in males and females. We concluded that endogenous brain Epo is a potent respiratory stimulant under normoxia and hypoxia in adult and newborn mice. Because sex-specific effects are different in newborn male and female, sex steroids secreted at different ages mice appear to modulate the effects of Epo on respiratory regulation in normoxia and in response to hypoxia.

Topics: Adaptation, Physiological; Age Factors; Animals; Animals, Newborn; Brain; Carbon Dioxide; Disease Models, Animal; Erythropoietin; Female; Hypoxia; Injections, Intraventricular; Male; Mice, Inbred C57BL; Oxygen Consumption; Pulmonary Ventilation; Receptors, Erythropoietin; Respiration; Respiratory Rate; Sex Factors; Signal Transduction; Tidal Volume; Time Factors

Adult animals that have been perinatally exposed to oxygen-rich atmospheres (hyperoxia), recalling those used for oxygen therapy in infants, exhibit a loss of hypoxic pulmonary vasoconstriction, whereas vasoconstriction elicited by depolarizing agents is maintained. Loss of pulmonary hypoxic vasoconstriction is not linked to alterations in oxygen-sensitive K(+) currents in pulmonary artery smooth muscle cells. Loss of hypoxic vasoconstriction is associated with early postnatal oxidative damage and corrected by an antioxidant diet. Perinatal hyperoxia damages carotid body chemoreceptor cell function and the antioxidant diet does not reverse it. The hypoxia-elicited increase in erythropoietin plasma levels is not affected by perinatal hyperoxia. The potential clinical significance of the findings in clinical situations such as pneumonia, chronic obstructive pulmonary disease or general anaesthesia is considered.. Adult mammalians possess three cell systems that are activated by acute bodily hypoxia: pulmonary artery smooth muscle cells (PASMC), carotid body chemoreceptor cells (CBCC) and erythropoietin (EPO)-producing cells. In rats, chronic perinatal hyperoxia causes permanent carotid body (CB) atrophy and functional alterations of surviving CBCC. There are no studies on PASMC or EPO-producing cells. Our aim is to define possible long-lasting functional changes in PASMC or EPO-producing cells (measured as EPO plasma levels) and, further, to analyse CBCC functional alterations. We used 3- to 4-month-old rats born and reared in a normal atmosphere or exposed to perinatal hyperoxia (55-60% O2 for the last 5-6 days of pregnancy and 4 weeks after birth). Perinatal hyperoxia causes an almost complete loss of hypoxic pulmonary vasoconstriction (HPV), which was correlated with lung oxidative status in early postnatal life and prevented by antioxidant supplementation in the diet. O2 -sensitivity of K(+) currents in the PASMC of hyperoxic animals is normal, indicating that their inhibition is not sufficient to trigger HPV. Perinatal hyperoxia also abrogated responses elicited by hypoxia on catecholamine and cAMP metabolism in the CB. An increase in EPO plasma levels elicited by hypoxia was identical in hyperoxic and control animals, implying a normal functioning of EPO-producing cells. The loss of HPV observed in adult rats and caused by perinatal hyperoxia, comparable to oxygen therapy in premature infants, might represent a previously unrecognized complication of such a medical intervention capable of aggravating medical conditions such as regional pneumonias, atelectases or general anaesthesia in adult life.

Topics: Animals; Antioxidants; Carotid Body; Erythropoietin; Female; Hyperoxia; Hypoxia; Pregnancy; Pulmonary Artery; Rats, Wistar; Vasoconstriction

This study aimed to observe the expression characteristics of the erythropoietin (EPO) gene in different tissues of Tibetan pigs and to explore the adaptation to hypoxic environments. The cDNA in heart, liver, lung, kidney, muscle, brain, and fat of Tibetan pigs was used as the template. Through the number of cycles of the polymerase chain reaction (PCR), annealing temperature, and system optimization, a stable and specific semi-quantitative PCR system was established. The EPO gene in different tissues of Tibetan pigs was detected using this system. The results showed that the EPO gene was expressed in heart, liver, lung, kidney, muscle, brain, and fat of the pigs. There were obvious differences in the expression in each tissue, and the expression sequence was as follows: kidney > muscle > lung > brain > liver > heart > fat. The results showed that the EPO gene was expressed in various tissues of Tibetan pigs. There were obvious differences in expression and each tissue may play a different regulatory role in the adaptation to hypoxia.

Topics: Adaptation, Physiological; Adipose Tissue; Animals; Brain; Erythropoietin; Gene Expression Regulation; Hypoxia; Kidney; Liver; Lung; Muscle, Skeletal; Myocardium; RNA, Messenger; Swine; Tibet

Erythropoietin (EPO), a regulator of red blood cell production, is endogenously expressed in the central nervous system. It is mainly produced by astrocytes under hypoxic conditions and has proven to have neuroprotective and neurotrophic effects. In the present study, we investigated the effect of midazolam on EPO expression in primary cultured astrocytes and the mouse brain. Midazolam was administered to 6-week-old BALB/c male mice under hypoxic conditions and pregnant C57BL/6N mice under normoxic conditions. Primary cultured astrocytes were also treated with midazolam under hypoxic conditions. The expression of EPO mRNA in mice brains and cultured astrocytes was studied. In addition, the expression of hypoxia-inducible factor (HIF), known as the main regulator of EPO, was evaluated. Midazolam significantly reduced the hypoxia-induced up-regulation of EPO in BALB/c mice brains and primary cultured astrocytes and suppressed EPO expression in the fetal brain. Midazolam did not affect the total amount of HIF proteins but significantly inhibited the nuclear expression of HIF-1α and HIF-2α proteins. These results demonstrated the suppressive effects of midazolam on the hypoxia-induced up-regulation of EPO both in vivo and in vitro.

Topics: Animals; Astrocytes; Basic Helix-Loop-Helix Transcription Factors; Brain; Cells, Cultured; Disease Models, Animal; Erythropoietin; Female; Fetal Hypoxia; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice, Inbred BALB C; Midazolam; Pregnancy; RNA, Messenger; Up-Regulation

Clinicians observed that the treatment of premature human newborns for anemia with erythropoietin (Epo) also improved their respiratory autonomy. This observation is in line with our previous in vitro studies showing that acute and chronic Epo stimulation enhances fictive breathing of brainstem-spinal cord preparations of postnatal day 3-4 mice during hypoxia. Furthermore, we recently reported that the antagonization of the cerebral Epo (by using the soluble Epo receptor; sEpoR) significantly reduced the basal ventilation and the hypoxic ventilatory response of 10 days old mice. In this study, we used transgenic (Tg21) mice to investigate the effect of the chronic cerebral Epo overexpression on the modulation of the normoxic and hypoxic ventilatory drive during the post-natal development. Ventilation was evaluated by whole body plethysmography at postnatal ages 3 (P3), 7 (P7), 15 (P15) and 21 (P21). In addition Epo quantification was performed by RIA and mRNA EpoR was evaluated by qRT-PCR. Our results showed that compared to control animals the chronic Epo overexpression stimulates the hypoxic (but not the normoxic) ventilation assessed as VE/VO2 at the ages of P3 and P21. More interestingly, we observed that at P7 and P15 the chronic Epo stimulation of ventilation was attenuated by the down regulation of the Epo receptor in brainstem areas. We conclude that Epo, by stimulating ventilation in brainstem areas crucially helps tolerating physiological (e.g., high altitude) and/or pathological (e.g., respiratory disorders, prematurity, etc.) oxygen deprivation at postnatal ages.

Topics: Age Factors; Analysis of Variance; Animals; Animals, Newborn; Cerebral Cortex; Erythropoietin; Female; Gene Expression; Hypoxia; Male; Mice; Mice, Transgenic; Plethysmography; Radioimmunoassay; Receptors, Erythropoietin; Sex Factors; Time Factors

A biomarker of hypoxic exposure would be useful in clinical diagnosis and prognosis. Acute hypoxia stimulates large increases in serum erythropoietin (EPO), and EPO induces formation of characteristic enlarged red blood cells (RBCs). The presence of large RBCs perturbs red cell distribution width (RDW).. Using a >2M patient medical claims database, the human pathome was scanned for diseases where RDW rose 0-50days following a new diagnosis. The course of RDW after selected diagnoses was visualized by registering RDW measurements by diagnosis date.. Acute hemorrhage, which provokes EPO-driven erythropoiesis, is followed by increases in RDW but not mean cell volume (MCV). Similar RDW increases follow many acute diseases with risk of hypoxia, including heart failure, pneumonia, atelectasis, pulmonary embolism, pneumothorax, and sepsis. Elevations reach maximum within 1month after onset and subside to pre-disease levels about 6months later. Unlike the case with iron-deficiency anemia (IDA), RDW elevations after hypoxia-associated diseases are unaccompanied by discernible change in average RBC size.. As predicted by a model risk pathway linking hypoxia to formation of enlarged RBCs via EPO, acute hypoxemia-related disease episodes induce change in RBC size distribution. Further study is needed to explore whether a more sensitive and specific signal can be extracted from the fine structure of the RBC size distribution routinely measured in automated hemocytometers.

Topics: Acute Disease; Biomarkers; Cell Size; Erythrocyte Indices; Erythrocytes; Erythropoietin; Humans; Hypoxia

Hepcidin is the main regulator of systemic iron homeostasis and its expression is modulated by iron status, hypoxia, erythroid factors and inflammation. The aim of our study was to examine a relationship between level of hepcidin and iron status, erythropoietic activity, hypoxia and inflammation in exacerbations and stable COPD patients. We hypothesized that hepcidin concentration is changed in COPD patients and is substantially influenced by inflammation and/or hypoxia.. The study included 40 COPD patients and 30 healthy subjects. We measured haemoglobin, serum level of hepcidin and parameters indicative for inflammation: interleukin-6 (IL-6) and C reactive protein (CRP); hypoxia: partial oxygen pressure and haemoglobin oxygen saturation; iron status: iron, total iron binding capacity (TIBC), transferring saturation and ferritin; and erythropoietic activity: soluble transferrin receptors, reticulocytes, and erythropoietin.. Hepcidin was elevated in exacerbations and in a stable phase compared to the control group and we found positive correlations of hepcidin with inflammatory markers IL-6 and CRP. Hepcidin also correlated positively with ferritin and inversely with TIBC. However, in COPD patients reticulocyte count was significantly reduced and negative correlation with hepcidin was established in exacerbation. No correlations were observed with iron, or indices of hypoxia. In the control group, positive associations were observed only with indices of iron status, positive with ferritin and negative one with TIBC.. Systemic inflammation and elevated values of IL-6 present in exacerbations and stabile COPD might be responsible for the observed increased hepcidin level.

Topics: Aged; Aged, 80 and over; C-Reactive Protein; Case-Control Studies; Disease Progression; Erythropoietin; Female; Gene Expression; Hemoglobins; Hepcidins; Humans; Hypoxia; Inflammation; Interleukin-6; Iron; Male; Middle Aged; Oxygen; Pulmonary Disease, Chronic Obstructive; Receptors, Transferrin; Reticulocytes; Transferrin

Topics: Erythropoietin; Humans; Hypoxia; Neuroprotective Agents

Anemia during pregnancy is a risk factor of disturbance in pre- and postnatal child's development. Hepcidin plays the key role in iron metabolism, as protein participating in the regulation of intestinal absorption of this element and its release from macrophages, and transport across the placenta. Maternal smoking during pregnancy can result in disturbances of iron homeostasis leading to a subclinical deficiency. The depletion of maternal iron can cause fetal hypoxia condition and decreased expression of hepcidin. The aim of the study was to evaluate the effect of smoking on the levels of hepcidin and erythropoietin (as an indicator of hypoxia) and their relationships in umbilical cord blood. The research material was the umbilical cord blood of 50 newborns born in the Department of Obstetrics and Gynecology (Institute of Mother and Child in Warsaw) in the years 2013-2014. Based on an interview and determination of cotinine in the blood of mothers, newborns were divided into following groups: children of smoking mothers (n=20) and children of tobacco abstinent mothers (n=30). Hepcidin and erythropoietin concentrations were determined by enzyme immunoassay using commercial kits (DRG, Germany). It has been shown that hepcidin concentrations were significantly lower in children of smoking mothers than in the group of tobacco abstinent (37.5 ng/mL vs 45.1 ng/mL, p<0.001). However, the level of erythropoietin was higher in children of smoking mothers than in children of non-smoking women (p<0.001). A negative correlation between the levels of hepcidin and erythropoietin (r = -0.41, p<0.05) and number of smoked cigarettes (r = -0.43, p<0.05) was observed. These results indicate that smoking during pregnancy significantly affects hepcidin levels in children born at term. Decrease of hepcidin concentration coexisting with high level of erythropoletin in umbilical cord blood in children of smoking pregnant women may be the cause of subclinical deficiency of iron in the newborn.

Topics: Adult; Erythropoietin; Female; Fetal Blood; Hepcidins; Humans; Hypoxia; Infant, Newborn; Pregnancy; Smoking; Umbilical Cord; Young Adult

Tibetan natives have lived on the Tibetan plateau (altitude ∼ 4,000 m) for at least 25,000 years, and as such they are adapted to life and reproduction in a hypoxic environment. Recent studies have identified two genetic loci, EGLN1 and EPAS1, that have undergone natural selection in Tibetans, and further demonstrated an association of EGLN1/EPAS1 genotype with hemoglobin concentration. Both genes encode major components of the hypoxia-inducible factor (HIF) transcriptional pathway, which coordinates an organism's response to hypoxia. Patients living at sea level with genetic disease of the HIF pathway have characteristic phenotypes at both the integrative-physiology and cellular level. We sought to test the hypothesis that natural selection to hypoxia within Tibetans results in related phenotypic differences. We compared Tibetans living at sea level with Han Chinese, who are Tibetans' most closely related major ethnic group. We found that Tibetans had a lower hemoglobin concentration, a higher pulmonary ventilation relative to metabolism, and blunted pulmonary vascular responses to both acute (minutes) and sustained (8 h) hypoxia. At the cellular level, the relative expression and hypoxic induction of HIF-regulated genes were significantly lower in peripheral blood lymphocytes from Tibetans compared with Han Chinese. Within the Tibetans, we found a significant correlation between both EPAS1 and EGLN1 genotype and the induction of erythropoietin by hypoxia. In conclusion, this study provides further evidence that Tibetans respond less vigorously to hypoxic challenge. This is evident at sea level and, at least in part, appears to arise from a hyporesponsive HIF transcriptional system.

Topics: Acclimatization; Adult; Altitude; Asian People; Basic Helix-Loop-Helix Transcription Factors; Cells, Cultured; China; Erythropoietin; Gene Expression Regulation; Haplotypes; Hemoglobins; Humans; Hypoxia; Hypoxia-Inducible Factor-Proline Dioxygenases; Male; Oxygen; Phenotype; Pulmonary Artery; Pulmonary Ventilation; Selection, Genetic; Tibet; Time Factors; Transcription, Genetic; Vasoconstriction; Young Adult

Exposure to intermittent hypoxia (IH) is associated with cognitive impairments and oxidative stress in brain regions involved in learning and memory. In earlier studies, erythropoietin (EPO) showed a neuroprotective effect in large doses. The aim of the present study was to explore the effect of smaller doses of EPO, such as those used in the treatment of anemia, on IH-induced cognitive deficits and hippocampal oxidative stress in young rats. The effect of concurrent EPO treatment (500 and 1,000 IU/kg/day ip) on spatial learning and memory deficits induced by long-term exposure to IH for 6 weeks was tested using the Morris water maze (MWM) test and the elevated plus maze (EPM) test. Moreover, the effect on hippocampal glutamate and oxidative stress were assessed. Exposure to IH induced a significant impairment of spatial learning and cognition of animals in both MWM and EPM performance parameters. Moreover, hippocampal glutamate and thiobarbituric acid reactive substances (TBARS) increased while antioxidant defenses (GSH and GSH-Px) decreased. EPO in the tested doses significantly reduced the IH-induced spatial learning deficits in both MWM and EPM tests and dose-dependently antagonized the effects of IH on hippocampal glutamate, TBARS, GSH levels, and GSH-Px activity. Treatment with EPO in moderate doses that used for anemia, concurrently with IH exposure can antagonize IH-induced spatial learning deficits and protect hippocampal neurons from IH-induced lipid peroxidation and oxidative stress-induced damage in young rats, possibly through multiple mechanisms involving a potential antioxidative effect.

Topics: Animals; Epoetin Alfa; Erythropoietin; Glutathione; Glutathione Peroxidase; Hippocampus; Hypoxia; Male; Maze Learning; Memory Disorders; Oxidative Stress; Rats; Recombinant Proteins

The cytokine erythropoietin (Epo) initiates adaptive cellular responses to both moderate environmental challenges and tissue damaging insults in various non-hematopoietic mammalian tissues including the nervous system. Neuroprotective and neuroregenerative functions of Epo in mammals are mediated through receptor-associated Janus kinase 2 and intracellular signaling cascades that modify the transcription of Epo-regulated genes. Signal transducers and activators of transcription (STAT) and phosphoinositol-3-kinase (PI3K) represent key components of two important Epo-induced transduction pathways. Our previous study on insects revealed neuroprotective and regenerative functions of recombinant human Epo (rhEpo) similar to those in mammalian nervous tissues. Here we demonstrate that rhEpo effectively rescues primary cultured locust brain neurons from apoptotic cell death induced by hypoxia or the chemical compound H-7. The Janus kinase inhibitor AG-490 and the STAT inhibitor sc-355797 abolished protective effects of rhEpo on locust brain neurons. In contrast, inhibition of PI3K with LY294002 had no effect on rhEpo-mediated neuroprotection. The results indicate that rhEpo mediates the protection of locust brain neurons through interference with apoptotic pathways by the activation of a Janus kinase-associated receptor and STAT transcription factor(s). The involvement of similar transduction pathways in mammals and insects for the mediation of neuroprotection and support of neural regeneration by Epo indicates that an Epo/Epo receptor-like signaling system with high structural and functional similarity exists in both groups of animals. Epo-like signaling involved in tissue protection appears to be an ancient beneficial function shared by vertebrates and invertebrates.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Apoptosis; Brain; Cell Survival; Cells, Cultured; Chromones; Enzyme Inhibitors; Erythropoietin; Grasshoppers; Humans; Hypoxia; Insect Proteins; Janus Kinase 2; Morpholines; Neurons; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; STAT Transcription Factors; Tyrphostins

Erythropoietin (EPO) is a hormone that induces red blood cell production. In its recombinant form, EPO is the one of most prescribed drugs to treat anemia, including that arising in cancer patients. In randomized trials, EPO administration to cancer patients has been associated with decreased survival. Here, we investigated the impact of EPO modulation on tumorigenesis. Using genetically engineered mouse models of breast cancer, we found that EPO promoted tumorigenesis by activating JAK/STAT signaling in breast tumor-initiating cells (TICs) and promoted TIC self renewal. We determined that EPO was induced by hypoxia in breast cancer cell lines, but not in human mammary epithelial cells. Additionally, we demonstrated that high levels of endogenous EPO gene expression correlated with shortened relapse-free survival and that pharmacologic JAK2 inhibition was synergistic with chemotherapy for tumor growth inhibition in vivo. These data define an active role for endogenous EPO in breast cancer progression and breast TIC self-renewal and reveal a potential application of EPO pathway inhibition in breast cancer therapy.

Topics: Animals; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Disease Progression; Disease-Free Survival; Endothelial Cells; Erythropoietin; Female; Gene Expression Regulation; Humans; Hypoxia; Mammary Neoplasms, Experimental; Mice; Neoplasm Transplantation; Neoplastic Stem Cells; Recurrence; Signal Transduction; Tetrazolium Salts; Thiazoles; Time Factors

Adaptation of cardiomyocytes to chronic hypoxia in cyanotic patients remains unclear. Mitochondrial biogenesis is enhanced in myocardium from cyanotic patients, which is possibly an adaptive response. Erythropoietin (EPO) in blood and its receptor (EPOR) on cardiomyocytes are upregulated by chronic hypoxia, suggesting that EPO-EPOR interaction is increased, which is inferred to positively regulate mitochondrial biogenesis through protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) signalling pathway. H9c2 cardiomyocytes were exposed to hypoxia (1% O(2)) for 1 week and treated with different doses of recombinant human erythropoietin (rhEPO). Mitochondrial number, mitochondrial DNA (mtDNA) copy number and peroxisome proliferator activated receptor gamma coactivator alpha (PGC-1α) mRNA expression increased in a dose-dependent manner induced by rhEPO. Akt and eNOS were significantly phosphorylated by rhEPO. Both blocking Akt with Wortmannin and silencing eNOS expression with shRNA plasmid decreased the mtDNA copy number and PGC-1α mRNA expression induced by rhEPO. Blocking Akt was associated with the decreased phosphorylation of Akt and eNOS. RNA interference led to a reduction in the total and phosphorylated proteins of eNOS. Thus EPO enhances mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia, at least partly through Akt/eNOS signalling, which might be an adaptive mechanism of cardiomyocytes associated with the increased EPO-EPOR interaction in patients with cyanotic congenital heart disease (CCHD).

Topics: Animals; Erythropoietin; Heart; Humans; Hypoxia; Mitochondria; Myocardium; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Transcription Factors; Up-Regulation

Gene therapy has been considered a promising approach for glioblastoma therapy. To avoid side effects and increase the specificity of gene expression, gene expression should be tightly regulated. In this study, glioma and hypoxia dual-specific plasmids (pEpo-NI2-SV-Luc and pEpo-NI2-SV-HSVtk) were developed by combining the erythropoietin (Epo) enhancer and nestin intron 2 (NI2). In the in vitro studies, pEpo-NI2-SV-Luc showed higher gene expression under hypoxia than normoxia in a glioblastoma-specific manner. The MTT and caspase assays demonstrated that pEpo-NI2-SV-HSVtk specifically induced caspase activity and cell death in hypoxic glioblastoma cells. For in vivo evaluation, subcutaneous and intracranial glioblastoma models were established. Dexamethasone-conjugated-polyethylenimine (PEI-Dexa) was used as a gene carrier, since PEI-Dexa efficiently delivers plasmid to glioblastoma cells and also has an antitumor effect due to the effect of dexamethasone. In the in vivo study in the subcutaneous and intracranial glioblastoma models, the tumor size was reduced more effectively in the pEpo-NI2-SV-HSVtk group than in the control and pSV-HSVtk groups. In addition, higher levels of HSVtk gene expression and TUNEL-positive cells were observed in the pEpo-NI2-SV-HSVtk group compared with the control and pSV-HSVtk groups, suggesting that pEpo-NI2-SV-HSVtk increased the therapeutic efficacy in hypoxic glioblastoma. Therefore, pEpo-NI2-SV-HSVtk/PEI-Dexa complex may be useful for glioblastoma-specific gene therapy.

Topics: Animals; Apoptosis; Blotting, Western; Cell Proliferation; Cells, Cultured; Dexamethasone; Drug Carriers; Electrophoretic Mobility Shift Assay; Enhancer Elements, Genetic; Epoetin Alfa; Erythropoietin; Flow Cytometry; Gene Transfer Techniques; Genes, Transgenic, Suicide; Glioblastoma; HEK293 Cells; Humans; Hypoxia; Immunoenzyme Techniques; Introns; Luciferases; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nestin; Plasmids; Polyethyleneimine; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Thymidine Kinase; Tissue Distribution; Transfection; Xenograft Model Antitumor Assays

Topics: 5' Untranslated Regions; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Oxygen; Response Elements

Ghrelin, a 28 amino acid peptide, has diverse effects in body organs. Erythropoietin is a key mediator in increasing the red blood cells during hypoxia. Previously, we have shown that ghrelin has a polycythemic effect. In the present study, we evaluated the effect of ghrelin on erythropoietin gene expression with the aim to find out the mechanism of its effect.. Thirty two adult male Wistar rats were divided randomly into four groups. The hypoxic condition was induced by placing the rats into the hypoxic chamber with 11% oxygen for two weeks. Saline- and ghrelin-treated control rats remained in room with a regular air conditions. Erythropoietin gene expression was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). Plasma erythropoietin was measured by enzyme linked immunosorbent assay (ELISA).. After 2-weeks of hypoxia, erythropoietin transcripts and erythropoietin plasma levels were significantly increased in hypoxic animals compared with control animals. Ghrelin treatment decreased both plasma erythropoietin and erythropoietin gene expression only in the hypoxic rats.. Our data indicate that ghrelin might induce polycythemia through an erythropoietin-independent manner. However, to confirm this hypothesis and to find out the precise mechanism of this phenomenon further investigations are needed.

Topics: Animals; Chronic Disease; Erythropoietin; Gene Expression; Ghrelin; Hematocrit; Hypoxia; Kidney; Male; Random Allocation; Rats; Rats, Wistar

Hypoxic ischemic encephalopathy continues to be a significant cause of death and disability worldwide. Erythropoietin (EPO) has the potential to lessen neurologic sequelae due to hypoxia-ischemia.. The in vitro effects of EPO on total embryonic development and brain VEGF receptor (VEGFR) expressions were investigated in 50 rat embryos at 9.5 days of gestation that were cultured in whole rat serum (WRS). According to the study protocol, the embryos were divided into two groups. The first group is comprised hypoxia, 100 and 50 U/ml EPO after hypoxia groups. Group 2 comprised control (WRS) and WRS+EPO. After 48-h culture, the embryos from each group were harvested to be analyzed according to a morphological scoring system and also genetically to measure brain VEGFR expression.. The mean morphological scores for the embryos grown in control, WRS+EPO, hypoxia, and in the presence of 100 and 50 U/ml EPO in hypoxic medium were 55.30±7.22, 52.10±5.27, 23.0±4.60, 36.20±5.07, and 19.70±5.07, respectively. Expressions of VEGFR-1, -2, -3 were significantly elevated in the 100U/ml EPO and WRS+EPO groups compared to the hypoxia group (p<0.05).. These results support the conclusion that (1) VEGFR-1, -2, -3 may increase with EPO treatment in hypoxic conditions, (2) VEGF and EPO may be part of a self-regulated physiological protection mechanism to prevent neuronal injury including in utero neural tube defects.

Topics: Animals; Brain; Embryo, Mammalian; Erythropoietin; Gene Expression Regulation, Developmental; Hypoxia; In Vitro Techniques; Organ Culture Techniques; Rats; Receptors, Vascular Endothelial Growth Factor; RNA, Messenger

Erythropoietin (EPO) is mainly synthesized within renal peritubular fibroblasts, and also other tissues such as the liver possess the ability. However, to what extent non-kidney produced EPO contributes to the hypoxia-induced increase in circulating EPO in adult humans remains unclear.. We aimed to quantify this by assessing the distribution of EPO glycoforms which are characterized by posttranslational glycosylation patterns specific to the synthesizing cell. The analysis was performed on samples obtained in seven healthy volunteers before, during and after 1 month of sojourn at 3,454 m altitude.. Umbilical cord (UC) plasma served as control. As expected a peak (p < 0.05) in urine (2.3 ± 0.5-fold) and plasma (3.3 ± 0.5-fold) EPO was observed on day 1 of high-altitude exposure, and thereafter the concentration decreased for the urine sample obtained after 26 days at altitude, but remained elevated (p < 0.05) by 1.5 ± 0.2-fold above the initial sea level value for the plasma sample. The EPO glycoform heterogeneity, in the urine samples collected at altitude, did not differ from values at sea level, but were markedly lower (p < 0.05) than the mean percent migrated isoform (PMI) for the umbilical cord samples.. Our studies demonstrate (1) UC samples express a different glycoform distribution as compared to adult humans and hence illustrates the ability to synthesis EPO in non-kidney cells during fetal development (2) as expected hypoxia augments circulating EPO in adults and the predominant source here for remains being kidney derived.

Topics: Adult; Altitude; Case-Control Studies; Erythropoietin; Glycosylation; Humans; Hypoxia; Kidney; Male; Protein Processing, Post-Translational

The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in pathogenesis. The bi-directionality of the interaction between both pathways has been shown on physiological and only recently also on molecular level. But the consequences of a disturbed circadian rhythm for the hypoxic response and the cardiovascular system have never been addressed in any organism. Here we show that the hypoxic response of animals subjected to chronodisruption is reduced by approximately 30%, as reflected by decreased expression levels of hypoxia inducible factor 1 and its down-stream target genes erythropoietin, responsible for the generation of red blood cells (RBC) and vascular endothelial growth factor, which is essential for proper vascularization. Beside malformations of their vascular beds, chronodisrupted animals surprisingly revealed elevated numbers of senescent erythrocytes under normoxic conditions, due to a reduced clearance rate via apoptosis. Over-aged erythrocytes in turn are characterized by decreased oxygen transport capacities and an increased tendency for aggregation, explaining the higher mortality of chronodisrupted animals observed in our study. The present study shows for the first time that chronodisruption strongly interferes with the hypoxic signalling cascade, increasing the cardiovascular risk in zebrafish due to elevated proportions of senescent erythrocytes. The results might shed new light on the etiology of the increased cardiovascular risk observed among shiftworkers.

Topics: Animals; Apoptosis; Cardiovascular Diseases; Cellular Senescence; Chronobiology Disorders; Circadian Rhythm; Erythrocytes; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Light; Photoperiod; Risk Factors; Time Factors; Vascular Endothelial Growth Factor A; Zebrafish; Zebrafish Proteins

Erythropoietin (EPO) is a glycoprotein hormone essential for the regulation of erythroid homeostasis. Although EPO production is prominent in the kidney and liver, its production in the central nervous system has also been detected. Tissue hypoxia due to systemic or local hypoxemia and acute anemia due to blood loss occurs frequently during various clinical settings, leading to a high possibility of elevated plasma EPO levels. However, it is largely unknown whether volatile anesthetic agents affect EPO production elicited by acute hypoxia in vivo. Male C57BL/6N CrSlc mice were exposed to a hypoxic insult as a result of bleeding-related anemia or hypoxemia while they were under anesthetized using various concentrations of isoflurane. EPO protein concentrations were assessed by enzyme-linked immunosorbent assay and mRNA levels were measured by quantitative real-time reverse transcriptase-polymerase chain reaction. Plasma EPO concentration was induced as early as 3h following acute anemic and hypoxemic hypoxia and suppressed by clinically relevant doses of isoflurane in a dose-dependent manner. Anemic hypoxia induced EPO mRNA and protein synthesis in the kidney. In the kidney, isoflurane inhibited EPO induction caused by anemia but not that caused by hypoxemia. On the other hand, in the brain hypoxemia-induced EPO production was suppressed by isoflurane. Finally, qRT-PCR studies demonstrate that isoflurane differentially inhibit HIF-1α and HIF-2α mRNA expression in brain and kidney, indicating the involvement of HIF-2 in the hypoxia-induced EPO expression and inhibition of the induction by isoflurane.

Topics: Anemia; Anesthetics, Inhalation; Animals; Basic Helix-Loop-Helix Transcription Factors; Brain Chemistry; Erythropoietin; Hemodynamics; Hypoxia; Isoflurane; Kidney; Male; Mice; Mice, Inbred C57BL

The aims of this study were to determine which of the two biomarkers of renal injury, kidney injury molecule-1 or cystatin C, is more sensitive and to evaluate whether erythropoietin protects kidneys injured by perinatal asphyxia.. Animals were split into three groups designated as follows: AE, pups that survived perinatal asphyxia and subsequently received 2.5 μg (0.1 ml) of darbepoetin-α (i.p.); A, the pups that survived perinatal asphyxia and received 0.1 ml of 0.9% NaCl; and C, control group. The pups were killed at different ages of life (6 h, 24 h, 48 h, 7 d, and 14 d of age; 10 rats in each subgroup). Immunohistopathological evaluation of kidneys was performed.. At 48 h and on days 7 and 14, absolute injury scores were significantly lower in group AE as measured by both biomarkers. Cystatin C expression was the most intensive 6 h after the hypoxic event (average value of absolute injury score was 2.82) and declined over time. Expression of kidney injury molecule-1 was less intensive, with the average value of absolute injury score being 2.02 at 6 h and 2.105 at 24 h; the peak value (2.155) was recorded 48 h after the hypoxic event.. Erythropoietin has a protective effect on hypoxic kidneys. Cystatin C is more sensitive as an early biomarker of acute kidney injury in comparison with kidney injury molecule-1.

Topics: Animals; Animals, Newborn; Asphyxia Neonatorum; Biomarkers; Cell Adhesion Molecules; Cystatin C; Darbepoetin alfa; Erythropoietin; Female; Hypoxia; Kidney; Male; Rats; Rats, Wistar; Time Factors; Treatment Outcome

Tissue hypoxia likely contributes to anemia-induced organ injury and mortality. Severe anemia activates hypoxia-inducible factor (HIF) signaling by hypoxic- and neuronal nitric oxide (NO) synthase- (nNOS) dependent mechanisms. However, organ-specific hemoglobin (Hb) thresholds for increased HIF expression have not been defined. To assess organ-specific Hb thresholds for tissue hypoxia, HIF-α (oxygen-dependent degradation domain, ODD) luciferase mice were hemodiluted to mild, moderate, or severe anemia corresponding to Hb levels of 90, 70, and 50 g/l, respectively. HIF luciferase reporter activity, HIF protein, and HIF-dependent RNA levels were assessed. In the brain, HIF-1α was paradoxically decreased at mild anemia, returned to baseline at moderate anemia, and then increased at severe anemia. Brain HIF-2α remained unchanged at all Hb levels. Both kidney HIF-1α and HIF-2α increased earlier (Hb ∼70-90 g/l) in response to anemia. Liver also exhibited an early HIF-α response. Carotid blood flow was increased early (Hb ∼70, g/l), but renal blood flow remained relatively constant, only increased at Hb of 50 g/l. Anemia increased nNOS (brain and kidney) and endothelia NOS (eNOS) (kidney) levels. Whereas anemia-induced increases in brain HIFα were nNOS-dependent, our current data demonstrate that increased renal HIFα was nNOS independent. HIF-dependent RNA levels increased linearly (∼10-fold) in the brain. However, renal HIF-RNA responses (MCT4, EPO) increased exponentially (∼100-fold). Plasma EPO levels increased near Hb threshold of 90 g/l, suggesting that the EPO response is sensitive. Collectively, these observations suggest that each organ expresses a different threshold for cellular HIF/NOS hypoxia responses. This knowledge may help define the mechanism(s) by which the brain and kidney maintain oxygen homeostasis during anemia.

Topics: Acute Disease; Anemia; Animals; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Brain; Cerebrovascular Circulation; Disease Models, Animal; Erythropoietin; Hemodilution; Hemodynamics; Hemoglobins; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Liver; Luciferases, Firefly; Mice; Mice, Transgenic; Monocarboxylic Acid Transporters; Muscle Proteins; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Recombinant Fusion Proteins; Renal Circulation; Severity of Illness Index

The aim of this study was to examine if erythropoietin (EPO) has the potential to act as a biological antioxidant and determine the underlying mechanisms.. The rate at which its recombinant form (rHuEPO) reacts with hydroxyl (HO˙), 2,2-diphenyl-1-picrylhydrazyl (DPPH˙) and peroxyl (ROO˙) radicals was evaluated in-vitro. The relationship between the erythopoietic and oxidative-nitrosative stress response to poikilocapneic hypoxia was determined separately in-vivo by sampling arterial blood from eleven males in normoxia and following 12 h exposure to 13% oxygen. Electron paramagnetic resonance spectroscopy, ELISA and ozone-based chemiluminescence were employed for direct detection of ascorbate (A(˙-) ) and N-tert-butyl-α-phenylnitrone spin-trapped alkoxyl (PBN-OR) radicals, 3-nitrotyrosine (3-NT) and nitrite (NO2-).. We found rHuEPO to be a potent scavenger of HO˙ (kr = 1.03-1.66 × 10(11) m(-1) s(-1) ) with the capacity to inhibit Fenton chemistry through catalytic iron chelation. Its ability to scavenge DPPH˙ and ROO˙ was also superior compared to other more conventional antioxidants. Hypoxia was associated with a rise in arterial EPO and free radical-mediated reduction in nitric oxide, indicative of oxidative-nitrosative stress. The latter was confirmed by an increased systemic formation of A˙(-) , PBN-OR, 3-NT and corresponding loss of NO2- (P < 0.05 vs. normoxia). The erythropoietic and oxidative-nitrosative stress responses were consistently related (r = -0.52 to 0.68, P < 0.05).. These findings demonstrate that EPO has the capacity to act as a biological antioxidant and provide a mechanistic basis for its reported cytoprotective benefits within the clinical setting.

Topics: Adult; Antioxidants; Electron Spin Resonance Spectroscopy; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Humans; Hypoxia; Luminescence; Male; Nitrosation; Oxidative Stress

Previous studies indicated that erythropoietin modulates central respiratory command in mice. Specifically, a one-hour incubation of the brainstems with erythropoietin attenuates hypoxia-induced central respiratory depression. Here, using transgenic mice constitutively overexpressing erythropoietin specifically in the brain (Tg21), we investigated the effect of chronic erythropoietin stimulation on central respiratory command activity during post-natal development. In vitro brainstem-spinal cord preparations from mice at 0 (P0) or 3 days of age (P3) were used to record the fictive inspiratory activity from the C4 ventral root. Our results show that erythropoietin already stimulates the hypoxic burst frequency at P0, and at P3, erythropoietin effectively stimulates the hypoxic burst frequency and amplitude. Because the maturation of the central respiratory command in mice is characterized by a decrease in the burst frequency with age, our results also suggest that erythropoietin accelerates the maturation of the newborn respiratory network and its response to hypoxia.

Topics: Animals; Animals, Newborn; Brain; Cervical Vertebrae; Erythropoietin; Female; Hypoxia; Male; Mice, Inbred C57BL; Mice, Transgenic; Microelectrodes; Respiration; Spinal Cord; Spinal Nerve Roots; Tissue Culture Techniques

It is well known that cobalt chloride (CoCl2) can enhance the stability of hypoxia-inducible factor (HIF)-1α. The aim of this study is to detect the effect of CoCl2 on the hypoxia tolerance of mice which were repeatedly exposed to autoprogressive hypoxia. Balb/c mice were randomly divided into groups of chemical pretreatment and normal saline (NS), respectively injected with CoCl2 and NS 3 h before exposure to hypoxia for 0 run (H0), 1 run (H1), and 4 runs (H4). Western Blot, electrophoretic mobility shift assay (EMSA), extracellular recordings population spikes in area cornus ammonis I (CA 1) of mouse hippocampal slices and real-time were used in this study. Our results demonstrated that the tolerance of mice to hypoxia, the changes of HIF-1α protein level and HIF-1 DNA binding activity in mice hippocampus, the mRNA level of erythropoietin (EPO) and vascular endothelial growth factor (VEGF), and the disappearance time of population spikes of hippocampal slices were substantially different between the control group and the CoCl2 group. Over-induction of HIF-1α by pretreatment with CoCl2 before hypoxia did not increase the hypoxia tolerance.

Topics: Animals; Cobalt; DNA; Erythropoietin; Hippocampus; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Models, Animal; Protein Binding; RNA, Messenger; Vascular Endothelial Growth Factor A

Hepcidin transcription is strongly down-regulated under hypoxic conditions, however whether hypoxia inhibits hepcidin directly or indirectly is still unknown. We investigated the time course of hypoxia-mediated hepcidin down-regulation in vivo in healthy volunteers exposed to hypobaric hypoxia at high altitude and, based on the hypothesis that circulating factors are implicated in hepcidin inhibition, we analyzed the effect of sera of these volunteers exposed to normoxia and hypoxia on hepcidin expression in Huh-7 cell lines. Hypoxia led to a significant hepcidin down-regulation in vivo that was almost complete within 72h of exposure and followed erythropoietin induction. This delay in hepcidin down-regulation suggests the existence of soluble factor/s regulating hepcidin production. We then stimulated HuH-7 cells with normoxic and hypoxic sera to analyze the effects of sera on hepcidin regulation. Hypoxic sera had a significant inhibitory effect on hepcidin promoter activity assessed by a luciferase assay, although the amount of such decrease was not as relevant as that observed in vivo. Cellular mRNA analysis showed that a number of volunteers' sera inhibited hepcidin expression, concurrently with ID1 inhibition, suggesting that inhibitory factor(s) may act through the SMAD-pathway.

Topics: Adult; Altitude; Biological Factors; Cell Line; Erythropoietin; Female; Gene Expression Regulation; Genes, Reporter; Hepatocytes; Hepcidins; Humans; Hypoxia; Inhibitor of Differentiation Protein 1; Luciferases; Male; Promoter Regions, Genetic; RNA, Messenger; Signal Transduction; Smad Proteins

Topics: Antioxidants; Erythropoietin; Humans; Hypoxia; Male; Oxidative Stress

Interest in erythropoietin (EPO) as a neuroprotective mediator has grown since it was found that systemically administered EPO is protective in several animal models of disease. However, given that the blood-brain barrier limits EPO entry into the brain, alternative approaches that induce endogenous EPO production in the brain may be more effective clinically and associated with fewer untoward side-effects. Astrocytes are the main source of EPO in the central nervous system. In the present study we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNFα) on hypoxia-induced upregulation of EPO in rat brain. Hypoxia significantly increased EPO mRNA expression in the brain and kidney, and this increase was suppressed by TNFα in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12 h, TNFα suppressed the hypoxia-induced increase in EPO mRNA expression in a concentration-dependent manner. TNFα inhibition of hypoxia-induced EPO expression was mediated primarily by hypoxia-inducible factor (HIF)-2α rather than HIF-1α. The effects of TNFα in reducing hypoxia-induced upregulation of EPO mRNA expression probably involve destabilization of HIF-2α, which is regulated by the nuclear factor (NF)-κB signaling pathway. TNFα treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNFα signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response.

Topics: Animals; Apoptosis; Astrocytes; Basic Helix-Loop-Helix Transcription Factors; Cell Hypoxia; Cells, Cultured; Cerebral Cortex; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Male; Neurons; Rats, Wistar; RNA, Messenger; Tumor Necrosis Factor-alpha

Low oxygen conditions, termed hypoxia, can affect cell survivals. Cells may adapt to hypoxic conditions through hypoxia response elements (HRE) such as erythropoietin enhancer or phosphoglycerate kinase element. Hypoxic conditions usually appear in solid tumors, and can cause resistance to radiotherapy or chemotherapy. In this study, a genetic approach based upon Tetrahymena group I ribozyme was developed, which can address the challenges induced by a hypoxic microenvironment. To this end, human telomerase reverse transcriptase (hTERT) targeting trans-splicing ribozymes whose expression and activity were induced by HRE under hypoxia were constructed. Luciferase reporter assay showed induction of the transgene to increase due to the hypoxia-inducible ribozymes through a specific trans-splicing reaction in hTERT-expressing cells under hypoxic conditions. Increase in the transgene expression was mainly due to the increased trans-splicing reaction through a concurrent increase of the ribozyme expression level. Moreover, hypoxia-inducible ribozyme with herpes simplex virus thymidine kinase as the 3'exon effectively induced cell death when treated with ganciclovir under both hypoxic and normoxic conditions. These results indicated that the trans-splicing ribozyme could be a target-specific and efficacious anti-cancer tool to overcome resistance to radio- and chemotherapy under hypoxic conditions.

Topics: 3' Untranslated Regions; Enhancer Elements, Genetic; Erythropoietin; Gene Expression; HEK293 Cells; Herpes Simplex; Humans; Hypoxia; Phosphoglycerate Kinase; RNA, Catalytic; Telomerase; Thymidine Kinase; Trans-Splicing; Transgenes; Viral Proteins

Iron and oxygen are essential substance for cellular activity in body tissues. Hypoxia-inducible factors (HIFs) can respond to available oxygen changes in the cellular environment and regulate the transcription of a series of target genes. The study was conducted to investigate the effects of iron supplementation on the expression of hypoxia-inducible factor-1 alpha (HIF-1α) and antioxidant status in rats exposed to high-altitude hypoxia environment. Forty rats were divided into control (CON), hypobaric hypoxia (HH), and hypobaric hypoxia plus ferrous sulfate (FeSO4) (9.93 mg/kg body weight (BW)/day) (HFS) and hypobaric hypoxia plus iron glycinate chelate (Fe-Gly) (11.76 mg/kg BW/day) (HFG) groups. Results showed that Fe-Gly effectively alleviated weight loss and intestinal mucosa damage induced by hypobaric hypoxia, whereas FeSO4 aggravated hypobaric hypoxia-induced weight loss, liver enlargement, spleen atrophy, and intestinal damage. Iron supplementation decreased liver superoxide dismutase (T-SOD) and catalase (CAT) activity (P < 0.01) and increased iron concentration in the liver compared to HH group (P < 0.001). Moreover, Fe-Gly upregulated liver transferrin expression in messenger RNA (mRNA) level (P < 0.05) and downregulated serum erythropoietin (EPO) concentration (P < 0.01) and liver HIF-1α expression level (P < 0.05 in mRNA level; P < 0.001 in protein level) compared to HH group. The study indicated that FeSO4 supplementation at high altitudes aggravated the oxidative damage of tissues and organs that could be mediated through production of malondialdehyde (MDA) and inhibition antioxidant enzyme activities. Fe-Gly can protect hypobaric hypoxia-induced tissues injury. Moreover, iron supplementation at high altitudes affected HIF-1α-mediated regulating expression of targeting genes such as EPO and transferrin. The study highlights that iron supplementation under hypobaric hypoxia environment has possible limitation, and efficient supplementation form and dosage need careful consideration.

Topics: Altitude; Animals; Antioxidants; Erythropoietin; Ferrous Compounds; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Liver; Male; Malondialdehyde; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transferrin

To evaluate the efficiency of treatment for renal anemia in patients with chronic glomerulonephritis (CGN), by using erythropoietin and its combination with hypoxic altitude chamber training (HACT).. Sixty-three patients (41 men and 22 women) (mean age 37.1 ± 3.3 years) with CGN during the predialysis phase of chronic kidney disease (CKD) complicated by anemia. Hemoglobin (Hb), packed cell volume (PCV), and red blood cell indices (mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC)), platelet count, serum iron, fibrinogen, C-reactive protein (CRP) and creatinine levels were determined in all the patients at baseline and during a prospective follow-up. Glomerular filtration rate (GFR) was measured using with the Rehberg-Tareev test. Along with standard renal protective therapy, all the patients received either epoetin beta (n=31; Group 1) or its combination with HACT (n=32; Group 2). In Group 1 patients (n=31), erythropoietin (EPO) was given in an initial dose of 20-50 IU/kg thrice daily, followed by the dose being adjusted until the target Hb level was reached. Group 2 patients (n=32) received HACT cycles by the standard procedure in combination with EPO given in lower doses (20-50 IU/kg once weekly). A prospective .follow-up of the patients was carried out during one year.. Following one year, the number of patients who had achieved the target Hb level was 74.1% in Group 1 and 87.5% in Group 2. Over time, there were increases in the concentration of Hb (from 108.6 ± 19.4 to 124.5 ± 14.09 g/l; p<0.05), PCV, and red blood cell indices (MCV, MCHC) in the patients receiving EPO (Group 1). Besides an'anti-anemic effect, there was a significant decrease in the concentrations of fibrinogen from 6655 (4884-7634) to 3776 (3330-4884) mg/dL; (p<0.05), serum creatinine from 159 (89--261) to 138 (79-258) pmol/I (p<0,05), proteinuria from 2.955 (1.024-6.745) to 2.069 (0.539-4.279) (p<0.05), which was accompanied by an increase in GFR from 62.3 (37.0 - 107.4) to 76.9 (46.0-96.0) mi/min (p<0.05). In Group 2, the rise in the concentration of Hb (from 114.1 ± 11.7 to 132.0 ± 16.5 g/I (p<0.05), PCV, MCV, and MCHC proved to be more pronounced than that in Group 1 (p<0.05) and accompanied by an elevation in the counts of platelets (from 222.7 ± 19.8.10(9)/1 to 249.3 ± 21.9.10(9)/1 (p<0:05)) and red blood cells (from 4.0 ± 0.4-10(12)/1 to 4.34 ± 0.3 X 10(12)/I (p<0.05)). There was a more marked reduction in the degree of proteinuria from 3.092 (0.764-7.694) g at baseline to 1.600 (0.677-4.078) g one year later (p<0.05) than that in Group 1 (p<0.05). The increase in GFR from 60.1 (46.0-96.0) to 79.4 (44.0-120.0) ml/min (p<0.05) and the fall in the concentration of fibrinogen from 5555 (4884-7770) to 4107 (3776-5328) mg/dL (p<0.05) and serum creatinine from 166 (92-273) to 147 (92-152) μmol/L (p<0.05), which were observed in Group 2, were comparable to those in Group 1.. Epoetin beta used in patients with CGN has an anti-anemic effect and leads to improved renal nitrogen-excretory function. Erythropoietin in combination with HACT used in CGN provides a higher anti-anemic efficacy and a more pronounced antiproteinuric effect.

Topics: Adult; Anemia; Atmosphere Exposure Chambers; Chronic Disease; Combined Modality Therapy; Erythropoietin; Female; Glomerulonephritis; Humans; Hypoxia; Male; Middle Aged; Respiratory Therapy; Treatment Outcome

Topics: Adult; Anemia, Sickle Cell; Antisickling Agents; Erythropoiesis; Erythropoietin; Female; Hemoglobins; Humans; Hydroxyurea; Hypoxia; Male; Middle Aged; Oxygen Inhalation Therapy; Pain; Reticulocyte Count; Retrospective Studies; Sleep Disorders, Intrinsic; Treatment Outcome; Young Adult

Exposure to high altitude is a well-known environmental stress with physiological and metabolic consequences, with the major stressor being hypobaric hypoxia. The disruption in cellular homeostasis elicits several acute and chronic adaptations designed to diminish the stress imposed by the hypoxic insult. Highly conserved cellular machinery protects the myocardium from damage under reduced oxygen tension. In the present study, adult Sprague-Dawley rats were exposed to an altitude of 9754 m in a decompression chamber and screened on the basis of the time taken for onset of gasping. The animals were grouped as susceptible (<10 min), normal (10-25 min), and tolerant (>25 min). Histologically, susceptible animals showed increased myocardial inflammation and infiltration and greater CK-MB activity. These animals showed a three-fold increase in reactive oxygen species levels and subsequent oxidative damage to proteins and lipids as compared to control unexposed group. In tolerant animals, the damage was minimal. The resistance to damage in these animals was possibly due to enhanced myocardial antioxidant enzymes, catalase and superoxide dismutase. A significantly higher expression of HIF-1α and its responsive genes, including EPO, HO-1, and GLUT1, was seen in tolerant animals, although VEGF expression was enhanced in the susceptible group. Cytoprotective chaperones, HSP70 and HSP90, were elevated in the tolerant animals. The differential expression of these hypoxia-responsive molecules may thus act as potential biochemical markers for screening and identifying individuals susceptible to environmental stress.

Topics: Altitude; Animals; Atmospheric Pressure; Catalase; Creatine Kinase, MB Form; Dyspnea; Endothelin-1; Erythropoietin; Heme Oxygenase-1; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Malondialdehyde; Myocarditis; Myocardium; Nitric Oxide; Oxidative Stress; Protein Carbonylation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Superoxide Dismutase; Time Factors; Up-Regulation; Vascular Endothelial Growth Factor A

To investigate the effect of recombine human erythropoietin (rhEPO) on apoptosis of neural cells in fetal rats after intrauterine hypoxic-ischemic injury.. Twenty SD rats on 19 days of pregnancy were divided into rhEPO (2500 U/kg, 5000 U/kg, 7500 U/kg) treated groups, ischemia-reperfusion (I/R) group and sham-operated group (4 rats in each group). Intrauterine hypoxic-ischemic injury of fetal rat was induced by bilateral occlusion of utero-ovarian artery for 20 min. rhEPO was injected into the rats in rhEPO treated group through the caudal vein 30 min before hypoxic-ischemic injury while saline was used in the other two groups. There was no hypoxic-ischemic injury in sham-operated group. The death rate of fetal rats was evaluated at 24 h after the operation, and then the brain samples of fetal rats were harvested. The expression of Caspase-3 protein was observed by immunohistochemistry. Neuroapoptosis was measured by TdT mediated dUTP-biotin nick end labeling (TUNEL) staining.. Death rates of fetal rats in rhEPO treated groups decreased compared with the I/R group (P < 0.05). Compared with the I/R group, there was less expression of copious Caspase-3 in rhEPO treated group (P < 0.01). The expression of Caspase-3 was decreased in the rhEPO treated groups with the increase of rhEPO dose (P < 0.01). Compared with the I/R group, the death rate of fetal rats in rhEPO treated groups decreased (P < 0.05), the number of apoptosis cells also decreased obviously (P < 0.01). The anti-apoptosis effect of 5000 U/kg rhEPO was similar to 7500 U/kg rhEPO, but better than 2500 U/kg rhEPO (P < 0.01).. rhEPO can inhibit the apoptosis of fetal rat brain cells after intrauterine hypoxic-ischemic injury.

Topics: Animals; Apoptosis; Brain; Caspase 3; Epoetin Alfa; Erythropoietin; Female; Fetus; Humans; Hypoxia; Neurons; Pregnancy; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury

To analyze the interconnection between erythropoiesis and iron metabolism, one of the issues raised in this study was to know iron bioavailability under physiopathological conditions. Our aim was to understand the functional axis response composed of erythropoietin (Epo)-hepcidin-ferroportin (FPN), when 2 dysfunctional states coexist, using an animal model of iron overload followed by hypoxia. FPN and prohepcidin were assessed by immunohistochemistry using rabbit anti-mouse FPN polyclonal and prohepcidin monoclonal antibodies. Goat-labeled polymer - horseradish peroxidase anti-rabbit EnVision + System (DAB) was used as the secondary antibody. Epo levels were measured by ELISA. Tissue iron was studied by Prussian blue iron staining. Erythropoietic response was assessed using conventional hematological tests. Iron overload increased prohepcidin that remained high in hypoxia, coexisting with high levels of Epo in hypoxia, with or without iron overload. In hypoxia, FPN was clearly evident in reticuloendothelial macrophages, more than in hypoxia with iron overload. Interestingly, duodenal FPN was clearly identified on the basolateral membrane in hypoxia, with or without iron overload. Our data indicate that 2 signals could induce the cell-specific response as follows: (i) iron signal, induced prohepcidin, which reduced reticuloendothelial FPN and reduced iron availability; and (ii) hypoxia signal, stimulated Epo, which affected iron absorption by stabilizing duodenal FPN and allowed iron supply to erythropoiesis independently of store size.

Topics: Animals; Biological Availability; Cation Transport Proteins; Duodenum; Enterocytes; Erythropoiesis; Erythropoietin; Female; Hepcidins; Hypoxia; Iron; Iron Overload; Macrophages; Mice; Spleen

The identity of the peritubular population of cells with mesenchymal phenotype thought responsible for producing erythropoietin in humans remains unclear. Here, renal CD133(+)/CD73(+) progenitor cells, isolated from the human renal inner medulla and described as a population of mesenchymal progenitors, released erythropoietin under hypoxic conditions. CD133(-) cells did not synthesize erythropoietin, and CD133(+) progenitor cells stopped producing erythropoietin when they differentiated and acquired an epithelial phenotype. Inhibition of prolyl hydroxylases, using either dimethyloxalylglycine or a small hairpin RNA against prolyl hydroxylase-2, increased both hypoxia-inducible factor-2α (HIF-2α) expression and erythropoietin transcription. Moreover, under hypoxic conditions, inhibition of prolyl hydroxylase significantly increased erythropoietin release by CD133(+) progenitors. Finally, blockade of HIF-2α impaired erythropoietin synthesis by CD133(+) progenitors. Taken together, these results suggest that it is the renal CD133(+) progenitor cells that synthesize and release erythropoietin under hypoxia, via the prolyl hydroxylase-HIF-2α axis, in the human kidney. In addition, this study provides rationale for the therapeutic use of prolyl hydroxylase inhibitors in the setting of acute or chronic renal injury.

Topics: 5'-Nucleotidase; AC133 Antigen; Antigens, CD; Erythropoietin; Glycoproteins; GPI-Linked Proteins; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Medulla; Peptides; Procollagen-Proline Dioxygenase; Stem Cells

Erythropoietin (Epo) is a key regulator of erythroid cell proliferation, differentiation and apoptosis. In the form of the recombinant protein, it is widely used to treat various types of anemias, including that associated with cancer and with the myelosuppressive effects of chemotherapy, particularly platinum-based regimens. Our previous studies confirmed the presence of Epo receptors (EpoRs) in ovarian adenocarcinoma cell lines and demonstrated that long-term Epo treatment of A2780 cells resulted in the development of a phenotype exhibiting both enhanced Epo signaling and increased paclitaxel resistance. In the present study, we carried out a series of experiments to analyze the pro-angiogenic potential of Epo-treated A2780 and SKOV-3 cells. Our studies revealed that conditioned media of Epo-treated A2780 cells had a stimulative effect on human umbilical vein endothelial cells (HUVECs). This effect was only seen when A2780 cells were incubated under hypoxic conditions. Furthermore, Epo increased the secretion of interleukin (IL)-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, GM-CSF and interferon-γ by A2780 cells that grew in hypoxic conditions. In this regard, conditioned media of hypoxic and Epo-treated A2780 cells induced a significant phosphorylation of STAT-5 in HUVECs. Our results may have important implications for ovarian cancer patients receiving Epo.

Topics: Adenocarcinoma; Blotting, Western; Cell Proliferation; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Epoetin Alfa; Erythropoietin; Female; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia; Neovascularization, Pathologic; Ovarian Neoplasms; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Intermittent hypoxia (IH) during sleep, such as occurs in obstructive sleep apnea (OSA), leads to degenerative changes in the hippocampus, and is associated with spatial learning deficits in adult mice. In both patients and murine models of OSA, the disease is associated with suppression of growth hormone (GH) secretion, which is actively involved in the growth, development, and function of the central nervous system (CNS). Recent work showed that exogenous GH therapy attenuated neurocognitive deficits elicited by IH during sleep in rats. Here, we show that administration of the Growth Hormone Releasing Hormone (GHRH) agonist JI-34 attenuates IH-induced neurocognitive deficits, anxiety, and depression in mice along with reduction in oxidative stress markers such as MDA and 8-hydroxydeoxyguanosine, and increases in hypoxia inducible factor-1α DNA binding and up-regulation of insulin growth factor-1 and erythropoietin expression. In contrast, treatment with a GHRH antagonist (MIA-602) during intermittent hypoxia did not affect any of the IH-induced deleterious effects in mice. Thus, exogenous GHRH administered as the formulation of a GHRH agonist may provide a viable therapeutic intervention to protect IH-vulnerable brain regions from OSA-associated neurocognitive dysfunction. Sleep apnea, characterized by chronic intermittent hypoxia (IH), is associated with substantial cognitive and behavioral deficits. Here, we show that administration of a GHRH agonist (JI-34) reduces oxidative stress, increases both HIF-1α nuclear binding and downstream expression of IGF1 and erythropoietin (EPO) in hippocampus and cortex, and markedly attenuates water maze performance deficits in mice exposed to intermittent hypoxia during sleep.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Cognition Disorders; Deoxyguanosine; Depression; Erythropoietin; Growth Hormone-Releasing Hormone; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Learning Disabilities; Lipid Peroxidation; Male; Maze Learning; Mice; Mice, Inbred C57BL; Oxidative Stress; Receptor, IGF Type 1; Sermorelin; Signal Transduction; Sleep

Gene therapy is suggested as a promising alternative strategy of hepatocellular carcinoma (HCC, also called hepatoma) therapy. To achieve a successful and safe gene therapy, tight regulation of gene expression is required to minimize side-effects in normal tissues. In this study, we developed a novel hypoxia and hepatoma dual specific gene expression vector. The constructed vectors were transfected into various cell lines using bio-reducible polymer, PAM-ABP. First, pAFPS-Luc or pAFPL-Luc vector was constructed with the alpha-fectoprotein (AFP) promoter and enhancer for hepatoma tissue specific gene expression. Then, pEpo-AFPL-Luc was constructed by insertion of the erythropoietin (Epo) enhancer for hypoxic cancer specific gene expression. In vitro transfection assay showed that pEpo-AFPL-Luc transfected hepatoma cell increased gene expression under hypoxic condition. To confirm the therapeutic effect of dual specific vector, herpes simplex virus thymidine kinase (HSV-TK) gene was introduced for cancer cell killing. The pEpo-AFPL-TK was transfected into hepatoma cell lines in the presence of ganciclovir (GCV) pro-drug. Caspase-3/7, MTT and TUNEL assays elucidated that pEpo-AFPL-TK transfected cells showed significant increasing of death rate in hypoxic hepatoma cells compared to controls. Therefore, the hypoxia/hepatoma dual specific gene expression vector with the Epo enhancer and AFP promoter may be useful for hepatoma specific gene therapy.

Topics: Acrylic Resins; alpha-Fetoproteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Dendrimers; DNA; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation, Neoplastic; Genes, Viral; Genetic Therapy; HEK293 Cells; Humans; Hypoxia; Luciferases; Plasmids; Promoter Regions, Genetic; Simplexvirus; Thymidine Kinase; Transfection

Haematological responses throughout 4 w of initial acclimation (IA) and three paradigms of re-acclimation (RA) to hypoxia (FI(O₂)) were examined in female mice. We hypothesised that (i) haematological responses would be increased during re-exposure, resulting in greater O₂-carrying capacity in RA compared to IA; and (ii) further improvements would occur when abbreviating the de-acclimation period to 1 w (RA↓DA) or extending the IA period to 8 w (RA↑IA). The serum [EPO] response was blunted in all RA groups compared to IA but the resulting reticulocyte response was similar in all experimental groups. The [Hb] response was the same in RA and RA↓DA as in IA but was blunted in RA↑IA due to a reduction in mean corpuscular Hb. The sensitivity of EPO-producing cells appears blunted but the sensitivity of erythroid precursors to EPO is enhanced by recent hypoxic exposure. Erythropoietic regulation is altered during RA in a manner that is dependent on the paradigm of initial exposure.

Topics: Acclimatization; Animals; Blood Cell Count; Enzyme-Linked Immunosorbent Assay; Erythropoiesis; Erythropoietin; Female; Hypoxia; Mice; Mice, Inbred C57BL; Reverse Transcriptase Polymerase Chain Reaction

Topics: Adult; Altitude; Antimicrobial Cationic Peptides; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Ethiopia; Female; Ferritins; Hemoglobins; Hepcidins; Humans; Hypoxia; Male; Oxyhemoglobins; Young Adult

Reduced insulin sensitivity might increase the susceptibility to acute mountain sickness (AMS). The diabetogenic side effects of dexamethasone should therefore be considered for AMS treatment. To examine whether reduced insulin sensitivity is predictive of AMS and how it is affected by dexamethasone at high altitude, we analyzed endocrine and metabolic parameters obtained from healthy mountaineers in Zurich (LA; 490 m), and 2 and 4 days after fast ascent to the Capanna Regina Margherita (HA2, HA4; 4559 m). 14 of 25 participants developed AMS and were treated with dexamethasone starting in the evening of HA2. Before and after ingestion of an 1800 kJ meal, plasma was analyzed for erythropoietin (EPO) and cholecystokinin (CCK). Insulin sensitivity (HOMA-S) and beta cell activity were calculated. HOMA-S (p<0.01) and EPO levels (p<0.05) were lower in Zurich in the group developing AMS and given dexamethasone, i.e., before treatment and exposure to hypoxia. CCK was lower (p<0.01) and glucose and insulin were higher on HA4 in the dexamethasone group compared to the untreated group. Individuals with low baseline insulin sensitivity and low baseline EPO levels were more susceptible to AMS. Reduced CCK may contribute to the beneficial effect of dexamethasone on high altitude anorexia. However, reduced insulin sensitivity questions the widespread use of dexamethasone to prevent/treat AMS.

Topics: Adult; Altitude; Altitude Sickness; Blood Glucose; Cholecystokinin; Dexamethasone; Energy Intake; Erythropoietin; Female; Glucocorticoids; Homeostasis; Humans; Hydrocortisone; Hypoxia; Insulin Resistance; Insulin-Secreting Cells; Interleukin-6; Islet Amyloid Polypeptide; Male; Middle Aged; Models, Biological; Oxygen; Retrospective Studies; Time Factors; Young Adult

In order to maintain normal cellular function, mammalian tissue oxygen concentrations must be tightly regulated within a narrow physiological range. The hormone erythropoietin (EPO) is essential for maintenance of tissue oxygen supply by stimulating red blood cell production and promoting their survival. In this study we compared the effects of 290 Torr atmospheric pressure on the kidney EPO protein levels in young (4-month-old) and aged (24-month-old) C57BL/6 mice. The mice were sacrificed after being anesthetized, and kidney samples were collected and processed by Western blot analysis. Relatively low basal expression of EPO during normoxia in young mice showed significant upregulation in hypoxia and stayed upregulated throughout the hypoxic period (threefold compared to normoxic control), showing a slight decline toward the third week. Whereas, a relatively higher normoxic basal EPO protein level in aged mice did not show significant increase until seventh day of hypoxia, but showed significant upregulation in prolonged hypoxia. Hence, we confirmed that there is a progressively increased accumulation of EPO during chronic hypoxia in young and aged mouse kidney, and the EPO upregulation during hypoxia showed a similarity with the pattern of increase in hematocrit, which we have reported previously.

Topics: Aging; Animals; Blotting, Western; Chronic Disease; Erythropoietin; Hematocrit; Hypoxia; Kidney; Male; Mice; Mice, Inbred C57BL; Oxygen; Up-Regulation

The purpose of the present study was to compare the acute hormonal response of angiogenic regulators to a short-term hypoxic exposure at different altitudes with and without exercise. 7 subjects participated in 5 experimental trials. 2 times subjects stayed in a sedentary position for 90 min at 2000 m or 4000 m, respectively. The same was carried out again in combination with exercise at the same relative intensity (2 mmol∙L(-1) of lactate). The fifth trial consisted of 90 min exercise at sea level. Venous blood samples were taken under resting conditions, 0 and 180 min after each condition to determine VEGF, EPO, IL-6, IL-8 and IGF-1 serum concentrations. EPO, VEGF, and IL-8 showed increases only, when hypoxia was combined with exercise. IL-6 was increased after exercise, independent of altitude. IGF-1 showed no changes in any intervention. The present study suggests that short term hypoxic exposure combined with low intensity exercise is able to up-regulate angiogenic regulators, which might be beneficial to induce angiogenesis and to improve endurance performance. However, in some cases high altitudes are needed, or it can be speculated that exercise intensity needs to be increased.

Topics: Altitude; Biomarkers; Cytokines; Erythropoietin; Exercise; Humans; Hypoxia; Insulin-Like Growth Factor I; Interleukin-6; Interleukin-8; Male; Vascular Endothelial Growth Factor A; Young Adult

Carbamylated erythropoietin (CEPO) is attracting widespread interest because of its neuroprotective effects without influencing erythropoiesis. Here we show that CEPO, unlike EPO, does not stimulate erythropoiesis. Both CEPO and EPO inhibit the death/apoptosis of neurons in the hypoxic model of primary neurons and induce neuron proliferation and differentiation in hypoxic mice. Hypoxic mice show apparent memory deficits at 3 and 30 days after hypoxia. The administration of CEPO/EPO significantly improves cognitive and behavioral defects after hypoxic insults. Further investigation shows that CEPO/EPO induces neuron proliferation and differentiation and promotes the generation of choline acetyltransferase (ChAT)(+) neurons in hypoxic mice. Phosphorylated AKT was colabeled with ChAT(+) neurons and coexpressed in bromodeoxyuridine-positive cells, suggesting that the PI3K/AKT pathway may play a pivotal role in CEPO/EPO-cholinergic neuron generation. These results reveal that CEPO/EPO ameliorates hypoxia-induced cognitive and behavioral defects possibly through the generation of ChAT-positive neurons.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Proliferation; Choline O-Acetyltransferase; Cognition Disorders; Erythropoietin; Flow Cytometry; Hypoxia; Immunohistochemistry; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents

Vascular endothelial growth factor (VEGF) and erythropoietin (EPO) exert neurotrophic and neuroprotective effects in the CNS. We recently demonstrated that VEGF, EPO and their receptors (VEGF-R2, EPO-R) are expressed in phrenic motor neurons, and that cervical spinal VEGF-R2 and EPO-R activation elicit long-lasting phrenic motor facilitation (pMF). Since VEGF, VEGF-R, EPO, and EPO-R are hypoxia-regulated genes, and repetitive exposure to acute intermittent hypoxia (rAIH) up-regulates these molecules in phrenic motor neurons, we tested the hypothesis that 4 weeks of rAIH (10 episodes per day, 3 days per week) enhances VEGF- or EPO-induced pMF. We confirm that cervical spinal VEGF and EPO injections elicit pMF. However, neither VEGF- nor EPO-induced pMF was affected by rAIH pre-conditioning (4 wks). Although our data confirm that spinal VEGF and EPO may play an important role in respiratory plasticity, we provide no evidence that rAIH amplifies their impact. Further experiments with more robust protocols are warranted.

Topics: Animals; Erythropoietin; Hypoxia; Male; Motor Neurons; Phrenic Nerve; Rats; Rats, Sprague-Dawley; Spinal Nerves; Vascular Endothelial Growth Factor A

Premature infants exhibit neurodevelopmental delay and reduced growth of the cerebral cortex. However, the underlying mechanisms have remained elusive. Therefore, we hypothesized that neurogenesis in the ventricular and subventricular zones of the cerebral cortex would continue in the third trimester of pregnancy and that preterm birth would suppress neurogenesis. To test our hypotheses, we evaluated autopsy materials from human fetuses and preterm infants of 16-35 gestational weeks (gw). We noted that both cycling and noncycling Sox2(+) radial glial cells and Tbr2(+) intermediate progenitors were abundant in human preterm infants until 28 gw. However, their densities consistently decreased from 16 through 28 gw. To determine the effect of premature birth on neurogenesis, we used a rabbit model and compared preterm [embryonic day 29 (E29), 3 d old] and term (E32, <2 h old) pups at an equivalent postconceptional age. Glutamatergic neurogenesis was suppressed in preterm rabbits, as indicated by the reduced number of Tbr2(+) intermediate progenitors and the increased number of Sox2(+) radial glia. Additionally, hypoxia-inducible factor-1α, vascular endothelial growth factor, and erythropoietin were higher in term than preterm pups, reflecting the hypoxic intrauterine environment of just-born term pups. Proneural genes, including Pax6 and Neurogenin-1 and -2, were higher in preterm rabbit pups compared with term pups. Importantly, neurogenesis and associated factors were restored in preterm pups by treatment with dimethyloxallyl glycine, a hypoxia mimetic agent. Hence, glutamatergic neurogenesis continues in the premature infants, preterm birth suppresses neurogenesis, and hypoxia-mimetic agents might restore neurogenesis, enhance cortical growth, and improve neurodevelopmental outcome of premature infants.

Topics: Adult; Animals; beta Catenin; Cell Count; Cerebral Ventricles; Erythropoietin; Female; Gestational Age; Glycine; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Immunohistochemistry; Infant, Newborn; Infant, Premature; Male; Nerve Tissue Proteins; Neural Stem Cells; Neurogenesis; Pregnancy; Pregnancy Trimester, Third; Premature Birth; Rabbits; Signal Transduction; Telencephalon; Vascular Endothelial Growth Factor A; Wnt Proteins

The involvement of erythropoietin in cardiac adaptation to acute and chronic (CHx) hypoxia was investigated in erythropoietin deficient transgenic (Epo-TAg(h)) and wild-type (WT) mice. Left (LV) and right ventricular functions were assessed by echocardiography and hemodynamics. HIF-1α, VEGF and Epo pathways were explored through RT-PCR, ELISA, Western blot and immunocytochemistry. Epo gene and protein were expressed in cardiomyocytes of WT mice in normoxia and hypoxia. Increase in blood hemoglobin, angiogenesis and functional cardiac adaptation occurred in CHx in WT mice, allowing a normal oxygen delivery (O2T). Epo deficiency induced LV hypertrophy, increased cardiac output (CO) and angiogenesis, but O2T remained lower than in WT mice. In CHx Epo-TAg(h) mice, LV hypertrophy, CO and O2T decreased. HIF-1α and Epo receptor pathways were depressed, suggesting that Epo-TAg(h) mice could not adapt to CHx despite activation of cardioprotective pathways (increased P-STAT-5/STAT-5). HIF/Epo pathway is activated in the heart of WT mice in hypoxia. Chronic hypoxia induced cardiac adaptive responses that were altered with Epo deficiency, failing to maintain oxygen delivery to tissues.

Topics: Adaptation, Physiological; Animals; Blotting, Western; Chronic Disease; Disease Models, Animal; Echocardiography; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Heart; Hemodynamics; Hypoxia; Male; Mice; Mice, Transgenic; Myocytes, Cardiac; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Topics: Animals; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Oxygen

Children and adults residing at high altitude (HA) compared to low altitude (LA) have larger lung volumes; however, it is unknown whether this response to chronic hypoxia begins early in life. Our objective was to determine whether infants and toddlers at HA have larger lung volumes compared to infants and toddlers at LA. Oxygen saturation (SaO2 ), functional residual capacity (FRC), as well as serum levels of vascular endothelial growth factor (VEGF) and erythropoietin (EPO) were measured in infants and toddlers from HA (N = 50; 3,440 m) and LA (N = 35; 440 m). There were no significant differences in somatic size for HA and LA subjects; however, HA subjects had significantly lower SaO2 (88.5% vs. 96.7%; P < 0.0001). Subjects at HA had significantly greater FRC compared to subjects at LA (group mean: 209 and 157 ml; P < 0.0001), adjusting for body length. Male infants at HA had a significantly greater FRC compared to males at LA (57 ml; P-value < 0.001); however, the increase in FRC for females at HA compared to LA was not significant (20 ml; P-value = 0.101). VEGF and EPO were significantly higher for subjects at HA compared to LA with no gender differences. In summary, infants and toddlers at HA have lower oxygen saturations, higher serum levels of VEGF and EPO, and higher FRC compared to subjects at LA; however, chronic hypoxia appears to generate a more robust response in lung growth in male compared to female infants early in life.

Topics: Altitude; Erythropoietin; Female; Functional Residual Capacity; Humans; Hypoxia; Infant; Lung; Lung Volume Measurements; Male; Oximetry; Vascular Endothelial Growth Factor A

In addition to its role in erythropoiesis, erythropoietin is also appreciated for its neuroprotective effects, and it has been suggested for treatment of some ischemic-hypoxic neurovascular diseases. The protective effects of endogenous erythropoietin in the brain give rise to the hypothesis that modulating erythropoietin expression might be a better way for treatment of ischemia-hypoxia neurovascular diseases. We have found that ethanol extract of Portulaca oleracea L. (EEPO) could increase erythropoietin expression in hypoxic mouse brain in our previous study. The present study is to investigate whether EEPO exerts its neuroprotective effects against hypoxia injury through regulating endogenous erythropoietin expression. The results demonstrated that EEPO decreased the serum neuron specific enolase level in hypoxia mice and the activity of caspase-3 in neuron, increased the neuron viability and attenuated the pathological damages caused by the hypoxia condition. Importantly, we also found that EEPO stimulated the endogenous erythropoietin expression at both mRNA and protein levels. Using the conditioned medium containing soluble erythropoietin receptor, we found that the neuroprotective effects of EEPO were dependent, at least partly, on erythropoietin expression. Although EEPO did not affect transcription of hypoxia inducible factor-1α (HIF-1α), it did stabilize expression of HIF-1α. It is concluded that EEPO has neuroprotective effects against hypoxia injury, which is at least partly through stimulating endogenous erythropoietin expression by stabilizing HIF-1α.

Topics: Animals; Caspase 3; Cells, Cultured; Erythropoiesis; Erythropoietin; Ethanol; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Ischemia, Brain; Mice; Mice, Inbred ICR; Neurons; Neuroprotective Agents; Plant Extracts; Portulaca; Receptors, Erythropoietin

Inhibition of the HIF regulating prolyl hydroxylation domain (PHDs) proteins prior to renal injury (preconditioning) has been shown to protect the kidney via activation of hypoxia-inducible transcription factors (HIF). Application of erythropoietin (EPO), one of the HIF target genes, has also been shown to be nephroprotective, and it remains unclear to what extent the effect of HIF induction is mediated by EPO. It is also unknown whether HIF activation after the onset of ischaemia (postconditioning) is still able to protect the kidney.. Using a rat model of renal ischaemia-reperfusion injury, animals were treated with the PHD inhibitor (PHD-I) 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA), vehicle (Veh) or recombinant human EPO (300 IU/kg) 6 h (ICA or Veh) or 30 min (EPO) prior to ischaemia (preconditioning) or with ICA prior to reperfusion (postconditioning). Renal function was assessed at baseline, 24 h and 72 h. After 72 h, kidneys were processed for histology and morphometric analysis. HIF immunohistochemistry and real-time polymerase chain reaction for HIF target genes, including EPO, were performed to evaluate ICA effects.. ICA treatment resulted in stabilization of HIF-1α and -2α and up-regulation of HIF target genes in a dose-dependent manner. Preconditional activation of HIF by ICA significantly improved serum creatinine levels and renal morphology in comparison to Veh (P < 0.05), while postconditional ICA treatment was ineffective. EPO therapy improved tissue morphology but had no impact on the course of serum creatinine.. These findings are in line with the concept that PHD-Is exert their protective effects through accumulation of HIF target gene products, with time requirements for increased transcription and translation of HIF-dependent genes, and suggest that their renoprotective effect is not predominately mediated by EPO.

Topics: Acute Kidney Injury; Animals; Enzyme Inhibitors; Epoetin Alfa; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoenzyme Techniques; Male; Procollagen-Proline Dioxygenase; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Reperfusion Injury; RNA, Messenger

Several polymorphisms of vasoactive hormones have been implicated in hypertension. Erythropoietin (EPO) interacts with vasoactive substances, such as angiotensin II. Previously detected single nucleotide polymorphisms in the hypoxia-responsive element of EPO might be associated with hypertension and hypertensive end organ damages.. 400 hypertensive patients and 200 age- and gender-matched normotensive controls were genotyped for an EPO polymorphism [cytosine (C)/thymine (T) single nucleotide polymorphism] at position 3434. Patients were grouped according to their genotype into the CC group (CC genotype) and the CT/TT group (CT and TT genotype). BP was measured by ambulatory BP monitoring.. The CC genotype was present in 87% of hypertensive patients and in 78.5% of controls (p = 0.007). In addition, patients with the CC genotype had higher BP levels compared with CT/TT genotypes (BPsys 143.7 ± 20.4 vs. 136.1 ± 13.5 mm Hg, p = 0.01, and BPdias 85.8 ± 11.6 vs. 82.4 ± 8.9, p = 0.043) despite a nearly identical number of antihypertensive drugs (2.3 ± 1.5 vs. 2.3 ± 1.6; p = 0.257). 100% of the small number of patients with end-stage renal disease (n = 15) had the CC genotype.. The CC genotype of the EPO gene at position 3434 is more frequently found in patients with hypertension and is associated with higher BP levels.

Topics: Adult; Aged; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Erythropoietin; Female; Genotype; Homozygote; Humans; Hypertension, Renal; Hypoxia; Male; Middle Aged; Myocardial Infarction; Polymorphism, Genetic; Stroke; Vascular Diseases

Hemoglobin mass (tHb) is considered to be a main factor for sea-level performance after "live high-train low" (LHTL) altitude training, but little research has focused on the persistence of tHb following cessation of altitude exposure. The aim of the case study was to investigate short-term effects of various hematological measures including tHb upon completion of a simulated altitude camp. Five female cyclists spent 26 nights at simulated altitude (LHTL, 16.6 ± 0.4 h/d, 3000 m in an altitude house) where tHb was measured at baseline, at cessation of the camp, and 9 d thereafter. Venous blood measures (hemoglobin concentration, hematocrit, %reticulocytes, serum erythropoietin, ferritin, lactate dehydrogenase, and haptoglobin) were determined at baseline; on day 21 during LHTL; and at days 2, 5, and 9 after LHTL. Hemoglobin mass increased by 5.5% (90% confidence limits [CL] 2.5 to 8.5%, very likely) after the LHTL training camp. At day 9 after simulated LHTL, tHb decreased by 3.0% (90%CL -5.1 to -1.0%, likely). There was a substantial decrease in serum EPO (-34%, 90%CL -50 to -12%) at 2 d after return to sea level and a rise in ferritin (23%, 90%CL 3 to 46%) coupled with a decrease in %reticulocytes (-23%, 90%CL -34 to -9%) between day 5 and 9 after LHTL. Our findings show that following a hypoxic intervention with a beneficial tHb outcome, there may be a high probability of a rapid tHb decrease upon return to normoxic conditions. This highlights a rapid component in red-cell control and may have implications for the appropriate timing of altitude training in relation to competition.

Topics: Adult; Altitude; Bicycling; Erythropoiesis; Erythropoietin; Female; Ferritins; Hemoglobins; Humans; Hypoxia; Oxygen; Physical Fitness; Young Adult

Enhanced erythropoietic drive and iron deficiency both influence iron homeostasis through the suppression of the iron regulatory hormone hepcidin. Hypoxia also suppresses hepcidin through a mechanism that is unknown. We measured iron indices and plasma hepcidin levels in healthy volunteers during a 7-day sojourn to high altitude (4340 m above sea level), with and without prior intravenous iron loading. Without prior iron loading, a rapid reduction in plasma hepcidin was observed that was almost complete by the second day at altitude. This occurred before any index of iron availability had changed. Prior iron loading delayed the decrease in hepcidin until after the transferrin saturation, but not the ferritin concentration, had normalized. We conclude that hepcidin suppression by the hypoxia of high altitude is not driven by a reduction in iron stores.

Topics: Adult; Altitude; Antimicrobial Cationic Peptides; beta-Thalassemia; Case-Control Studies; Erythropoiesis; Erythropoietin; Ferritins; Gene Expression Regulation; Growth Differentiation Factor 15; Hepcidins; Homeostasis; Humans; Hypoxia; Iron; Iron Metabolism Disorders; Iron, Dietary; Transferrin

Hypoxia inducible factor (HIF)-1 plays an important role in cellular adaptation to hypoxia by activating oxygen-regulated genes such as vascular endothelial growth factor (VEGF) and erythropoietin. Sputum VEGF levels are reported to be decreased in COPD, despite hypoxia. Here we show that patients with COPD fail to induce HIF-1α and VEGF under hypoxic condition because of a reduction in histone deacetylase (HDAC) 7.. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with moderate to severe COPD (n = 21), smokers without COPD (n = 12), and nonsmokers (n = 15). PBMCs were exposed to hypoxia (1% oxygen, 5% CO(2), and 94% N(2)) for 24 h, and HIF-1α and HDAC7 protein expression in nuclear extracts were determined by sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE)/Western blotting.. HIF-1α was significantly induced by hypoxia in each group when compared with the normoxic condition (12-fold induction in nonsmokers, 24-fold induction in smokers without COPD, fourfold induction in COPD), but induction of HIF-1α under hypoxia was significantly lower in patients with COPD than in nonsmokers and smokers without COPD (P < .05 and P < .01, respectively). VEGF messenger RNA detected by quantitative real-time polymerase chain reaction was correlated with HIF-1α protein in nuclei (r = 0.79, P < .05), and HDAC7 protein expression was correlated with HIF-1α protein in nuclei (r = 0.46, P < .05). HDAC7 knockdown inhibited hypoxia-induced HIF-1α activity in U937 cells, and HIF-1α nuclear translocation and HIF-1α binding to the VEGF promoter in A549 cells.. HDAC7 reduction in COPD causes a defect of HIF-1α induction response to hypoxia with impaired VEGF gene expression. This poor cellular adaptation might play a role in the pathogenesis of COPD.

Topics: Adaptation, Physiological; Aged; Cell Hypoxia; Cells, Cultured; Erythropoietin; Female; Gene Expression; Histone Deacetylases; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Middle Aged; Monocytes; Promoter Regions, Genetic; Pulmonary Disease, Chronic Obstructive; RNA, Messenger; Statistics as Topic; Transcriptional Activation; Vascular Endothelial Growth Factor A

Cell surface proteins Hfe, Tfr2, hemojuvelin and Tmprss6 play key roles in iron homeostasis. Hfe and Tfr2 induce transcription of hepcidin, a small peptide that promotes the degradation of the iron transporter ferroportin. Hemojuvelin, a co-receptor for bone morphogenic proteins, induces hepcidin transcription through a Smad signaling pathway. Tmprss6 (also known as matriptase-2), a membrane serine protease that has been found to bind and degrade hemojuvelin in vitro, is a potent suppressor of hepcidin expression. In order to examine if Hfe and Tfr2 are substrates for Tmprss6, we generated mice lacking functional Hfe or Tfr2 and Tmprss6. We found that double mutant mice lacking functional Hfe or Tfr2 and Tmprss6 exhibited a severe iron deficiency microcytic anemia phenotype mimicking the phenotype of single mutant mice lacking functional Tmprss6 (Tmprss6msk/msk mutant) demonstrating that Hfe and Tfr2 are not substrates for Tmprss6. Nevertheless, the phenotype of the mice lacking Hfe or Tfr2 and Tmprss6 differed from Tmprss6 deficient mice alone, in that the double mutant mice exhibited much greater erythropoiesis. Hfe and Tfr2 have been shown to play important roles in the erythron, independent of their role in regulating liver hepcidin transcription. We demonstrate that lack of functional Tfr2 and Hfe allows for increased erythropoiesis even in the presence of high hepcidin expression, but the high levels of hepcidin levels significantly limit the availability of iron to the erythron, resulting in ineffective erythropoiesis. Furthermore, repression of hepcidin expression by hypoxia was unaffected by the loss of functional Hfe, Tfr2 and Tmprss6.

Topics: Anemia, Hypochromic; Animals; Erythropoiesis; Erythropoietin; Female; Hemochromatosis Protein; Histocompatibility Antigens Class I; Hypoxia; Male; Membrane Proteins; Mice; Mice, 129 Strain; Mice, Inbred AKR; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Receptors, Transferrin; Reticulocytosis; Serine Endopeptidases

In humans, acute erythroid expansion can lead to maturation of red blood cell (RBC) precursors containing fetal hemoglobin (F red cells). This can occur in patients after recovery from bone marrow transplantation, or in individuals affected by sickle cell or thalassemic syndromes. An accelerated erythroid lineage expansion is also a hallmark of the adaptive response to high altitude hypoxia. To explore the possible effect of this environment on F red cell production, we analyzed RBCs from five subjects during and after 17 days spent at high altitude and investigated the expression of fetal hemoglobin by different methodological approaches. By flow cytometry, we found a moderate increase of circulating F red cells during and after the hypoxia exposure, with respect to control cells analyzed before a stay at high altitude. The increased expression of γ-globin (as the specific subunit contained in F hemoglobin together with α-globin) was further confirmed by immunoblotting of young RBC hemolysates and quantitative RT-PCR of transcripts purified from a reticulocyte-enriched RBC fraction. Thus, in healthy adults the exposure to high altitude hypoxia induces maturation of F red cells at a level higher than under normal condition. The effect appears reduced after return to normoxia.

Topics: Adult; Altitude; Erythrocytes; Erythropoietin; Female; Fetal Hemoglobin; gamma-Globins; Gene Expression; Humans; Hypoxia; Male; Middle Aged; RNA, Messenger

Erythropoietin (EPO) preserves arterial oxygen content by controlling red blood cell and plasma volumes. Synthesis of EPO was long thought to relate inversely to renal oxygenation, but in knockout mice, brain and skin have been identified as essential for the acute hypoxic EPO response. Whether these findings apply to humans remains unknown. We exposed healthy young subjects to hypoxia (equivalent to 3800 m) and measured EPO in arterial and jugular venous plasma and in cerebrospinal fluid. To examine the role of the skin for EPO production during hypoxia, subjects were exposed to 8 h of hypobaric hypoxia with or without breathing oxygen-enriched air to ensure systemic normoxemia. With 9 h of hypoxia, arterial EPO increased (from 6.0±2.2 to 22.0±6.0 mU/ml, n=11, P<0.0001) and jugular venous EPO displayed a similar response (to 22.2±6.0 mU/ml, n=11). Thus, the arterio-jugular venous EPO difference was unaffected by hypoxia and also in cerebrospinal fluid EPO remained stable following hypoxic exposure (0.33±0.15 mU/ml, n=9 in normoxia vs. 0.41±0.20 mU/ml, n=9 in hypoxia, P=0.40). No change in plasma EPO was observed when only skin was exposed to hypobaric hypoxia (n=8). Thus, neither dermal oxygen exposure nor cerebral EPO production appears to be important for the systemic EPO response to acute hypoxia in healthy humans.

Topics: Acute Disease; Adult; Brain; Erythropoietin; Female; Humans; Hypoxia; Male; Skin

Topics: Altitude; Animals; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Mice; Mitochondria; Neoplasms; Signal Transduction

Topics: Erythropoietin; Humans; Hypoxia; Oxygen; Oxygen Inhalation Therapy

Topics: Doping in Sports; Erythropoietin; Humans; Hypoxia; Oxygen

Clinical use of erythropoietin in adult and newborn patients has revealed its involvement in neuroprotection, neurogenesis, and angiogenesis. More recently, we showed in adult mouse, that brain erythropoietin interacts with the major brainstem centers associated with respiration to enhance the ventilatory response to acute and chronic conditions of physiological hypoxia (e.g., as occurring at high altitude). However, whether brain erythropoietin is involved in breathing regulation in newborns remains unknown. In this study, en bloc brainstem-spinal cord preparations were obtained from mice at postnatal day 4. After various periods (30, 60, or 90 min) of incubation with 0, 25, or 250 U of erythropoietin, preparations were superfused with artificial cerebrospinal fluid bubbled with normoxic or hypoxic gas mixtures. The electrophysiological fictive breathing produced by axons at the C4 ventral root was next recorded. Our results show that erythropoietin attenuates the hypoxia-mediated decrease of the central respiratory activity and improves post-hypoxic recovery. Additional analysis revealed that the soluble erythropoietin receptor (the endogenous erythropoietin antagonist) dramatically decreases neural hypoxic respiratory activity, confirming the specific erythropoietin effect on respiratory drive. These results imply that erythropoietin exerts main modulation and maintenance of respiratory motor output during hypoxic and post-hypoxic challenges in 4-days old mice.

Topics: Altitude; Animals; Animals, Newborn; Brain Stem; Cerebrospinal Fluid; Erythropoietin; Humans; Hypoxia; Male; Mice; Mice, Inbred C57BL; Respiration; Spinal Cord

Erythropoietin (EPO) is known to improve exercise performance by increasing oxygen blood transport and thus inducing a higher maximum oxygen uptake (VO2max). Furthermore, treatment with (or overexpression of) EPO induces protective effects in several tissues, including the myocardium. However, it is not known whether EPO exerts this protective effect when present at physiological levels. Given that EPO receptors have been identified in skeletal muscle, we hypothesized that EPO may have a direct, protective effect on this tissue. Thus, the objectives of the present study were to confirm a decrease in exercise performance and highlight muscle transcriptome alterations in a murine EPO functional knock-out model (the EPO-d mouse).. We determined VO2max peak velocity and critical speed in exhaustive runs in 17 mice (9 EPO-d animals and 8 inbred controls), using treadmill enclosed in a metabolic chamber. Mice were sacrificed 24h after a last exhaustive treadmill exercise at critical speed. The tibialis anterior and soleus muscles were removed and total RNA was extracted for microarray gene expression analysis.. The EPO-d mice's hematocrit was about 50% lower than that of controls (p<0.05) and their performance level was about 25% lower (p<0.001). A total of 1583 genes exhibited significant changes in their expression levels. However, 68 genes were strongly up-regulated (normalized ratio>1.4) and 115 were strongly down-regulated (normalized ratio<0.80). The transcriptome data mining analysis showed that the exercise in the EPO-d mice induced muscle hypoxia, oxidative stress and proteolysis associated with energy pathway disruptions in glycolysis and mitochondrial oxidative phosphorylation.. Our results showed that the lack of functional EPO induced a decrease in the aerobic exercise capacity. This decrease was correlated with the hematocrit and reflecting poor oxygen supply to the muscles. The observed alterations in the muscle transcriptome suggest that physiological concentrations of EPO exert both direct and indirect muscle-protecting effects during exercise. However, the signaling pathway involved in these protective effects remains to be described in detail.

Topics: Adenosine Triphosphate; Animals; Biological Transport; Cytoskeleton; Down-Regulation; Erythropoietin; Female; Hematocrit; Hypoxia; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Muscular Atrophy; Oligonucleotide Array Sequence Analysis; Oxygen Consumption; Physical Conditioning, Animal; Proteolysis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Up-Regulation

In rodents, exposure to intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Such findings are markedly attenuated in rodents exposed to sustained hypoxia 9SH) of similar magnitude. The hypoxia-sensitive gene erythropoietin (EPO) has emerged as a major endogenous neuroprotectant, and could be involved in IH-induced neuronal dysfunction.. IH induced only transiently increased expression of EPO mRNA in hippocampus, which was continued in (SH)-exposed mice. IH, but not SH, adversely affected two forms of spatial learning in the water maze, and increased markers of oxidative stress. However, on a standard place training task, mice treated with exogenously administered EPO displayed normal learning, and were protected from the spatial learning deficits observed in vehicle-treated (C) littermates exposed to IH. Moreover, anxiety levels were increased in IH as compared to normoxia, while no changes in anxiety emerged in EPO-treated mice. Additionally, C mice, but not EPO-treated IH-exposed mice had significantly elevated levels of NADPH oxidase expression, as well as increased MDA and 8-OHDG levels in cortical and hippocampal lysates.. The oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by imbalances between EPO expression and increased NADPH oxidase activity, and thus pharmacological agents targeting EPO expression in CNS may provide a therapeutic strategy in sleep-disordered breathing.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Analysis of Variance; Animals; Cells, Cultured; Cerebral Cortex; Cognition Disorders; Deoxyguanosine; Disease Models, Animal; Embryo, Mammalian; Erythropoietin; Escape Reaction; Gene Expression Regulation; Humans; Hypoxia; Injections, Intraperitoneal; Lipid Peroxidation; Male; Malondialdehyde; Maze Learning; Memory; Mice; Mice, Inbred C57BL; NADPH Oxidases; Neurons; Phosphopyruvate Hydratase; Sleep Apnea Syndromes; Swimming; Time Factors

Hypoxia-inducible factor-1 (HIF-1) regulates the expression of hypoxia-inducible genes by binding erythropoietin (EPO) enhancer fragments. Of these genes, HIF-1 upregulates voltage-gated K+1.2 channels (Kv1.2) in rat PC12 cells. Whether HIF-1 regulates hypoxia-induced Kv channel expression in cultured pulmonary artery smooth muscle cells (PASMCs), however, has not been determined. In this study, we investigated the effects of hypoxia on the expression of Kv1.2 Kv1.5, Kv2.1, and Kv9.3 channels in PASMCs and examined the direct role of HIF-1 by transfecting either wild type or mutant EPO enhancer fragments. Our results showed that 18 h exposure to hypoxia significantly increased the expression of Kv1.2, Kv1.5, Kv2.1, and Kv9.3; and this hypoxia-induced upregulation was completely inhibited after transfection with the wild type but not mutant EPO enhancer fragment. These results indicate that HIF-1 regulates hypoxia-stimulated induction of Kv1.2 Kv1.5, Kv2.1, and Kv9.3 channels in cultured PASMCs.

Topics: Animals; Base Sequence; DNA; Enhancer Elements, Genetic; Erythropoietin; Gene Expression; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kv1.2 Potassium Channel; Kv1.5 Potassium Channel; Muscle, Smooth, Vascular; Mutant Proteins; PC12 Cells; Potassium Channels, Voltage-Gated; Pulmonary Artery; Rats; RNA, Messenger; Shab Potassium Channels; Transfection

Intermittent hypoxia treatment (IHT) is gaining attention as a clinical modality due to its capacity to protect cells, tissues, organs, and the whole organism from more intense and/or sustained hypoxia, ischemia and other stresses, to enhance physical and mental capacity. Circulating hematopoietic stem and progenitor cells (HSPC) play an important role in immune response to hypoxia ensuring tissue reparation processes, formation of all types of blood cells etc. There exist considerable individual differences in the capability to mobilize HSPC. This study was designed to compare the effects of IHT on HSPC, various factors of natural resistance and main humoral and cellular components of adaptive immunity in peripheral blood of subjects with normal and reduced tolerance to hypoxic load. Ten healthy male volunteers (age 30,9 +/- 0,6 y.o.) participated in the study and were divided into two groups with reduced (RT, 5 subjects) and normal (NT, 5 subjects) hypoxic tolerance. Criterion for reduced hypoxic tolerance detection was the exceeding deviation of arterial blood pressure, minute ventilation, SaO2 and/or pathological changes in ECG during sustained hypoxia test (breathing with 10% oxygen, 10 min). All subjects were studied before and after a 14 day IHT program consisting of four 5 min bouts/d of breathing 10% O2, with intervening 5 min room air exposures. Immunofluorescence detected HSPCs as CD45+CD34+ cells in peripheral blood. Phagocytic and bactericidal activities of neutrophils, circulating immunoglobulins (IgM, IgG, IgA), immune complexes, complement, and cytokines (EPO, TNF-alpha, IL-4, IFN-gamma) were measured. It was shown that NT subjects had higher hemoglobin and erythrocytes level, hematocrit and physical working capacity, but leukocytes, lymphocytes, CD8+-cells contents and level of IgA were lower than in RT. These differences were preserved after IHT course. CD45+34+cells content was the same in both groups before IHT, but RT subjects demonstrated twice decrease in HSPCs content after IHT as opposed to NT who did not show distinct reactions. A decrease in HSPCs is probably associated with the change of their migration capacity. However, it remains unclear whether there is an inhibition of HSPCs migration into circulation or an activation ofHSPCs escape from circulation. In both cases tissues could accumulate more HSPCs which in turn could enhance hematopoiesis and general regenerative potential. RT group also had lower complement, induced and

Topics: Adaptation, Physiological; Adult; Antigen-Antibody Complex; Blood Pressure; Cell Count; Cell Movement; Complement System Proteins; Cytokines; Electrocardiography; Erythropoietin; Hematopoietic Stem Cells; Hemoglobins; Humans; Hypoxia; Immunity, Cellular; Immunity, Humoral; Male; Oxygen; Phagocytosis; Pulmonary Gas Exchange; Respiration

Autophagy is a self-degradative process that involves turnover and recycling of cytoplasmic components in healthy and diseased tissue. Autophagy has been shown to be protective at the early stages of programmed cell death but it can also promote apoptosis under certain conditions. Earlier we demonstrated that oxygen contributes to the pathogenesis of neonatal brain damage, which can be ameliorated by intervention with recombinant human erythropoietin (rhEpo). Extrinsic- and intrinsic apoptotic pathways are involved in oxygen induced neurotoxicity but the role of autophagy in this model is unclear. We analyzed the expression of autophagy activity markers in the immature rodent brain after exposure to elevated oxygen concentrations. We observed a hyperoxia-exposure dependent regulation of autophagy-related gene (Atg) proteins Atg3, 5, 12, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), LC3A-II, and LC3B-II which are all key autophagy activity proteins. Interestingly, a single injection with rhEpo at the onset of hyperoxia counteracted these oxygen-mediated effects. Our results indicate that rhEpo generates its protective effect by modifying the key autophagy activity proteins.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Proteins; Beclin-1; Brain; Epoetin Alfa; Erythropoietin; Humans; Hypoxia; Microtubule-Associated Proteins; Models, Animal; Peptide Synthases; Proteins; Rats; Rats, Wistar; Recombinant Proteins; Signal Transduction

Heat shock protein 72 (HSP72) performs vital roles within the body at rest and during periods of stress. In vitro, research demonstrates HSP72 induction in response to hypoxia. Recently, in vivo, an acute hypoxic exposure (75 min at 2,980 m) was sufficient to induce significant increases in monocyte expressed HSP72 (mHSP72) and a marker of oxidative stress in healthy human subjects. The purpose of the current study was to identify the impact of 10 consecutive days of hypoxic exposures (75 min at 2,980 m) on mHSP72 and erythropoietin (EPO) expression, markers of oxidative stress, and maximal oxygen consumption in graded incremental aerobic exercise. Eight male subjects were exposed to daily normobaric hypoxic exposures for 75 min at 2,980 m for 10 consecutive days, commencing and ceasing at 0930 and 1045, respectively. This stressor was sufficient to induce significant increases in mHSP72, which was significantly elevated from day 2 of the hypoxic exposures until 48 h post-final exposure. Notably, this increase had an initial rapid (30% day on day compared to baseline) and final slow phase (16% day on day compared to baseline) of expression. The authors postulate that 7-day hypoxic exposure in this manner would be sufficient to induce near maximum hypoxia-mediated basal mHSP72 expression. Elevated levels of mHSP72 are associated with acquired thermotolerance and provide cross tolerance to non-related stressors in vivo, the protocol used here may provide a useful tool for elevating mHSP72 in vivo. Aside from these major findings, significant transient daily elevations were seen in a marker of oxidative stress, alongside sustained increases in EPO expression. However, no physiologically significant changes were seen in maximal oxygen consumption or time to exhaustion.

Topics: Adolescent; Cells, Cultured; Erythropoietin; HSP72 Heat-Shock Proteins; Humans; Hypoxia; Male; Monocytes; Oxidative Stress; Oxygen Consumption; Up-Regulation; Young Adult

Erythropoietin (EPO) and soluble EPO receptors (sEPOR) have been proposed to play a central role in the ventilatory acclimatisation to continuous hypoxia in mice. In this study, we demonstrated for the first time in humans (n = 9) that sEPOR is downregulated upon daytime exposure to 4 days of intermittent hypoxia (IH; 6 h·day⁻¹, cycles of 2 min of hypoxia followed by 2 min of reoxygenation; peak end-tidal oxygen tension (P(ET,O₂)) 88 Torr, nadir P(ET,O₂)) 45 Torr), thereby allowing EPO concentration to rise. We also determined the strength of the association between these haematological adaptations and alterations in the acute hypoxic ventilatory response (AHVR). We observed a nadir in sEPOR on day 2 (-70%), concomitant with the peak in EPO concentration (+50%). Following exposure to IH, tidal volume (V(T)) increased, respiratory frequency remained unchanged, and minute ventilation (V'(E)) was increased. There was a negative correlation between EPO and sEPOR (r = -0.261; p = 0.05), and between sEPOR and V(T) (r = -0.331; p = 0.02). EPO was positively correlated with V'(E) (r = 0.458; p = 0.001). In conclusion, the downregulation of sEPOR by IH modulates the subsequent EPO response. Furthermore, the alterations in AHVR and breathing pattern following IH appear to be mediated, at least in part, by the increase in EPO.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Deoxyguanosine; Down-Regulation; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Male; Oxidative Stress; Oxygen; Receptors, Erythropoietin; Respiration; Ventilation

Hypoxia at high altitudes can lead to increased production of red blood cells through the hormone erythropoietin (EPO). In this study, we observed how the EPO-unresponsive hematopoietic stem cell (HSC) compartment responds to high-altitude hypoxic environments and contributes to erythropoiesis.. Using a mouse model at simulated high altitude, the bone marrow (BM) and spleen lineage marker(-)Sca-1(+)c-Kit(+) (LSK) HSC compartment were observed in detail. Normal LSK cells were then cultured under different conditions (varying EPO levels, oxygen concentrations, and BM supernatants) to investigate the causes of the HSC responses.. Hypoxic mice exhibited a marked expansion in BM and spleen LSK compartments, which were associated with enhanced proliferation. BM HSCs seemed to play a more important role in erythropoiesis at high altitude than spleen HSCs. There was also a lineage fate change of BM HSCs in hypoxic mice that was manifested in increased megakaryocyte-erythrocyte progenitors and periodically reduced granulocyte-macrophage progenitors in the BM. The LSK cells in hypoxic mice displayed upregulated erythroid-specific GATA-1 and downregulated granulocyte-macrophage-specific PU.1 messenger RNA expression, as well as the capacity to differentiate into more erythroid precursors after culture. BM culture supernatant from hypoxic mice (but not elevated EPO or varying O(2) tension) could induce expansion and erythroid-priority differentiation of the HSC population, a phenomenon partially caused by increasing interleukin-3 and interleukin-6 secretion in the BM.. The present study suggests a new EPO-independent HSC mechanism of high-altitude erythrocytosis.

Topics: Altitude; Animals; Base Sequence; Cells, Cultured; Cytokines; Disease Models, Animal; DNA Primers; Erythropoietin; Flow Cytometry; Hematopoietic Stem Cells; Hypoxia; Male; Mice; Mice, Inbred BALB C; Oxygen; Polycythemia; Reverse Transcriptase Polymerase Chain Reaction

Diabetic retinopathy (DR) is a leading cause of acquired blindness among the people at working age. Although remarkable advances have been made in the treatment of the proliferative form of DR, there is still no effective treatment for the most prevalent early form of DR. The exact etiology and molecular pathogenesis of the early DR are not fully understood, but the selective loss of pericytes is believed to play a major role in the pathological process of DR. Herein we propose a novel approach to the treatment of early form of DR, using erythropoietin (EPO). We hypothesize that EPO reduces the loss of retinal pericytes, and therefore can be used as a novel therapeutic agent for early form of DR, which is based on its antioxidant, anti-inflammatory, and neuroprotective properties. If successful, future studies based on this hypothesis may also help shield the lights on the molecular mechanisms of early DR.

Topics: Animals; Apoptosis; Diabetic Retinopathy; Erythropoietin; Female; Humans; Hypoxia; Male; Mice; Models, Theoretical; Neurons; Pericytes; Retina; Treatment Outcome

Iron is tightly connected to oxygen homeostasis and erythropoiesis. Our aim was to better understand how hypoxia regulates iron acquisition for erythropoiesis in humans, a topic relevant to common hypoxia-related disorders. Forty-seven healthy volunteers participated in the HIGHCARE project. Blood samples were collected at sea level and after acute and chronic exposure to high altitude (3400-5400 m above sea level). We investigated the modifications in hematocrit, serum iron indices, erythropoietin, markers of erythropoietic activity, interleukin-6, and serum hepcidin. Hepcidin decreased within 40 hours after acute hypoxia exposure (P < .05) at 3400 m, reaching the lowest level at 5400 m (80% reduction). Erythropoietin significantly increased (P < .001) within 16 hours after hypoxia exposure followed by a marked erythropoietic response supported by the increased iron supply. Growth differentiation factor-15 progressively increased during the study period. Serum ferritin showed a very rapid decrease, suggesting the existence of hypoxia-dependent mechanism(s) regulating storage iron mobilization. The strong correlation between serum ferritin and hepcidin at each point during the study indicates that iron itself or the kinetics of iron use in response to hypoxia may signal hepcidin down-regulation. The combined and significant changes in other variables probably contribute to the suppression of hepcidin in this setting.

Topics: Antimicrobial Cationic Peptides; Down-Regulation; Erythropoiesis; Erythropoietin; Female; Ferritins; Hematocrit; Hepcidins; Humans; Hypoxia; Iron; Male

Rhodiolae Crenulatae Radix et Rhizoma (Rhodiola), the root and rhizome of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, has been used as a traditional Chinese medicine (TCM) to increase the body resistance against hypoxia in mountain sickness. The mechanism of this adaptogenic property deriving from Rhodiola, however, has not been revealed. Erythropoietin (EPO) is an erythrocyte-specific hematopoietic hormone that increases the production of red blood cells: this hormone is a crucial factor in regulating the body balance in responding to hypoxia. In cultured kidney fibroblasts (HEK293T), application of water extract deriving from Rhodiola induced the expression of EPO both in mRNA and protein levels. The activation of the Hypoxia Response Element (HRE) located on the promoter region of the EPO gene is one of the mechanisms accounting for transcriptional activation. In addition, the Rhodiola-induced EPO expression was triggered by an increase of hypoxia-inducible factor-1 α (HIF-1 α) protein, via the reduction of HIF-1 α degradation but not the induction of HIF-1 α mRNA. Moreover, the same EPO induction effect by Rhodiola was also observed in cultured liver cells since liver is another vital organ to provide EPO regulation apart from the kidney. These results therefore elucidate one of the molecular mechanisms of this herb in mediating the anti-hypoxia function.

Topics: Cells, Cultured; Deferoxamine; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Erythropoietin; Fibroblasts; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Mixed Function Oxygenases; Plant Roots; Recombinant Fusion Proteins; Response Elements; Rhizome; Rhodiola; RNA, Messenger; Siderophores; Transcriptional Activation

Topics: Animals; Astrocytes; Erythropoietin; Humans; Hypoxia

Acute lung exposure to low oxygen results in pulmonary vasoconstriction and redistribution of blood flow. We used human microvascular endothelial cells from lung (HMVEC-L) to study the acute response to oxygen stress. We observed that hypoxia and erythropoietin (EPO) increased erythropoietin receptor (EPOR) gene expression and protein level in HMVEC-L. In addition, EPO dose- and time-dependently stimulated nitric oxide (NO) production. This NO stimulation was evident despite hypoxia induced reduction of endothelial NO synthase (eNOS) gene expression. Western blot of phospho-eNOS (serine1177) and eNOS and was significantly induced by hypoxia but not after EPO treatment. However, iNOS increased at hypoxia and with EPO stimulation compared to normal oxygen tension. In accordance with our previous results of NO induction by EPO at low oxygen tension in human umbilical vein endothelial cells and bone marrow endothelial cells, these results provide further evidence in HMVEC-L for EPO regulation of NO production to modify the effects of hypoxia and cause compensatory vasoconstriction.

Topics: Blotting, Western; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular; Erythropoietin; Humans; Hypoxia; Lung; Microvessels; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Reverse Transcriptase Polymerase Chain Reaction

Angiogenesis is an important mechanism of airway remodeling in lung disease. We previously demonstrated that serum vascular endothelial growth factor (VEGF) is elevated in cystic fibrosis (CF) patients and declines with therapy for pulmonary exacerbation. We hypothesized that VEGF is elevated early in the course of CF and is associated with markers of tissue hypoxia. A prospective, single-visit evaluation of thirty stable infants and children with CF was performed. Serum was analyzed for VEGF and for other markers of tissue hypoxia (erythropoietin (EPO), insulin-like growth factor binding protein-1 (IGFBP-1)) and for inflammatory mediators (IL-1 beta, IL-6, IL-8, and tumor necrosis factor alpha (TNFα)) using Meso Scale multi-spot serum immunoassays. Measurements were correlated between assay groups; and with age in months and pulmonary function (FEV0.5 or FEV1). VEGF, EPO, TNFα and IL-8 were elevated compared to published normative values. VEGF levels were not significantly correlated with any inflammatory mediators. However, VEGF correlated with EPO (r=0.505; P<0.05). There was no correlation between lung function and markers of inflammation or tissue hypoxia. VEGF is elevated in young, stable infants and children suggesting angiogenesis as a contributing mechanism for early lung disease in CF. VEGF elevation does not show significant correlation with inflammatory mediators known to be increased in CF, but is significantly correlated with EPO levels. We propose that VEGF elevation and angiogenesis contribute to early lung disease and may result from a direct tissue hypoxia pathway in CF.

Topics: Age Factors; Child; Child, Preschool; Cohort Studies; Cystic Fibrosis; Cytokines; Erythropoietin; Female; Humans; Hypoxia; Inflammation Mediators; Lung; Male; Nutritional Status; Prospective Studies; Respiratory Function Tests; Vascular Endothelial Growth Factor A

Despite enhancing cardiopulmonary and muscular fitness, the effect of hypoxic exercise training (HE) on hemorheological regulation remains unclear. This study investigates how HE modulates erythrocyte rheological properties and further explores the underlying mechanisms in the hemorheological alterations. Twenty-four sedentary males were randomly divided into hypoxic (HE; n = 12) and normoxic (NE; n = 12) exercise training groups. The subjects were trained on 60% of maximum work rate under 15% (HE) or 21% (NE) O(2) condition for 30 min daily, 5 days weekly for 5 wk. The results demonstrated that HE 1) downregulated CD47 and CD147 expressions on erythrocytes, 2) decreased actin and spectrin contents in erythrocytes, 3) reduced erythrocyte deformability under shear flow, and 4) diminished erythrocyte volume changed by hypotonic stress. Treatment of erythrocytes with H(2)O(2) that mimicked in vivo prooxidative status resulted in the cell shrinkage, rigidity, and phosphatidylserine exposure, whereas HE enhanced the eryptotic responses to H(2)O(2). However, HE decreased the degrees of clotrimazole to blunt ionomycin-induced shrinkage, rigidity, and cytoskeleton breakdown of erythrocytes, referred to as Gardos effects. Reduced erythrocyte deformability by H(2)O(2) was inversely related to the erythrocyte Gardos effect on the rheological function. Conversely, NE intervention did not significantly change resting and exercise erythrocyte rheological properties. Therefore, we conclude that HE rather than NE reduces erythrocyte deformability and volume regulation, accompanied by an increase in the eryptotic response to oxidative stress. Simultaneously, this intervention depresses Gardos channel-modulated erythrocyte rheological functions. Results of this study provide further insight into erythrocyte senescence induced by HE.

Topics: Adult; Cell Count; Cell Separation; Erythrocyte Aging; Erythrocyte Deformability; Erythrocytes; Erythropoietin; Exercise; Exercise Test; Flow Cytometry; Humans; Hydrogen Peroxide; Hypoxia; Intermediate-Conductance Calcium-Activated Potassium Channels; Male; Oxidative Stress; Physical Fitness; Reticulocytes; Rheology; Sedentary Behavior

Periventricular leukomalacia (PVL) is the predominant pathology in premature infants, characterized by prominent cerebral white matter injury, and commonly caused by hypoxia-ischemia and inflammation. Activated microglia trigger white matter damage and play a major role in the development of PVL. Erythropoietin (EPO) and its derivative carbamylated erythropoietin (CEPO) have been shown to be neuroprotective in several brain disease models. Here we investigated whether EPO and CEPO could provide protection in mouse models of PVL induced by hypoxia-ischemia or hypoxia-ischemia-inflammation. We administered EPO or CEPO to mice with PVL, and found that both EPO and CEPO treatments decreased microglia activation, oligodendrocyte damage and myelin depletion. We also noted improved performance in neurological function assays. Inhibited disease progression in PVL mice by EPO or CEPO treatment was associated with decreased poly-(ADP-ribose) polymerase-1 (PARP-1) activity. PARP-1 activity was increased dramatically in activated microglia in untreated mice with PVL. Furthermore, we demonstrated that the neuroprotective properties of EPO and CEPO were diminished after PARP-1 gene depletion. The therapeutic doses of EPO and CEPO used in this study did not interfere with normal oligodendrocyte maturation and myelination. Together, our data demonstrate that EPO and CEPO are neuroprotective in cerebral white matter injury via a novel microglial PARP-1 dependent mechanism, and hold promise as a future treatment for PVL and other hypoxic-ischemic/inflammatory white matter diseases.

Topics: Animals; Brain; Brain Ischemia; Disease Models, Animal; Erythropoietin; Humans; Hypoxia; Infant, Newborn; Inflammation; Leukomalacia, Periventricular; Mice; Microglia; Myelin Sheath; Neurons; Neuroprotective Agents; Oligodendroglia

Critical limb ischemia causes severe damage to the skeletal muscle. This study develops a reproducible model of myotube ischemia by simulating, in vitro, the critical parameters that occur in skeletal muscle ischemia. Monolayers of C2C12 myoblasts were differentiated into mature myotubes and exposed to nutrition depletion, hypoxia and hypercapnia for variable time periods. A range of culture media and gas mixture combinations were used to obtain an optimum ischemic environment. Nuclear staining, cleaved caspase-3 and lactate dehydrogenase (LDH) release assay were used to assess apoptosis and myotube survival. HIF-1α concentration of cell lysates, pH of conditioned media as well as partial pressures of oxygen (PO₂) and carbon dioxide (PCO₂) in the media were used to confirm ischemic simulation. Culturing myotubes in depleted media, in a gas mixture containing 20% CO+80% N₂ for 6-12 h increased the PCO₂ and decreased the pH and PO₂ of culture media. This attempts to mimic the in vivo ischemic state of skeletal muscle. These conditions were used to study the potential tissue-protective effects of erythropoietin (EPO) in C2C12 myotubes exposed to ischemia. EPO (60 ng/ml) suppressed LDH release, decreased cleaved caspase-3 and reduced the number of apoptotic nuclei, suggesting significantly decreased ischemia-induced apoptosis in myotubes (P<0.01) and a potential role in tissue protection. Additional therapeutic agents designed for tissue protection can also be evaluated using this model.

Topics: Animals; Apoptosis; Carbon Dioxide; Caspase 3; Cell Differentiation; Cell Line; Cell Nucleus; Chromatin; Erythropoietin; Hydrogen-Ion Concentration; Hypercapnia; Hypoxia; Ischemia; L-Lactate Dehydrogenase; Mice; Models, Biological; Muscle Fibers, Skeletal; Myoblasts, Skeletal; Oxygen; Receptors, Erythropoietin

Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1 (+/-) ) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1 (+/-) mice relative to wild-type mice. Endothelial cells from Becn1 (+/-) mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1 (+/-) cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1 (+/-) endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; Basic Helix-Loop-Helix Transcription Factors; Beclin-1; Blotting, Western; Cell Movement; Cell Proliferation; Endothelial Cells; Erythropoietin; Heterozygote; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lung; Melanoma; Mice; Neoplasm Transplantation; Neovascularization, Pathologic; Protein Stability; RNA, Small Interfering; Stress, Physiological

In amyotrophic lateral sclerosis (ALS), respiratory muscle weakness causes ventilatory insufficiency and tissue hypoxia, which induces a number of metabolic pathways, and in particular increases erythropoietin (EPO) synthesis. EPO is a glycoprotein with neuroprotective properties that stimulates erythropoiesis. Here, EPO plasma level in a large population of ALS patients, with and without respiratory failure, was measured. Plasma EPO level of patients with ALS (n = 98), controls with other neuromuscular diseases (n = 58) and healthy controls (n = 20) has been quantified by ELISA. No significant difference was found between ALS patients and the two control groups. EPO level was not different between bulbar- and spinal-onset patients and was not correlated with disease duration or functional impairment. However, in the ALS group EPO level was higher in females (p = 0.0006) and correlated positively with age (p = 0.006). The subgroup of ALS patients with respiratory failure had higher plasma levels of EPO compared with ALS patients with preserved respiratory function (p = 0.016), but short-term non-invasive ventilation did not change EPO level. In conclusion, EPO levels were found to be significantly higher in ALS patients with respiratory impairment representing preservation of this homeostatic mechanism.

Topics: Amyotrophic Lateral Sclerosis; Animals; Erythropoietin; Female; Humans; Hypoxia; Male; Respiratory Muscles; Ventilation

Hypoxia frequently associated with certain physiologic and pathologic conditions influences numerous cellular functions. Because the effects of short-term hypoxia are incompletely understood, we examined phagocytosis and cytokine production as well as the activation of the transcription factors HIF-1 and NFκB in peripheral blood cells of healthy volunteers exposed to an oxygen concentration equivalent to that found at a height of 5500 m. Furthermore, we analysed plasma HIF-1 and serum concentrations of various HIF-1-dependent genes. Results showed that short-term hypoxia increased phagocytosis in neutrophils without affecting monocyte phagocytosis. Hypoxia decreased basal TNFα concentration in monocytes and basal interferon γ concentration in CD4(+) T lymphocytes. In contrast, plasma HIF and serum VEGF concentrations were not affected by hypoxia, although serum EPO concentration was raised. In PBMC, hypoxia increased cytosolic HIF-1 concentration without affecting nuclear HIF-1 concentration and led to a rise in the nuclear NFκB in PBMC. Our results show that short-term hypoxia affects immune functions in healthy individuals. Furthermore, we speculate that the effects of hypoxia are not due to HIF-1, but are caused by the activation of NFκB .

Topics: Adult; Atmospheric Pressure; CD4-Positive T-Lymphocytes; Cytokines; Erythropoietin; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lactic Acid; Male; Monocytes; Neutrophils; NF-kappa B p50 Subunit; Phagocytosis; Transcription Factor RelA; Vascular Endothelial Growth Factor A; Young Adult; Zymosan

Cerebral malaria (CM) is associated with high mortality and risk of sequelae, and development of adjunct therapies is hampered by limited knowledge of its pathogenesis. To assess the role of cerebral hypoxia, we used two experimental models of CM, Plasmodium berghei ANKA in CBA and C57BL/6 mice, and two models of malaria without neurologic signs, P. berghei K173 in CBA mice and P. berghei ANKA in BALB/c mice. Hypoxia was demonstrated in brain sections using intravenous pimonidazole and staining with hypoxia-inducible factor-1α-specific antibody. Cytopathic hypoxia was studied using poly (ADP-ribose) polymerase-1 (PARP-1) gene knockout mice. The effect of erythropoietin, an oxygen-sensitive cytokine that mediates protection against CM, on cerebral hypoxia was studied in C57BL/6 mice. Numerous hypoxic foci of neurons and glial cells were observed in mice with CM. Substantially fewer and smaller foci were observed in mice without CM, and hypoxia seemed to be confined to neuronal cell somas. PARP-1-deficient mice were not protected against CM, which argues against a role for cytopathic hypoxia. Erythropoietin therapy reversed the development of CM and substantially reduced the degree of neural hypoxia. These findings demonstrate cerebral hypoxia in malaria, strongly associated with cerebral dysfunction and a possible target for adjunctive therapy.

Topics: Animals; Brain; Disease Models, Animal; Erythropoietin; Female; Fluorescent Dyes; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Malaria, Cerebral; Mice; Nitroimidazoles; Parasitemia; Plasmodium berghei; Poly(ADP-ribose) Polymerases; Survival Analysis; Treatment Outcome

Topics: Erythropoietin; Humans; Hypoxia; Oxygen

Sleep disordered breathing (SDB), which is characterized by intermittent hypoxia (IH) during sleep, causes substantial cardiovascular and neurocognitive complications and has become a growing public health problem. SDB is associated with suppression of growth hormone (GH) secretion, the latter being integrally involved in the growth, development, and function of the CNS. Since GH treatment is able to attenuate neurocognitive deficits in a hypoxic-ischemic stroke model, GH, GH receptor (GHR) mRNA expression, and GH protein expression were assessed in rat hippocampus after exposures to chronic sustained hypoxia (CH, 10% O(2)) or IH (10% O(2) alternating with 21% O(2) every 90 s). In addition, the effect of GH treatment (50 μg/kg daily s.c. injection) on erythropoietin (EPO), vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1), and GLUT-1 mRNA expression and neurobehavioral function was assessed. CH significantly increased GH mRNA and protein expression, as well as insulin-like growth factor-1 (IGF-1). In contrast, IH only induced a moderate increase in GH mRNA and a slight elevation in GH protein at day 1, but no increases in IGF-1. CH, but not IH, up-regulated GHR mRNA in the hippocampus. IH induced marked neurocognitive deficits compared with CH or room air (RA). Furthermore, exogenous GH administration increased hippocampal mRNA expression of IGF-1, EPO, and VEGF, and not only reduced IH-induced hippocampal injury, but also attenuated IH-induced cognitive deficits. Thus, exogenous GH may provide a viable therapeutic intervention to protect IH-vulnerable brain regions from SDB-associated neuronal loss and associated neurocognitive dysfunction.

Topics: Animals; Caspase 3; Cognition Disorders; Disease Models, Animal; Erythropoietin; Glucose Transporter Type 1; Growth Hormone; Heme Oxygenase-1; Hippocampus; Humans; Hypoxia; Insulin-Like Growth Factor I; Male; Maze Learning; Rats; Rats, Sprague-Dawley; Receptors, Somatotropin; Vascular Endothelial Growth Factor A

Although hypoxia and ischemia are known to be involved in the pathogenesis of cardiovascular disease, specific therapeutic targets still remain elusive. To address this important issue, we have performed 2 series of experimental studies, aiming at erythropoietin (Epo)/Epo receptor (EpoR) based on the following backgrounds. Epo has long been regarded as a hematopoietic hormone that acts exclusively in the proliferation and differentiation of erythroid progenitors. Although recent studies have demonstrated that EpoR is expressed in the cardiovascular system, the potential protective role of the vascular Epo/EpoR system in vivo remains to be examined. We hypothesized that the vascular Epo/EpoR system plays an important protective role against the development of cardiovascular disease. Using vascular EpoR-deficient mouse, we demonstrated that the vascular Epo/EpoR system plays a crucial role for endothelial function and vascular homeostasis. The vascular Epo/EpoR system is important for the activation of the vascular endothelial growth factor/vascular endothelial growth factor receptor-2 system, inhibits hypoxia-induced pulmonary endothelial damage and promotes ischemia-induced angiogenesis in vivo. These results indicate that the vascular Epo/EpoR system plays an important protective role against hypoxia/ischemia, demonstrating that this system is a novel therapeutic target in cardiovascular medicine.

Topics: Animals; Cardiovascular Agents; Cardiovascular Diseases; Drug Delivery Systems; Endothelium, Vascular; Erythropoietin; Homeostasis; Humans; Hypoxia; Mice; Mice, Knockout; Myocardial Ischemia; Receptors, Erythropoietin

Anemia because of insufficient production of and/or response to erythropoietin (Epo) is a major complication of chronic kidney disease and cancer. The mechanisms modulating the sensitivity of erythroblasts to Epo remain poorly understood. We show that, when cultured with Epo at suboptimal concentrations, the growth and clonogenic potential of erythroblasts was rescued by transferrin receptor 1 (TfR1)-bound polymeric IgA1 (pIgA1). Under homeostatic conditions, erythroblast numbers were increased in mice expressing human IgA1 compared to control mice. Hypoxic stress of these mice led to increased amounts of pIgA1 and erythroblast expansion. Expression of human IgA1 or treatment of wild-type mice with the TfR1 ligands pIgA1 or iron-loaded transferrin (Fe-Tf) accelerated recovery from acute anemia. TfR1 engagement by either pIgA1 or Fe-Tf increased cell sensitivity to Epo by inducing activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. These cellular responses were mediated through the TfR1-internalization motif, YXXΦ. Our results show that pIgA1 and TfR1 are positive regulators of erythropoiesis in both physiological and pathological situations. Targeting this pathway may provide alternate approaches to the treatment of ineffective erythropoiesis and anemia.

Topics: Anemia; Animals; Cell Proliferation; Cells, Cultured; Erythroblasts; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Immunoglobulin A; Mice; Mice, Inbred NOD; Mice, SCID; Mice, Transgenic; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Receptors, Transferrin; Signal Transduction; Transferrin

Inflammatory microglia modulate a host of cellular processes in the central nervous system that include neuronal survival, metabolic fluxes, foreign body exclusion, and cellular regeneration. Elucidation of the pathways that oversee microglial survival and integrity may offer new avenues for the treatment of neurodegenerative disorders. Here we demonstrate that erythropoietin (EPO), an emerging strategy for immune system modulation, prevents microglial early and late apoptotic injury during oxidant stress through Wnt1, a cysteine-rich glycosylated protein that modulates cellular development and survival. Loss of Wnt1 through blockade of Wnt1 signaling or through the gene silencing of Wnt1 eliminates the protective capacity of EPO. Furthermore, endogenous Wnt1 in microglia is vital to preserve microglial survival since loss of Wnt1 alone increases microglial injury during oxidative stress. Cellular protection by EPO and Wnt1 intersects at the level of protein kinase B (Akt1), the mammalian target of rapamycin (mTOR), and p70S6K, which are necessary to foster cytoprotection for microglia. Downstream from these pathways, EPO and Wnt1 control "anti-apoptotic" pathways of microglia through the modulation of mitochondrial membrane permeability, the release of cytochrome c, and the expression of apoptotic protease activating factor-1 (Apaf-1) and X-linked inhibitor of apoptosis protein (XIAP). These studies offer new insights for the development of innovative therapeutic strategies for neurodegenerative disorders that focus upon inflammatory microglia and novel signal transduction pathways.

Topics: Analysis of Variance; Animals; Antibodies; Apoptotic Protease-Activating Factor 1; Cell Death; Cell Line, Transformed; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression Regulation; Glucose; Humans; Hypoxia; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Mice; Mitochondria; Neuroglia; Phosphatidylserines; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Wnt1 Protein; X-Linked Inhibitor of Apoptosis Protein

Erythropoietin (EPO), originally identified as a hematopoietic growth factor produced in the kidney and fetal liver, is also endogenously expressed in the central nervous system (CNS). EPO in the CNS, mainly produced in astrocytes, is induced under hypoxic conditions in a hypoxia-inducible factor (HIF)-dependent manner and plays a dominant role in neuroprotection and neurogenesis. We investigated the effect of general anesthetics on EPO expression in the mouse brain and primary cultured astrocytes.. BALB/c mice were exposed to 10% oxygen with isoflurane at various concentrations (0.10-1.0%). Expression of EPO mRNA in the brain was studied, and the effects of sevoflurane, halothane, nitrous oxide, pentobarbital, ketamine, and propofol were investigated. In addition, expression of HIF-2α protein was studied by immunoblotting. Hypoxia-induced EPO mRNA expression in the brain was significantly suppressed by isoflurane in a concentration-dependent manner. A similar effect was confirmed for all other general anesthetics. Hypoxia-inducible expression of HIF-2α protein was also significantly suppressed with isoflurane. In the experiments using primary cultured astrocytes, isoflurane, pentobarbital, and ketamine suppressed hypoxia-inducible expression of HIF-2α protein and EPO mRNA.. Taken together, our results indicate that general anesthetics suppress activation of HIF-2 and inhibit hypoxia-induced EPO upregulation in the mouse brain through a direct effect on astrocytes.

Topics: Anesthetics, General; Animals; Brain; Cells, Cultured; Erythropoietin; Hypoxia; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Up-Regulation

Topics: Animals; Doping in Sports; Erythropoietin; Humans; Hypoxia; Oxygen Consumption; Recombinant Proteins

HIF-1 alpha (hypoxia-inducible factor-1 alpha) mediates the responses of mammalian cells to hypoxia/ischemia by inducing the expression of adaptive gene products (e.g., vascular endothelial growth factor (VEGF) and erythropoietin (EPO)). Persistent pulmonary hypertension of the newborn (PPHN) and cyanotic congenital heart disease (CCHD) are common neonatal diseases considered as paradigms of hypoxemia. Since the expression HIF-1 alpha, VEGF and EPO in newborns diagnosed with these diseases has yet to be studied, we set out to define the expression of these genes in peripheral blood from newborn infants diagnosed with PPHN and CCHD.. The mRNA transcripts encoding HIF-1 alpha, VEGF and EPO were measured by RT-PCR in healthy newborn infants and infants diagnosed with PPHN and CCHD.. An important increase in HIF-1 alpha expression was observed in both pathological conditions, accompanied by significant increases in VEGF and EPO expression when compared to healthy infants.. HIF-1 alpha mRNA expression increases in newborn infants with PPHN or CCHD, as does the expression of its target genes VEGF and EPO.

Topics: Erythropoietin; Heart Diseases; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Infant; Infant, Newborn; Persistent Fetal Circulation Syndrome; Vascular Endothelial Growth Factor A

Topics: Animals; Doping in Sports; Erythropoietin; Humans; Hypoxia; Oxygen Consumption; Rats; Recombinant Proteins

In a comparative study on erythrocytes (RBCs) drawn from mountaineers before and after a high-altitude stay, we observed that upon returning to sea level, their RBCs displayed a senescent-like phenotype as indicated by their density and the partial loss of membrane proteins which are shed by ageing RBCs. The aim of this study was to investigate possible changes in the membrane skeleton of these RBCs and to compare them with pathological RBCs. We analysed the proteins of RBC ghosts obtained from our subjects before and after returning to sea level by two-dimensional electrophoresis and mass spectrometry. We observed lower expression and fragmentation of beta-actin after exposure to hypoxia. This suggested an alteration in membrane skeleton structure, which was confirmed by beta-actin release in cell lysates during ghost preparation. We observed a similar actin fragmentation and release in RBC lysates from beta-thalassaemic patients. In conclusion, these results indicate that after exposure to hypoxia, RBCs display a modification of their actin and cytoskeleton instability.

Topics: Acclimatization; Actins; Adult; Altitude; Electrophoresis, Gel, Two-Dimensional; Erythrocyte Aging; Erythrocyte Membrane; Erythrocytes; Erythropoietin; Female; Flow Cytometry; Humans; Hypoxia; In Vitro Techniques; Male; Mountaineering; Oxidative Stress; Peptide Fragments; Phenotype; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The role of vascular endothelial growth factor (VEGF) and erythropoietin (EPO) in mediating hypoxic preconditioning under the acute intermittent hypoxic condition (AIH) was investigated in this study. Male Wistar rats were randomly assigned and kept in normoxic conditions, (Nx) or in AIH conditions and subjected to brief cycles hypoxia/reoxygenation. Hearts were isolated, perfused, and subjected to in vitro global ischemia followed by reperfusion. During and at the end of reperfusion, left ventricular developed pressure (LVDP); LV end diastolic pressure (LVEDP); rate pressure product (RPP); peak left ventricular pressure rise (DeltaP/Deltat (max) ) and heart rate (HR) were measured. Hearts subjected to AIH displayed a significant higher LVDP (P < .001), RPP (P < .001), and DeltaP/Deltat ( max) (P < .001). Expression of VEGF and EPO were significantly increased at 3, 8, and 24 hours after AIH. Hypoxic training could provide a new approach to enhance endogenous cardioprotective mechanisms.

Topics: Adaptation, Physiological; Animals; Erythropoietin; Hypoxia; Immunohistochemistry; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Male; Myocardial Reperfusion Injury; Myocytes, Cardiac; Rats; Rats, Wistar; Vascular Endothelial Growth Factor A

Topics: Animals; Apoptosis; Disease Models, Animal; Erythropoietin; Hypoxia; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitric Oxide Synthase; Oxidative Stress; Oxygen Consumption; Shock, Septic; Survival Rate

Topics: Animals; Doping in Sports; Erythropoietin; Humans; Hypoxia; Recombinant Proteins

Erythropoietin (Epo)-induced polycythemia is the main factor of adaptation to hypoxia. In this study, we analysed the effects of Epo deficiency on intrinsic functional properties of slow and fast twitch muscles in a model of erythropoietin deficient mice (Epo-TAg(h)) exposed to hypoxia. We hypothesised that Epo deficiency would be deleterious for skeletal muscle structure and phenotype, which could change its functional properties and alters the adaptive response to ambient hypoxia. Wild-type (WT) and Epo-TAg(h) mice were left in hypobaric chamber at 420 mm Hg pressure for 14 days. Soleus (SOL) and extensor digitorum longus (EDL) were analysed in vitro by mechanical measurements, immunohistological and biochemical analyses. The results were compared to those obtained in corresponding muscles of age-matched normoxic groups. Our data did not show any difference between the groups whatever the Epo deficiency and/or hypoxic conditions for twitch force, tetanic force, fatigue, typology and myosin heavy chain composition. Normoxic Epo-TAg(h) mice exhibit improved capillary-to-fibre ratio compared to WT mice in both SOL and EDL whereas no angiogenic effects of hypoxia or combined Epo-deficiency/hypoxia were observed. These results suggest that skeletal muscles possess a great capacity of adaptation to Epo deficiency. Then Epo deficiency is not a sufficient factor to modify intrinsic functional properties of skeletal muscles.

Topics: Animals; Erythropoietin; Hypoxia; Male; Mice; Mice, Knockout; Muscle Contraction; Muscle Fatigue; Muscle, Skeletal

It is not clear whether blood hemoglobin concentration ([Hb]) increases with an increase in the exposure period of intermittent hypoxia (IHx) and reaches a constant level. Furthermore, it is not known whether plasma erythropoietin concentration ([EPO]) also increases with an increase in the exposure period. Using a rat model, first, we evaluated changes in [EPO] every hour after single exposure of 10% O(2) for 120 min in order to determine a peak level of [EPO]. Second, we evaluated the effect of IHx of 10% O(2), 120 min/day for 0 (control), 1, 2, 3, 4, 6 and 8 weeks on [Hb], arterial blood pressure (BP), heart rate (HR), arterial blood gases (ABGs) and [EPO]. [EPO] increased after cessation of the single hypoxic exposure, reached a peak at 1 h, and decreased gradually to the control levels within 18 h. IHx of 10% O(2), 120 min/day, produced a time-dependent increase in [Hb], and [Hb] reached a constant level after the exposure for 6 weeks. BP increased after the exposure for 4 weeks and remained elevated. There was no significant difference in HR and ABGs. [EPO] increased significantly and remained elevated at the same level for 1-3 weeks, however, the peak level of [EPO] declined markedly after [Hb] reached a constant level.

Topics: Animals; Blood Pressure; Erythropoietin; Hemoglobins; Hypoxia; Male; Polycythemia; Rats; Rats, Sprague-Dawley

Topics: Antioxidants; Doping in Sports; Erythropoietin; Humans; Hypoxia; Muscle, Skeletal; Oxygen; Plasma Volume

Central sleep apnoea (CSA) and increased serum erythropoietin (EPO) concentration have each been associated with adverse prognosis in heart failure (HF) patients. The aim of this study was to examine the relationship between nocturnal hypoxaemia due to CSA and the serum EPO concentration in patients with HF.. Heart failure subjects (n = 33) and healthy controls (n = 18) underwent polysomnography (PSG) for diagnosis of CSA and identification and quantification of hypoxaemia. Blood collection for measurement of EPO was performed immediately post-PSG. For the analysis, HF subjects were dichotomized into subgroups defined by the presence or absence of CSA and by HF severity. Multivariate analyses were performed to evaluate the relationships of hypoxaemia and advanced HF to EPO concentration. Mean EPO concentration was 62% higher for HF subjects with CSA than for healthy controls (P = 0.004). The magnitude of nocturnal hypoxaemia was significantly and positively related to EPO concentration (r = 0.45, P = 0.02). Advanced HF was also significantly and positively related to EPO concentration (r = 0.43, P = 0.02). On multivariate analysis, the presence of combined nocturnal hypoxaemia and advanced HF yielded greater correlation to EPO concentration than either factor alone (r = 0.57, P = 0.04 and P = 0.05, respectively). Linear regression demonstrated that the combination of New York Heart Association Class and CSA was strongly associated with EPO concentration (P < 0.0001).. In non-anaemic HF patients, advanced HF and hypoxaemia due to CSA may each be independently associated with increased serum EPO concentration.

Topics: Aged; Biomarkers; Case-Control Studies; Disease Progression; Erythropoietin; Female; Health Status Indicators; Heart Failure; Humans; Hypoxia; Linear Models; Male; Middle Aged; Multivariate Analysis; Oxygen Consumption; Polysomnography; Prognosis; Risk Factors; Severity of Illness Index; Sleep Apnea, Central; Statistics as Topic

Training and hypoxia-associated changes in maximal oxygen uptake are mediated by different blood adaptations. Training increases blood volume because of plasma and red cell volume expansion, resulting in increased cardiac output, whereas hypoxia increases only red cell volume, leading to increased hemoglobin concentration and oxygen transport capacity. Blood doping mimics the altitude effects, however, by far exceeding its magnitude.

Topics: Adaptation, Physiological; Altitude; Blood Volume; Doping in Sports; Erythropoietin; Hematocrit; Hemoglobins; Humans; Hypoxia; Oxygen Consumption; Physical Education and Training; Recombinant Proteins

The level of circulating erythropoietin (EPO) in response to a fixed level of hypoxia shows substantial inter-individual variability, the source of which is undetermined. Arterial PO(2) at altitude is regulated in part by the hypoxic ventilatory response, which also shows a wide inter-individual variability. We asked if the ventilatory response to hypoxia is related to the magnitude of EPO release at moderate altitude. Twenty-six national class US distance runners (17 M, 9 F) participated in a test of isocapnic hypoxic ventilatory response (HVR) at sea level, 2-7 days prior to departure to altitude. EPO measures were obtained at sea level and after 20 h at 2500 m. HVR for all subjects was 0.21±0.16 L min⁻¹ %SaO₂⁻¹ (range 0.01-0.61 L min⁻¹ %SaO₂⁻¹), with no significant difference between men and women. EPO was significantly increased from pre-altitude (8.6±2.6 ng ml(-1), range 4.0-14.6 ng ml⁻¹) to acute altitude (16.6±4.4 ng ml⁻¹, range 5.0-27.0 ng ml⁻¹), an increase of 92.2±70.1%. There was no significant sex difference in the EPO increase. ΔEPO for all subjects was not correlated with HVR (r=-0.17). Similarly, a statistically or physiologically significant correlation was not present between ΔEPO and HVR within the group of men (r=-0.22) or women (r=-0.19). The variability in the acute EPO response to moderate altitude is not explained by differences in peripheral chemoresponsiveness in elite distance runners. These results suggest that factors acting downstream from the lung influence the magnitude of the acute EPO response to altitude.

Topics: Adult; Altitude; Breath Tests; Erythropoietin; Female; Humans; Hypoxia; Male; Respiratory Physiological Phenomena; Running; Sex Factors; Young Adult

Hypoxia-inducible expression of the erythropoietin (EPO) gene is mediated principally by hypoxia-inducible factor 2alpha (HIF-2alpha) in Hep3B cells under physiologic conditions. How/whether p300/CBP and the members of p160 coactivator family potentiate hypoxic induction of endogenous EPO and other HIF-2alpha and hypoxia-inducible factor 1alpha (HIF-1alpha) target genes remains unclear.. We demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that the histone acetyl transferase (HAT) coactivators p300, SRC-1 and SRC-3 are recruited to the 3' enhancer of the EPO gene upon hypoxic stimulation, and that each associates with the enhancer in a periodic fashion. Hypoxia induced acetylation of the EPO gene 5' promoter at histone 4 and lysine 23 of histone 3. Knocking down SRC-3, but not SRC-1 or SRC-2, using short interfering RNAs (siRNAs), reduced EPO transcriptional activity. Knocking down p300 resulted in dramatic down-regulation of hypoxic stimulation of EPO gene transcription, negated recruitment of RNA polymerase II to the gene's promoter, and eliminated hypoxia-stimulated acetylation at the promoter and recruitments of SRC-1 and SRC-3 to the enhancer. The inhibitory effects of knocking down p300 and the chromatin remodeling coactivator, Brm/Brg-1, on EPO transcription were additive, suggesting that p300 and Brm/Brg-1 act independently. p300 was also required for hypoxia induced transcription of the HIF-1alpha target gene, VEGF, but was dispensable for induction of two other HIF-1alpha target genes, PGK and LDHA. Knocking down CBP, a homolog of p300, augmented hypoxic induction of VEGF, LDHA and PGK. Different HIF target genes also exhibited different requirements for members of the p160 coactivator family.. p300 plays a central coactivator role in hypoxic induction of EPO. The coactivators exhibit different specificities for different HIF target genes and each can behave differently in transcriptional regulation of different target genes mediated by the same transcription factor.

Topics: Cell Line; Enhancer Elements, Genetic; Erythropoietin; Histone Acetyltransferases; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Nuclear Receptor Coactivator 3; p300-CBP Transcription Factors; Protein Binding; Protein Transport; Transcriptional Activation

Mandarin voles (Lasiopodomys mandarinus) spend almost all their lives underground and must have evolved remarkable physiological adaptations to subterranean hypoxic stress. To understand the response to hypoxia in blood system of this rodent in a region with lower altitude, we tested and compared the responding characteristics between Mandarin vole and Kunming mouse (Mus musculus) under chronic normobaric hypoxic treatment (10.0% O(2), 4 w). The results showed that: 1) as for responses to chronic hypoxia, HIF-1 alpha, EPO and VEGF exhibited similar patterns in two species. The expression of HIF-1 alpha and VEGF significantly increased while EPO decreased significantly, and HIF-1 alpha showed a greater increase at 10.0% oxygen level in Mandarin voles; 2) both rodents responded to chronic hypoxia mainly by increasing MCH, though KM mouse responded more acutely; 3) the change in MCHC in Mandarin vole was ignorable though it is significantly higher than that in KM mouse whose MCHC changed extensively and 4) both before and after hypoxic treatment, the capillary density in Mandarin vole was significantly higher than that in KM mouse, and it increased sharply in KM mouse after treatment. Our results indicated that, compared to KM mice, Mandarin voles did respond effectively to hypoxia stress after long-term adaptation to subterranean life environment.

Topics: Adaptation, Physiological; Animals; Arvicolinae; Capillaries; Erythropoietin; Female; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Vascular Endothelial Growth Factor A

Preliminary findings of various types of globin expressed in the respiratory bronchiolar and alveolar epithelium prompted us to compare the expression of erythropoietin (Epo) and its receptor (EpoR) in normal (healthy) human lung tissues with that in malignant lung tissues. The expression of Epo and EpoR was examined at the transcriptional and protein levels in normal and malignant lung tissues by reverse transcription-PCR, western blot, and immunohistochemical analyses. EpoR mRNA, but not Epo mRNA, was detected in all samples. In normal tissues, EpoR was detected in the mesothelium, chondrocytes, alveolar cells, vascular endothelial cells, smooth muscle fibers, macrophages, and neutrophils, while in malignant foci, the cancer cells of five malignant types showed various intensities of EpoR immunoreactivity. The pattern of staining of EpoR protein was generally stronger in the malignant tissues than in the normal samples. Phosphorylation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK-ERK1/2) was frequently seen in malignant cells, but not in the normal tissues, with the exception of macrophages. Based on the expression of Epo and EpoR mRNA with the EpoR in almost all cell components in normal tissues, we suggest that the normal lung may produce various types of globin through the autocrine and/or paracrine role of Epo. When the Epo signal is upregulated by hypoxic stress, the normal cells appear to transform into malignant cells and proliferate through activated MAPK signaling.

Topics: Autocrine Communication; Blotting, Western; Cell Transformation, Neoplastic; Erythropoietin; Globins; Humans; Hypoxia; Immunohistochemistry; Lung; Lung Neoplasms; MAP Kinase Signaling System; Paracrine Communication; Receptors, Erythropoietin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

Erythropoietin (Epo) and vascular growth factor (VEGF) are known to be involved in the regulation of cellular activity when oxygen transport is reduced as in anaemia or hypoxic conditions. Because it has been suggested that Epo could play a role in skeletal muscle development, regeneration, and angiogenesis, we aimed to assess Epo deficiency in both normoxia and hypoxia by using an Epo-deficient transgenic mouse model (Epo-TAg(h)). Histoimmunology, ELISA and real time RT-PCR did not show any muscle fiber atrophy or accumulation of active HIF-1alpha but an improvement of microvessel network and an upregulation of VEGFR2 mRNA in Epo-deficient gastrocnemius compared with Wild-Type one. In hypoxia, both models exhibit an upregulation of VEGF120 and VEGFR2 mRNA but no accumulation of Epo protein. EpoR mRNA is not up-regulated in both Epo-deficient and hypoxic gastrocnemius. These results suggest that muscle deconditioning observed in patients suffering from renal failure is not due to Epo deficiency.

Topics: Analysis of Variance; Animals; Erythropoietin; Histocytochemistry; Hypoxia; Hypoxia-Inducible Factor 1; Male; Mice; Mice, Transgenic; Microvessels; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Neovascularization, Physiologic; Receptors, Erythropoietin; Sarcomeres; Statistics, Nonparametric; Up-Regulation; Vascular Endothelial Growth Factors

Hypoxia and toxic metals are two common stressors found in the estuarine environment. To date little information is available on the combined effects of these stressors on early larval development in fish. We investigated the effect of cadmium and hypoxia exposure alone as well in combination on larval Cyprinodon variegatus. The LC(10) for cadmium was determined to be 0.3 ppm in a 96 h acute exposure. This concentration was used in all studies. Cadmium in larvae increased significantly with exposure time (1, 3, 5 and 7 days post-hatch). The increase was proportional to body weight and not affected by hypoxia. Cadmium responsive genes were identified by suppression subtractive hybridization (SSH) in Cyprinodon variegatus larvae after exposure to cadmium for 1, 3, 5 and 7 days. We obtained over 700 sequences from the cadmium cDNA library. Blast search of ESTs suggested that cadmium modulates multiple physiological processes. Pertinent to this study, cadmium was found to down-regulate both embryonic alpha and beta globin, which are expressed in erythrocytes generated during the first, or primitive, wave of erythropoiesis in teleosts. Hemoglobin (Hb) and erythropoietin (Epo) (the hormone that promotes red blood cell production) are known hypoxia-inducible genes. To explore the possibility that cadmium might offset the hypoxia-induced expression of Hb and Epo, we investigated the expression of both genes following hypoxia, cadmium and combined exposures for 1, 3, 5 and 7 days post-hatch. Since Epo had not yet been identified in C. variegatus we first successfully cloned a partial coding sequence of the C. variegatus hormone. Subsequent studies revealed that expression levels of Hb and Epo remained unchanged in the normoxic controls during the time course of the study. Hypoxia increased Epo expression relative to normoxic controls, on days 3, 5 and 7, while cadmium in hypoxia inhibited the increase. Only the changes on days 5 and 7 were statistically significant. Hypoxia also lead to a modest, but significant induction of Hb after 5 days. However, in spite of the Cd-induced down-regulation of Epo on day 5, Cd did not affect the hypoxia-induced expression of embryonic Hb at this time point. It appears therefore that Epo has only limited effect on primitive erythropoiesis in C. variegatus.

Topics: Amino Acid Sequence; Animals; Cadmium; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Hemoglobins; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Killifishes; Larva; Lethal Dose 50; Molecular Sequence Data; Sequence Alignment; Water Pollutants, Chemical

The source of circulating erythropoietin (EPO), the mediators and the mechanisms involved in the upregulation of EPO gene expression during acute-phase reaction are still poorly understood. Acute-phase reaction was induced by either intramuscular turpentine oil (TO) or intraperitoneal lipopolysaccharide (LPS) administration into wild-type and interleukin (IL)-6 knockout (KO) mice. Animals were killed at different time points and blood, liver and muscle tissue were collected. Serum levels of EPO were measured by enzyme-linked immunoadsorbent assay; liver and injured muscle samples were processed for RNA isolation and for protein analysis. EPO, hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) mRNA were analyzed by RT-PCR and the protein levels were analyzed by western blot and electrophoretic mobility shift assay. HIF-1alpha and HIF-2alpha localization was performed through immunofluorescence staining. EPO, HIF-1 and HIF-2 gene and protein expression levels were also analyzed in isolated mouse hepatocytes after stimulation with IL-6. In the wild-type animals, EPO serum levels increased dramatically at 12 h after the insults together with the hepatic gene expression. In TO-treated animals, the EPO gene expression reached an 8.2-fold increase at 12 h, and in LPS-treated mice a similar induction was recorded at 6 h (about 4.5-fold increase). In the IL-6KO strain, the upregulation after the inflammatory stimuli was much lower (only 2.0-fold increase). A progressive upregulation of HIF-1alpha and HIF-2alpha was detectable until 6 h after the insults, but only HIF-1alpha upregulation was reduced in IL-6KO mice. In isolated hepatocytes, stimulation with a single dose of IL-6 induced a nuclear accumulation of HIF-1alpha, in parallel with an increase of EPO mRNA. No effect on HIF-2alpha expression was found. IL-6 appears to be the main regulator of EPO gene expression and a major contributor for HIF-1alpha induction in hepatocytes and Kupffer cells during acute-phase response. The increase of HIF-2alpha, predominantly expressed in endothelial cells and fibroblast-like cells, seems not to be affected by the lack of IL-6.

Topics: Acute-Phase Reaction; Animals; Enzyme Assays; Erythropoietin; Gene Expression; Genes; Hepatocytes; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-6; Kupffer Cells; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Turpentine; Up-Regulation; Vascular Endothelial Growth Factor A

Altitude poses physiological challenges to the sports participant in excess of those encountered at sea level. The main problem is hypoxia and the reduction in oxygen transport capacity, which is linked to the fall in alveolar oxygen tension. Training at altitude is imperative as preparation for competing there in aerobic events. The acute adaptations on exposure to moderate altitudes may be beneficial for subsequent performance at sea level, and this has led to the use of altitude training camps, though their advantages have not been conclusively demonstrated. The benefits of altitude training depend on a variety of factors, including individual characteristics and environmental training variables.

Topics: Adaptation, Physiological; Altitude; Erythropoietin; Exercise; Humans; Hypoxia; Oxygen Consumption; Running; Sports

In the present study, the zebrafish breakdance mutant (bre) was used to assess the role of blood flow in development because it has been previously shown that bre larvae have a chronically reduced cardiac output as a result of ventricular contraction following only every second atrial contraction in addition to an atrial bradycardia. We confirmed a 50% reduction compared with control fish and further showed that blood flow in the caudal part of the dorsal aorta decreased by 80%. Associated with these reductions in blood flow were indications of developmental retardation in bre mutants, specifically delayed hatching, reduced cell proliferation, and a transiently decreased growth rate. Surprisingly, an increased red blood cell concentration and an earlier appearance of trunk vessels in bre larvae indicated some compensation to convective oxygen transport, although in previous studies it has been shown that zebrafish larvae at this stage obtain oxygen by bulk diffusion. In bre animals immunohistochemical analyses showed a significant increase in hypoxia inducible factor 1 (HIF)-α protein expression, comparable with wild-type larvae that were raised under hypoxic conditions. Accordingly, the expression of some hif downstream genes was affected. Furthermore, Affymetrix microarray analyses revealed a large number of genes that were differently expressed comparing control and bre larvae, and the number even increased with proceeding development. The results showed that a chronic reduction in blood flow generated hypoxic molecular signals despite partial compensation by increased oxygen carrying capacity and transiently slowed the overall development of zebrafish bre larvae.

Topics: Animals; Biological Transport; Cardiac Output; Cell Cycle Proteins; CLOCK Proteins; Cyclin B1; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Larva; Oligonucleotide Array Sequence Analysis; Oxygen; Polymerase Chain Reaction; Vascular Endothelial Growth Factor A; Zebrafish; Zebrafish Proteins

A hypoxia-inducible VEGF expression system with the oxygen-dependent degradation (ODD) domain was constructed and tested to be used in gene therapy for ischemic myocardial disease.. Luciferase and VEGF expression vector systems were constructed with or without the ODD domain: pEpo-SV-Luc (or pEpo-SV-VEGF) and pEpo-SV-Luc-ODD (or pEpo-SV-VEGF-ODD). In vitro gene expression efficiency of each vector type was evaluated in HEK 293 cells under both hypoxic and normoxic conditions. The amount of VEGF protein was estimated by ELISA. The VEGF expression vectors with or without the ODD domain were injected into ischemic rat myocardium. Fibrosis, neovascularization, and cardiomyocyte apoptosis were assessed using Masson's trichrome staining, α-smooth muscle actin (α-SMA) immunostaining, and the TUNEL assay, respectively.. The plasmid vectors containing ODD significantly improved the expression level of VEGF protein in hypoxic conditions. The enhancement of VEGF protein production was attributed to increased protein stability due to oxygen deficiency. In a rat model of myocardial ischemia, the pEpo-SV-VEGF-ODD group exhibited less myocardial fibrosis, higher microvessel density, and less cardiomyocyte apoptosis compared to the control groups (saline and pEpo-SV-VEGF treatments).. An ODD-mediated VEGF expression system that facilitates VEGF-production under hypoxia may be useful in the treatment of ischemic heart disease.

Topics: Animals; Apoptosis; Cell Line; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Genetic Therapy; Genetic Vectors; Humans; Hypoxia; Immunohistochemistry; In Situ Nick-End Labeling; Luciferases; Male; Myocardial Ischemia; Myocardium; Oxygen; Plasmids; Protein Stability; Rats; Rats, Sprague-Dawley; Simian virus 40; Transfection; Vascular Endothelial Growth Factor A

The kidney is the main physiologic source of erythropoietin (EPO) in the adult and responds to decreases in tissue oxygenation with increased EPO production. Although studies in mice with liver-specific or global gene inactivation have shown that hypoxia-inducible factor 2 (Hif-2) plays a major role in the regulation of Epo during infancy and in the adult, respectively, the contribution of renal HIF-2 signaling to systemic EPO homeostasis and the role of extrarenal HIF-2 in erythropoiesis, in the absence of kidney EPO, have not been examined directly. Here, we used Cre-loxP recombination to ablate Hif-2α in the kidney, whereas Hif-2-mediated hypoxia responses in the liver and other Epo-producing tissues remained intact. We found that the hypoxic induction of renal Epo is completely Hif-2 dependent and that, in the absence of renal Hif-2, hepatic Hif-2 takes over as the main regulator of serum Epo levels. Furthermore, we provide evidence that hepatocyte-derived Hif-2 is involved in the regulation of iron metabolism genes, supporting a role for HIF-2 in the coordination of EPO synthesis with iron homeostasis.

Topics: Anemia; Animals; Basic Helix-Loop-Helix Transcription Factors; Erythropoiesis; Erythropoietin; Hypoxia; Iron; Kidney; Liver; Mice; Mice, Knockout; Transcription Factors

The aim of this study was to determine the effects of intermittent hypoxia training (IHT), with and without fatty-diet, on bodyweight, serum glucose, leptin, insulin, and their receptors, and to test whether erythropoietin (EPO) mediates these effects.. Kunming mice were divided into four groups. 1: untreated control; 2: IHT; 3: fatty-diet; 4: fatty-diet and IHT. After 40 days exposure to IHT, the bodyweight, serum glucose, serum leptin, insulin and EPO were measured by ELISA. Liver leptin and insulin receptors were quantified. A separate set of mice were treated with several doses of EPO (0-320 U/kg i.p.) for 5 days. In addition, human hepatic cell lines were treated with EPO for 24h and the expression of genes OB-Ra, OB-Rb and IR were measured using RT-PCR.. IHT reduced bodyweight and serum glucose, with corresponding increases in the serum levels of leptin, insulin, EPO and expression of leptin and insulin receptors in liver. Repeated EPO treatment increased serum leptin concentration, but had no effects on insulin levels. The expression of the genes OB-Ra, OB-Rb and IR were increased after EPO treatment.. We postulate that, in mice, IHT reduces bodyweight and serum glucose by increasing EPO synthesis which secondarily increases leptin and insulin production in liver.

Topics: Animals; Blood Glucose; Body Weight; Cell Line; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Humans; Hypoxia; Leptin; Liver; Mice; Receptor, Insulin; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction

The main cause of anaemia of prematurity is low erythropoietin levels. A few years ago hypoxia-inducible factor/HIF/gene transcriptor was established, regulating not only the synthesis of erythropoietin /EPO/, but also other growth factors as well as enzymes of anaerobic glycolysis, activated by hypoxia.. The aim of the study is to establish in clinical practice the role of hypoxia, respectively, activated HIF during treatment with erythropoietin by analyzing variations in hematological values; to examine blood lactate levels as an indicator of activated HIF and anaerobic glycolysis with Hb values 110-120 g/l; to analyze the number and impact of red blood cells transfusions on different categories of babies. STUDY DESIGN; The study includes 112 premature infants born before 34 weeks of gestation and below 1400 g. 90 babies, treated with EPO (700-1000 E/kg weekly dose in 2-3 applications, for 2-4 weeks), values of Hb g/l, Ht%, Ret%, Platelets 109/l were followed and compared. Treated babies were divided in two groups: group I--treatment (starting at Hb below 106 g/l, Ht less than 31%); group II--late prophylaxis (starting at Hb > or = 106 g/l, Ht > or = 31%). Blood lactate was examined in 22 non oxygen dependent premature babies without EPO treatment, with Hb 110-120 g/l, Ht 29-32%.. We found that in group II during the first 7-10 days Hb decreases to 105.6 (+/- 9.4) g/l, rising up afterwards to 113.5 (+/-11.0) g/l at day 25-30. Ret reach maximal values at day 15-20 when Hb drops below 110 g/l and Ht below 31%. In group I at day 25-30 of treatment is observed a rise in Hb up to 117.3 (+/-11.3) and of Ht up to 32.7% (+/- 2.6) and no decrease of Hb and Ht values during the first 7-10 days, while Ret rise up to maximal values 6.5% (+/- 3.6) at day 7-10. With Hb levels of 116.4 (+/- 4.6) g/l we found an increase in blood lactate levels up to 2.6 (+/- 0.7) mmol/l as an indicator of relative hypoxia and activated HIF. Mean number of blood transfusions in group I is 3.01(+/- 1.7), versus 2.15 (+/- 1.7) in group II (statistically non-significant). In 29 infants in group II treatment was started at Hb 110-120 g/l and the mean number of red blood cell transfusion is 1.8 (1.5)--statistically significant difference with group I. In 32% from the treated infants we found platelets count rising above 500 x 109/l.. The presence of hypoxia at low levels of Hb and Ht leads to more rapid activation of erythropoiesis. Nevertheless, these babies need more red blood cell transfusions due to clinical symptoms of hypoxia. Normoxia after red blood cell transfusion leads to decrease of reticulocytes count by 30% and platelets by 35% in spite of treatment. The presence of relative hypoxia with Hb 110-120 g/l u Ht 31-32% is optimal for starting treatment with EPO--levels, low enough for activation of HIF and high enough to avoid blood transfusions.

Topics: Anemia, Neonatal; Erythrocyte Transfusion; Erythropoietin; Hematocrit; Hemoglobinometry; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Infant, Newborn; Infant, Premature; Lactic Acid; Recombinant Proteins

Effect of erythropoietin (EPO) preparation (epocrine) on the blood oxygen transport in rats exposed to cold (120 min in a water-cooled box at 19 degrees C) and then rewarmed (next 120 min at a mean heating rate of 0.06 degrees C/min) has been studied. The administration of EPO reduced the body temperature fall at the end of cold exposure and enhanced its rise during the rewarming stage. The effect of EPO in tested rats is associated with a decrease in the hemoglobin affinity to oxygen, which increases the oxygen supply of tissues and improves the organism adaptability to cold.

Topics: Animals; Biological Transport; Erythropoietin; Hypothermia; Hypothermia, Induced; Hypoxia; Male; Oxygen; Rats; Recombinant Proteins; Rewarming

Topics: Erythropoietin; Heart Failure; Humans; Hypoxia; Prognosis; Pulmonary Disease, Chronic Obstructive; Risk Factors

The aim of this study was to investigate the associations between alleles of the hypoxia-inducible factor 1A (HIF1A) C1772T polymorphism and several physiological responses to hypoxia, including the hypoxic ventilatory response (HVR), and serum erythropoietin (EPO), arterial oxygen saturation (Sao2), and acute mountain sickness (AMS) responses during 8 hours of exposure to normobaric hypoxia.. A total of 76 males participated in the study; 52 participants completed an 8-hour exposure to 12.7% oxygen, during which time Sao2, EPO concentrations, and AMS scores were measured, while 62 individuals took part in an HVR trial (in total 38 individuals completed both protocols). DNA was obtained from leukocytes, and a 346-bp fragment of the HIF1A gene containing the C1772T polymorphism was amplified using polymerase chain reaction. Fragments were sequenced to reveal individual genotypes, and the associations between HIF1A genotype and EPO, Sao2, AMS responses to hypoxia and HVR were examined.. The magnitude of the hypoxic responses was highly variable between individuals. The increase in participants' EPO responses ranged from 89% to 388% of baseline values following hypoxia, while Sao2 values during the exposure ranged from 71% to 89%. The HVR ranged from -0.04 to +2.18 L x min(-1) x Sao2 %(-1) among participants. No significant differences in EPO, Sao2, AMS, or HVR results were observed between the HIF1A CC genotype and the combined CT/TT genotype group.. In this study, the HIF1A C1772T polymorphism does not appear to influence EPO, Sao2, or AMS responses during acute hypoxic exposure, or the magnitude of the HVR.

Topics: Adult; Alleles; Altitude Sickness; Analysis of Variance; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Polymerase Chain Reaction; Polymorphism, Genetic; Pulmonary Ventilation; Surveys and Questionnaires; Young Adult

Apart from enhancing the production of red blood cells, erythropoietin (Epo) alters the ventilatory response when oxygen supply is reduced. We recently demonstrated that Epo's beneficial effect on the ventilatory response to acute hypoxia is sex dependent, with female mice being better able to cope with reduced oxygenation. In the present work, we hypothesized that ventilatory acclimatization to chronic hypoxia (VAH) in transgenic female mice (Tg6) harboring high levels of Epo in the brain and blood will also be improved compared with wild-type (WT) animals. Surprisingly, VAH was blunted in Tg6 female mice. To define whether this phenomenon had a central (brain stem respiratory centers) and/or peripheral (carotid bodies) origin, a bilateral transection of carotid sinus nerve (chemodenervation) was performed. This procedure allowed the analysis of the central response in the absence of carotid body information. Interestingly, chemodenervation restored the VAH in Tg6 mice, suggesting that carotid bodies were responsible for the blunted response. Coherently with this observation, the sensitivity to oxygen alteration in arterial blood (Dejour test) after chronic hypoxia was lower in transgenic carotid bodies compared with the WT control. As blunted VAH occurred in female but not male transgenic mice, the involvement of sex female steroids was obvious. Indeed, measurement of sexual female hormones revealed that the estradiol serum level was 4 times higher in transgenic mice Tg6 than in WT animals. While ovariectomy decreased VAH in WT females, this treatment restored VAH in Tg6 female mice. In line with this observation, injections of estradiol in ovariectomized Tg6 females dramatically reduced the VAH. We concluded that during chronic hypoxia, estradiol in carotid bodies suppresses the Epo-mediated elevation of ventilation. Considering the increased application of recombinant Epo for a variety of disorders, our data imply the need to take the patient's hormonal status into consideration.

Topics: Acclimatization; Analysis of Variance; Animals; Carotid Body; Erythropoietin; Estradiol; Female; Hypoxia; Immunohistochemistry; Mice; Mice, Transgenic; Ovariectomy; Oxygen Consumption; Plethysmography; Pulmonary Ventilation; Radioimmunoassay; Respiration

Acute systemic hypoxia induces delayed cardioprotection against ischaemia-reperfusion injury in the heart. As cobalt chloride (CoCl₂) is known to elicit hypoxia-like responses, it was hypothesized that this chemical would mimic the preconditioning effect and facilitate acclimatization to hypobaric hypoxia in rat heart.. Male Sprague-Dawley rats treated with distilled water or cobalt chloride (12.5 mg Co/kg for 7 days) were exposed to simulated altitude at 7622 m for different time periods (1, 2, 3 and 5 days).. Hypoxic preconditioning with cobalt appreciably attenuated hypobaric hypoxia-induced oxidative damage as observed by a decrease in free radical (reactive oxygen species) generation, oxidation of lipids and proteins. Interestingly, the observed effect was due to increased expression of the antioxidant proteins hemeoxygenase and metallothionein, as no significant change was observed in antioxidant enzyme activity. Hypoxic preconditioning with cobalt increased hypoxia-inducible factor 1α (HIF-1α) expression as well as HIF-1 DNA binding activity, which further resulted in increased expression of HIF-1 regulated genes such as erythropoietin, vascular endothelial growth factor and glucose transporter. A significant decrease was observed in lactate dehydrogenase activity and lactate levels in the heart of preconditioned animals compared with non-preconditioned animals exposed to hypoxia.. The results showed that hypoxic preconditioning with cobalt induces acclimatization by up-regulation of hemeoxygenase 1 and metallothionein 1 via HIF-1 stabilization.

Topics: Acclimatization; Animals; Cardiotonic Agents; Cobalt; DNA-Binding Proteins; Erythropoietin; Heart; Heme Oxygenase-1; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Metallothionein; Myocardium; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Up-Regulation; Vascular Endothelial Growth Factor A

Nonviral ex vivo local gene therapy systems consisting of regulated gene expression vectors and cellular delivery platforms represent a novel strategy for tissue repair and regeneration. We introduced a hypoxia-regulated plasmid-based system into mouse neural stem cells (NSCs) as an efficient gene expression and delivery platform for rapid, robust and persistent hypoxic/ischemic-regulated gene expression in the spinal cord.. A synthetic hypoxia-responsive erythropoietin (Epo) enhancer, the SV40 minimal promoter and the luciferase (Luc) reporter gene were incorporated in a DsRed-expressing double-promoter plasmid for cell lipofection and Zeocin-selection to establish a hypoxia-regulated stable NSC line (NSC-Epo-SV-Luc). A nonhypoxia-regulated stable NSC line (NSC-SV-Luc) was also established as a control.. Under the transcriptional regulation of the Epo enhancer, in vitro luciferase expression in NSC-Epo-SV-Luc, but not in NSC-SV-Luc, was sensitively augmented according to the strength and duration of the hypoxic stimulus and was quickly down-regulated to a low basal level after reoxygenation of the hypoxic cells. Furthermore, deoxygenation of the reoxygenated cells clearly enhanced the luciferase activity again. After transplantation into a rat spinal cord injury (SCI) model, only NSC-Epo-SV-Luc showed ischemic injury-specific luciferase expression Notably, the engineered NSC lines kept the neural differentiation potential and retained the hypoxia-regulated luciferase expression after differentiation.. We propose that NSCs engineered with the Epo-SV-therapeutic gene will be valuable for developing a controllable stem cell-mediated nonviral gene therapy for SCI or other central nervous system diseases accompanied with chronic or episodic hypoxic/ischemic stresses.

Topics: Animals; Cell Line; Erythropoietin; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Hypoxia; Mice; Neural Stem Cells; Simian virus 40; Transfection

Animals respond to hypoxia by increasing synthesis of the glycoprotein hormone erythropoietin (Epo) which in turn stimulates the production of red blood cells. The gene encoding Epo has been recently cloned in teleost fishes such as the pufferfish Takifugu rubripes (fugu) and zebrafish (Danio rerio). It has been shown that the transcription levels of Epo in teleost fishes increase in response to anemia or hypoxia in a manner similar to its human ortholog. However, the cis-regulatory element(s) mediating the hypoxia response of Epo gene in fishes has not been identified. In the present study, using the human hepatoma cell line (Hep3B), we have identified and characterized a hypoxia response element (HRE) in the fugu Epo locus. The sequence of the fugu HRE (ACGTGCTG) is identical to that of the HRE in the human EPO locus. However, unlike the HRE in the mammalian Epo locus, which is located in the 3' region of the gene, the fugu HRE is located in the 5' flanking region and on the opposite strand of DNA. This HRE is conserved in other teleosts such as Tetraodon and zebrafish in a similar location. A 365-bp fragment containing the fugu HRE was able to drive GFP expression in the liver of transgenic zebrafish. However, we could not ascertain if the expression of transgene is induced by hypoxia in vivo due to the low and variable levels of GFP expression in transgenic zebrafish. Our investigations also revealed that the Epo locus has experienced extensive rearrangements during vertebrate evolution.

Topics: Animals; Base Sequence; beta-Galactosidase; Cell Line, Tumor; Conserved Sequence; DNA Primers; Erythropoietin; Green Fluorescent Proteins; Humans; Hypoxia; Luciferases; Molecular Sequence Data; Real-Time Polymerase Chain Reaction; Regulatory Elements, Transcriptional; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Sequence Analysis, DNA; Takifugu; Zebrafish

To analyse the correlation between production of angiogenic [vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL-8)] and lymphangiogenic factors (VEGF-C and D) and adaptation to high altitude (>8000 m). Erythropoietin (EPO) served as a positive control.. We analysed the percentage of oxygen saturation and the plasmatic contents of VEGF-A, C, D, IL-8 and EPO in seven mountaineers and four Sherpas during an expedition to Mount Everest. Acute mountain sickness was also evaluated using the Lake Louise score.. Whereas VEGF-A, IL-8, VEGF-C and EPO were transiently up-regulated at 5000 m and decreased at the highest altitudes, VEGF-D remained elevated throughout the ascent. Sherpas had increased basal levels of VEGF-A, C, IL-8 and EPO and up-regulation of all the tested factors when they passed the altitude at which they lived.. Our data suggest that expression of angiogenic and lymphangiogenic factors is up-regulated directly or indirectly by altitude-dependent hypoxia. Both factors could be involved in a mechanism of adaptation to high altitudes.

Topics: Acclimatization; Adult; Altitude; Altitude Sickness; Angiogenic Proteins; Erythropoietin; Female; Humans; Hypoxia; Interleukin-8; Lymphangiogenesis; Middle Aged; Mountaineering; Neovascularization, Physiologic; Oxygen; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor C; Vascular Endothelial Growth Factor D

The aim of this study was to investigate the relations among reactive oxygen species (ROS), hypoxia inducible factor (HIF-1 alpha) gene expression, HIF-1 alpha target gene erythropoietin (EPO), and vascular endothelium growth factor (VEGF) in humans. Five healthy men (32+/-7 years, mean+/-SD) were exposed to 12 h of sustained poikilocapnic hypoxia (P(ET)O(2)=60 mmHg). DNA oxidation (8-hydroxy-2'-deoxyguanosine, 8-OHdG), advanced oxidation protein products (AOPP), EPO, and VEGF were measured in plasma and HIF-1 alpha mRNA was assessed in leukocytes before and after 1, 2, 4, 6, 8, 10, and 12 h of exposure to hypoxia. HIF-1 alpha mRNA amount increased during the first two hours of hypoxic exposure and then returned to baseline levels. The findings reveal an up-regulation of HIF-1 alpha (+68%), VEGF (+46%), and EPO (+74%). AOPP increased continuously from 4 h (+69%) to 12 h (+216%) of hypoxic exposure while 8-OHdG increased after 6 h (+78%) and remained elevated until 12 h. During the "acute" increase phase of HIF-1 alpha (between 0 and 2 h), 8-OHdG was positively correlated with HIF-1 alpha (r=0.55). These findings suggest that hypoxia induces oxidative stress via an overgeneration of reactive oxygen species (ROS). Finally, this study in humans corroborates the previous in vitro findings demonstrating that ROS is involved in HIF-1 alpha transcription.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Deoxyguanosine; DNA; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Leukocytes, Mononuclear; Male; Oxidation-Reduction; Oxidative Stress; RNA, Messenger; Transcriptional Activation; Vascular Endothelial Growth Factor A

Anemia and hypoxia in rats result in an increase in factors potentially involved in cerebral angiogenesis. Therefore, the aim of this study was to assess the effect of chronic anemia and/or chronic hypoxia on cerebral cellular responses and angiogenesis in wild-type and anemic transgenic mice. These studies were done in erythropoietin-deficient mice (Epo-TAg(h)) in normoxia and following acute (one day) and chronic (14 days, barometric pressure = 420 mmHg) hypoxia. In normoxia, Epo-TAg(h) mice showed an increase in transcript and protein levels of hypoxia-inducible factor 1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), erythropoietin receptors (EpoR), phospho-STAT-5/STAT-5 ratio, and neuronal neuronal nitric oxide synthase (nNOS) along with a higher cerebral capillary density. In wild-type (WT) mice, acute hypoxia increased all of the studied factors, while in chronic hypoxia, HIF-1alpha, EpoR, phospho-STAT-5/STAT-5 ratio, nNOS, and inducible NOS remained elevated, with an increase in capillary density. Surprisingly, in Epo-TAg(h) mice, chronic hypoxia did not further increase any factor except the nitric oxide metabolites, while HIF-1alpha, EpoR, and phospho-STAT-5/STAT-5 ratio were reduced. Normoxic Epo-TAg(h) mice developed cerebral angiogenesis through the HIF-1alpha/VEGF pathway. In acute hypoxia, WT mice up-regulated all of the studied factors, including cerebral NO. Polycythemia and angiogenesis occurred with acclimatization to chronic hypoxia only in WT mice. In Epo-TAg(h), the decrease in HIF-1alpha, VEGF proteins, and phospho-STAT-5 ratio in chronic hypoxia suggest that neuroprotective and angiogenesis pathways are altered.

Topics: Anemia; Animals; Body Weight; Brain; Cerebral Cortex; Chronic Disease; Erythropoietin; Hemoglobins; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoassay; Immunohistochemistry; Male; Mice; Mice, Inbred CBA; Mice, Knockout; Nitric Oxide; Receptors, Erythropoietin; Reverse Transcriptase Polymerase Chain Reaction; RNA; STAT5 Transcription Factor; Vascular Endothelial Growth Factor A

Acetazolamide has been recognized as an effective treatment for acute mountain sickness. The efficacy of acetazolamide is related to metabolic acidosis, which promotes chemoreceptors to respond to hypoxic stimuli at altitude. In this study, adult male Sprague-Dawley rats were treated with acetazolamide (100mg/kg or 50mg/kg, I.P.) for 3 days. Primary cultured cortical neurons and PC12 cell lines were exposed to acidosis-permissive (pH 6.5) or standard (pH 7.2) media for 20h. HIF-1alpha and its target genes were assayed by Western blot, real-time PCR, HIF-1 DNA-binding assay and chloramphenicol acetyltransferase reporter gene assay. HIF-1alpha protein level and HIF-1 DNA-binding activities were increased in cerebral cortices of rats treated with acetazolamide. Moreover, the mRNA levels of erythropoietin, vascular endothelial growth factor, and glucose transporter-1 also increased. The HIF-1alpha protein level and activity of HIF-driven chloramphenicol acetyltransferase reporters of cortical neurons and PC12 cells treated with acidosis media were significantly enhanced. We conclude that the normoxic induction of HIF-1alpha and HIF-1 mediated genes by acetazolamide may mediate the effect of acetazolamide in the reduction of symptoms of acute mountain sickness.

Topics: Acetazolamide; Acidosis, Respiratory; Altitude Sickness; Animals; Carbonic Anhydrase Inhibitors; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; DNA-Binding Proteins; Erythropoietin; Glucose Transporter Type 1; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Oxygen; PC12 Cells; Rats; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation; Vascular Endothelial Growth Factor A

Our objective was to assess the regulation of the hypoxia response of angiogenic and inflammatory factors from 76 cerebrospinal fluids (CSF) of sporadic amyotrophic lateral sclerosis (ALS) patients with different respiratory status. We first analysed the hypoxia response capacity by measuring CSF levels of angiogenin (ANG), VEGF, angiopoietin-2 (ANG-2) and PGE-2 in 40 ALS patients according to their hypoxaemia level and compared it with 40 neurological controls. We then compared the ANG, VEGF, EPO and ANG-2 CSF levels of 36 other ALS patients, divided into three groups with either 1) normoxaemia, 2) intermittent desaturation in the absence of hypoxaemia, or 3) chronic hypoxaemia with or without desaturation. We demonstrated a lack of up-regulation of both ANG and VEGF during hypoxaemia in ALS, compared with hypoxaemic controls. In contrast, PGE-2 and ANG-2 levels were increased in both hypoxaemic ALS patients and controls. ANG and VEGF levels did not increase in patients with long disease durations and with intermittent or chronic hypoxaemia. ANG-2 and EPO levels were up-regulated early in intermittent hypoxaemia and late in chronic hypoxaemia, respectively. Our results suggest alteration of the HIF-1alpha-mediated response to hypoxia during sporadic ALS, whereas the NFK-B pathway seems early activated.

Topics: Aged; Amyotrophic Lateral Sclerosis; Angiopoietin-2; Dinoprostone; Erythropoietin; Humans; Hypoxia; Inflammation; Middle Aged; Oxygen; Respiration; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A

Hepcidin, a key regulator of iron metabolism, is a small antimicrobial peptide produced by the liver that regulates intestinal iron absorption and iron recycling by macrophages. Hepcidin is stimulated when iron stores increase and during inflammation and, conversely, is inhibited by hypoxia and augmented erythropoiesis. In many pathologic situations, such as in the anemia of chronic disease (ACD) and iron-loading anemias, several of these factors may be present concomitantly and may generate opposing signaling to regulate hepcidin expression. Here, we address the question of dominance among the regulators of hepcidin expression. We show that erythropoiesis drive, stimulated by erythropoietin but not hypoxia, down-regulates hepcidin in a dose-dependent manner, even in the presence of lipopolysaccharide (LPS) or dietary iron-loading, which may act additively. These effects are mediated through down-regulation of phosphorylation of Stat3 triggered by LPS and of Smad1/5/8 induced by iron. In conclusion, hepcidin expression levels in the presence of opposing signaling are determined by the strength of the individual stimuli rather than by an absolute hierarchy among signaling pathways. Our findings also suggest that erythropoietic drive can inhibit both inflammatory and iron-sensing pathways, at least in part, via the suppression of STAT3 and SMAD4 signaling in vivo.

Topics: Anemia, Iron-Deficiency; Animals; Antimicrobial Cationic Peptides; Erythropoiesis; Erythropoietin; Female; Hepcidins; Hypoxia; Interleukin-6; Iron, Dietary; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Phosphorylation; Signal Transduction; Smad1 Protein; Smad4 Protein; Smad5 Protein; Smad8 Protein; STAT3 Transcription Factor

Oxygen-carrying capacity of blood is reduced in anemic infants because of low hemoglobin levels. Red blood cell transfusions become necessary if low hematocrit causes tissue hypoxia. No reliable parameters exist for detecting chronic tissue hypoxia. Vascular endothelial growth factor is upregulated by hypoxia; hence, elevated vascular endothelial growth factor levels may be a marker for tissue hypoxia and may indicate the need for red blood cell transfusions.. In a prospective study, plasma vascular endothelial growth factor levels were measured in 3 groups of infants suspected of requiring red blood cell transfusions to find a vascular endothelial growth factor cutoff value indicative of tissue hypoxia. The 3 groups were acute anemic (an episode of acute bleeding [hematocrit drop > 5%] per day); chronic anemic (hematocrit drop < 5% per day); and nontransfused (hematocrit drop < 5% per day) but not meeting clinical criteria for a transfusion. Blood was sampled before transfusion and again 48 hours after transfusion if required. Plasma vascular endothelial growth factor and erythropoietin concentrations were measured.. Vascular endothelial growth factor concentrations were lower in acutely anemic compared with chronically anemic infants, whereas erythropoietin levels did not differ between these groups. The vascular endothelial growth factor concentration was <140 pg/mL in all acutely anemic infants, and this was deemed the threshold level indicating sufficient tissue oxygenation in subsequent analysis. We found that 30% of chronically anemic and 43% of nontransfused infants had vascular endothelial growth factor levels of >140 pg/mL. In transfused infants, with elevated vascular endothelial growth factor levels, red blood cell transfusion resulted in lowering of vascular endothelial growth factor concentrations.. Vascular endothelial growth factor concentrations of >140 pg/mL may indicate insufficient oxygen delivery to tissues and may serve as a marker of the need for transfusion or of tissue hypoxia in other diseases.

Topics: Anemia, Neonatal; Biomarkers; Erythrocyte Transfusion; Erythropoietin; Female; Hematocrit; Hemorrhage; Humans; Hypoxia; Infant, Newborn; Infant, Premature, Diseases; Male; Predictive Value of Tests; Prospective Studies; Reference Values; Vascular Endothelial Growth Factor A

Erythropoietin (EPO), long appreciated as the chief endocrine regulator of red blood cell formation, is now recognized to exert many additional functions outside the bone marrow. Thus, the quest is on to define the full range of EPO functions in the physiology and pathology of non-hematopoietic tissues. Two recent studies in man and mice have highlighted the importance of the mammalian skin as one peripheral tissue with a previously unsuspected role in EPO biology; both, as a target and as a source of EPO. In addition, the skin has been proposed to function as an oxygen sensor. The present hypothesis essay critically reviews the currently available evidence for this and provides a unifying theoretical scenario for intracutaneous EPO functions and for a potential role of the skin in the control of EPO production. Mainly, we propose that the skin itself directly contributes to the up-regulation of EPO plasma levels in response to hypoxia.

Topics: Animals; Erythropoietin; Humans; Hypoxia; Oxygen; Skin

Acclimatization to hypoxic exposure relies on an elevated ventilation and erythropoietic activity. We recently proposed that erythropoietin (Epo) links both responses: apart from red blood cell production, cerebral and plasma Epo interact with the central and peripheral respiratory centers. Knowing that women cope better than men with reduced oxygen supply (as observed at high altitude), we analyzed the hypoxic ventilatory response in Epo-overexpressing transgenic male and female mice with high Epo levels in brain and plasma (Tg6) or in wild-type animals injected with recombinant human Epo (rhEpo). Exposure to moderate and severe hypoxia as well as to hyperoxia and injection of domperidone, a potent peripheral ventilatory stimulant, revealed that the presence of transgenic or rhEpo extensively increased the hypoxic ventilatory response in female mice compared with their corresponding male siblings. Alterations of catecholamines in the brain stem's respiratory centers were also sex dependent. In a proof-of-concept study, human volunteers were intravenously injected with 5,000 units rhEpo and subsequently exposed to 10% oxygen. Compared with men, the hypoxic ventilatory response was significantly increased in women. We conclude that Epo exerts a sex-dependent impact on hypoxic ventilation improving the response in female mice and in women that most probably involves sexual hormones. Our data provides an explanation as to why women are less susceptible to hypoxia-associated syndromes than men.

Topics: Adaptation, Physiological; Adult; Animals; Brain Stem; Catecholamines; Disease Models, Animal; Domperidone; Dopamine Antagonists; Erythropoietin; Female; Humans; Hyperoxia; Hypoxia; Injections, Intravenous; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pulmonary Ventilation; Recombinant Proteins; Respiratory Mechanics; Sex Factors; Young Adult

Hypoxia and inflammation often develop concurrently in numerous diseases, and both hypoxia-inducible factor (HIF)-1alpha and nuclear factor-kappaB (NF-kappaB) are key transcription factors of stress response genes. An NF-kappaB inhibitor, inhibitor of NF-kappaB alpha (IkappaB alpha), was found to interact with factor inhibiting HIF (FIH) and to be hydroxylated by FIH. However, FIH did not functionally regulate IkappaB alpha, and the consequence of the FIH-IkappaB alpha interaction thus remains uncertain. In the present study, we tested the possibility that IkappaB alpha regulates FIH. FIH-IkappaB alpha binding was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. Functionally, IkappaB alpha expression further enhanced the transcriptional activity of HIF-1alpha under hypoxic conditions. Furthermore, IkappaB alpha knockdown repressed HIF-1alpha activity. Mechanistically, IkappaB alpha derepressed HIF-1alpha activity by inhibiting the FIH-mediated Asn803 hydroxylation of HIF-1alpha. It was also found that IkappaB alpha activated HIF-1alpha by sequestering FIH from HIF-1alpha. However, the effect of IkappaB alpha on HIF-1alpha activity was only observed in atmospheres containing 1% or more of oxygen. After tumor necrosis factor-alpha treatment, IkappaB alpha downregulation, Asn803 hydroxylation and HIF-1alpha inactivation all occurred up to 8 h, but subsided later. On the basis of these results, we propose that IkappaB alpha plays a positive regulatory role during HIF-1-mediated gene expression. Therefore, IkappaB alpha, owing to its interactions with NF-kappaB and HIF-1alpha, may play a pivotal role in the crosstalk between the molecular events that underlie inflammatory and hypoxic responses.

Topics: Animals; Asparagine; Cell Line; Erythropoietin; Gene Expression Regulation; Genes, Reporter; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; I-kappa B Proteins; Mixed Function Oxygenases; NF-kappa B; NF-KappaB Inhibitor alpha; Oxygen; Repressor Proteins; RNA, Small Interfering; Tumor Necrosis Factor-alpha; Two-Hybrid System Techniques

Erythropoietin receptor (EpoR) binding mediates neuroprotection by endogenous Epo or by exogenous recombinant human (rh)Epo. The level of EpoR gene expression may determine tissue responsiveness to Epo. Thus, harnessing the neuroprotective power of Epo requires an understanding of the Epo-EpoR system and its regulation. We tested the hypothesis that neuronal expression of EpoR is required to achieve optimal neuroprotection by Epo. The ventral limbic region (VLR) in the rat brain was used because we determined that its neurons express minimal EpoR under basal conditions, and they are highly sensitive to excitotoxic damage, such as occurs with pilocarpine-induced status epilepticus (Pilo-SE). We report that (i) EpoR expression is significantly elevated in nearly all VLR neurons when rats are subjected to 3 moderate hypoxic exposures, with each separated by a 4-day interval; (ii) synergistic induction of EpoR expression is achieved in the dorsal hippocampus and neocortex by the combination of hypoxia and exposure to an enriched environment, with minimal increased expression by either treatment alone; and (iii) rhEpo administered after Pilo-SE cannot rescue neurons in the VLR, unless neuronal induction of EpoR is elicited by hypoxia before Pilo-SE. This study thus demonstrates using environmental manipulations in normal rodents, the strict requirement for induction of EpoR expression in brain neurons to achieve optimal neuroprotection. Our results indicate that regulation of EpoR gene expression may facilitate the neuroprotective potential of rhEpo.

Topics: Animals; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Gene Expression Regulation; Hypoxia; Male; Neurons; Neuroprotective Agents; Pilocarpine; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Status Epilepticus

The carotid body (CB) plays important roles in cardiorespiratory changes in intermittent hypoxia (IH). Erythropoietin (EPO), a hypoxia-inducible factor (HIF)-1 target gene, is present in the chemoreceptive type-I cells in the CB but its expression and role in IH resembling sleep apnoeic conditions are not known. We hypothesized that IH upregulates the expression of EPO and its receptor (EPOr) in the rat CB. The CB expressions of EPO and EPOr were examined in rats breathing 10% O(2) (in isobaric chamber for CH, 24 hour/day) or in IH (cyclic between air and 5% O(2) per minute, 8 hour/day) for 3-28 days. Immunohistochemical studies revealed that the EPO and EPOr proteins were localized in CB glomic clusters. The proportional amount of cells with positive staining of EPO immunoreactivities was significantly increased in both IH and CH groups when compared with the normoxic control. The EPO expression was more markedly increased in the CH than that of the IH groups throughout the time course, reaching a peak level at day 14. The positive EPOr immunostaining was increased significantly in the 3-day CH group. By day 14, the EPOr expression elevated considerably at peak levels in both IH and CH rats, whereas the elevation was greater in the CH rats. These results suggest an upregulation of EPO and its receptor expression in the rat CB under IH and CH conditions, presumably mediated by the activation of HIF-1 pathway. The increased EPO binding to its receptor might play a role in the enhancement of CB excitability during the early pathogenesis in patients with sleep-disordered breathing.

Topics: Animals; Carotid Body; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Oxygen; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Respiration; Signal Transduction; Time Factors; Up-Regulation

Recent publications reflect the anti-doping authorities' concern about the use of altitude simulator systems as violating the spirit of sport criterion (Levine 2006; Loland and Murray 2007; Spriggs 2005). The aim of our study was to determine whether intermittent hypoxic treatments could modify the hemoglobin, hematocrit, reticulocytes, and erythropoietic stimulation index (OFF-Hr Score) values after administration of rHuEPO-alpha. Although these hematological parameters are of secondary nature some international sport federations currently exclude athletes who show aberrant values of these parameters from competition. Ten young male Wistar rats were treated, three times a week for 2 weeks, with 500 IU of rHuEPO-alpha. After the treatment, the animals were randomly divided into two groups: normoxic and hypoxic. The normoxic group was maintained at 21% O(2) 24 h a day for 23 days. The hypoxic group was maintained 12 h at 21% O(2) and 12 h at 12% O(2) (~4,000 m) the same time period. After the rHuEPO-alpha treatment, the hypoxic group of animals had a faster recovery rate in the reticulocyte count, elevated concentrations of hemoglobin and hematocrit and a significant increase in the endogenous EPO levels when compared with the normoxic group of animals. These changes led to significant modifications in the OFF-Hr Score between the hypoxic and normoxic animals. Intermittent hypoxic treatments after rHuEPO administration can significantly modify the main hematological parameters tested by the anti-doping authorities. Our results in an animal model suggest checking the described phenomena in humans in order to reach major conclusions.

Topics: Administration, Cutaneous; Algorithms; Animals; Doping in Sports; Erythropoietin; Hematologic Tests; Hemoglobins; Humans; Hypoxia; Male; Oxygen Consumption; Periodicity; Rats; Rats, Wistar; Recombinant Proteins; Reticulocyte Count

Endothelial progenitor cells (EPCs, defined as sca-1(+)flk-1(+)lin(-) mononuclear blood cells) contribute to vascular repair. The role of hypoxia and reactive oxygen species (ROS) in mobilization and function of these cells is incompletely understood.. We studied the contribution of the NADPH oxidase Nox2, an important vascular source of ROS in this context.. Hypoxia (10% oxygen) induced the mobilization of EPCs in wild-type (WT) and Nox1 but not in Nox2 knockout (Nox2(y/-)) mice. As erythropoietin (EPO) is known to induce EPC mobilization, we focused on this hormone. EPO induced the mobilization of EPCs in WT and Nox1(y/-) but not Nox2(y/-) animals. Transplantation of bone marrow from Nox2(y/-) mice into WT-mice blocked mobilization in response to hypoxia and EPO, whereas transplantation of WT bone marrow into Nox2(y/-) mice restored mobilization. Reendothelialization of the injured mouse carotid artery was enhanced by hypoxia as well as by EPO, and this effect was not observed in Nox2(y/-) mice or after transplantation of Nox2(y/-) bone marrow. In cultured EPCs from WT but not Nox2(y/-) mice, EPO induced ROS production, migration, and proliferation. EPO signaling involves the STAT5 transcription factor. EPO-induced STAT5-dependent reporter gene expression was absent in Nox2-deficient cells. siRNA against the redox-sensitive phosphatase SHP-2 restored EPO-mediated STAT5 induction and inhibition of SHP-2 restored EPO-induced migration in Nox2-deficient cells. We conclude that Nox2-derived ROS inactivate SHP-2 and thereby facilitate EPO signaling in EPCs to promote hypoxia-induced mobilization and vascular repair by these cells.

Topics: Animals; Antigens, Ly; Bone Marrow Transplantation; Carotid Arteries; Carotid Artery Injuries; Cells, Cultured; Endothelial Cells; Erythropoietin; Gene Expression Regulation; Hypoxia; Leukocytes, Mononuclear; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Knockout; NADH, NADPH Oxidoreductases; NADPH Oxidase 1; NADPH Oxidase 2; NADPH Oxidases; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Reactive Oxygen Species; Signal Transduction; STAT5 Transcription Factor; Stem Cells; Vascular Endothelial Growth Factor Receptor-2; Wound Healing

To investigate the effect of polycythemia on hypoxia induced pulmonary hypertension and pulmonary vascular remodeling in rats.. The healthy female Sprague-Dawley rats were randomly divided into 3 groups: normoxia control group (C group), hypoxia group (H group), hypoxia + different doses of human recombine hemopoietin (rEPO) group. All rats in hyoxia groups were exposed to hypoxia, 8 hours every day, for 21 days. The rEPO groups were injected sc with different doses of rEPO (300 U/kg, 600 U/kg, 900 U/kg, 1200 U/kg) thrice weekly. Blood samples were taken for the measurement of RBC, Hb, Hct, plasma EPO concentration, whole blood/plasma viscosities, the animals were then catheterized to record mean pulmonary arterial pressure (mPAP) and demised to calculate the ratio [RV/(LV+S)]. Percentage of vascular wall thickness and muscularization of non-muscular pulmonary arteriole were examined microscopically.. (1) As the dosage of exogenous rEPO increased, blood concentration of EPO increased correspondingly, as RBC, Hb, Hct and whole blood/plasma viscosities increased in various degrees. (2) There was positive correlation between whole blood viscosity and Hct at both high and low shears and linear correlation between mPAP and whole blood viscosity at high shear. (3) The degree of pulmonary hypertension, reflected by mPAP increased in accordance to rEPO dosage increment. However, the extent of pulmonary vascular remodeling alleviated somehow as the rEPO dose increased and so did right ventricular hypertrophy.. Polycythemia induced by exogenous EPO increases the blood viscosity and the pulmonary vascular resistance, which contributes to the formation of hypoxia induced pulmonary hypertension.

Topics: Animals; Erythropoietin; Female; Hypertension, Pulmonary; Hypoxia; Polycythemia; Pulmonary Artery; Random Allocation; Rats; Rats, Sprague-Dawley

In vitro studies have indicated that reactive oxygen species modifying cellular redox status are involved in hypoxia-induced erythropoietin (EPO) production. However, the effects of redox balance on hypoxia-induced EPO production in vivo are still not fully understood. To investigate the effect of the change in cellular redox status on EPO generation, we determined whether glutathione (GSH) depletion has a significant influence on hypoxia-induced EPO production in rats. For the inhibition of GSH synthesis, DL-buthionine-[S,R]-sulfoximine (BSO) was employed by intraperitoneal injection. Twenty male rats were assigned to one of four experimental groups: (1) normoxic placebo, (2) normoxic BSO, (3) hypoxic placebo, and (4) hypoxic BSO. Hypoxic groups were exposed to a simulated normobaric hypoxic condition (4,500 m above sea level) for 12 hours. BSO treatment resulted in a significant depletion of total GSH levels in kidney and plasma in both conditions. However, the hypoxia-induced elevation in serum EPO concentration was not completely affected by the inhibition of GSH synthesis. These data demonstrate that GSH depletion in the kidney is not involved in the increase in serum EPO concentration in response to systemic hypoxia. It is also conceivable that the cellular redox changes could not function as a primary regulator of hypoxia-induced renal erythropoietin formation in vivo.

Topics: Animals; Buthionine Sulfoximine; Erythropoietin; Glutathione; Hypoxia; Injections, Intraperitoneal; Kidney; Male; Oxidation-Reduction; Rats; Rats, Wistar

To investigate the effects of intermittent hypoxic exposure and normoxic convalescence on the parameter of erythrocyte and serum hypoxia inducible factor-1 alpha (HIF-1alpha) and erythropoietin (EPO) levels.. Rat models of intermittent hypoxic exposure were established, combined with the clinical research on volunteers experiencing the intermittent plateau work. Blood samples for red blood cell (RBC) counts, hemoglobin (Hb) and hematocrit (HCT) were collected, serum HIF-1alpha and EPO levels were measured using enzyme linked immunosorbent assay.. RBC counts, Hb concentration and HCT were significantly higher than the normoxic group (P < 0.05), after exposure of rats to hypoxia from 7 to 28 days. Compared with the normoxic group, serum HIF-1alpha levels were higher in the group of IH3, 7, 14 days, and EPO had a corresponding increase in the group of IH3, 7 days. Then, a decrease was observed in parameter of erythrocyte and serum HIF-1alpha and EPO levels after 14 days normoxic convalescence treat. In volunteers studies, RBC counts in 8 months group and Hb concentration in 2 years group were significantly higher than the plain group (P < 0.05). Compared with the plain group, EPO had no significant differences in any of plateau group.. Intermittent hypoxic exposure can enhance serum hypoxia inducible factor-1 alpha and erythropointin levels and the generation of red blood cells, which leads to an increase in hemoglobin concentration and hematocrit. The results have changed with the hypoxic exposure period prolonged. Normoxic convalescence after intermittent hypoxic exposure can make the related indexes reduced, and contribute to the organism recovery.

Topics: Adolescent; Adult; Animals; Erythrocytes; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Rats; Rats, Wistar; Time Factors; Young Adult

To investigate the protective effects of ginkgolide B and hypoxic preconditioning against acute hypoxia injury in mice.. Ordinary pressure acute hypoxia model in mice was adopted to observe the ethology, the duration of the death and the degree of brain edema. Meanwhile the expression of RTP801 mRNA and erythropoietin (EPO) were measured by RT-PCR and Western blot, respectively.. Ginkgolide B and hypoxic preconditioning could both prolong the survival time of hypoxia under ordinary pressure,and significantly decreased the degree of brain edema. Besides ginkgolide B and hypoxic preconditioning could both up-regulate the expression of RTP801mRNA and EPO.. Ginkgolide B has the similar effect to hypoxic preconditioning against acute hypoxia. Both of these protective effects may be associated with the up-regulation of the expression of RTP801 mRNA and EPO.

Topics: Animals; Brain; Brain Edema; Erythropoietin; Female; Ginkgolides; Hypoxia; Ischemic Preconditioning; Lactones; Male; Mice; Mice, Inbred ICR; Reperfusion Injury; Repressor Proteins; RNA, Messenger; Transcription Factors; Up-Regulation

Hypoxia-inducible factor-1 (HIF-1) regulates the expression of neuroprotective genes such as erythropoietin (EPO). We investigated the mechanism by which zinc, an excitotoxin-like metal, regulates HIF-1 under hypoxic conditions in astrocytes. In hypoxic LN215 cells, HIF-1alpha stabilized and accumulated in the nucleus, resulting in an increase in its DNA-binding activity to the EPO enhancer. Zinc inhibited hypoxia-induced increases in HIF-1 DNA-binding activity and the HIF-1-dependent mRNA expression of EPO. Zinc did not affect hypoxic stabilization of HIF-1alpha. Nuclear migration of HIF-1alpha upon hypoxia was reduced by zinc. Complete blockade of hypoxia-induced assembly of HIF-1alpha-HIF-1beta complex was observed after treatment of zinc. These findings suggest that zinc hampers hypoxia-stimulated HIF-1 activation in astrocytes by inhibiting nuclear HIF-1alpha translocation and subsequently disrupting HIF-1 heterodimerization.

Topics: Antineoplastic Agents; Astrocytoma; Cell Line, Tumor; Cell Nucleus; Dose-Response Relationship, Drug; Drug Interactions; Electrophoretic Mobility Shift Assay; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Proline; Protein Transport; Thiocarbamates; Time Factors; Zinc Sulfate

Treatment with recombinant human erythropoietin (rhEpo) induces a rise in blood oxygen-carrying capacity (CaO(2)) that unequivocally enhances maximal oxygen uptake (VO(2)max) during exercise in normoxia, but not when exercise is carried out in severe acute hypoxia. This implies that there should be a threshold altitude at which VO(2)max is less dependent on CaO(2). To ascertain which are the mechanisms explaining the interactions between hypoxia, CaO(2) and VO(2)max we measured systemic and leg O(2) transport and utilization during incremental exercise to exhaustion in normoxia and with different degrees of acute hypoxia in eight rhEpo-treated subjects. Following prolonged rhEpo treatment, the gain in systemic VO(2)max observed in normoxia (6-7%) persisted during mild hypoxia (8% at inspired O(2) fraction (F(I)O(2)) of 0.173) and was even larger during moderate hypoxia (14-17% at F(I)O(2) = 0.153-0.134). When hypoxia was further augmented to F(I)O(2) = 0.115, there was no rhEpo-induced enhancement of systemic VO(2)max or peak leg VO(2). The mechanism highlighted by our data is that besides its strong influence on CaO(2), rhEpo was found to enhance leg VO(2)max in normoxia through a preferential redistribution of cardiac output toward the exercising legs, whereas this advantageous effect disappeared during severe hypoxia, leaving augmented CaO(2) alone insufficient for improving peak leg O(2) delivery and VO(2). Finally, that VO(2)max was largely dependent on CaO(2) during moderate hypoxia but became abruptly CaO(2)-independent by slightly increasing the severity of hypoxia could be an indirect evidence of the appearance of central fatigue.

Topics: Cardiac Output; Drug Administration Schedule; Erythrocyte Volume; Erythropoietin; Humans; Hypoxia; Inhalation; Iron; Male; Oxygen Consumption; Physical Fitness; Recombinant Proteins; Reference Values

Topics: Acclimatization; Adult; Altitude; Erythropoietin; Hematocrit; Humans; Hypoxia; Male; Pulmonary Edema; Time Factors

Preconditioning, such as by brief hypoxic exposure, has been shown to protect hearts against severe ischaemia. Here we hypothesized that hypoxic preconditioning (HPC) protects injured hearts by mobilizing the circulating progenitor cells. Ischaemia-reperfusion (IR) injury was induced by left coronary ligation and release in rats kept in room air or preconditioned with 10% oxygen for 6 weeks. To study the role of erythropoietin (EPO), another HPC + IR group was given an EPO receptor (EPOR) antibody via a subcutaneous mini-osmotic pump 3 weeks before IR induction. HPC alone gradually increased haematocrit, cardiac and plasma EPO, and cardiac vascular endothelial growth factor (VEGF) only in the first two weeks. HPC improved heart contractility, reduced ischaemic injury, and maintained EPO and EPOR levels in the infarct tissues of IR hearts, but had no significant effect on VEGF. Interestingly, the number of CD34(+)CXCR4(+) cells in the peripheral blood and their expression in HPC-treated hearts was higher than in control. Preconditioning up-regulated cardiac expression of stromal derived factor-1 (SDF-1) and prevented its IR-induced reduction. The EPOR antibody abolished HPC-mediated functional recovery, and reduced SDF-1, CXCR4 and CD34 expression in IR hearts, as well as the number of CD34(+)CXCR4(+) cells in blood. The specificity of neutralizing antibody was confirmed in an H9c2 culture system. In conclusion, exposure of rats to moderate hypoxia leads to an increase in progenitor cells in the heart and circulation. This effect is dependent on EPO, which induces cell homing by increased SDF-1/CXCR4 and reduces the heart susceptibly to IR injury.

Topics: Animals; Antibodies; Antigens, CD34; Chemokine CXCL12; Creatine Kinase, MB Form; Erythropoietin; Gene Expression; Heart; Hypoxia; Ischemic Preconditioning, Myocardial; Male; Models, Biological; Myocardium; Myocytes, Cardiac; Rats; Rats, Wistar; Receptors, CXCR4; Receptors, Erythropoietin; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Vascular Endothelial Growth Factor A; Ventricular Function, Left

To investigate the anti-inflammatory effect of erythropoietin (EPO) pretreatment on cardiomyocytes exposed to hypoxia/reoxygenation injury (H/R) and explore the possible mechanism.. The cultured neonatal rats?ventricular cardiomyocytes were divided randomly into 4 groups, control group (C group), EPO pretreatment group (E group), EPO and pyrrolidine dithiocarbamate (PDTC) pretreatment group (EP group) and PDTC pretreatment group (P group). After 24 hours?pretreatment, the cardiomyocytes were exposed to H/R. After pretreatment and H/R, the expression of tumor necrosis factor-alpha(TNF-alpha) gene in all the groups was detected by RT-PCR and Western blot. The nuclear factor-kappa B (NF-kappa B) activity was detected by electrophoretic mobility shift assay (EMSA) and the inhibitor-kappa B alpha (I-kappa B alpha) protein level was detected by Western blot.. The decrement of I-kappa B alpha protein and the increasing NF-kappa B activity were found in cardiomyocytes pretreated with EPO before H/R compared to other groups (t equal to 3.321, 4.183, P less than 0.01). However, after H/R, NF-kappa B activity and expression of TNF-alphagene were significantly reduced, I-kappa B alpha protein expression was increased in cardiomyocytes of E group compared to other groups (t=3.425, 3.687, 3.454, P less than 0.01). All theses changes caused by EPO pretreatment were eliminated by the intervention of PDTC (an antagonist to NF-kappa B) during pretreatment.. EPO pretreatment can inhibit the activation of NF-kappa B and upregulation of TNF-alpha gene in cardiomyocytes exposed to H/R through a negative feedback of NF-kappa B signaling pathway, and thus produces the anti-inflammatory effect. This might be one of the ways EPO produces the anti-inflammatory effect.

Topics: Analysis of Variance; Animals; Animals, Newborn; Antioxidants; Blotting, Western; Cells, Cultured; Electrophoretic Mobility Shift Assay; Erythropoietin; Hypoxia; Inflammation; Myocytes, Cardiac; NF-kappa B; Pyrrolidines; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Thiocarbamates; Tumor Necrosis Factor-alpha

Circulating erythropoietin (EPO) is mainly produced by the kidneys and mediates erythrogenesis in bone marrow and nonhematopoietic cell survival. EPO is also produced in other tissues where it functions as a paracrine. Moreover, the hypoxic induction of EPO is known to be mediated by HIF-1alpha and HIF-2alpha, but it remains obscure as to which of these two mediators mainly contributes to EPO expression. Thus, we designed in vivo experiments to evaluate the contributions made by HIF-1alpha and HIF-2alpha to EPO expression. In mice exposed to mild whole body hypoxia, HIF-1alpha and HIF-2alpha were both induced in all tissues examined. However, EPO mRNA was expressed in kidney and brain, but not in liver and lung. Likewise, chromatin immunoprecipitation (CHIP) analyses demonstrated that HIF-1alpha or HIF-2alpha binding to the EPO gene increased under hypoxic conditions only in kidney and brain. A comparison of CHIP data and EPO mRNA levels suggested that, during mild hypoxia, renal EPO transcription is induced equally by HIF-1alpha and HIF-2alpha, but that brain EPO is mainly induced during hypoxia by HIF-2alpha. Thus, HIF-1alpha and HIF-2alpha appear to contribute to EPO expression tissue specifically.

Topics: Animals; Chromatin Immunoprecipitation; Erythropoietin; Gene Expression Regulation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Male; Mice; Mice, Inbred ICR; Organ Specificity; RNA, Messenger; Transcription Factors; Transcription, Genetic

Uncontrolled expression of vascular endothelial growth factor (VEGF) in vivo may cause unexpected side effects, such as brain hemangioma or tumor growth. Because hypoxia-inducible factor-1 (HIF-1) is upregulated during cerebral ischemia and regulates gene expression by binding to a cis-acting hypoxia-responsive element (HRE), we therefore used a novel HRE, originating in the 3'-end of the erythropoietin (Epo) gene, to control gene expression in the ischemic brain. A concatemer of nine copies (H9) of the consensus sequence of HRE was used to mediate hypoxia induction. Three groups of adult CD-1 mice received AAVH9-VEGF, AAVH9-lacZ or saline injection, and then underwent 45 min of transient middle cerebral artery occlusion (tMCAO). Results show that HIF-1 was persistently expressed in the ischemic brain. VEGF was overexpressed in the ischemic perifocal region in AAVH9-VEGF-transduced mice. Double-labeled immunostaining showed that VEGF expressed in neurons and astrocytes but not endothelial cells, suggesting that adeno-associated virus (AAV) vectors transduced neurons and astrocytes predominantly. The total number of microvessels/enlarged microvessels was greatly increased in the AAVH9-VEGF-transduced mice (180+/-29/27+/-4) compared to the AAVH9-lacZ (118+/-19/14+/-3) or saline-treated (119+/-20/14+/-2) mice after tMCAO (P<0.05). Cell proliferation examination demonstrated that these microvessels were newly formed. Regional cerebral blood flow recovery in the AAVH9-VEGF-transduced mice was also better than in AAVH9-lacZ or saline-treated mice (P<0.05). Our data indicated that HRE is a novel trigger for the control of VEGF expression in the ischemic brain. VEGF overexpression through AAVH9-VEGF gene transfer showed stable focal angiogenic effects in post-ischemic repair process, providing an opportunity to rebuild injured brain tissue.

Topics: 3' Untranslated Regions; Animals; Astrocytes; Brain Ischemia; Cerebrovascular Circulation; Dependovirus; Erythropoietin; Gene Expression; Genetic Therapy; Genetic Vectors; Hypoxia; Hypoxia-Inducible Factor 1; Immunohistochemistry; Mice; Neovascularization, Physiologic; Neurons; Transduction, Genetic; Vascular Endothelial Growth Factor A

Relationship between haemoglobin levels and tumour oxygenation has been already reported. The purpose of this work was to compare in human malignant glioma-bearing mice the sensitivity of two well established techniques of tumour hypoxia assessment, especially their ability to detect expected weak variations of tumour oxygenation status associated to haemoglobin level modifications. The relationship between tumour hypoxia and glucose metabolism was also investigated. Experiments were performed on a human malignant glioma (GBM Nan1) xenografted into nude mice. Twenty-four hours after tumour implantation, animals were randomized into three groups: 'Anaemia' for mice subjected to repeated blood samplings, 'Control', and 'rHuEPO' for mice receiving recombinant human erythropoietin. Once the tumours reached a volume of 300+/-100 mm(3), tumour hypoxia was assessed both using the pO(2)-Histograph, Eppendorftrade mark and the pimonidazole binding assay. Glucose metabolism was evaluated by (18)F-FDG autoradiography and compared with the pimonidazole binding distribution pattern. Repeated blood samplings significantly reduced mean haemoglobin levels (10.9+/-2.0 g/dl), inducing chronic anaemia in mice, while daily administration of rHuEPO led to increase of haemoglobin levels (15.8+/-2.0 g/dl). Oxygenation status evaluated by a microelectrode was worsened in anaemic mice (mean pO(2) in tumour = 6.9+/-0.8 mmHg) and improved in rHuEPO-treated animals (mean pO(2)in tumour = 11.4+/-1.2 mmHg). No correlation was observed between the oxygen-sensitive probe and pimonidazole labelling results: both techniques give different but complementary information about tumour hypoxia. Areas of high pimonidazole binding and areas of high (18)F-FDG uptake superimposed well. Present results confirm that modification of haemoglobin levels leads to alteration of tumour oxygenation status. These variations were detectable using the oxygen-sensitive electrode but not the pimonidazole binding assay. The strong correlation between pimonidazole labelling and (18)F-FDG uptake suggests a positive relationship between hypoxia and increased glucose metabolism in this tumour model.

Topics: Anemia; Animals; Autoradiography; Electrodes; Erythropoietin; Fluorodeoxyglucose F18; Glioma; Hemoglobins; Humans; Hypoxia; Mice; Mice, Nude; Muscle, Skeletal; Neoplasm Transplantation; Nitroimidazoles; Recombinant Proteins; Transplantation, Heterologous

Hypoxia-induced increases in red blood cell production have been found in both altitude-adapted populations and acclimatized lowlanders. This process is mediated by erythropoietin (EPO) released mainly by the hypoxic kidney. We have previously observed high hemoglobin concentrations in elite breath-hold divers and our aim was to investigate whether apnea-induced hypoxia could increase EPO concentration. Ten healthy volunteers performed 15 maximal duration apneas, divided into three series of five apneas, each series separated by 10 min of rest. Apneas within series were separated by 2 min and preceded by 1 min of hyperventilation to increase apnea duration and arterial oxygen desaturation. When EPO concentration after serial apneas was compared to baseline values, an average maximum increase of 24% was found (P < 0.01). No changes in EPO concentration were observed during a control day without apnea, eliminating possible effects of a diurnal rhythm or blood loss. We therefore conclude that serial apneas increase circulating EPO concentration in humans.

Topics: Adult; Apnea; Bradycardia; Carbon Dioxide; Diving; Erythropoietin; Female; Humans; Hypoxia; Male; Oxygen; Physical Fitness; Spirometry

In response to anemia, erythropoietin (Epo) gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ectopic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type-specific expression of the Epo gene.

Topics: Amino Acid Motifs; Anemia; Animals; Erythropoietin; GATA2 Transcription Factor; GATA3 Transcription Factor; Gene Expression Regulation; Hypoxia; Kidney; Liver; Mice; Mice, Transgenic; Neurons; Promoter Regions, Genetic; Tissue Distribution

Erythropoietin (Epo), a hormone known to stimulate bone marrow erythrocyte production, is widely used to treat anemia in patients at risk for vascular disease. However, the effects of Epo on angiogenesis are not well defined. We studied the role of Epo in a mouse model of retinopathy characterized by oxygen-induced vascular loss followed by hypoxia-induced pathological neovascularization. Without treatment, local retinal Epo levels were suppressed during the vessel loss phase. Administration of exogenous Epo prevented both vessel dropout and subsequent hypoxia-induced neovascularization. Early use of Epo also protected against hypoxia-induced retinal neuron apoptosis. In contrast, retinal Epo mRNA levels were highly elevated during the retinopathy neovascular phase. Exogenous late Epo treatment did not protect the retina, but rather enhanced pathological neovascularization. Epo's early protective effect occurred through both systemic retinal recruitment of proangiogenic bone marrow-derived progenitor cells and activation of prosurvival NF-kappaB via Epo receptor activation on retinal vessels and neurons. Thus early retinal Epo suppression contributed to retinal vascular instability, and elevated Epo levels during the proliferation stage contributed to neovascularization and disease. Understanding the role of Epo in angiogenesis is critical to timing its intervention in patients with retinopathy or other diseases in which pathological angiogenesis plays a significant role.

Topics: Animals; Apoptosis; Erythropoietin; Hypoxia; Mice; Neovascularization, Pathologic; Neurons; NF-kappa B; Oxygen; Receptors, Erythropoietin; Retina; Retinal Diseases; Retinal Vessels; Stem Cells

Topics: Anemia; Erythropoiesis; Erythropoietin; History, 19th Century; History, 20th Century; Humans; Hypoxia; Neoplasms; Recombinant Proteins; Uremia

Hyperbaric oxygen preconditioning (HBO-PC) has been shown to be effective in preventing hypoxic injuries in many animal models. The aim of the present study was to examine the hypoxic tolerance induced by HBO-PC and to explore the role of hypoxia-inducible factor-1alpha (HIF-1alpha) in a global hypoxia model. Male mice received HBO-PC before hypoxia exposure and swimming. HBO-PC significantly prolonged the survival time and the tolerance time of swimming under normobaric hypoxia. HBO-PC increased the protein content of HIF-1alpha and erythropoietin (EPO) in the cerebral cortex and hippocampus and prevented the changes of blood brain barrier (BBB) permeability and brain edema caused by hypoxia exposure. The results suggested that HBO-PC induced hypoxic tolerance in mice via up-regulation of HIF-1alpha and its downstream genes.

Topics: Analysis of Variance; Animals; Behavior, Animal; Brain Edema; Cerebral Cortex; Disease Models, Animal; Erythropoietin; Hippocampus; Hyperbaric Oxygenation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning; Male; Mice; Mice, Inbred Strains; Survival Analysis; Time Factors; Up-Regulation

We have previously shown that 2 weeks of hypoxia protect the right ventricle of the rat heart from subsequent ischemia and reperfusion (I/R). In the present study, we examined the following: (1) Do shorter periods of hypoxia protect from subsequent I/R? (2) Does intermittent normoxia increase the cardioprotective effect? (3) Is hypoxia-inducible factor-1alpha (HIF-1alpha), erythropoietin (EPO), or vascular endothelial growth factor (VEGF) involved in the protective effects? Preischemic cardiac work was followed by global ischemia, reperfusion, and postischemic cardiac work (15 min each). External heart work was determined at the end of both work phases. Four groups of hearts were investigated: hearts from normoxic rats (n=8), hearts from rats after 24 h of continuous hypoxia (10.5% inspired oxygen, n=7), hearts from rats after 24 h hypoxia with a single intermission of 30 min normoxia (n=9), and hearts from rats after 24 h hypoxia and multiple intermissions of 30 min normoxia (n=7). Protein levels of HIF-1alpha and mRNA levels of EPO and VEGF were determined in right ventricular tissue of normoxic and hypoxic hearts. Postischemic right heart recovery was better in all three hypoxic groups compared with normoxic hearts (61.8 +/- 5.9%, 65.6 +/- 3.0%, and 75.7 +/- 2.6% vs. 46.0 +/- 3.9%, p < 0.01). Hypoxia with multiple normoxic intermissions further improved right heart recovery compared to continuous hypoxia (p < 0.05). HIF-1alpha protein levels were 80.3 +/- 2.5 pg/microg in normoxic hearts and 108.0 +/- 10.3 pg/microg in hypoxic hearts (p = 0.02). No differences in EPO and VEGF mRNA levels were found between normoxic and hypoxic hearts. Twenty-four hours of continuous hypoxia protect the isolated working right heart from subsequent ischemia and reperfusion. When preceding hypoxia is interrupted by multiple reoxygenation periods, there is a further significant increase in cardiac functional recovery. HIF-1alpha may be involved in the protective effect.

Topics: Animals; Erythropoietin; Heart Ventricles; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning, Myocardial; Male; Oxygen; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Time Factors; Vascular Endothelial Growth Factor A

To observe change of binding activity of HIF-1 with erythropoietin (EPO) hypoxia response element (HRE) in the hippocampus of mice preconditioned to hypoxia and explore relationship between the changes and the preconditioning.. The hippocampus was removed from mice exposed to hypoxia for 0 run (control group), 1 run (H1 group) and 4 runs(H4 group). Electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP)and real time PCR were used to detect the change of activity of HIF-1 on HRE of EPO.. Both in vitro and in vivo binding tests showed that the HIF-1 DNA-binding activities were increased in group H1 and markedly increased in group H4.. The increase of HIF-1 and HRE of EPO binding activities is thought be involved in hypoxic preconditioning.

Topics: Animals; Erythropoietin; Hippocampus; Hypoxia; Hypoxia-Inducible Factor 1; Male; Mice; Mice, Inbred BALB C; Response Elements

Animal studies have highlighted the potentially neuroprotective role of vascular endothelial growth factor (VEGF). Low levels of this growth factor have been found in the cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS). VEGF (and other proteins, such as erythropoietin (EPO)) are produced in response to hypoxia via a common pathway involving a specific transcription factor (hypoxia-inducible factor, HIF) and a hypoxia responsive element (HRE) in the respective genes' promoter regions. In this study, we report finding the expected, high levels of VEGF and EPO in CSF from hypoxemic neurological controls, whereas EPO (but not VEGF) levels are high in the CSF from hypoxemic ALS patients. Hence, the VEGF levels in CSF from patients with ALS were significantly lower than those seen in hypoxemic controls. There was a trend towards higher CSF levels of EPO in hypoxemic ALS patients than in hypoxemic controls. Our results suggest that VEGF may not be produced in sufficient amounts in chronically hypoxic ALS patients and that this dysfunction may participate in the pathogenesis of the disease. The high EPO levels in hypoxemic ALS patients nevertheless suggest an intact common oxygen-sensor pathway.

Topics: Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Erythropoietin; Female; Humans; Hypoxia; Male; Middle Aged; Oxygen Consumption; Vascular Endothelial Growth Factor A

The aim of this work was to investigate the effects of different recombinant human erythropoietin (rhEPO) doses on the infarction development and if rhEPO could protect against the deleterious consequences induced by a previous intermittent hypoxia (IH, FiO(2) 5%, 4h). First, isolated rat hearts were submitted to an ischemia-reperfusion. rhEPO was infused before or after ischemia, at different doses. Secondly, rats were exposed to of IH. Twenty-four hours later, hearts underwent the ischemia-reperfusion protocol. For some hearts, 5Uml(-1) rhEPO was infused as previously. We observed that rhEPO has a dose-dependant effect on infarct size since it was significantly reduced by rhEPO infusions before or after ischemia. We also showed that 4h of IH induced a higher sensitivity to the infarction which was prevented by rhEPO. In conclusion, rhEPO administration before or after ischemia can protect isolated rat myocardium in a dose dependent manner and efficiently prevents the higher sensitivity to the infarction induced by previous intermittent hypoxia.

Topics: Animals; Coronary Circulation; Erythropoietin; Heart Diseases; Heart Rate; Hypoxia; In Vitro Techniques; Male; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Wistar; Recombinant Proteins; Ventricular Function, Left

Topics: Acclimatization; Altitude; Erythrocyte Count; Erythropoiesis; Erythropoietin; Exercise; Hematocrit; Hemoglobins; Humans; Hypoxia; Research Design

Topics: Acclimatization; Altitude; Erythrocyte Count; Erythropoiesis; Erythropoietin; Exercise; Hematocrit; Hemoglobins; Humans; Hypoxia; Research Design

Yoga induces long-term changes in respiratory function and control. We tested whether it represents a successful strategy for high-altitude adaptation. We compared ventilatory, cardiovascular and hematological parameters in: 12 Caucasian yoga trainees and 12 control sea-level residents, at baseline and after 2-week exposure to high altitude (Pyramid Laboratory, Nepal, 5,050 m), 38 active lifestyle high-altitude natives (Sherpas) and 13 contemplative lifestyle high-altitude natives with practice of yoga-like respiratory exercises (Buddhist monks) studied at 5,050 m. At baseline, hypoxic ventilatory response (HVR), red blood cell count and hematocrit were lower in Caucasian yoga trainees than in controls. After 14 days at altitude, yoga trainees showed similar oxygen saturation, blood pressure, RR interval compared to controls, but lower HVR (-0.44 +/- 0.08 vs. -0.98 +/- 0.21 l/min/m/%SaO(2), P < 0.05), minute ventilation (8.3 +/- 0.9 vs. 10.8 +/- 1.6 l/min, P < 0.05), breathing rate (indicating higher ventilatory efficiency), and lower red blood cell count, hemoglobin, hematocrit, albumin, erythropoietin and soluble transferrin receptors. Hypoxic ventilatory response in monks was lower than in Sherpas (-0.23 +/- 0.05 vs. -0.63 +/- 0.09 l/min/m/%SaO(2), P < 0.05); values were similar to baseline data of yoga trainees and Caucasian controls, respectively. Red blood cell count and hematocrit were lower in monks as compared to Sherpas. In conclusion, Caucasian subjects practicing yoga maintain a satisfactory oxygen transport at high altitude, with minimal increase in ventilation and with reduced hematological changes, resembling Himalayan natives. Respiratory adaptations induced by the practice of yoga may represent an efficient strategy to cope with altitude-induced hypoxia.

Topics: Acclimatization; Adult; Altitude; Blood Pressure; Breathing Exercises; Buddhism; Erythrocyte Count; Erythropoietin; Female; Heart Rate; Hematocrit; Hemoglobins; Humans; Hypoxia; Male; Mountaineering; Nepal; Oxygen; Pulmonary Ventilation; Receptors, Transferrin; Respiration; Respiratory Mechanics; Serum Albumin; Time Factors; Yoga

To determine if posthypoxia treatment with erythropoietin (EPO) has protective effects against subsequent susceptibility to seizure related neuronal injury in rat pups subjected to acute hypoxia at P10.. Four groups of rats were manipulated at P10, as described below, then all received kainic acid (KA) (10 mg/kg i.p.) at P29: Hypoxia-NS-KA group (n = 11): subjected to acute hypoxia (down to 4% O2), and then immediately received saline i.p. Hypoxia-EPO-KA group (n = 10): subjected to acute hypoxia and then immediately received EPO (1,000 U/Kg i.p.). Normoxia-NS-KA group (n = 11): sham manipulated and injected with saline. Normoxia-EPO-KA group (n = 10): sham manipulated then immediately injected with EPO (1000 U/Kg i.p.). After receiving KA at P29, all rats were monitored using videotape techniques, and were sacrificed at P31. TUNEL and Hoechst stains to assess for apoptosis, and regular histology for hippocampal cell counts were performed.. Administration of the single dose of erythropoietin directly after an acute hypoxic event at P10 resulted at P29 in increased latency to forelimb clonus seizures, reduced duration of these seizures, protection against hippocampal cell loss, and decreased hippocampal apoptosis in the Hypoxia-EPO-KA group as compared to the Hypoxia-NS-KA group.. These data support the presence of favorable protective effects of erythropoietin against the long-term consequences of acute hypoxia in the developing brain and raise the possibility of its investigation as a potential neuroprotective agent after human neonatal hypoxic encephalopathy.

Topics: Animals; Animals, Newborn; Cell Count; Disease Models, Animal; Disease Susceptibility; Dose-Response Relationship, Drug; Erythropoietin; Excitatory Amino Acid Agonists; Hippocampus; Humans; Hypoxia; Hypoxia, Brain; Kainic Acid; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Seizures

Adaptive transcriptional responses to oxygen deprivation (hypoxia) are mediated by the hypoxia-inducible factors (HIFs), heterodimeric transcription factors composed of two basic helix-loop-helix-PAS family proteins. The transcriptional activity of HIF is determined by the hypoxic stabilization of the HIF-alpha proteins. HIF-1alpha and HIF-2alpha exhibit high sequence homology but have different mRNA expression patterns; HIF-1alpha is expressed ubiquitously whereas HIF-2alpha expression is more restricted to certain tissues, e.g., the endothelium, lung, brain, and neural crest derivatives. Germ-line deletion of either HIF subunit is embryonic lethal with unique features suggesting important roles for both HIF-alpha isoforms. Global deletion of Hif-2alpha results in distinct phenotypes depending on the mouse strain used for the mutation, clearly demonstrating an important role for HIF-2alpha in mouse development. The function of HIF-2alpha in adult life, however, remains incompletely understood. In this study, we describe the generation of a conditional murine Hif-2alpha allele and the effect of its acute postnatal ablation. Under very stringent conditions, we ablate Hif-2alpha after birth and compare the effect of acute global deletion of Hif-2alpha and Hif-1alpha. Our results demonstrate that HIF-2alpha plays a critical role in adult erythropoiesis, with acute deletion leading to anemia. Furthermore, although HIF-1alpha was first purified and cloned based on its affinity for the human erythropoietin (EPO) 3' enhancer hypoxia response element (HRE) and regulates Epo expression during mouse embryogenesis, HIF-2alpha is the critical alpha isoform regulating Epo under physiologic and stress conditions in adults.

Topics: Anemia; Animals; Basic Helix-Loop-Helix Transcription Factors; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Hypoxia; Mice; Mice, Knockout; Phenotype; Protein Isoforms; Response Elements; Stress, Physiological

Tissue hypoxia induces a variety of functional changes including enhanced transcriptional activity associated with high transmethylation activity (e.g. mRNA cap methylation) in the nucleus. It is well known that the kidney responds to hypoxia with enhanced transcription of erythropoietin (EPO) in the interstitial cells. Since S-adenosylhomocysteine (AdoHcy)-hydrolase regulates most S-adenosylmethionine (AdoMet) dependent transmethylation reactions by hydrolyzing the potent feedback inhibitor AdoHcy to adenosine and homocysteine we studied the effect of hypoxia by carbon monoxide (CO) inhalation (1200 ppm) on AdoHcy-hydrolase gene expression and its localization in rat kidneys.. CO lowered renal AdoHcy-hydrolase mRNA expression by 64% whereas AdoHcy-hydrolase activity was not changed during 4h of CO exposure 0.7+/-0.04 mU/mg (control) vs. 0.75+/-0.06 mU/mg protein. Using two-channel immunofluorescence confocal laser scanning microscope AdoHcy-hydrolase was visualized in different cells of the hypoxic rat kidney. A very bright immunofluorescence of AdoHcy-hydrolase was observed in the nuclei of single interstitial cells of renal cortex and outer medulla which respond to hypoxia with increased EPO secretion indicating translocation of AdoHcy-hydrolase from the cytosol to the nucleus.. These data suggest that AdoHcy-hydrolase accumulation in the nucleus of adult mammalian cells is involved in maintaining efficient transmethylation reactions in transcriptionally active cells by removing the product inhibitor AdoHcy.

Topics: Adenosylhomocysteinase; Animals; Carbon Monoxide; Cell Nucleus; Erythropoietin; Hypoxia; Immunohistochemistry; In Situ Hybridization; Kidney; Male; Methylation; Rats; Rats, Sprague-Dawley; Transcription, Genetic

This study tested the hypothesis that maximal oxygen uptake (VO(2max)) and performance increase upon altitude acclimatization at moderate altitude. Eight elite cyclists were studied at sea level, and after 1 (Day 1), 7 (Day 7), 14 (Day 14) and 21 (Day 21) days of exposure to 2340 m. Capillary blood samples were taken on these days before performing two consecutive maximal exercise trials. Acclimatization increased hemoglobin concentration and arterial oxygen content. On Day 1, VO(2max) and time to exhaustion (at 80% of sea-level maximal power output) decreased by 12.8% (P<0.05) and 25.8% (P<0.05), respectively, compared with the corresponding sea-level values. Subsequently, these parameters increased by 3.2% (P<0.05) and 6.0% (P<0.05) from Days 1 to 7, by 4.8% (P<0.05) and 5.7% (P<0.05) from Days 7 to 14, followed by 0.7% (P>0.05) and 1.4% (P>0.05) from Days 14 to 21, respectively. These data suggest that endurance athletes competing at altitudes around 2340 m should expose themselves to this altitude at least 14 days before competition.

Topics: Acclimatization; Adult; Altitude; Anthropometry; Bicycling; Doping in Sports; Erythropoietin; Exercise; Hemoglobins; Humans; Hypoxia; Male; Muscle Contraction; Muscle, Skeletal; Physical Endurance; Prospective Studies; Task Performance and Analysis; Time Factors

High-dose recombinant erythropoietin (rEpo) is neuroprotective in neonatal animal models of brain injury, but the long-term consequences of neonatal exposure have not been studied.. We hypothesized that multiple injections of high-dose rEpo during the neonatal period would be safe, and would improve neurologic outcomes after exposure to neonatal hypoxia or hypoxic-ischemic injury.. Three experimental groups of Sprague-Dawley rats were assessed: (1) normoxia, (2) hypoxia and (3) hypoxia-ischemia. Groups 1 and 2 were given 0, 2,500 or 5,000 U/kg rEpo subcutaneously for the first 5 days of life (P1-P5). Group 2 animals also underwent 2 h of hypoxia (8% O(2)) daily from P1-P3. Group 3 animals underwent right carotid artery ligation followed by hypoxia (8% O(2) x 90 min) on P7, followed by either vehicle or rEpo (2,500 U/kg subcutaneously QD x3). We evaluated short- and long-term physiologic and behavioral outcomes. Major organs were evaluated grossly and histologically.. rEpo treatment transiently raised hematocrit, prevented hypoxia-induced delays in geotaxis and growth, improved forelimb strength, promoted liver growth in males, lowered the adult platelet count, but did not alter other CBC indices or histology. rEpo prevented hypoxia-ischemia-induced learning impairment and substantia nigra neuron loss.. Repeated treatment of newborn rats with high-dose rEpo was safe under all conditions tested. rEpo treatment improved the development of hypoxia-exposed newborns and prevented the learning impairment and dopamine neuron loss due to unilateral hypoxic-ischemic brain injury.

Topics: Animals; Animals, Newborn; Avoidance Learning; Behavior, Animal; Brain; Dopamine; Drug-Related Side Effects and Adverse Reactions; Erythropoietin; Hematocrit; Hypoxia; Hypoxia-Ischemia, Brain; Liver; Models, Animal; Neurons; Neuroprotective Agents; Organ Size; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Weight Gain

Recently a family of O(2)-dependent prolyl hydroxylase domain-containing enzymes (PHD) has been identified as a cellular oxygen-sensing mechanism. Reduced prolyl hydroxylase activity initiates a signalling cascade that includes the accumulation, as well as the activation, of hypoxia-inducible factor (HIF-1alpha). In turn the transcription factor HIF-1alpha, and other targets of the PHD, elicit a myriad of incompletely understood cellular responses. In these studies we have tested: (1) whether a small-molecule prolyl hydroxylase inhibitor (PHI) can effectively activate the oxygen-sensing pathway when administered systemically to mice, and (2) whether the activation of the PHD signalling pathway at the cellular level results in whole-animal hypoxic tolerance.. Mice received daily injections of the PHI, ethyl-3,4 dihydroxybenzoate (EDHB, 100-250 mg kg(-1)) or vehicle. Tissue levels of HIF-1alpha and the serum levels of the HIF-inducible gene, erythropoietin (EPO), were measured to evaluate PHD-pathway activation. To evaluate hypoxic tolerance, the endurance and survival ability of these animals was tested in sublethal (8% O(2)) and lethal hypoxia (5% O(2)) respectively.. Systemic treatment of mice with the PHD inhibitor, EDHB, leads to elevated levels of HIF-1alpha in liver and HIF-inducible EPO in serum, indicating activation of the cellular oxygen-sensing pathway. Animals treated with EDHB display significantly increased viability and enhanced exercise performance in hypoxia.. These results demonstrate a novel pharmacological strategy to induce hypoxic tolerance and are the first to demonstrate that the activation of the PHD oxygen-sensing pathway at the cellular level is sufficient to produce a hypoxic-tolerant phenotype at the physiological level of the whole animal.

Topics: Amino Acids, Dicarboxylic; Animals; Enzyme Inhibitors; Erythropoietin; Hematocrit; Hydroxybenzoates; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Injections, Intraperitoneal; Liver; Male; Mice; Mice, Inbred Strains; Oxygen; Physical Conditioning, Animal; Physical Endurance; Procollagen-Proline Dioxygenase; Signal Transduction

According to recent data erythropoietin receptor (EPOR) is expressed not only by bone marrow erythroid progenitors but by endothelial- and cancer cells and it was suggested that erythropoietin (EPO) may affect their functions as well. We have analyzed the effects of recombinant human erythropoietin-alpha (rHuEPOalpha) on radiation sensitivity of EPOR+ human epidermoid carcinoma (A431) xenograft model. In vivo rHuEPOalpha treatment was started after tumor cell inoculation into SCID mice. 5 Gy irradiation was performed on day 14, the effect of which was measured on day 22. Hemoglobin level, tumor-associated microvessels and HIF-1alpha expression of the xenograft were monitored during the experiment. rHuEPOalpha administration prevented the development of tumor-induced anemia of SCID mice and reduced the level of HIF-1alpha expression of the xenograft tumor without affecting tumor growth. We have found that rHuEPOalpha treatment significantly increased the efficacy of antitumor effect of irradiation which was partly mediated by complex effects on tumor-associated microvessels. In vitro rHuEPOalpha did not affect proliferation of A431 cells but enhanced the antiproliferative and proapoptotic effects of irradiation. We concluded that rHuEPOalpha administration positively modulated the antitumoral effects of irradiation at least by two pathways, decreasing systemic hypoxia resulting in decreased tumoral HIF-1alpha expression and enhancing direct effects on tumor-associated microvessels.

Topics: Anemia, Hypochromic; Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Epoetin Alfa; Erythropoietin; Hematinics; Hemoglobins; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, SCID; Microcirculation; Polymerase Chain Reaction; Radiation-Sensitizing Agents; Recombinant Proteins; Transplantation, Heterologous

Nucleated red blood cells (NRBCs) in fetal circulation have been proposed as a marker of chronic hypoxia in fetuses with intrauterine growth restriction (IUGR). We sought to determine the effects of chronic hypoxia, chronic nitric oxide inhibition with N(G)-nitro-L-arginine methyl ester (L-NAME), or both on NRBC counts, erythropoietin levels, and pathologic changes in an animal model of IUGR.. We assigned timed pregnant adult Sprague Dawley rats to the following groups: (1) 21% oxygen + saline solution (n = 7); (2) 21% oxygen + L-NAME (n = 8); (3) 10% oxygen + saline solution (n = 6); and (4) 10% oxygen + L-NAME (n = 6). We inserted osmotic pumps that were prefilled with saline solution or L-NAME subcutaneously on day 17 of gestation. The animals were placed in a Plexiglas hypoxic chamber, which ensured a constant hypoxic environment. The animals were killed on day 21 of gestation before the onset of spontaneous labor. We collected maternal and fetal blood for measurement of NRBC and erythropoietin levels. The results were interpreted in relationship to maternal arterial blood gases and hemoglobin and hematocrit levels. Fetuses were examined for gross abnormalities and histological abnormalities that are characteristic of vascular disruptions by a blind examiner to experimental manipulation.. Nitric oxide inhibition induced IUGR with maximal effect when both L-NAME and hypoxia treatments were combined. Inhibition of nitric oxide synthesis, but not chronic hypoxia, increased the number of fetal NRBCs and generalized hemorrhagic diathesis in utero. These features were aggravated significantly when the treatments were combined. Moreover, chronic hypoxia induced significant maternal metabolic acidosis and increased hematocrit and erythropoietin levels in maternal and fetal blood. Nitric oxide inhibition increased maternal hematocrit levels while decreasing maternal erythropoietin levels without significantly altering the maternal acid-base status. In contrast with chronic hypoxia, nitric oxide inhibition increased fetal NRBCs without affecting erythropoietin levels.. Our findings indicate that the number of NRBCs in fetal circulation does not serve as a specific marker of chronic hypoxia that accompanies IUGR or of elevated erythropoietin levels but are an epiphenomenon that is related to the inhibition of nitric oxide.

Topics: Animals; Chronic Disease; Disease Models, Animal; Erythroblasts; Erythrocyte Count; Erythropoietin; Female; Fetal Blood; Fetal Growth Retardation; Hypoxia; Nitric Oxide Synthase; Placental Insufficiency; Pregnancy; Rats; Rats, Sprague-Dawley

Ischemia/reperfusion (I/R) injury is closely associated with tissue damage in various organs, as well as in kidney transplants. Erythropoietin (EPO) has been shown to have a cytoprotective effect against hypoxia. We examined the effect of EPO against renal I/R injury and the underlying mechanism.. Human umbilical vein endothelial cells and human renal proximal tubular epithelial cells were cultured under hypoxic conditions with various EPO concentrations at 37 degrees C and examined the mechanism of cell proliferation by EPO. Moreover, to demonstrate the renoprotective effect in vivo, we treated Sprague-Dawley rats with 100 IU/kg EPO every 2 days for 2 weeks (a total of 6 doses). One day after the last injection, the operations to produce renal I/R injury (bilateral renal occlusion for 60 min) were done, and rats were killed at the end of the reperfusion period (24 hr and 72 hr after reperfusion began).. First, we demonstrated in vitro that EPO increased hypoxia inducible factor-1alpha (HIF-1alpha) expression and stimulated proliferation of both cells under hypoxic conditions. Next, we demonstrated in vivo that EPO treatment increased the number of HIF-1alpha-positive cells, and markedly induced the expression of vascular endothelial growth factor messenger RNA. Using pimonidazole, a molecular probe that detects hypoxia, we found that EPO markedly attenuated tubular hypoxia, and reduced the number of terminal transferase dUTP nick end labeling-positive apoptotic cells and alpha-smooth muscle actin-positive interstitial cells.. We suggested a novel HIF-1alpha induction pathway by EPO under hypoxic conditions. Thus, EPO may protect the kidneys against ischemia reperfusion injury by activating HIF-1alpha.

Topics: Cell Division; Cell Line; Cells, Cultured; Endothelium, Vascular; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; In Situ Nick-End Labeling; Kidney; Reperfusion Injury; RNA, Messenger; Umbilical Veins; Vascular Endothelial Growth Factor A

Erythropoietin primarily serves as an essential growth factor for erythrocyte precursor cells. However, there is increasing evidence that erythropoietin (EPO)/EPO receptor (EPO-R) signaling operates as a potential tissue-protective system outside the bone marrow. Arguing that growing hair follicles (HF) are among the most rapidly proliferating tissues, we have here explored whether human HFs are sources of EPO and targets of EPO-R-mediated signaling. Human scalp skin and microdissected HFs were assessed for EPO and EPO-R expression, and the effects of EPO on organ-cultured HFs were assessed in the presence/absence of a classical apoptosis-inducing chemotherapeutic agent. Here, we show that human scalp HFs express EPO on the mRNA and protein level in situ, up-regulate EPO transcription under hypoxic conditions, and express transcripts for EPO-R and the EPO-stimulatory transcriptional cofactor hypoxia-inducible factor-1alpha. Although EPO does not significantly alter human hair growth in vitro, it significantly down-regulates chemotherapy-induced intrafollicular apoptosis and changes the gene expression program of the HFs. The current study points to intriguing targets of EPO beyond the erythropoietic system: human HFs are an extrarenal site of EPO production and an extrahematopoietic site of EPO-R expression. They may recruit EPO/EPO-R signaling e.g., for modulating HF apoptosis under conditions of hypoxia and chemotherapy-induced stress.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Cyclophosphamide; Erythropoietin; Gene Expression Profiling; Hair Follicle; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Keratinocytes; Kidney; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Organ Culture Techniques; Receptors, Erythropoietin; Scalp; Signal Transduction

The hypoxia-inducible factor (HIF) pathway is crucial in mitigating the deleterious effects of oxygen deprivation. HIF-alpha is an essential component of the oxygen-sensing mechanisms and under normoxic conditions is targeted for degradation via hydroxylation by HIF-prolyl hydroxylases. Several HIF-prolyl hydroxylase inhibitors (PHIs) induced erythropoietin (epo) expression in vitro and in mice, with peak epo expression ranging from 5.6- to 207-fold above control animals. Furthermore, several PHIs induced fetal hemoglobin (HbF) expression in primary human erythroid cells in vitro, as determined by flow cytometry. One PHI, FG-2216, was further tested in a nonhuman primate model without and with chronic phlebotomy. FG-2216 was orally bioavailable and induced significant and reversible Epo induction in vivo (82- to 309-fold at 60 mg/kg). Chronic oral dosing in male rhesus macaques was well tolerated, significantly increased erythropoiesis, and prevented anemia induced by weekly phlebotomy. Furthermore, modest increases in HbF-containing red cells and reticulocytes were demonstrated by flow cytometry, though significant increases in HbF were not demonstrated by high-pressure liquid chromatography (HPLC). HIF PHIs represent a novel class of molecules with broad potential clinical application for congenital and acquired anemias.

Topics: Administration, Oral; Animals; Carcinoma, Hepatocellular; Chronic Disease; Enzyme Inhibitors; Erythrocytes; Erythroid Cells; Erythroid Precursor Cells; Erythropoietin; Fetal Hemoglobin; Flow Cytometry; Humans; Hypoxia; Liver Neoplasms; Macaca mulatta; Male; Mice; Oxygen; Phlebotomy; Polycythemia; Procollagen-Proline Dioxygenase; Reticulocytes

Hypoxia-inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease.. ARPE-19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme-linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real-time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques.. HIF-1 alpha was readily produced by ARPE-19 cells under hypoxia, but HIF-2 alpha and HIF-3 alpha could not be detected even after HIF-1 alpha silencing. HIF-1 alpha protein levels showed an increasing trend for the first 24 h while HIF-1 alpha mRNA levels fluctuated during this time. After 36 h HIF-1 alpha protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF-1 alpha significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level.. HIF-1 alpha appears to be the main isoform of HIF functioning in ARPE-19 cells. Under hypoxia, HIF-1 alpha levels are likely self-regulated by a feedback loop that involves both transcriptional and post-translational mechanisms. VEGF production by human RPE cells is regulated by HIF-1 alpha. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina are responsible for its production.

Topics: Cells, Cultured; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Isomerism; Pigment Epithelium of Eye; Procollagen-Proline Dioxygenase; RNA, Messenger; Vascular Endothelial Growth Factor A

Erythropoietin (Epo) and its cognate receptor (EpoR) are required for maintaining adequate levels of circulating erythrocytes during embryogenesis and adulthood. Here, we report the functional characterization of the zebrafish epo and epor genes. The expression of epo and epor was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, revealing marked parallels between zebrafish and mammalian gene expression patterns. Examination of the hypochromic mutant, weissherbst, and adult hypoxia-treated hearts indicate that zebrafish epo expression is induced by anemia and hypoxia. Overexpression of epo mRNA resulted in severe polycythemia, characterized by a striking increase in the number of cells expressing scl, c-myb, gata1, ikaros, epor, and betae1-globin, suggesting that both the erythroid progenitor and mature erythrocyte compartments respond to epo. Morpholino-mediated knockdown of the epor caused a slight decrease in primitive and complete block of definitive erythropoiesis. Abrogation of STAT5 blocked the erythropoietic expansion by epo mRNA, consistent with a requirement for STAT5 in epo signaling. Together, the characterization of zebrafish epo and epor demonstrates the conservation of an ancient program that ensures proper red blood cell numbers during normal homeostasis and under hypoxic conditions.

Topics: Amino Acid Sequence; Anemia; Animals; Conserved Sequence; DNA, Complementary; Embryo, Nonmammalian; Erythroid Cells; Erythropoiesis; Erythropoietin; Gene Expression Regulation, Developmental; Humans; Hypoxia; Molecular Sequence Data; Receptors, Erythropoietin; Sequence Alignment; Signal Transduction; STAT5 Transcription Factor; Zebrafish

Vascular endothelial growth factor (VEGF) has been investigated as a therapy for many disorders and injuries involving ischemia. In this report, we constructed and evaluated a hypoxia-inducible VEGF expression system as a treatment for spinal cord injury (SCI).. The hypoxia-inducible VEGF plasmid was constructed using the erythropoietin (Epo) enhancer with the Simian virus 40 (SV40) promoter (pEpo-SV-VEGF) or the RTP801 promoter (pRTP801-VEGF). The expression of VEGF in vitro was evaluated after transfection into N2A cells. The plasmids were then injected into rat spinal cords with contusion injuries. The expression of VEGF in vivo was measured using reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Locomotor recovery in the rats was evaluated using the Basso, Beattie and Bresnahan (BBB) scale for locomotor analysis.. In vitro transfection showed that pEpo-SV-VEGF or pRTP801-VEGF induced VEGF expression under hypoxic conditions, whereas pSV-VEGF did not. The VEGF level was higher in the pEpo-SV-VEGF and pRTP801-VEGF groups than in the control group. The VEGF expression was detected in neurons and astrocytes of the spinal cord. Locomotor recovery was improved in the pEpo-SV-VEGF and pRTP801-VEGF groups, and BBB scores were higher than in the control group. Staining using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling showed that the number of apoptotic cells decreased in the plasmid-injected groups compared with the control group, and significant differences were observed between the hypoxia-responsive groups and the pSV-VEGF group.. These results suggest that the hypoxia-inducible VEGF expression system may be useful for gene therapy of SCI.

Topics: Animals; Apoptosis; Astrocytes; Cell Line, Tumor; Enhancer Elements, Genetic; Erythropoietin; Gene Expression; Genetic Therapy; Genetic Vectors; Hypoxia; Male; Mice; Motor Activity; Neurons; Plasmids; Rats; Rats, Sprague-Dawley; Simian virus 40; Spinal Cord; Spinal Cord Injuries; Transfection; Vascular Endothelial Growth Factor A

Apart from enhancing red blood cell production, erythropoietin (Epo) has been shown to modulate the ventilatory response to reduced oxygen supply. Both functions are crucial for the organism to cope with increased oxygen demand. In the present work, we analyzed the impact of Epo and the resulting excessive erythrocytosis in the neural control of normoxic and hypoxic ventilation. To this end, we used our transgenic mouse line (Tg6) that shows high levels of human Epo in brain and plasma, the latter leading to a hematocrit of approximately 80%. Interestingly, while normoxic and hypoxic ventilation in Tg6 mice was similar to WT mice, Tg6 mice showed an increased respiratory frequency but a decreased tidal volume. Knowing that Epo modulates catecholaminergic activity, the altered catecholaminergic metabolism measured in brain stem suggested that the increased respiratory frequency in Tg6 mice was related to the overexpression of Epo in brain. In the periphery, higher response to hyperoxia (Dejours test), as well as reduced tyrosine hydroxylase activity in carotid bodies, revealed a higher chemosensitivity to oxygen in transgenic mice. Moreover, in line with the decreased activity of the rate-limiting enzyme for dopamine synthesis, the intraperitoneal injection of a highly specific peripheral ventilatory stimulant, domperidone, did not stimulate hypoxic ventilatory response in Tg6 mice. These results suggest that high Epo plasma levels modulate the carotid body's chemotransduction. All together, these findings are relevant for understanding the cross-talk between the ventilatory and erythropoietic systems exposed to hypoxia.

Topics: Acclimatization; Animals; Domperidone; Dopamine Antagonists; Erythropoietin; Female; Gene Expression; Humans; Hypoxia; Male; Mice; Mice, Transgenic; Oxygen; Respiration

We used transgenic mice constitutively over-expressing erythropoietin ("tg6" mice) and wild-type (wt) mice to investigate whether the high hematocrit (hct), consequence of Epo over-expression affected: (1) the normoxic ventilation (V (E)) and the acute hypoxic ventilatory response (HVR) and decline (HVD), (2) the increase in ventilation observed after chronic exposure to hypobaric hypoxia (430mmHg for 21 days), (3) the respiratory "blunting", and (4) the erythrocythemic response induced by chronic hypoxia exposure. V (E) was found to be similar in tg6 and wt mice in normoxia (FIO2=0.21). Post-acclimation V (E) was significantly elevated in every time point in wt mice at FIO2=0.10 when compared to pre-acclimation values. In contrast, tg6 mice exhibited a non-significant increase in V (E) throughout acute hypoxia exposure. Changes in V (E) are associated with adjustments in tidal volume (V(T)). HVR and HVD were independent of EE in tg6 and wt mice before chornic hypoxia exposure. HVR was significantly greater in wt than in tg6 mice after chronic hypoxia. After acclimation, HVD decreased in tg6 mice. Chronic hypoxia exposure caused hct to increase significantly in wt mice, while only a marginal increase occurred in the tg6 group. Although pre-existent EE does not appear to have an effect on HVR, the observation of alterations on V(T) suggests that it may contribute to time-dependent changes in ventilation and in the acute HVR during exposure to chronic hypoxia. In addition, our results suggest that EE may lead to an early "blunting" of the ventilatory response.

Topics: Acclimatization; Animals; Erythropoietin; Female; Hematocrit; Hemoglobins; Hypoxia; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oxygen Consumption; Polycythemia; Pulmonary Ventilation; Tidal Volume

Previous studies have yielded conflicting results as to whether extracellular adenosine generation and signaling contributes to hypoxia-induced increases in renal erythropoietin (EPO) secretion. In this study, we combined pharmacological and genetic approaches to elucidate a potential contribution of extracellular adenosine to renal EPO release in mice. To stimulate EPO secretion, we used murine carbon monoxide exposure (400 and 750 parts per million CO, 4 h), ambient hypoxia (8% oxygen, 4 h), or arterial hemodilution. Because the ecto-5-nucleotidase (CD73, conversion of AMP to adenosine) is considered the pacemaker of extracellular adenosine generation, we first tested the effect of blocking extracellular adenosine generation with the specific CD73-inhibitor adenosine 5'-(alpha,beta-methylene) diphosphate (APCP) or by gene-targeted deletion of cd73. These studies showed that neither APCP-treatment nor targeted deletion of cd73 resulted in changes of stimulated EPO mRNA or serum levels, although the increases of adenosine levels in the kidney following CO exposure were attenuated in mice with APCP treatment or in cd73(-/-) mice. Moreover, pharmacological studies using specific inhibitors of individual adenosine receptors (A1 AR, DPCPX; A 2A AR, DMPX; A 2B AR, PSB 1115; A3AR, MRS 1191) showed no effect on stimulated increases of EPO mRNA or serum levels. Finally, stimulated EPO secretion was not attenuated in gene-targeted mice lacking A1A(-/-, A2A AR-/-, A2BAR(-/-), or A3AR-/-. Together, these studies combine genetic and pharmacological in vivo evidence that increases of EPO secretion during limited oxygen availability are not affected by extracellular adenosine generation or signaling.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Diphosphate; Animals; Arteries; Carbon Monoxide; Erythropoietin; Extracellular Fluid; Hemodilution; Hypoxia; Kidney; Mice; Mice, Inbred C57BL; Mice, Knockout; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; RNA, Messenger; Signal Transduction

This study is aimed to determine whether short or prolonged residency at high altitude (HA) elicits erythropoietin (EPO) secretion effectively in subjects who were able to acclimatize and those who were not able to acclimatize and suffered from acute mountain sickness (AMS) and high altitude pulmonary edema (HAPE).. Plasma EPO was measured in 16 lowland residents (LLR) at sea level (SL) and during 11 d of their sojourn at an altitude of 3450 m. Identical studies were also conducted in LLR acclimatized to HA (LLR-accl), high altitude natives (HAN) and in patients of AMS and HAPE.. In LLR at SL, the mean +/- SD EPO levels were 8.93 +/- 3.75 mU x ml(-1), increased significantly after 8 h (20.0 +/- 11.06) of arrival at HA, peaked by day 1 (27.91 +/- 10.74 mU x ml(-1)), and started declining thereafter. The hemoglobin and hematocrit also increased after 8 h of arrival at HA and the increased levels were maintained during sojourn at high altitude. The EPO levels in LLR-accl were found to be significantly higher than the LLR SL values, but were not significantly different in HAN. The EPO levels in patients of AMS were not significantly different than the LLR values during the initial 2 d after arrival at HA but were found to be increased in patients of HAPE.. Short or prolonged residency at HA is associated with increased secretion of EPO. The EPO response to hypoxia is not significantly altered in AMS but is markedly enhanced in HAPE, which may be due to exaggerated hypoxemia in these patients.

Topics: Acclimatization; Adult; Altitude; Analysis of Variance; Erythropoietin; Hematocrit; Humans; Hypoxia; Male; Time Factors

Erythropoietin supports the survival of erythroblasts. We previously demonstrated that 24 malignant human cell lines expressed erythropoietin and its receptor and that erythropoietin secretion was enhanced under anoxia. In this study, we examined the viability of 22 of these cell lines excluding two leukemia cell lines under anoxia.. Twenty-two cancer cell lines of various origins were cultured under anoxia or normoxia for 4 days, and their viability was examined at 1-day intervals. The levels of lactate and ATP were measured. The expressions of hypoxia-inducible transcription factor 1alpha (HIF-1alpha) and Bcl-2 family proteins were examined by western blotting analysis. The cellular and mitochondrial features were examined by microscopy.. Eleven of the 22 cancer cell lines examined showed 80% to 100% cell viability after 4 days under anoxia; 2 cell lines showed similar viability for 3 days, 3 cell lines showed similar viability for 2 days, and 6 cell lines showed similar viability for 1 day or less. These 11 death-resistant cell lines, which secrete various amounts of erythropoietin under anoxia, produced significantly more lactate during 2 days under anoxia than under normoxia, with ATP levels about 60% of those before anoxia. ATP returned to the normal level when normoxia was restored after 4 days of anoxia. However, the nonresistant cell lines responded to anoxia by yielding significantly more lactate without a reduction of the ATP level. The expression patterns of Bcl-2 family proteins revealed that apoptosis-inhibiting signals predominated over proapoptotic signals in the death-resistant cells under anoxia.. The majority of the cancer cell lines examined survived under anoxia in vitro, through the Pasteur effect, in a dormant state without direct support of erythropoietin.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Erythropoietin; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit

We investigated erythropoietin (Epo) response in a cohort of diabetic patients with various types of anemia to approach the pathogenesis of some cases of "unexplained" anemia encountered among diabetics. Serum Epo levels were determined totally in 747 evaluable subjects with normal renal and hepatic function, of whom 694 had anemia. Among anemic patients, 237 were diabetics, while among the 53 nonanemic persons, there were also 21 diabetics. Diabetic and nondiabetic subjects were uniformly balanced in relation to their demographic features and were categorized according to the etiology of their anemia. Hemoglobin (Hb) did not differ between diabetic and nondiabetic subjects in all the etiological groups and in the whole population. Diabetic patients had significantly lower serum Epo levels as compared to nondiabetics (36.5+/-61 vs 69.4+/-191 IU/ml, p<0.0001), and this was true for all etiologic groups of anemia with the exception of patients with myeloproliferative disorders and those with megaloblastic anemia. The natural logarithmic (ln)-EpoxHb component was used as an index of response to anemia and was found to be significantly decreased in almost all subgroups of diabetic patients. Serum Epo levels were also negatively correlated with the percentage of glycosylated Hb, HbA1(C) (r=-0.446), and the correlation was stronger with the ln of serum Epo (r=-0.638, p<0.001). Inappropriately low serum Epo level is a uniform feature in patients with type II diabetes mellitus and may represent a constitutive blunted response to anemia or an altered metabolic rate of Epo, probably as a result of abnormal glycosylation of the cytokine.

Topics: Adult; Aged; Aged, 80 and over; Anemia; Cytokines; Diabetes Mellitus, Type 2; Erythropoietin; Female; Glycated Hemoglobin; Glycosylation; Hemoglobins; Humans; Hypoxia; Male; Middle Aged

Amphotericin B (AmB) is widely used for treating severe systemic fungal infections. However, long-term AmB treatment is invariably associated with adverse effects such as anemia. The erythropoietin (EPO) suppression by AmB has been proposed to contribute to the development of anemia. However, the mechanism whereby EPO is suppressed remains obscure. In this study, we investigated the possibility that AmB inhibits the transcription of the EPO gene by inactivating HIF-1, which is a known key transcription factor and regulator of EPO expression. EPO mRNA levels were markedly attenuated by AmB treatment both in rat kidneys and in Hep3B cells. AmB inactivated the transcriptional activity of HIF-1alpha, but did not affect the expression or localization of HIF-1 subunits. Moreover, AmB was found to specifically repress the C-terminal transactivation domain (CAD) of HIF-1alpha, and this repression by AmB required Asn803, a target site of the factor-inhibiting HIF-1 (FIH); moreover, this repressive effect was reversed by FIH inhibitors. Furthermore, AmB stimulated CAD-FIH interaction and inhibited the p300 recruitment by CAD. We propose that this mechanism underlies the unexplained anemia associated with AmB therapy.

Topics: Amphotericin B; Anemia; Animals; Antifungal Agents; Cell Line; Down-Regulation; E1A-Associated p300 Protein; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Male; Mycoses; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Repressor Proteins; Transcription, Genetic

Renal (peritubular) tissue hypoxia is a well-known physiological trigger for erythropoietin (EPO) production. We investigated the effect of rebound relative hypoxia after hyperoxia obtained under normo- and hyperbaric oxygen breathing conditions. A group of 16 healthy volunteers were investigated before and after a period of breathing 100% normobaric oxygen for 2 h and a period of breathing 100% oxygen at 2.5 ATA for 90 min (hyperbaric oxygen). Serum EPO concentration was measured using a radioimmunoassay at various time points during 24-36 h. A 60% increase (P < 0.001) in serum EPO was observed 36 h after normobaric oxygen. In contrast, a 53% decrease in serum EPO was observed at 24 h after hyperbaric oxygen. Those changes were not related to the circadian rhythm of serum EPO of the subjects. These results indicate that a sudden and sustained decrease in tissue oxygen tension, even above hypoxia thresholds (e.g., after a period of normobaric oxygen breathing), may act as a trigger for EPO serum level. This EPO trigger, the "normobaric oxygen paradox," does not appear to be present after hyperbaric oxygen breathing.

Topics: Blood Gas Monitoring, Transcutaneous; Erythropoietin; Female; Humans; Hyperbaric Oxygenation; Hyperoxia; Hypoxia; Male; Oxygen; Time Factors

Recent studies have suggested that endogenous erythropoietin (Epo) plays an important role in the mobilization of bone marrow-derived endothelial progenitor cells (EPCs). However, it remains to be elucidated whether the Epo system exerts protective effects on pulmonary hypertension (PH), a fatal disorder encountered in cardiovascular medicine.. A mouse model of hypoxia-induced PH was used for study. We evaluated right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in mice lacking the Epo receptor (EpoR) in nonerythroid lineages (EpoR(-/-) rescued mice) after 3 weeks of exposure to hypoxia. Those mice lack EpoR in the cardiovascular system but not in the hematopoietic system. The development of PH and pulmonary vascular remodeling were accelerated in EpoR(-/-) rescued mice compared with wild-type mice. The mobilization of EPCs and their recruitment to the pulmonary endothelium were significantly impaired in EpoR(-/-) rescued mice. By contrast, reconstitution of the bone marrow with wild-type bone marrow cells ameliorated PH in the EpoR(-/-) rescued mice. Hypoxia enhanced the expression of EpoR on pulmonary endothelial cells in wild-type but not EpoR(-/-) rescued mice. Finally, hypoxia activated endothelial nitric oxide synthase in the lungs in wild-type mice but not in EpoR(-/-) rescued mice.. These results indicate that the endogenous Epo/EpoR system plays an important role in the recruitment of EPCs and prevents the development of PH during chronic hypoxia in mice in vivo, suggesting the therapeutic importance of the system for the treatment of PH.

Topics: Animals; Bone Marrow Transplantation; Cell Movement; Cells, Cultured; Chronic Disease; Endothelial Cells; Endothelium; Endothelium, Vascular; Enzyme Activation; Erythroid Precursor Cells; Erythropoietin; GATA1 Transcription Factor; Heart Failure; Hematopoietic Stem Cells; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Lung; Male; Mice; Mice, Knockout; Mice, Transgenic; Muscle, Smooth, Vascular; Nitric Oxide Synthase Type III; Organ Specificity; Radiation Chimera; Receptor, TIE-2; Receptors, Erythropoietin; Systole; Ventricular Dysfunction, Right

The concomitant manifestations of proximal renal tubular dysfunction and anemia with erythropoietin (Epo) deficiency observed in chronic cadmium (Cd) intoxication, such as Itai-itai disease, suggest a close local correlation between the Cd-targeted tubular cells and Epo-producing cells in the kidney. Therefore, we investigated the local relationship between hypoxia-induced Epo production and renal tubular injury in rats injected with Cd at 2 mg/kg twice a week for 8 months. Anemia due to insufficient production of Epo was observed in Cd-intoxicated rats. In situ hybridization detected Epo mRNA expression in the proximal renal tubular cells of hypoxic rats without Cd intoxication, and the Cd-intoxicated rats showed atrophy of Epo-expressing renal tubules and replacement of them with fibrotic tissue. A single dose of cisplatin at 8 mg/kg, which can induce clinical manifestations similar to those of Cd including renal tubular damage along with Epo-deficient anemia, resulted in Epo-expressing renal tubule destruction on day 4. These data indicate that Cd and cisplatin would induce anemia through the direct injury of the proximal renal tubular cells that are responsible for Epo production.

Topics: Anemia, Hypochromic; Animals; Antineoplastic Agents; Body Weight; Cadmium Chloride; Cisplatin; Environmental Pollutants; Erythropoietin; Female; Hematologic Tests; Hypoxia; In Situ Hybridization; Kidney Tubules, Proximal; Liver; Organ Size; Rats; Rats, Wistar; RNA, Messenger; Spleen; Time Factors

Topics: Animals; Cell Differentiation; Erythropoietin; Genomic Instability; Humans; Hydroxylation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Mitochondria; Models, Biological; Neoplasm Metastasis

Erythropoietin (EPO) is a glycoprotein hormone that regulates the proliferation and differentiation of erythroid progenitor cells in mammals. Although EPO has been identified in fish, the specific function and effects of hypoxia have not been investigated previously. In this study, we have demonstrated a relationship between increases in renal EPO levels and decreases in spleen EPO levels and spleen-somatic index (SSI), with increases in haemoglobin (Hb) concentration in the blood during hypoxia exposure in rainbow trout. Splenic contraction and the subsequent red blood cell release accounts for the initial increase in Hb concentration in the blood, whereas EPO action probably accounts for the later increases in hemoglobin concentration in the blood. Our data indicate that fish and mammalian erythropoietic systems are similar in response to hypoxia, in that erythropoiesis in fish is influenced by EPO.

Topics: Animals; Erythropoietin; Hydrocortisone; Hypoxia; Kidney; Oncorhynchus mykiss; Spleen

Erythropoietin is present in human amniotic fluid and has been suggested as a marker of fetal hypoxia. The objectives of the present study were to determine whether erythropoietin is present in ovine amniotic fluid, fetal urine, and/or lung liquid and whether concentrations in these compartments change in parallel with endogenous fetal plasma erythropoietin concentration when the latter is increased experimentally.. In late gestation chronically catheterized fetal sheep, samples of amniotic fluid and plasma, urine and plasma, lung liquid, amniotic fluid, and plasma were collected before and up to 7 days after induction of 4 types of fetal hypoxia: (1) acute anemic hypoxia that was induced by a single fetal hemorrhage, (2) progressive anemic hypoxia that was induced by daily exchange transfusion, (3) acute hypoxic hypoxia that was induced by the reduction of maternal inspired oxygen content, or (4) chronic placental insufficiency that was induced by daily umbilicoplacental embolization for 4 days. Erythropoietin concentrations were determined by radioimmunoassay. Statistical testing included analysis of variance and least squares regression.. Under basal, nonhypoxic conditions, amniotic fluid erythropoietin concentration averaged 33.2% +/- 1.6% (SE) of fetal plasma erythropoietin concentration, and basal fetal urine and lung liquid erythropoietin concentrations ranged from low (<10% of plasma concentration) to nondetectable. Unlike the strong correlation in humans, basal amniotic fluid and plasma erythropoietin concentrations were correlated only weakly (r = 0.259; r2 = 6.7%; P = .0027; n = 132). Amniotic fluid erythropoietin concentration approximately doubled after 12 hours of severe hypoxic hypoxia or after 24 hours of embolization-induced severe hypoxia but was unchanged after 12 hours of mild-moderate hypoxic hypoxia or 24 hours of anemic hypoxia. Concomitant fetal plasma erythropoietin concentrations increased to 28.1 +/- 5.3, 12.5 +/- 2.7, 10.8 +/- 4.6, and 10.0 +/- 1.3 times basal values, respectively. During progressive fetal anemia, urinary erythropoietin concentration increased almost 10-fold (P = .0023) but remained a small fraction (3.7% +/- 0.4%) of plasma concentration; at 12 hours of hypoxic hypoxia, lung liquid erythropoietin concentration did not vary with the severity of the hypoxia and remained low relative to plasma concentration (4.2% +/- 2.1%).. Erythropoietin is present in ovine amniotic fluid, urine, and lung liquid. With only 3 potential sources, the fetal membranes appear to be the primary source of amniotic fluid erythropoietin in the nonhypoxic ovine fetus because basal urine and lung liquid erythropoietin concentrations are much lower than amniotic fluid concentrations. Although unchanged during mild-to-moderate fetal hypoxia, amniotic fluid erythropoietin concentration increases modestly during severe fetal hypoxia. In sheep, fetal urinary erythropoietin may contribute to this rise in amniotic fluid erythropoietin concentration during severe hypoxia, because fetal urinary and plasma concentrations increase in parallel during anemia.

Topics: Amniotic Fluid; Animals; Erythropoietin; Extraembryonic Membranes; Fetus; Hematocrit; Hypoxia; Lung; Multivariate Analysis; Sheep

Erythropoietin (Epo) is used commonly to treat cancer and/or therapy-related anemia. Until recently, Epo was considered to be a specific stimulator of erythropoiesis, acting via its receptor, EpoR. It becomes clear, however, that EpoR is expressed in a variety of cell types other than hematopoietic cells, and that Epo is a potent cytoprotective cytokine increasing cell survival under hypoxic conditions. Epo and EpoR are also expressed in various malignant tumors, and EpoR expression shows association with tumor invasion and progression. Recently, a functional Epo autocrine signaling mechanism was also detected in human melanoma cells. In this study, we examined the hypothesis that Epo activates the Akt signaling pathway in human melanoma cells and thus promotes the survival of tumor cells. The Akt signaling pathway in response to Epo was examined in melanoma. Similar to Epo, the expression of EpoR was up-regulated in response to hypoxia and Epo stimulation in melanoma cells. Melanoma cells constitutively expressed Akt with variable expression of mammalian target of rapamycin, and Epo dose-dependently induced their activity. Epo increased Akt kinase activity, which was abrogated by co-treatment with LY294002, a specific blocker of phosphoinositide 3-kinase. LY294002 also inhibited the cytoprotective effects of Epo in melanoma cells under both normoxic and hypoxic conditions. Our results suggest that Epo promotes melanoma cell survival by activating an Akt-dependent signaling pathway.

Topics: Cell Proliferation; Cells, Cultured; Enzyme Activation; Erythropoietin; Glycogen Synthase Kinase 3; Humans; Hypoxia; Melanocytes; Melanoma; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Erythropoietin; Recombinant Proteins; Signal Transduction; Skin Neoplasms

Hepcidin, the principal iron regulatory hormone, regulates the absorption of iron from the diet and the mobilization of iron from stores. Previous studies indicated that hepcidin is suppressed during anemia, a response that would appropriately increase the absorption of iron and its release from stores. Indeed, in the mouse model, hepcidin-1 was suppressed after phlebotomy or erythropoietin administration but the suppression was reversed by inhibitors of erythropoiesis. The suppression of hepcidin necessary to match iron supply to erythropoietic demand thus requires increased erythropoiesis and is not directly mediated by anemia, tissue hypoxia, or erythropoietin.

Topics: Anemia; Animals; Antimicrobial Cationic Peptides; Erythropoiesis; Erythropoietin; Female; Hepcidins; Hypoxia; Iron; Iron, Dietary; Male; Mice

It has been hypothesized that autacoids, such as endothelin-1 (ET), may modulate erythropoietin (Epo) secretion. Therefore, we studied the effect of ET-1 infusion and of a nonselective ET(A/B) receptor antagonist on Epo secretion under carbon monoxide (CO) exposure. Anesthetized rats were supplied with room temperature air containing increasing concentrations of CO by an aerating cap. A CO-Epo dose-response curve over the range of 0.02-0.14 vol% CO was conducted. Subpressor doses of ET-1 (3 pmol/min/kg BW) and the ET(A/B) receptor antagonist LU302872 (LU; 30 mg/kg) were applied to anaesthetized rats under normoxia (controls CON, ET, LU) and following hypoxia (CO exposure; H-CON, H-ET, H-LU). Mean arterial blood pressure (MAP), glomerular filtration rate (GFR, inulin clearance), Epo and ET-1 serum concentrations (ELISA) and renal Epo mRNA (Light Cycler) were determined. The EC50 value for CO was 0.1 vol% with a 70-fold increase in Epo serum concentrations. CO exposure increased Epo serum and Epo mRNA concentrations in the expected range in all groups. None of the treatments with ET or LU influenced the effect of hypoxia on Epo serum concentrations and renal Epo mRNA content. Under hypoxia, administration of ET-1 as well as LU prevented the hypoxia-induced decrease in MAP (p<0.05). Under hypoxia, GFR was reduced by 50% except for H-LU with values comparable to normoxia. Taken together, the influence of hypoxia exceeds by far the effect of ET-1 on Epo production, irrespective of the presence or absence of exogenous ET-1. Thus, ET-1 does not appear to be a major modulator of Epo production.

Topics: Animals; Blood Pressure; Carbon Monoxide; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Erythropoietin; Glomerular Filtration Rate; Hypoxia; Kidney; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger

Gene therapy with the vascular endothelial growth factor (VEGF) gene is a potential treatment for many disorders or injuries with ischemia. However, unregulated expression of VEGF may induce pathological angiogenesis, promoting tumor growth, diabetic proliferative retinopathy and rupture of atherosclerotic plaque. Therefore, the effective regulation of the gene expression is one of the requirements for the VEGF gene therapy. In this research, we evaluated the hypoxia-inducible gene expression system with the erythropoietin (Epo) enhancer and the Epo 3'-untranslated region (UTR). The luciferase plasmids were constructed with the Epo enhancer (pEpo-SV-Luc), the Epo 3'-UTR (pSV-Luc-EpoUTR) or both (pEpo-SV-Luc-EpoUTR). The polyethylenimine/plasmid complexes were transfected to 293 or A7R5 cells and the cells were incubated under normoxia or hypoxia. The results showed that the Epo enhancer or Epo 3'-UTR increased the target gene expression under hypoxia. pEpo-SV-Luc-EpoUTR showed the highest luciferase expression. The VEGF expression plasmid with the Epo enhancer and 3'-UTR was also constructed. The VEGF expression by pEpo-SV-VEGF-EpoUTR showed the highest specificity of the gene expression in the hypoxic cells. The results suggest that the VEGF plasmid with the Epo enhancer and the Epo 3'-UTR may be useful for gene therapy for ischemic diseases.

Topics: 3' Untranslated Regions; Animals; Cell Line; Cloning, Molecular; Enhancer Elements, Genetic; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Gene Expression; Genetic Therapy; Humans; Hypoxia; Luciferases; Plasmids; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Vascular Endothelial Growth Factor A

Ischemia and chronic hypoxia (CH) trigger a variety of adverse effects arising from metabolic stress that injures cells. In response to reduced O2, hypoxia-inducible factor 1alpha (HIF-1alpha) activates erythropoietin (Epo) as well as many other target genes that counteract the effects of O2 deficiency. Epo produced by the kidney stimulates erythrocyte production, leading to decreased HIF-1alpha production by improved tissue O2 delivery. However, Epo is produced by many other tissues, and it is currently unclear to what extent, if any, locally produced Epo modulates HIF-1alpha expression. Derivatives of Epo that possess tissue-protective activities but do not stimulate erythropoiesis [e.g., carbamylated Epo (CEpo)] are useful tools with which to determine whether exogenous Epo modulates HIF-1alpha in the absence of changes in hemoglobin concentration. We compared the effects of CH (6.5% O2 for 10 days) with or without CEpo administered by daily s.c. injection (10 microg/kg of body weight). CEpo administration did not alter the survival rate, weight loss, or increased hemoglobin concentration associated with CH. Therefore, CEpo does not directly suppress HIF-mediated erythropoiesis. CEpo does, however, prevent CH-induced neuronal increases of HIF-1alpha and Epo receptor-associated immunoreactivity (a measure of stress) while reducing the apoptotic index. In contrast, the myocardium did not exhibit increased HIF-1alpha expression during CH, although CEpo did reduce the apoptotic index. These observations therefore demonstrate that CEpo administration reduces the metabolic stress caused by severe CH, resulting in improved cellular survival independent of erythrocyte production.

Topics: Animals; DNA Nucleotidylexotransferase; Erythropoietin; Hypoxia; Male; Mice; Mice, Inbred ICR; Receptors, Erythropoietin; Stress, Physiological

Physiologically, hypoxia induces the expression of erythropoietin (Epo) in adult kidney cells. Epo, in turn, acts on the Epo receptor (EpoR) in RBC precursors to stimulate growth and prevent apoptosis. Because hypoxia plays a major role in the malignant progression of tumors and Epo and its receptors have also been detected in malignant tumors, we investigated the expression of Epo and EpoR and their relationship with hypoxia, proliferation, apoptosis, and clinicopathologic variables in cervical cancer.. Intratumoral oxygen measurement and needle biopsies of the tumors were done in 48 patients with cervical cancer. The obtained tissue was analyzed by immunohistochemistry with antibodies against Epo, EpoR, and Ki-67 as well as by terminal deoxynucleotidyl transferase-mediated deoxyuracil triphosphate nick-end labeling assays.. Epo and EpoR were expressed in 88% and 92% of samples, respectively. Cervical cancers with higher Epo expression showed a significantly reduced overall survival (3 years, 50.0% versus 80.6%; P = 0.0084). Epo and EpoR expression correlated significantly with apoptosis (r = 0.49, P = 0.001 and r = 0.36, P = 0.021). Furthermore, EpoR expression correlated significantly with tumor size (r = 0.32, P = 0.032) and was significantly associated with the presence of lymphovascular space involvement (P = 0.037). However, we observed no correlation between Epo or EpoR expression and intratumoral hypoxia, although in well-oxygenated tumors, EpoR localized significantly more often to the invasion front (P = 0.047).. This study analyzes Epo/EpoR expression and their relationship with intratumoral pO(2) levels as well as with survival in patients with cervical cancer. The data suggest a critical role of the endogenous Epo/EpoR system in cervical cancer.

Topics: Adult; Aged; Apoptosis; Cell Proliferation; Disease Progression; Disease-Free Survival; Erythropoietin; Female; Follow-Up Studies; Humans; Hypoxia; Immunohistochemistry; Kaplan-Meier Estimate; Middle Aged; Neoplasm Staging; Predictive Value of Tests; Receptors, Erythropoietin; Recurrence; Survival Rate; Treatment Outcome; Uterine Cervical Neoplasms

Hypoxia and biological responses to hypoxia are commonly encountered in both normal and pathologic cellular processes. Here we report that extracellular superoxide dismutase (EC-SOD) plays a major role in regulating the magnitude of hypoxia-induced erythropoietin (Epo) gene expression, thus implicating superoxide as an intermediary signal transduction molecule critical to this process. We found that mice which have the EC-SOD gene inactivated show a marked more than 100-fold elevation in hypoxia-induced Epo gene expression, compared with wild-type controls, which was both dose and time dependent. These mice also showed a significant increase in serum Epo levels after 1 d hypoxia. Interestingly, despite elevated Epo levels, reciprocal changes in hematocrit and reticulocyte counts were not found, suggesting that this newly synthesized Epo lacks functional hematopoietic effects. When EC-SOD was overexpressed in Hep3B cells, we found a significant reduction in Epo gene induction by both CoCl2 (50 microM) and hypoxia (1% O2). Similar findings were noted with another hypoxia-inducible gene, carbonic anhydrase IX. We conclude that EC-SOD functions as a major repressor of hypoxia-induced Epo gene expression, which implicates superoxide as a signaling intermediate whose downstream effects, at least in part, may be mediated by HIF-1alpha.

Topics: Animals; Blotting, Western; DNA; Enzyme-Linked Immunosorbent Assay; Erythropoiesis; Erythropoietin; Gene Expression; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Kidney; Mice; Mice, Knockout; RNA, Messenger; Superoxide Dismutase; Transcription Factors

Blind subterranean mole rats of the Spalax ehrenbergi superspecies in Israel have evolved multiple adaptive strategies to face underground hypoxia. Hypoxia-inducible factor-1alpha (HIF-1alpha) and erythropoietin (Epo) are key factors in the development of normal erythropoiesis and angiogenesis. Here, we demonstrate via real-time polymerase chain reaction (PCR) quantification that Spalax fetal liver and kidney express higher levels of Epo mRNA than Rattus, generating reinforcement of fetal erythropoiesis underground and adapting it to life underground in an atmosphere of abrupt and sharp fluctuations of O2 supply. In neonates, Rattus liver and kidney express higher Epo levels than Spalax under both normoxia and hypoxia, probably due to Rattus ineffective erythropoiesis during embryonic life and its birth in a poorly ventilated breeding nest under ground. Adult Rattus kidney and liver, and adult Spalax liver express similar levels of Epo mRNA under normoxia and hypoxia. However, adult Spalax hypoxic kidney, the major site of erythropoietin production in adult mammals, shows levels that were twice as high as that of Rattus. Spalax expresses remarkably higher levels of HIF-1alpha mRNA than Rattus at all developmental stages studied, which peaked in neonates, as an adaptation against hypoxia.

Topics: Animals; Animals, Newborn; DNA-Binding Proteins; Embryo, Mammalian; Erythropoietin; Gene Expression Regulation; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Liver; Mole Rats; Nuclear Proteins; Rats; Transcription Factors

Erythropoiesis in the adult mammal depends critically on erythropoietin, an inducible cytokine with pluripotent effects. Erythropoietin gene expression increases under conditions associated with lowered oxygen content such as anemia and hypoxia. HIF-1alpha, the founding member of the hypoxia-inducible factor (HIF) alpha class, was identified by its ability to bind and activate the hypoxia-responsive enhancer in the erythropoietin regulatory region in vitro. The existence of multiple HIF alpha members raises the question of which HIF alpha member or members regulates erythropoietin expression in vivo. We previously reported that mice lacking wild-type HIF-2alpha, encoded by the EPAS1 gene, exhibit pancytopenia. In this study, we have characterized the etiology of this hematopoietic phenotype. Molecular studies of EPAS1-null kidneys reveal dramatically decreased erythropoietin gene expression. EPAS1-null as well as heterozygous mice have impaired renal erythropoietin induction in response to hypoxia. Treatment of EPAS1-null mice with exogenous erythropoietin reverses the hematopoietic and other defects. We propose that HIF-2alpha is an essential regulator of murine erythropoietin production. Impairments in HIF signaling, involving either HIF-1alpha or HIF-2alpha, may play a prominent role in conditions involving altered hematopoietic or erythropoietin homeostasis.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Erythropoietin; Female; Gene Expression; Hematopoiesis; Hypoxia; Kidney; Male; Mice; Mice, Mutant Strains; Pancytopenia; Phenotype; RNA, Messenger; Trans-Activators

Central nervous and hematopoietic systems share developmental features. We report that thrombopoietin (TPO), a stimulator of platelet formation, acts in the brain as a counterpart of erythropoietin (EPO), a hematopoietic growth factor with neuroprotective properties. TPO is most prominent in postnatal brain, whereas EPO is abundant in embryonic brain and decreases postnatally. Upon hypoxia, EPO and its receptor are rapidly reexpressed, whereas neuronal TPO and its receptor are down-regulated. Unexpectedly, TPO is strongly proapoptotic in the brain, causing death of newly generated neurons through the Ras-extracellular signal-regulated kinase 1/2 pathway. This effect is not only inhibited by EPO but also by neurotrophins. We suggest that the proapoptotic function of TPO helps to select for neurons that have acquired target-derived neurotrophic support.

Topics: Animals; Apoptosis; Brain; Brain Chemistry; Erythropoietin; Gene Expression Regulation; Hematopoietic Cell Growth Factors; Hypoxia; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Nerve Growth Factors; Neurons; Prosencephalon; Rhombencephalon; Thrombopoietin

Erythropoietin, an oxygen-regulated glycoprotein hormone, is a hematopoietic cytokine that stimulates erythropoiesis by binding to its cellular receptor [erythropoietin receptor (EPOR)]. The recombinant form of human erythropoietin is used to prevent or treat anemia in cancer patients. However, in a recent randomized, placebo-controlled trial involving patients receiving curative radiotherapy for squamous cell carcinoma of the head and neck, erythropoietin treatment was associated with poorer locoregional progression-free survival. The purpose of our study was to determine whether EPOR and its ligand erythropoietin are expressed in primary head and neck cancer. We also investigated the hypothesis that erythropoietin expression in malignant cells may be associated with the presence of tumor hypoxia, an important factor involved in resistance to radiation treatment, tumor aggressiveness, and poor prognosis.. Twenty-one patients received an i.v. infusion of the hypoxia marker pimonidazole hydrochloride before multiple tumor biopsies. Contiguous sections from 74 biopsies were analyzed by immunohistochemistry for EPOR and erythropoietin expression and pimonidazole binding.. EPOR expression was present in tumor cells in 97% of the biopsies. Coexpression of erythropoietin was observed in 90% of biopsies. Erythropoietin and pimonidazole adduct staining did not always colocalize within tumors, but there was a significant positive correlation between levels of microregional erythropoietin expression and pimonidazole binding.. The coexpression of erythropoietin and EPOR in tumor cells suggests that erythropoietin may potentially function as an autocrine or paracrine factor in head and neck cancer. The expression of the hypoxia-inducible protein erythropoietin in tumor cells correlates with levels of tumor hypoxia.

Topics: Disease-Free Survival; Erythropoietin; Head and Neck Neoplasms; Humans; Hypoxia; Immunohistochemistry; Ligands; Nitroimidazoles; Prognosis; Radiation-Sensitizing Agents; Receptors, Erythropoietin; Recombinant Proteins; Time Factors

We hypothesized that cord blood erythropoietin (EPO), a marker of fetal hypoxia, relates to gestational factors and not solely those associated with delivery. We investigated the association between birth weight SD score (SDS) and cord blood EPO in 290 twins (145 pairs), assessing the influence of gestational versus perinatal factors by comparing the association in those who were delivered by elective cesarean (CS) with that in other delivery modes. Blood EPO values were skewed, so geometric means are presented and log EPO values were used in statistical models. The birth size-EPO association was estimated in mixed-effects models that included terms that represented difference in log EPO and mean log EPO for each twin pair. Within-pair estimates of the association were unconfounded by maternal factors (because these were perfectly controlled). Geometric mean EPO was higher in boys versus girls (24.4 versus 17.0 IU/L; p = 0.0001) and increased with gestational age (p = 0.0003) but was similar after elective CS versus other delivery modes. The negative birth size-EPO association was stronger in infants who were delivered by elective CS than by other delivery modes [beta for log(2) EPO: -0.56 (95% CI, -0.77 to -0.36) versus -0.27 (-0.42 to -0.12), respectively; p = 0.02 for interaction). Because the association was seen after elective CS delivery, cord blood EPO must relate to factors during gestation, not just perinatal factors. There was no evidence of an association between birth weight SDS and pair mean log EPO, indicating that the association is entirely due to fetus-specific rather than pair-specific factors.

Topics: Cesarean Section; Delivery, Obstetric; Erythropoietin; Female; Fetal Blood; Gestational Age; Humans; Hypoxia; Infant, Newborn; Labor, Obstetric; Linear Models; Male; Models, Theoretical; Pregnancy; Twins; Twins, Dizygotic; Twins, Monozygotic

Although the structure and function of erythropoietin (Epo) are well documented, the mechanisms of the regulation of the renal synthesis of Epo are still poorly understood. Especially, the description of the localization and function of the O(2)-sensitive sensor regulating the renal synthesis of Epo is insufficient. A body of evidence suggests that extrarenal O(2)-sensitive sensors, localized particularly in the brain stem, play an important role in this connection. To support this concept, high cerebral pressure with consecutive hypoxia of the brain stem was generated by insufflation of synthetic cerebrospinal fluid into the catheterized cisterna magna of rats. When the cerebral pressure of the rats was above the level of their mean arterial blood pressure or the high cerebral pressure persisted for a longer period (>/=10 min), the Epo plasma concentration increased significantly. Bilateral nephrectomy or hypophysectomy before initiation of high intracranial pressure abolished this effect. Systemic parameters (heart rate, blood pressure, Pa(O(2)), Pa(CO(2)), arterial pH, renal blood flow, glucose concentration in blood) were not affected. Other stressors, like restricting the mobility of the rats, had no effect on Epo production. Hence, the effect of high cerebral pressure on renal synthesis of Epo seems to be specific. Increasing cerebral hydrostatic pressure leads to increased renal synthesis of Epo. Obviously, during hypoxia, cerebral O(2)-sensitive sensors release humoral factors, triggering the renal synthesis of Epo. The structure and function of these "Epo-releasing-factors" will have to be characterized in future experiments.

Topics: Animals; Brain Stem; Erythropoietin; Homeostasis; Hypophysectomy; Hypoxia; Intracranial Pressure; Kidney; Male; Models, Biological; Oxygen; Rats; Rats, Sprague-Dawley

Inter-individual variations in sea level performance after altitude training have been attributed, at least in part, to an inter-individual variability in hypoxia induced erythropoiesis. The aim of the present study was to examine whether the variability in the increase in total haemoglobin mass after training at moderate altitude could be predicted by the erythropoietin response after 4 h exposure to normobaric hypoxia at an ambient Po(2) corresponding to the training altitude.. Erythropoietin levels were measured in 16 elite junior swimmers before and after 4 h exposure to normobaric hypoxia (Fio(2) 0.15, approximately 2500 m) as well as repeatedly during 3 week altitude training (2100-2300 m). Before and after the altitude training, total haemoglobin mass (CO rebreathing) and performance in a stepwise increasing swimming test were determined.. The erythropoietin increase (10-185%) after 4 h exposure to normobaric hypoxia showed considerable inter-individual variation and was significantly (p<0.001) correlated with the acute erythropoietin increase during altitude training but not with the change in total haemoglobin mass (significant increase of approximately 6% on average). The change in sea level performance after altitude training was not related to the change in total haemoglobin mass.. The results of the present prospective study confirmed the wide inter-individual variability in erythropoietic response to altitude training in elite athletes. However, their erythropoietin response to acute altitude exposure might not identify those athletes who respond to altitude training with an increase in total haemoglobin mass.

Topics: Adolescent; Altitude; Erythrocyte Volume; Erythropoiesis; Erythropoietin; Exercise Test; Female; Hemoglobins; Humans; Hypoxia; Male; Prospective Studies; Swimming

Although erythropoietin (Epo) is a known stimulator of erythropoiesis, recent evidence suggests that its biological functions are not confined to hematopoietic cells. To elucidate the role of Epo and erythropoietin receptor (EpoR) in melanoma, we examined the expression and function of these proteins in melanocytes and melanoma cells. We found increased expression of Epo in melanoma cells compared to melanocyte in vitro. EpoR was also strongly expressed in all of the melanoma cell lines and two of the three melanocyte cell lines examined. Epo expression was significantly higher in melanoma than in benign nevi as determined by immunohistochemistry. Although melanoma cells secreted Epo in normoxic condition in vitro, hypoxia and CoCl(2) treatment increased Epo secretion. EpoR in melanoma cells was functional, because exogenous Epo increased melanoma resistance to hypoxic stress, pretreatment of melanoma cells with Epo significantly increased resistance to dacarbazine treatment, and Epo increased the phosphorylation of EpoR, RAF, and MEK. In conclusion, we demonstrated constitutive expression of Epo and EpoR as well as autonomous secretion of Epo by melanoma cells, indicating a novel autocrine loop of Epo in melanoma. The results suggest that the autocrine and paracrine functions of Epo might play a role in malignant transformation of melanocytes and in the survival of melanoma cells in hypoxia and other adverse conditions.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Cobalt; Coloring Agents; Culture Media, Conditioned; Disease Progression; Dose-Response Relationship, Drug; Erythropoietin; Humans; Hypoxia; Immunohistochemistry; MAP Kinase Signaling System; Melanocytes; Melanoma; Membrane Potentials; Mitochondria; Phosphorylation; Receptors, Erythropoietin; Tetrazolium Salts; Thiazoles; Trypan Blue

The cytokine erythropoietin (Epo) is an essential factor promoting the survival, proliferation, and differentiation of erythroid progenitor cells. Epo expression and the initial phase of definitive erythropoietic differentiation in the fetal liver (E9-E12) are compromised in mouse embryos lacking the retinoic acid receptor RXRalpha. Our previous work demonstrated that the Epo gene is a direct target of retinoic acid action, via a retinoic acid receptor binding site in the Epo gene enhancer. However, Epo expression and erythropoietic differentiation become normalized in RXRalpha mutants from E12. In this study, we have investigated the molecular mechanisms underlying the transition in Epo gene regulation from RXRalpha-dependence to RXRalpha-independence. We find that three independent regulatory components are required for high level Epo expression in the early fetal liver: ligand-activated retinoic acid receptors, the hypoxia-regulated factor HIF1, and GATA factors. By E11.5, the fetal liver is no longer hypoxic, and retinoic acid signaling is no longer active; Epo expression from E11.5 onward is enhancer-independent, and is driven instead by basal promoter elements that provide a sufficient level of expression to support further erythropoietic differentiation.

Topics: Aldehyde Oxidoreductases; Animals; Cell Differentiation; Cells, Cultured; Cytochrome P-450 Enzyme System; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; Erythropoiesis; Erythropoietin; Female; Fetus; Gene Expression Regulation, Developmental; Gestational Age; Hepatocyte Nuclear Factor 4; Hepatocytes; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Liver; Mice; Mice, Knockout; Nuclear Proteins; Phosphoproteins; Pregnancy; Protein Isoforms; Retinal Dehydrogenase; Retinoic Acid 4-Hydroxylase; Retinoid X Receptor alpha; Signal Transduction; Transcription Factors; Tretinoin

Topics: Anemia; Animals; Disease Models, Animal; Erythropoietin; Humans; Hypoxia; Kidney; Kidney Failure, Chronic; Neovascularization, Physiologic; Rats; Recombinant Proteins

In this study we examined the effects of intermittent hypoxia exposure (IHE) in a group of professional cyclists (n = 6; age 26 +/- 1 yr) competing in the 2001 Vuelta a España. After each daily stage, treated subjects received four 5-min bouts of normobaric IHE (mean O2 concentration of 12.6%, simulating a mean altitude of 4,000 m) interspersed with 5-min bouts of breathing hotel room air (normoxia) until completing a total IHE of 20-min duration. The primary outcome, compared to a control group of similar characteristics not receiving IHE (n = 5; age 25 +/- 1 yr), was the % increase in erythropoietin (Epo) from the beginning to the end of the Vuelta. Statistical analysis showed that Epo increase tended to be higher (p = 0.052) in the IHE group than in controls (37.4 +/- 5.8% vs. -4.4 +/- 19.5%, respectively). However IHE had no effect on reticulocytes or erythrocyte count (p > 0.05).

Topics: Adult; Altitude; Anaerobic Threshold; Bicycling; Erythrocyte Count; Erythropoiesis; Erythropoietin; Ferritins; Heart Rate; Hematocrit; Hemoglobins; Humans; Hypoxia; Nutritional Physiological Phenomena; Oxygen Consumption; Physical Endurance; Reticulocytes; Time Factors

Pulmonary oxygen toxicity is believed to play a prominent role in the lung injury that leads to the development of bronchopulmonary dysplasia (BPD). To determine whether human recombinant erythropoietin (rhEPO) treatment reduces the risk of developing BPD, we investigated the effect of rhEPO treatment on the histopathologic changes seen in hyperoxia-induced lung injury of BPD. Twenty-five rat pups were divided into four groups: air-exposed control group (n = 5), hyperoxia-exposed placebo group (n = 7), hyperoxia-exposed rhEPO-treated group (n = 6), and air-exposed rhEPO-treated group (n = 7). Measurement of alveolar surface area, quantification of secondary crest formation, microvessel count, evaluation of alveolar septal fibrosis, and smooth muscle actin immunostaining were performed to assess hyperoxia-induced changes in lung morphology. Treatment of hyperoxia-exposed animals with rhEPO resulted in a significant increase in the mean alveolar area, number of secondary crests formed, and the microvessel count in comparison with hyperoxia-exposed placebo-treated animals. There was significantly less fibrosis in rhEPO-treated animals. However, treatment of hyperoxia-exposed animals with rhEPO did not result in a significant change in smooth muscle content compared with hyperoxia-exposed placebo treated animals. Our results suggest treatment with rhEPO during hyperoxia exposure is associated with improved alveolar structure, enhanced vascularity, and decreased fibrosis. Therefore, we conclude that treatment of preterm infants with EPO might reduce the risk of developing BPD.

Topics: Animals; Animals, Newborn; Body Weight; Erythropoietin; Fibrosis; Hypoxia; Immunohistochemistry; Lung; Lung Injury; Microcirculation; Neovascularization, Pathologic; Oxygen; Pulmonary Alveoli; Rats; Rats, Wistar; Recombinant Proteins

This study was performed to investigate the effects of intermittent hypoxic exposure on blood and exercise parameters. Eight sea level residents were exposed to 2 h daily stimulus to 4100 m altitude in a hypobaric chamber for a total of 14 days. Exercise performance was evaluated at sea level before and after the hypoxic stimulation. Blood samples were obtained before, during, and at time points up to 14 days after the hypoxic exposure. No changes were observed in haemoglobin, haematocrit, reticulocytes, serum transferrin receptors, or EPO levels in the blood. Submaximal cycle (150 W) ergometer exercise corresponded to a oxygen uptake of 1.9+/-0.1 and 1.9+/-0.1 L min(-1) before and after the intermittent altitude exposure, respectively. At maximal exercise the workloads attained were 343+/-17 and 354+/-27 W before and after the exposure, with corresponding oxygen uptakes of 4.0+/-0.2 and 4.2+/-0.2 L min(-1). It is concluded that intermittent hypoxic exposure to 4100 m altitude for 2 h daily and a total of 14 days does not affect exercise capacity.

Topics: Adult; Altitude; Blood Chemical Analysis; Erythrocyte Count; Erythropoietin; Exercise; Ferritins; Hemoglobins; Humans; Hypoxia; Male; Oxygen Consumption; Pulmonary Gas Exchange

Hypoxic preconditioning is an endogenous protection against subsequent lethal hypoxia, but the mechanism involved is not understood. Hypoxia is followed by reactive oxygen species (ROS) production and induces hypoxia-inducible factor (HIF) and its downstream factor erythropoietin (Epo), which is associated with neuroprotection. We hypothesized that these endogenous processes may contribute to hypoxic preconditioning.. We used a mouse neuronal culture model, with 2 hours of hypoxia as preconditioning followed by 15 hours of hypoxic insult, and examined the expression of HIF-1alpha, Epo, and their downstream proteins by Western blotting. Copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) mice were used to detect the effect of ROS. Cell survival and apoptosis were detected by mitogen-activated protein 2 quantification, apoptotic-related DNA fragmentation, and caspase-3 fragmentation. Antisense Epo was used to block endogenously produced Epo.. Hypoxic preconditioning was protective in wild-type (Wt) neurons but not in neurons obtained from SOD1 Tg mice. In Wt neurons, HIF-1alpha and Epo expression showed a greater increase after hypoxia compared with Tg neurons and reached a higher level with preconditioned hypoxia, followed by pJak2, pStat5, and nuclear factor kappaB (NF-kappaB) expression. Antisense Epo decreased these downstream proteins and the neuroprotection of hypoxic preconditioning.. Hypoxic preconditioning induces ROS, which may downregulate the threshold for production of HIF-1alpha and Epo expression during subsequent lethal hypoxia, thus exerting neuroprotection through the Jak2-Stat5 and NF-kappaB pathways.

Topics: Animals; Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Survival; Cells, Cultured; DNA Fragmentation; Erythropoietin; Heterozygote; Hypoxia; Hypoxia-Inducible Factor 1; Ischemic Preconditioning; Janus Kinase 2; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Neurons; Neuroprotective Agents; NF-kappa B; Oligonucleotides, Antisense; Oxidative Stress; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Reactive Oxygen Species; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; STAT5 Transcription Factor; Superoxide Dismutase

Ten-day-old rat pups (P10) subjected to acute hypoxia (down to 4% O2) had as adults increased aggression (handling test), memory impairment (water maze test), and decreased CA1 cell counts. Pups subjected to chronic hypoxia (10% O2 from P0 to P21) had increased aggression, hyperactivity (open-field test), and decreased CA1 cell counts. Chronic hypoxia with superimposed acute hypoxia resulted in consequences that were not different from those of chronic hypoxia.

Topics: Analysis of Variance; Animals; Animals, Newborn; Behavior, Animal; Cell Count; Cell Death; Chronic Disease; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Exploratory Behavior; Hippocampus; Hypoxia; In Situ Nick-End Labeling; Maze Learning; Rats; Rats, Sprague-Dawley; Time; Time Factors

Erythropoietin (Epo) is used for managing anemia in cancer patients. However, recent studies have raised concerns for this practice. We investigated the expression and function of Epo and the erythropoietin receptor (EpoR) in tumor biopsies and cell lines from human head and neck cancer. Epo responsiveness of the cell lines was assessed by Epoetin-alpha-induced tyrosine phosphorylation of the Janus kinase 2 (JAK2) protein kinase. Transmigration assays across Matrigel-coated filters were used to examine the effects of Epoetin-alpha on cell invasiveness. In 32 biopsies, we observed a significant association between disease progression and expression of Epo and its receptor, EpoR. Expression was highest in malignant cells, particularly within hypoxic and infiltrating tumor regions. Although both Epo and EpoR were expressed in human head and neck carcinoma cell lines, only EpoR was upregulated by hypoxia. Epoetin-alpha treatment induced prominent JAK2 phosphorylation and enhanced cell invasion. Inhibition of JAK2 phosphorylation reduced both basal and Epo-induced invasiveness. Our findings support a role for autocrine or paracrine Epo signaling in the malignant progression and local invasiveness of head and neck cancer. This mechanism may also be activated by recombinant Epo therapy and could potentially produce detrimental effects in rhEpo-treated cancer patients.

Topics: Biopsy; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Erythropoietin; Gene Expression Regulation; Head and Neck Neoplasms; Humans; Hypoxia; Immunohistochemistry; Immunoprecipitation; Janus Kinase 2; Laminin; Models, Statistical; Neoplasm Invasiveness; Phosphorylation; Protein-Tyrosine Kinases; Proteoglycans; Proto-Oncogene Proteins; Receptors, Erythropoietin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tyrosine

We studied the dynamics of erythropoietin content and erythropoietic activity of the serum during hypoxia of different genesis and severity. Our results show that humoral factors play an important role in the regulation of erythropoiesis during oxygen deficiency. Serum erythropoietic activity underwent similar changes in different types of hypoxia associated with similar hematological shifts. A discrepancy was observed between erythropoietic activity and serum erythropoietin concentration.

Topics: Adaptation, Physiological; Animals; Erythropoiesis; Erythropoietin; Hypoxia; Mice; Mice, Inbred CBA

Erythropoietin (EPO), a hematopoietic growth factor, has been shown to be neuroprotective when administered as either a pretreatment or posttreatment. This study tested the hypothesis that one of the mechanisms of protection afforded by posttreatment with recombinant human EPO (rh-EPO) is an anti-inflammatory effect via inhibition of interleukin (IL)-1beta.. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 90 minutes of hypoxia (8% O2 at 37 degrees C). Pups were divided into the following groups: control, hypoxia/ischemia, and hypoxia/ischemia plus rh-EPO. In the rh-EPO-treated pups, rh-EPO (5 U/g body weight IP) was administered starting 24 hours after the insult and then for 2 additional days. Samples were collected at 3, 7, 14, and 21 days after the insult. IL-1beta mRNA and protein levels were determined by quantitative real-time reverse transcription-polymerase chain reaction and ELISA. Tumor necrosis factor (TNF)-alpha mRNA levels were determined by colorimetric microplate assay.. rhEPO attenuated brain injury, as assessed by brain weight, and attenuated both the hypoxia/ischemia-induced increases in IL-1beta mRNA and protein levels. TNF-alpha mRNA levels did not increase at 3 to 14 days after the hypoxic/ischemic insult.. Administration of exogenous rh-EPO starting 24 hours after a hypoxic/ischemic insult is neuroprotective in the neonatal rat. This neuroprotective activity prevented the secondary, delayed rise in IL-1beta and attenuated the infiltration of leukocytes into the ipsilateral hemisphere.

Topics: Animals; Animals, Newborn; Brain; Carotid Arteries; Erythropoietin; Growth Substances; Humans; Hypoxia; Hypoxia-Ischemia, Brain; Inflammation; Interleukin-1; Ischemia; Leukocytes; Organ Size; Oxygen; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Temperature; Time Factors

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of the cellular hypoxic response. We previously showed that HIF-1 activation is essential for heat acclimation (AC) in Caenorhabditis elegans. Metabolic changes in AC rat hearts indicate HIF-1alpha activation in mammals as well. Here we characterize the HIF-1alpha profile and the transcriptional activation of its target genes following AC and following heat stress (HS) in hearts from nonacclimated (C; 24 degrees C) and AC (34 degrees C, 1 mo) rats. We used Western blot and immunohistochemistry to measure HIF-1alpha levels and EMSA and RT-PCR/quantitative RT-PCR to detect expression of the HIF-1alpha-targeted genes, including vascular endothelial growth factor (Vegf), heme oxygenase-1 (HO1), erythropoietin (Epo), and Epo receptor (EpoR). EpoR and Epo mRNA levels were measured to determine systemic effects in the kidneys and cross-tolerance effects in C and AC ischemic hearts (Langendorff, 75% ischemia, 40 min). The results demonstrated that 1) after AC, HIF-1alpha protein levels were increased, 2) HS alone induced transient HIF-1alpha upregulation, and 3) VEGF and HO1 mRNA levels increased after HS, with greater magnitude in the AC hearts. Epo mRNA in AC kidneys and EpoR mRNA in AC hearts were also elevated. In AC hearts, EpoR expression was markedly higher after HS or ischemia. Hearts from AC rats were dramatically protected against infarction after ischemia-perfusion. We conclude that HIF-1 contributes to the acclimation-ischemia cross-tolerance mechanism in the heart by induction of both chronic and inducible adaptive components.

Topics: Animals; Blotting, Western; Body Temperature; Caenorhabditis elegans; Cell Nucleus; Cytosol; Erythropoietin; Heart; Heme Oxygenase-1; Hot Temperature; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Immunoprecipitation; Ischemia; Kidney; Male; Microscopy, Confocal; Microscopy, Fluorescence; Models, Statistical; Myocardium; Rats; Receptors, Erythropoietin; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Subcellular Fractions; Temperature; Time Factors; Transcription, Genetic; Transcriptional Activation; Up-Regulation; Vascular Endothelial Growth Factor A

Apart from its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions in brain, retina and heart upon ischaemic injury. However, the physiological non-erythroid functions of Epo remain unclear. Here we use a transgenic mouse line (Tg21) constitutively overexpressing human Epo in brain to investigate Epo's impact on ventilation upon hypoxic exposure. Tg21 mice showed improved ventilatory response to severe acute hypoxia and moreover improved ventilatory acclimatization to chronic hypoxic exposure. Furthermore, following bilateral transection of carotid sinus nerves that uncouples the brain from the carotid body, Tg21 mice adapted their ventilation to acute severe hypoxia while chemodenervated wild-type (WT) animals developed a life-threatening apnoea. These results imply that Epo in brain modulates ventilation. Additional analysis revealed that the Epo receptor (EpoR) is expressed in the main brainstem respiratory centres and suggested that Epo stimulates breathing control by alteration of catecholaminergic metabolism in brainstem. The modulation of hypoxic pattern of ventilation after i.v. injection of recombinant human Epo in WT mice and the dense EpoR immunosignal observed in carotid bodies showed that these chemoreceptors are sensitive to plasma levels of Epo. In summary, our results suggest that Epo controls ventilation at the central (brainstem) and peripheral (carotid body) levels. These novel findings are relevant to understanding better respiratory disorders including those occurring at high altitude.

Topics: Adaptation, Physiological; Animals; Atmosphere Exposure Chambers; Carotid Arteries; Carotid Body; Denervation; Erythropoietin; Hypoxia; Immunohistochemistry; Mice; Mice, Transgenic; Norepinephrine; Oxygen Consumption; Receptors, Erythropoietin; Receptors, Neurokinin-1; Recombinant Proteins; Respiration; Respiratory Center; Tyrosine 3-Monooxygenase

Chronic pulmonary disease and progressive tissue hypoxia are major causes of morbidity and mortality in cystic fibrosis (CF). Normally the body adapts to tissue hypoxia by increasing the red cell mass and decreasing the Hb-O(2) affinity. These adaptations are commonly observed in patients with cyanotic heart disease and individuals living at high altitude. However, patients with CF not only have an impaired erythroid response to hypoxia, but also are frequently anaemic.. In order to evaluate erythroid marrow activity and tissue oxygenation in 37 patients with CF we measured: the haematological and blood chemistry parameters; including red cell indices, ferritin, erythropoietin (Epo) and soluble transferrin receptors (sTfR) levels; arterial blood gases, P(50) and oxygen release to the tissues (O(2)(R)) and the 2,3-BPG levels.. The main results showed that a) patients with CF have a mild degree of tissue hypoxia which is expressed by the moderately decreased of P(50) and O(2)(R) values and the relative increase of Epo level, b) 2,3-BPG synthesis in patients with CF is normal and c) sTfR levels are significantly increased (3-fold normal) in patients with CF compared to normal controls.. The above observations indicate that erythroid marrow activity in patients with CF is increased.

Topics: Adolescent; Adult; Biomarkers; Blood Gas Analysis; Child; Child, Preschool; Cystic Fibrosis; Erythrocytes; Erythropoietin; Female; Ferritins; Humans; Hypoxia; Infant; Male; Oxygen Consumption; Receptors, Transferrin; Severity of Illness Index

To examine the utility of red blood cell (RBC) zinc protoporphyrin/heme ratio (ZnPP/H) as an indicator of fetal iron status, because unfavorable neurodevelopmental outcomes have been associated with poor iron status at birth, as indicated by low serum ferritin, and because few reliable indicators of fetal and early neonatal iron status exist.. Consecutively studied preterm and term fetuses at delivery included the following groups: (1) control nonhypoxic, (2) fetuses with intrauterine growth retardation (IUGR), and (3) fetuses of insulin-treated mothers (FDM). We hypothesized (1) that rapid growth velocity associated with an accelerated erythropoiesis among normal fetuses will lead to reduced iron delivery to a rapidly expanding RBC mass and higher umbilical cord blood RBC ZnPP/H and (2) that fetuses that are exposed to pathologic hypoxemia will experience an additional increase in erythropoiesis and higher cord ZnPP/H. ZnPP/H was determined on saline-washed cord blood erythrocytes by hematofluorometry and was examined for its relationship with clinical factors and cord blood laboratory measurements indicative of tissue oxygenation (plasma erythropoietin [EPO] and reticulocyte count) and iron status (plasma ferritin and erythrocyte indices). Statistical testing included 1-way analysis of variance, 2-way analysis of variance with covariates, simple linear regression, and multiple regression analysis.. Among control group subjects, gestational age at birth was inversely correlated with RBC ZnPP/H and reticulocyte count and positively correlated with ferritin and EPO. Relative to control subjects, IUGR and FDM fetuses at specified gestational age groupings had higher ZnPP/H, lower plasma ferritin, and higher plasma EPO. Statistical modeling of the relationship between ZnPP/H and plasma ferritin among all study groups demonstrated significant impacts of gestational age, plasma EPO, maternal hypertension, and maternal smoking.. The inverse association of fetal ZnPP/H with gestational age at birth among control subjects is attributable to erythropoietic stimulation likely as a result of increasing growth velocity at the earliest gestational ages. The relatively higher ZnPP/H observed among fetuses in the IUGR and FDM groups likely is attributable to increased erythropoietic activity secondary to pathologic hypoxemia. Decreased placental iron transfer may also have limited iron availability and contributed to elevated ZnPP/H in the IUGR group. These data support the concept that increased erythropoietic activity and/or limited iron transport may place infants of diabetic mothers and infants with growth retardation at risk for developing systemic iron deficiency later in infancy and in early childhood.

Topics: Erythrocyte Indices; Erythrocytes; Erythropoietin; Female; Ferritins; Fetal Blood; Fetal Diseases; Fetal Growth Retardation; Gestational Age; Heme; Humans; Hypoxia; Infant, Newborn; Infant, Premature; Insulin; Pregnancy; Pregnancy in Diabetics; Protoporphyrins; Reticulocyte Count

Erythropoietin (Epo) is the primary regulator of erythropoiesis, controlling the proliferation, maturation, and survival of erythroid progenitor cells. The functions of Epo are mediated through its specific receptor (EpoR) expressed mainly on the surface of erythroid progenitor cells, and the expression of both responds to hypoxia. The subterranean mole rat (Spalax) is a unique model system to study the molecular mechanisms for adaptation to hypoxia. Here, we cloned two forms of Spalax EpoR: a complete EpoR cDNA as well as a novel truncated bone marrow specific EpoR form. In the full-length Spalax EpoR (sEpoR), two out of the eight conserved tyrosine- phosphorylation sites were substituted (Y481F and Y499G), suggesting that Spalax Epo signaling pathways may be modulated. The level of the sEpoR mRNA in the spleen and in bone marrow was relatively low and similar in Spalax newborns and adults, with no significant response to hypoxia. The truncated sEpoR was not detected in the spleen and comprised only approximately 1% of the sEpoR expressed in the bone marrow. In Rattus, the truncated EpoR form was approximately 15% of the total expressed receptor. The level of Rattus EpoR in newborn spleens was three- to fourfold higher than in Spalax newborns and decreased toward adulthood. Severe hypoxia induces a significant increase in adult Rattus EpoR. Our data provide further insight into the adaptive mechanisms of Spalax to the extreme conditions of hypoxia in its subterranean environment.

Topics: Alternative Splicing; Amino Acid Sequence; Animals; Bone Marrow; Cloning, Molecular; DNA, Complementary; Erythropoietin; Gene Expression Regulation; Hypoxia; Mole Rats; Molecular Sequence Data; Oxygen; Phosphorylation; Plasmids; Rats; Receptors, Erythropoietin; RNA; RNA, Messenger; Sequence Homology, Amino Acid; Signal Transduction; Species Specificity; Spleen; Tyrosine

Topics: Diabetic Retinopathy; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Humans; Hypoxia; Vascular Endothelial Growth Factor A; Vitreous Body

Apart from its hematopoietic function, erythropoietin (Epo) exerts neuroprotective functions in brain hypoxia and ischemia. To examine the mechanisms mediating Epo's neuroprotective activity in vivo, we made use of our transgenic mouse line tg21 that constitutively expresses human Epo in brain without inducing excessive erythrocytosis. We show that human Epo is expressed in tg21 brains and that cortical and striatal neurons carry the Epo receptor. After middle cerebral artery occlusion, human Epo potently protected brains of tg21 mice against ischemic injury, both when severe (90 min) and mild (30 min) ischemia was imposed. Histochemical studies revealed that Epo induced an activation of JAK-2, ERK-1/-2, and Akt pathways in the ischemic brain. This activation was associated with elevated Bcl-XL and decreased NO synthase-1 and -2 levels in neurons. Intracerebroventricular injections of selective inhibitors of ERK-1/-2 (PD98059) or Akt (wortmannin) pathways revealed that both ERK-1/-2 and Akt were required for Epo's neuroprotective function, antagonization of either pathway completely abolishing tissue protection. On the other hand, ERK-1/-2 and Akt blockade did not reverse the neuronal NO synthase-1/-2 inhibition, indicating that Epo down-regulates these NO synthases in an ERK-1/-2 and Akt independent manner. On the basis of our data, the dual activation of ERK-1/-2 and Akt is crucial for Epo's neuroprotective activity.

Topics: Androstadienes; Animals; bcl-X Protein; Brain; Brain Ischemia; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Flavonoids; Humans; Hypoxia; Immunohistochemistry; Ischemia; Janus Kinase 2; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Biological; Neurons; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Erythropoietin; Signal Transduction; Time Factors; Wortmannin

Anemia is a major secondary symptom in chronic renal disorder (CRD), but the precise cause of insufficient production of erythropoietin (EPO) remains unclear owing to the controversial localization of EPO-producing cells in the kidneys. The ICR-derived glomerulonephritis (ICGN) mouse, a new hereditary nephrotic mouse, is an appropriate model of anemia associated with CRD. By using an amplified in situ hybridization technique, we detected and counted the renal EPO-producing cells under both normoxic and hypoxic conditions. The expression levels of renal EPO mRNA were quantified and oxygen gradients were also assessed immunohistochemically. Amplified in situ hybridization clarified that EPO-producing cells were peritubular interstitial cells in the middle region of renal cortex in both ICR and ICGN mice. Hypoxia (7% O2) induced low oxygen tension in proximal tubular epithelial cells of renal cortex, and increased the expression of EPO mRNA and the number of EPO-producing cells in both ICR and ICGN mice. However, hypoxia did not increase the serum EPO levels in ICGN mice. The ICGN mouse is a good model for anemia associated with CRD, and the suppression of EPO protein production in the renal EPO-producing cells is considered to be a potential cause of anemia associated with CRD.

Topics: Analysis of Variance; Anemia; Animals; Erythropoietin; Hypoxia; Immunohistochemistry; In Situ Hybridization; Kidney; Kidney Failure, Chronic; Mice; Mice, Inbred ICR; RNA, Messenger

Anemia of patients with malignancy might have various reasons. No matter if its background is the underlying tumorous disease or chemo- and/or radiotherapy, it can cause fatigue, malaise, it certainly decreases the patients' quality of life and, furthermore, shortens their survival. Chronic hypoxia caused by anemia promotes tumor progression by several mechanisms e.g. by enhancing angioneogenesis by the production of VEGF. At the same time it decreases the efficacy of chemo- and radiotherapy. Therefore, prevention and/or correction of chemo/radiotherapy-induced anemia is a major goal of modern oncotherapy.

Topics: Anemia; Antineoplastic Agents; Chemotherapy, Adjuvant; Erythropoietin; Hematinics; Humans; Hypoxia; Neoplasms; Radiotherapy, Adjuvant; Survival Rate

Acute alveolar hypoxia causes pulmonary vasoconstriction that matches lung perfusion to ventilation to optimize gas exchange. Chronic alveolar hypoxia induces pulmonary hypertension, characterized by increased muscularization of the pulmonary vasculature and right ventricular hypertrophy. Elevated erythropoietin (EPO) plasma levels increase hematocrit and blood viscosity and may affect structure and function of the pulmonary circulation. To differentiate between the direct effects of hypoxia and those linked to a hypoxia-induced increase in EPO/hematocrit levels, we investigated the lung vasculature in transgenic mice constitutively over-expressing EPO (termed tg6) upon exposure to normoxia and chronic hypoxia. Despite increased hematocrit levels (approximately 0.86),tg6 mice kept in normoxia did not develop selective right ventricular hypertrophy. The portion of vessels with a diameter of 51-95 microm and >155 microm was increased whereas the portion of small vessels (30-50 microm) was decreased. Pulmonary vascular resistance and the strength of hypoxic vasoconstriction measured in isolated perfused lungs were decreased. Vasoconstrictions induced by the thromboxane mimetic U46619 tended to be reduced. After chronic hypoxia (FiO2 = 0.10, 21 days), vascular resistance and vasoconstrictor responses to acute hypoxia and U46619 were reduced in tg6 mice compared to wildtype controls. Chronic hypoxia increased the degree of pulmonary vascular muscularization in wildtype but not in tg6 mice that already exhibited less muscularization in normoxia. In conclusion, congenital over-expression of EPO exerts an "anti-pulmonary hypertensive" effect, both structurally and functionally, particularly obvious upon chronic hypoxia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aspirin; Blood Pressure; Blood Vessels; Blood Viscosity; Cyclooxygenase Inhibitors; Erythropoietin; Hematocrit; Hypertension, Pulmonary; Hypertrophy, Left Ventricular; Hypertrophy, Right Ventricular; Hypoxia; Lung; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Smooth, Vascular; Nitric Oxide Synthase; omega-N-Methylarginine; Up-Regulation; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents

The use of erythropoietin in head and neck squamous cell carcinoma (HNSCC) has been associated with poor survival. This study examines the protein and mRNA expression of erythropoietin and erythropoietin receptor in HNSCC and their relation to hypoxia, hemoglobin (Hb), and clinical outcome.. The immunohistochemical expression of erythropoietin and erythropoietin receptor was assessed in 151 cases of HNSCC. Expression was compared with the hypoxia-dependent proteins hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase-9 (CA-9) and correlated with clinical outcome. The mRNA expression of erythropoietin and erythropoietin receptor was measured in paired samples of HNSCC.. Erythropoietin and erythropoietin receptor were expressed in 95% and 99% of tumors, respectively. Using a weighed expression score, there was a positive correlation between erythropoietin and erythropoietin receptor expression (r = 0.18, P = 0.03). HIF-1alpha (r = 0.38, P < 0.01) and CA-9 (r = 0.26, P = 0.002) correlated with erythropoietin expression, but there was no correlation with erythropoietin receptor. No correlation was found between Hb and erythropoietin (r = 0.07, P = 0.36) or erythropoietin receptor (r = -0.02, P = 0.8), and no survival difference between high and low erythropoietin or erythropoietin receptor expression (P = 0.59 and P = 0.98, respectively). The mRNA expression of erythropoietin (P = 0.03) but not erythropoietin receptor (P = 0.62) was significantly increased in 11 paired samples of HNSCC.. In vivo, the HIF pathway regulates erythropoietin at the mRNA level but not erythropoietin receptor expression in HNSCC. Anemia does not seem to influence the hypoxic microenvironment of tumors sufficiently to alter the expression of erythropoietin. The effects of exogenous erythropoietin may be acting via receptors expressed on tumor cells in vivo, or on vascular cells, which also express the pathway.

Topics: Anemia; Antigens, Neoplasm; Carbonic Anhydrase IX; Carbonic Anhydrases; Carcinoma, Squamous Cell; Cytoplasm; Disease-Free Survival; DNA, Complementary; Erythropoietin; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Hemoglobins; Humans; Hypoxia; Immunohistochemistry; Male; Protein Array Analysis; Receptors, Erythropoietin; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Transcription, Genetic; Treatment Outcome

To identify anti-hypoxia ingredients extracted from Portulaca oleracea and to find out the possible mechanism of its anti-hypoxia actions.. Seventy mice were randomly divided into seven groups which were untreated (normal saline), ginsenosides-treated, polysaccharide-treated, acidic components-treated, basic components-treated, alkaloids-treated and flavones-treated groups, and the ingredients of polysaccharide, acidic components, basic components, alkaloids and flavones were extracted from Portulaca olerace. The mice in each group were fed with corresponding ingredients for one week respectively. Then the survival time of mice in hypoxic conditions was observed. Another 90 mice were divided into 3 groups: untreated (normal saline), ginsenosides-treated and flavones-treated groups. The mice in each of these 3 groups were divided into 3 subgroups according to 12-, 24- and 36-hour exposure to hypoxia (10% oxygen and 90% nitrogen), respectively. After exposure to hypoxia, the red blood cell count (RBC), hemoglobin (Hb) concentration and hematocrit (HCT) in mice were determined. The plasma erythropoietin (EPO) levels of mice were detected by enzyme-linked immunosorbent assay (ELISA) and the relative values of EPO mRNA in renal tissue and pallium of mice were determined by reverse transcriptase-polymerase chain reaction (RT-PCR).. The survival time of mice in hypoxic conditions in flavones-treated group was significantly longer than that in the untreated group. The RBC, Hb concentration, HCT, plasma EPO level and the relative values of EPO mRNA in renal tissue and pallium of mice were significantly higher in the flavones-treated group than those in the untreated group.. The anti-hypoxia ingredients extracted from Portulaca oleracea are flavones and the anti-hypoxia effects may be obtained by improving the expression level of EPO and accelerating the generations of erythrocyte and Hb.

Topics: Animals; Erythrocytes; Erythropoietin; Flavones; Hematocrit; Hemoglobins; Hypoxia; Male; Mice; Mice, Inbred ICR; Portulaca; Random Allocation

We previously demonstrated that chronic hypoxia has pivotal roles in the progression of tubulointerstitial injury from the early stage of the uninephrectomized Thy1 nephritis model. We have also shown that pretreatment of cobalt confers renoprotection in the ischemia/reperfusion (I/R) injury, in association with the up-regulation of hypoxia-inducible factor (HIF)-regulated genes. Here, we tested the hypothesis that cobalt administration not only attenuates acute ischemic insult, but also ameliorates tubulointerstitial injury secondary to chronic hypoxia.. We applied sustained cobalt treatment to the uninephrectomized Thy1 nephritis model at 3 to 5 weeks, when tubular hypoxia appeared. Histologic evaluation, including glomerular and peritubular capillary networks, was made at 8 weeks. HIF activation was confirmed by real-time polymerase chain reaction (PCR) analyses for HIF-regulated genes, such as erythropoietin (EPO), vascular endothelial growth factor (VEGF), and heme oxygenase 1 (HO-1). Up-regulation of HIF-1alpha and HIF-regulated genes was also verified by Western blotting analysis. To elucidate responsible mechanisms of cobalt in the amelioration of tubuloniterstitial injury, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining was conducted at 5 weeks. A combination therapy with angiotensin receptor blocker (ARB), olmesartan, was also challenged.. Although the intervention did not change glomerular structural damage or urinary protein excretion rate, tubulointerstitial injury was improved in cobalt-treated animals when compared with the vehicle-treated group. The amelioration was associated with the parallel up-regulation of renoprotective, HIF-regulated gene expression. TUNEL staining revealed that the number of apoptotic cells was reduced in the cortex by cobalt administration, suggesting that renoprotection was achieved partly through its antiapoptotic properties. Furthermore, it was demonstrated that cobalt treatment exerts additional renoprotective effects with the ARB treatment in this model.. Maneuvers to activate HIF in the ischemic tubulointerstitium will be a new direction to future therapeutic strategies.

Topics: Animals; Antibodies; Antimutagenic Agents; Apoptosis; Capillaries; Cobalt; Erythropoietin; Gene Expression Regulation; Glomerulonephritis; Glomerulosclerosis, Focal Segmental; Heme Oxygenase-1; Hypoxia; Hypoxia-Inducible Factor 1; In Situ Nick-End Labeling; Isoantibodies; Kidney Tubules; Male; Nephrectomy; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System; Reperfusion Injury; Vascular Endothelial Growth Factor A

In order to determine the initial values and dynamic changes of EPO (erythropoietin) after therapy, 57 consecutively presenting, typical COPD (chronic obstructive pulmonary disease) patients with chronic hypoxia and acute exacerbated serum EPO levels were serially measured. Initial mean EPO levels were slightly above the normal range (41.4 +/- 83.5 units/l), but in the majority of patients the initial EPO levels were significantly reduced. Following the correction of hypoxaemia, mean EPO levels decreased to 14.1 +/- 16.9 units/l (P=0.0093). However, not all COPD patients showed this pattern; in an important subset of patients (36.8%), who had initially lower EPO levels and lower erythrocyte count, EPO levels were significantly increased (by more than 60%; P=0.0028) on the second day of treatment, despite correction of the hypoxaemia. This finding was unexpected and paradoxical when compared with physiological studies addressing the same issue. The data presented support previous reports of variable EPO levels in severely hypoxic COPD patients and suggest that the haematological response is already hampered at an early stage, at the level of EPO production, and much less likely at later steps in the haemopoietic response by failure to respond to elevated EPO levels. Our data are consistent with recent discoveries that the O2 sensing and regulation of EPO production is a complex process in which multiple factors, including cytokines and therapeutic agents, play a role by enhancing or inhibiting the response. We believe that further studies on this clinical condition are complementary to basic physiological research and may help to elucidate the role of cytokines and other individual factors in complex clinical hypoxic situations.

Topics: Acute Disease; Aged; Carbon Dioxide; Erythropoietin; Female; Hemoglobins; Humans; Hypoxia; Male; Middle Aged; Oxygen; Partial Pressure; Prospective Studies; Pulmonary Disease, Chronic Obstructive; Respiratory Insufficiency

Erythropoietin (Epo) exerts neuroprotection against neuronal death induced by ischemia and hypoxia in vitro and in vivo. Recent studies suggest that the neuroprotective effects of Epo may depend upon different mechanisms, including the inhibition of nitric oxide (NO). We recently demonstrated that Epo exerts neuroprotection in a model of neonatal hypoxic-ischemic brain damage. In the present study, we directly determined whether systemic administration of recombinant Epo modulates cerebral NO production in a neonatal rat model of hypoxic-ischemic brain injury. Seven-day-old Wistar rat pups were subjected to left carotid artery occlusion followed by 2.5 h of hypoxic exposure. Brain nitrite levels were evaluated in both hemispheres (carotid ligated or nonligated) by Griess reagent 72 h after the hypoxic-ischemic insult. Our results show that hypoxic-ischemic insult results a significant increase in NO production as compared with NO levels in hypoxic hemispheres and control animals. A single dose of Epo treatment (1,000 U/kg i.p.) significantly decreased NO overproduction in the hypoxic-ischemic hemisphere, whereas no significant change appeared in hypoxia alone or in controls. These data suggest that the selective inhibitory effect of Epo on NO overproduction could have a neuroprotective effect in neonatal hypoxic-ischemic brain injury.

Topics: Animals; Brain Chemistry; Brain Diseases; Carotid Arteries; Disease Models, Animal; Erythropoietin; Hypoxia; Hypoxia-Ischemia, Brain; Ligation; Nitric Oxide; Nitrites; Rats; Rats, Wistar; Recombinant Proteins

Pulmonary vascular remodeling during chronic hypoxia may be the result of either oxygen deprivation or erythrocytosis. To separate experimentally the effects of hypoxia and erythrocytosis, we analyzed transgenic mice that constitutively overexpress the human erythropoietin gene in an oxygen-independent manner. These mice are characterized by polycythemia but have normal blood pressure, heart rate, and cardiac output. In transgenic mice, pulmonary artery pressure (PAP) was increased in vivo but was reduced in blood-free perfused lungs. The thromboxane receptor agonist U46619 caused a smaller rise in PAP in isolated transgenic lungs than in lungs from wild-type mice. The transgenic pulmonary vasculature was characterized by elevated prostacyclin production, stronger endothelial nitric oxide synthase expression, and reduced pulmonary vascular smooth muscle thickness. The fact that transgenic polycythemic mice have marked pulmonary hypertension in vivo but not in vitro suggests that their pulmonary hypertension is due to the increased blood viscosity, thus supporting an independent role of polycythemia in the development of pulmonary hypertension. In addition, our findings indicate that the lungs of transgenic animals adapt to the high PAP by elevated synthesis of vasodilators and reduced vascular smooth muscle thickness that tend to reduce vascular tone and vascular responsiveness.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Analysis of Variance; Animals; Blood Viscosity; Erythropoietin; Hypertension, Pulmonary; Hypoxia; Immunohistochemistry; Lung; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Polycythemia; Pulmonary Artery; Vasoconstrictor Agents

To explore the effects of acute hypoxia and intermittent hypoxic acclimatization on gene expression of erythropoietin (EPO) and hypoxia-inducible factor-1alpha (HIF-1alpha) in rat hepatic and renal tissues.. Twenty-four rats (weight was 180 to 220 g) were divided into three groups: normal control group (NC), intermittent hypoxic acclimatization (IH) and acute hypoxia groups (AH). Gene expression of EPO and HIF-1alpha were examined with Northern dot blot method.. As compared with NC group, the levels of EPO gene expression in rat hepatic and renal tissues in AH group were both significantly elevated (P<0.05 or P<0.01). In IH group, the contents of EPO mRNA in hepatic and renal tissues were apparently decreased versus AH group (P<0.01), whilst were not differently elevated in comparison with group NC (P>0.05). In hepatic tissue from AH group, the content of HIF-1alpha mRNA were more than those in NC group and IH group, while the levels of gene expression of HIF-1alpha were no substantial change in renal tissues from different groups (all P>0.05).. Hypoxic acclimatization can inhibit the increment of EPO gene expression induced by acute hypoxia in rat hepatic and renal tissues, in which HIF-1alpha may play important roles.

Topics: Adaptation, Physiological; Animals; Blotting, Northern; DNA-Binding Proteins; Erythropoietin; Female; Gene Expression; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Liver; Male; Nuclear Proteins; Rats; Rats, Wistar; RNA, Messenger; Transcription Factors

We reported previously that 17-beta estradiol (E2-beta) attenuates hypoxic induction of erythropoietin (EPO) synthesis in rats. We hypothesized this attenuation is mediated by increased nitric oxide (NO) bio-availability. To investigate this hypothesis, ovariectomized estrogen-depleted rats were instrumented with arterial and venous catheters and treated with either E2-beta (20 microg/24 hrs) or vehicle (polypropylene glycol) for 7 days. Rats were placed in Plexiglas boxes and administered a bolus of either the NO synthase inhibitor, Nomega-nitro-L-arginine (l-NNA, 15 mg/kg) or saline. Following this bolus, saline or l-NNA was continuously infused (15 mg/kg/h) throughout the 8 hours of hypoxic exposure (12% O2). Hypoxia increased plasma NO metabolites (NOx) in both saline groups but more in E2-beta-treated rats. l-NNA prevented this increase in both groups. Renal endothelial NO synthase (NOS) expression was unaltered by hypoxia, l-NNA, or E2-beta. Despite preventing increases in plasma NOx during hypoxia, l-NNA did not affect E2-beta attenuation of EPO synthesis. We conclude that E2-beta independently attenuates hypoxic induction of EPO and augments hypoxic increases in NO synthesis.

Topics: Animals; Blood Pressure; Erythropoietin; Estradiol; Female; Heart Rate; Hypoxia; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Ovariectomy; Rats; Rats, Sprague-Dawley

Erythropoietin (Epo) is the humoral regulator of red blood-cell production. Low oxygen tension increases the Epo levels by enhancing transcription, through the hypoxia-inducible factor (HIF)-1, a transcriptional modulator in oxygen-regulated gene expression. In the present work, a cooperative interaction between hypoxia, mediated by the HIF-1 complex, and transforming growth factor-beta (TGF-beta), mediated by Smad3/4, was revealed in the Epo gene. This cooperation is due to physical interaction between Smad3/4 and HIF-1alpha. The Smad3/4 binding site is located within the 3' Epo enhancer, downstream from the HRE consensus, and immediately adjacent to the orphan hepatic nuclear factor receptor (HNF-4). HNF-4 is interacting also with Smad3 and the HIF-1 complex, to potentiate further the cooperative effect between both factors. Moreover, Sp1 has been identified as the factor binding the promoter necessary for the full hypoxia inducibility of the EPO gene. However, this full induction is achieved only if the TGF-beta pathway is mediating a cross-talk between promoter (Sp1) and enhancer (HIF-1alpha) regions through Smad3. We show that Sp1 binding to the proximal promoter is relevant for Epo transcription, and contributes to the Epo induction by hypoxia. A functional cooperation among the transcription factors mediating hypoxia (HIF-1, Sp1), the TGF-beta pathway (Smad3/4), and tissue-specific HNF-4 is proposed for the regulation of the Epo gene. In this model, the physical contact between the upstream promoter and the 3' downstream enhancer is mediated by Sp1 and Smad3 factors, and would occur upon bending of the DNA intervening sequences. Thus, Sp1 would reinforce the promoter/enhancer contact, while Smad3 would stabilize the multifactorial complex by interacting with HIF-1/Sp1/HNF-4 and the coactivator CBP/p300. This model may be extended to other genes where collaboration between TGF-beta and hypoxia takes place.

Topics: Animals; Cell Line; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation; Genes, Reporter; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Promoter Regions, Genetic; Protein Binding; Signal Transduction; Smad3 Protein; Smad4 Protein; Sp1 Transcription Factor; Trans-Activators; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Two-Hybrid System Techniques

Extracellular superoxide dismutase (SOD3) is the primary extracellular enzymatic scavenger of superoxide ((.)O(2)(-)). SOD3's expression is highest in the kidney, but its distribution and biologic functions there are unknown. To investigate the function of renal SOD3, we colocalized it with erythropoietin (EPO) to proximal tubules using in situ hybridization and immunohistochemistry. We then exposed wild-type (Wt) and SOD3 knock-out (KO) mice to hypoxia and found a late hematocrit response in the KO strain. EPO mRNA expression was attenuated in KO mice during the first 6 hours of hypoxia preceded at 2 hours by less accumulation of nuclear hypoxia-inducible transcription factor 1 alpha (HIF-1 alpha) protein. Meanwhile KO mice exposed to hypoxia showed increases in renal mRNA for superoxide-producing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4) and early significant increases in glutathione disulfide (GSSG)/glutathione (GSH), a marker of oxidative stress, compared with Wt mice. Plasma nitrite/nitrate and renal 3-nitrotyrosine (3-NTyr), indicating peroxynitrite formation, increased later in hypoxia, and renal endothelial nitric oxide synthase protein induction was similar in both strains. These data show that hypoxic activation of HIF-1 alpha and its target gene EPO in mouse kidney is regulated closely by the oxidant/antioxidant equilibrium involving SOD3, thus identifying renal SOD3 as a regulatory element in the body's innate adaptation to hypoxia.

Topics: Animals; Erythropoietin; Gene Expression Regulation; Glutathione; Glutathione Disulfide; Hematocrit; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Kidney Tubules, Proximal; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Oxidase 4; NADPH Oxidases; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Oxidative Stress; RNA, Messenger; Superoxide Dismutase; Transcription Factors; Transcription, Genetic

S-adenosylhomocysteine (AdoHcy), a by-product and inhibitor of S-adenosylmethionine (AdoMet)-dependent methylation reactions, is removed by AdoHcy hydrolase. The ratio of AdoMet and AdoHcy, also termed methylation potential (MP), is a metabolic indicator for cellular methylation status. In the present study, we have investigated the influence of hypoxia and inhibition of AdoHcy hydrolase on MP in HepG2 cells. Furthermore, we studied the impact of deviations in MP on mRNA and DNA methylation and the expression of selected genes: erythropoietin, VEGF-A, AdoHcy hydrolase, cyclophilin, and HIF-1alpha. Under hypoxic conditions, the MP raised from 53.4 +/- 3.3 to 239.4 +/- 24.8, which is the result of increased AdoMet and decreased AdoHcy levels. Inhibition of AdoHcy hydrolase by adenosine-2',3'-dialdehyde leads to a 40-fold reduction of the MP under both normoxic and hypoxic conditions. Hypoxia increases erythropoietin (2.7-fold) and VEGF-A (5-fold) mRNA expression. During a reduced MP erythropoietin mRNA expression is lowered under normoxia and hypoxia by 70%, whereas VEGF-A mRNA expression is only reduced under hypoxic conditions by 60%. The mRNA expression of AdoHcy hydrolase, HIF-1alpha, and cyclophilin is insensitive to an altered MP. Furthermore, decreased MP leads to a highly significant decrease in overall mRNA methylation. Our results show that the mRNA levels of the studied genes respond differentially to changes in MP. This implies that genes with a slower transcription rate and mRNAs with a slower turnover are insensitive to short-term changes in MP.

Topics: Adenosine; Adenosylhomocysteinase; Cell Line, Tumor; Cyclophilins; DNA Methylation; Down-Regulation; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Methylation; RNA Processing, Post-Transcriptional; RNA, Messenger; S-Adenosylhomocysteine; S-Adenosylmethionine; Transcription Factors; Up-Regulation; Vascular Endothelial Growth Factor A

Topics: Altitude; Blood Platelets; Erythropoietin; Female; Hepatocytes; Humans; Hypoxia; Platelet Count; Purpura, Thrombocytopenic, Idiopathic; Thrombopoietin; Time Factors

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcription factor that regulates expression of several hypoxia-inducible genes, including erythropoietin (EPO), by binding to hypoxia response elements (HREs) in their promoters/enhancers. Previously, we have shown that 17-beta estradiol (E2-beta) attenuates hypoxic induction of EPO in rats. We hypothesized that this response is mediated by E2-beta-induced attenuation of HIF-1alpha activity/expression. To test this hypothesis, we performed reporter gene assays in Hep3B cells to assess E2-beta effects on hypoxia-induced activity of a reporter gene driven by the HRE from a cloned EPO-enhancer element. Immunocytochemistry and Western blots were additionally used to determine effects of E2-beta on hypoxic increases in HIF-1alpha and EPO immunoreactivity. Finally, we examined potential influences of E2-beta on HIF-1alpha mRNA levels by real-time PCR. Consistent with our hypothesis, E2-beta (100 pM) inhibited hypoxic increases in HRE-mediated reporter gene activity. Furthermore, the estrogen-receptor antagonist ICI 182,780 (25 microM) eliminated these inhibitory effects of E2-beta. E2-beta similarly attenuated hypoxic induction of both EPO and HIF-1alpha protein in an estrogen-receptor dependent manner, but was without effect on HIF-1alpha mRNA expression. These findings suggest a role for E2-beta to attenuate EPO expression by interfering with hypoxic increases in HIF-1alpha protein through an estrogen receptor-dependent mechanism.

Topics: Animals; Blotting, Western; Cells, Cultured; Erythropoietin; Estradiol; Fluorescent Antibody Technique; Genes, Reporter; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Polymerase Chain Reaction; Rats; Transcription Factors

Erythropoietin (EPO), a hypoxia-inducible cytokine, is required for survival, proliferation, and differentiation of erythroid progenitor cells. EPO can also stimulate proliferation and angiogenesis of endothelial cells that express EPO receptors (EPORs). In this study we investigated the EPO response of vascular endothelial cells at reduced oxygen tension (5% and 2%), in particular the effect of EPO on nitric oxide (NO) release. Endothelial nitric oxide synthase (eNOS) produces NO, which maintains blood pressure homeostasis and blood flow. We find that EPOR is inducible by EPO in primary human endothelial cells of vein (HUVECs) and artery (HUAECs) and cells from a human bone marrow microvascular endothelial line (TrHBMEC) to a much greater extent at low oxygen tension than in room air. We found a corresponding increase in eNOS expression and NO production in response to EPO during hypoxia. Stimulation of NO production was dose dependent on EPO concentration and was maximal at 5 U/mL. NO activates soluble guanosine cyclase to produce cyclic guanosine monophosphate (cGMP), and we observed that EPO induced cGMP activity. These results suggest that low oxygen tension increases endothelial cell capacity to produce NO in response to EPO by induction of both EPOR and eNOS. This effect of EPO on eNOS may be a physiologically relevant mechanism to counterbalance the hypertensive effects of increased hemoglobin-related NO destruction resulting from hypoxia-induced increased red cell mass.

Topics: Cardiovascular Diseases; Cell Differentiation; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Erythropoietin; Hemoglobins; Humans; Hypoxia; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitrites; Oligonucleotides; Oxygen; Receptors, Erythropoietin; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Time Factors; Ultraviolet Rays

Systemic hypoxia is a common complication in stroke patients and may exacerbate ischemic brain damage. Expression of the hypoxia-inducible cytokine erythropoietin (Epo) is upregulated in the brain in both stroke patients and in animal stroke models and exerts local neuroprotective effects in the ischemic brain. Epo is also well known to stimulate red blood cell (RBC) production. The purpose of the present study was to evaluate whether poststroke systemic hypoxia is present in the rat model and whether it is associated with increased peripheral Epo and RBC production.. Wistar rats underwent 1-hour transient middle cerebral artery occlusion (MCAO) under mechanical ventilation, followed by reperfusion without further ventilation. Groups of MCAO and sham-operated animals were evaluated at extended times after reperfusion for assessment of arterial blood gases, plasma Epo, and complete blood count.. Arterial oxygen saturation was significantly lower in the infarct group between 6 and 24 hours after reperfusion (P=0.0005), and plasma Epo levels were increased 6 hours after reperfusion (P<0.05). RBC counts and hematocrit were transiently increased 2 to 7 days after reperfusion in animals with MCAO compared with sham. Maximal increases were seen at day 7 (22% and 16% increases of RBC count and hematocrit, respectively; P<0.001). In contrast, the white blood cell counts in animals with MCAO decreased by >30% in the same time period.. Plasma Epo levels, RBC counts, and hematocrit are all increased in response to systemic hypoxia after cerebral ischemia in rats.

Topics: Animals; Disease Models, Animal; Erythrocyte Count; Erythropoiesis; Erythropoietin; Hematocrit; Homeostasis; Hypoxia; Infarction, Middle Cerebral Artery; Male; Oxygen; Rats; Rats, Wistar; Stroke

Blind subterranean mole rats (Spalax, Spalacidae) evolved adaptive strategies to cope with hypoxia that climaxes during winter floods in their burrows. By using real-time PCR, we compared gene expression of erythropoietin (Epo), a key regulator of circulating erythrocytes, and hypoxia-inducible factor 1 alpha (HIF-1 alpha), Epo expression inducer, in the kidneys of Spalax and white rats, Rattus norvegicus. Our results show significantly higher, quicker, and longer responses to different O(2) levels in Spalax compared with Rattus. (i) In normoxia, both Spalax and Rattus kidneys produce small amounts of Epo. Maximal expression of Rattus Epo is noticed after a 4-h hypoxia at 6% O(2). Under these conditions, Spalax Epo levels are 3-fold higher than in Rattus. After 24 h of 10% O(2), Spalax Epo reaches its maximal expression, remarkably 6-fold higher than the maximum in Rattus; (ii) the HIF-1 alpha level in normoxia is 2-fold higher in Spalax than in Rattus. Spalax HIF-1 alpha achieves maximal expression after 4-h hypoxia at 3% O(2), a 2-fold increase compared with normoxia, whereas no significant change was detected in Rattus HIF-1 alpha at any of the conditions studied; (iii) at 6% O(2) for 10 h, in which Rattus cannot survive, Epo and HIF-1 alpha levels in Spalax galili, living in heavily flooded soils, are higher than in Spalax judaei, residing in light aerated soil. We suggest that this pattern of Epo and HIF-1 alpha expression is a substantial contribution to the adaptive strategy of hypoxia tolerance in Spalax, evolved during 40 million years of evolution to cope with underground hypoxic stress.

Topics: Animals; Blindness; Cloning, Molecular; Erythropoietin; Gene Expression Profiling; Gene Expression Regulation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Mole Rats; Molecular Sequence Data; Oxygen; Protein Structure, Tertiary; Rats; RNA, Messenger; Stress, Physiological; Transcription Factors; Vascular Endothelial Growth Factor A

Topics: Adult; Analysis of Variance; Circadian Rhythm; Erythropoietin; Female; Hematocrit; Humans; Hypoxia; Male; Middle Aged; Oxygen; Respiration; Time Factors

Activation of the hypoxia-inducible factor alpha-subunits, HIF-1alpha and HIF-2alpha, seems to be subject to similar regulatory mechanisms, and transgene approaches suggested partial functional redundancy. Here, we used RNA interference to determine the contribution of HIF-1alpha vs. HIF-2alpha to the hypoxic gene induction. Surprisingly, most genes tested were responsive only to the HIF-1alpha siRNA, showing no effect by HIF-2alpha knock-down. The same was found for the activation of reporter genes driven by hypoxia-responsive elements (HREs) from the erythropoietin (EPO), vascular endothelial growth factor, or phosphoglycerate kinase gene. Interestingly, EPO was the only gene investigated that showed responsiveness only to HIF-2alpha knock-down, as observed in Hep3B and Kelly cells. In contrast to the EPO-HRE reporter, the complete EPO enhancer displayed dependency on HIF-2alpha regulation, indicating that additional cis-acting elements confer HIF-2alpha specificity within this region. In 786-0 cells lacking HIF-1alpha protein, the identified HIF-1alpha target genes were regulated by HIF-2alpha. Overexpression of the HIFalpha subunits in different cell lines also led to a loss of target gene specificity. In conclusion, we found a remarkably restricted target gene specificity of the HIFalpha subunits, which can be overcome in cells with perturbations in the pVHL/HIF system and under forced expression.

Topics: Basic Helix-Loop-Helix Transcription Factors; Cell Line; Cell Line, Tumor; Enhancer Elements, Genetic; Erythropoietin; Genes, Reporter; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Protein Subunits; Response Elements; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sensitivity and Specificity; Trans-Activators; Transcription Factors; Transcriptional Activation; Transfection

Living at 2300-m altitude combined with intermittent training at 3500 m leads to cardiovascular alterations in dogs, including increase in systemic and pulmonary artery pressure. Despite moderate to marked hypoxemia at these altitudes, erythrocytosis does not develop. To study humoral mechanisms of acclimatisation to high altitude, erythropoietin (EPO), endothelin-1 (ET-1), big endothelin (Big-ET) and vascular endothelial growth factor (VEGF) were measured in dogs living at 2300 m and intermittently ascending to 3500 m, and compared to the values obtained in control dogs living at 700-900 m. While the median EPO and ET-1 level in dogs at 2300 m did not differ from the one measured at 700-900 m, exposure from 2300 to 3500 m resulted in significantly elevated EPO and ET-1 levels. Big-ET levels were significantly higher at 2300 and 3500 m compared to dogs at low altitude, but did not differ between 2300 and 3500 m. VEGF was significantly elevated in dogs at 2300 m compared to dogs at low altitude. The increases in EPO, VEGF, ET-1 and Big-ET are thought to reflect the effect of hypoxia on a cellular level in these dogs. Obviously, the mild elevation of EPO levels observed at 3500 m was not sufficient to cause erythrocytosis. Elevations of the vasoconstrictors Big-ET and ET-1 may play some, but not a central role in hypoxic vasoconstriction in these dogs. Finally, serum VEGF measurement may be a sensitive and useful test to assess hypoxic stress in dogs.

Topics: Acclimatization; Altitude; Anaerobiosis; Animals; Dogs; Endothelin-1; Erythropoietin; Hypoxia; Physical Conditioning, Animal; Vascular Endothelial Growth Factor A

Upon hypoxia, the human erythropoietin (EPO) gene is transactivated by the heterodimeric hypoxia-inducible factor 1 (HIF-1). Mammalian SWI/SNF is a chromatin-remodeling complex involved in the modulation of gene expression. We demonstrate that Brahma (Brm) and Brahma/SWI2-related gene 1 (Brg-1), alternative ATPase subunits of SWI/SNF, potentiate reporter gene activation mediated by HIF-1 in an ATPase-dependent manner. Brm was more potent than Brg-1 in the reporter gene assays. Simultaneous depletion of both Brm and Brg-1 by small interfering RNAs significantly compromised the transcription of the endogenous EPO gene triggered by hypoxia. Whereas knocking down Brm alone resulted in a moderate reduction in transcription of the EPO gene, depletion of Brg-1 resulted in an augmentation of transcription of both the EPO gene and the Brm gene, indicating that Brm can compensate for loss of Brg-1. Chromatin immunoprecipitation (ChIP) and sequential ChIP (re-ChIP) analysis showed that both Brm and Brg-1 associate with the enhancer region of the EPO gene in vivo in a hypoxia-dependent fashion and that each is present in a complex with HIF-1. Brm and Brg-1 were also recruited to the promoter of the vascular endothelial growth factor (VEGF) gene in a hypoxia-dependent fashion, although hypoxic induction of VEGF transcription was not affected by depletions of either or both Brm and Brg-1. Together these studies reveal a novel role for SWI/SNF in the activation of transcription of the EPO gene, indicate an important communication and compensation between Brm and Brg-1, and suggest that the requirement for SWI/SNF during hypoxic induction is gene-specific.

Topics: 3' Untranslated Regions; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Chromatin; Cross-Linking Reagents; Dimerization; DNA Helicases; Drosophila Proteins; Enhancer Elements, Genetic; Erythropoietin; Genes, Reporter; Genetic Vectors; Histones; Humans; Hypoxia; Immunoprecipitation; Luciferases; Models, Genetic; Nuclear Proteins; Oxygen; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Vascular Endothelial Growth Factor A

The glycoprotein hormone Erythropoietin (EPO) stimulates red cell production and maturation. EPO is produced by the kidneys and the fetal liver in response to hypoxia (HOX). Recently, EPO expression has also been observed in the central nervous system where it may be neuroprotective. It remained unclear, however, whether EPO is expressed in the peripheral nervous system and, if so, whether a neuronal phenotype is required for its regulation. Herein, we report that EPO expression was induced by HOX and a HOX mimetic in two cell lines derived from neuroblastoma (NB), a tumor of the peripheral nervous system. Both cell lines with inducible EPO expression, SH-SY5Y and Kelly cells, expressed typical neuronal markers like neuropeptide Y (NPY), growth-associated protein-43 (GAP-43), and neuron-specific enolase (ENO). NB cells with a more epithelial phenotype like SH-SHEP and LAN-5 did not show HOX inducible EPO gene regulation. Still, oxygen sensing and up-regulation of hypoxia-inducible factor-1 (HIF-1) were intact in all cell lines. We found that CpG methylation of the HIF binding site (HBS) in the EPO gene 3' enhancer was only present in the SH-SHEP and LAN-5 cells but not in SH-SY5Y and Kelly cells with regulated EPO expression. The addition of recombinant EPO to all NB cells, both under normoxic and hypoxic conditions, had no effect on cell proliferation. We conclude that the ability to respond to HOX with an increase in EPO expression in human NB may depend on CpG methylation and the differentiation status of these embryonic tumor cells but does not affect the proliferative characteristics of the cells.

Topics: Binding Sites; Blotting, Western; Cell Differentiation; CpG Islands; DNA Methylation; DNA-Binding Proteins; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Gene Expression Regulation; Helix-Loop-Helix Motifs; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Neuroblastoma; Nuclear Proteins; Peripheral Nervous System Neoplasms; Receptors, Erythropoietin; Transcription Factors; Tumor Cells, Cultured

We tested the hypothesis that repetitive severe hypoxemia resulting from obstructive sleep apnea would increase serum erythropoietin, and that this increase would be attenuated by effective treatment of obstructive sleep apnea.. We studied healthy untreated patients with obstructive sleep apnea (18 severe and 10 very mild) before and after acute treatment with continuous positive airway pressure, and 12 healthy control subjects free of obstructive sleep apnea.. Baseline erythropoietin levels before sleep were similar in the obstructive sleep apnea and control groups. However, erythropoietin levels increased (by 20%, P =.037) in patients with severe obstructive sleep apnea after 3.5 hours untreated (lowest O2, 77% +/- 3%), and decreased after 4 hours of continuous positive airway pressure treatment (P =.001). Erythropoietin responses in patients with severe obstructive sleep apnea were different (F = 4.0, P =.03) from controls, in whom erythropoietin levels remained stable throughout the night (P =.94). Erythropoietin responses were similar in very mild obstructive sleep apnea and controls (P =.58).. Our results indicate that untreated severe obstructive sleep apnea results in increased erythropoietin, which decreases after continuous positive airway pressure treatment. Increased erythropoietin may be a potential reversible mechanism to explain the association between obstructive sleep apnea and cardiovascular disease.

Topics: Adult; Cardiovascular Diseases; Continuous Positive Airway Pressure; Erythropoietin; Female; Humans; Hypoxia; Male; Middle Aged; Severity of Illness Index; Sleep Apnea, Obstructive

Apigenin, a plant-derived flavone, is a potent inhibitor of cell proliferation and angiogenesis, but the mechanisms leading to the pathological anti-angiogenic effects of apigenin are still unclear. In this study, we found that apigenin inhibited the hypoxia-induced expression of vascular endothelial growth factor (VEGF) mRNA in human umbilical artery endothelial cells. Apigenin also suppressed the expression of erythropoietin mRNA, which is a typical hypoxia-inducible gene, via the degradation of hypoxia-inducible factor 1 (HIF-1) alpha. We investigated the effect of apigenin on the interaction of HIF-1alpha with heat shock protein 90 (Hsp90), which is reported to be important for the stabilization of HIF-1alpha, and found that VEGF expression was inhibited via degradation of HIF-1alpha through interference with the function of Hsp90.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Apigenin; Cell Line; Cobalt; Endothelial Cells; Endothelium, Vascular; Erythropoietin; Gene Expression Regulation; HSP90 Heat-Shock Proteins; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Transcription Factors; Vascular Endothelial Growth Factor A

The purpose of this study was to test for the presence of liver hypoxia and recovery after reperfusion when blood alcohol levels (BAL) are high. Male rats were fed ethanol intragastrically at a constant rate for 1 month. The pO(2) levels were then measured on the liver surface of these rats, in vivo during laparatomy under isoflurane anesthesia. To measure the response to acute hypoxia, the hepatic blood flow was clamped off at the porta hepatis. When the clamp was released, recovery from hypoxia was measured. A number of hypoxic-inducible genes in the liver were analyzed by means of quantitative RT-PCR as a measure of increased activation of hypoxia initiated transcription. The mRNA levels of genes for adrenomedullin, adrenergic receptor alpha, 1a and 1d, CDK inhibitor 1a, and erythropoietin were all significantly higher at the peaks than troughs. Expression of these same genes in the livers of control rats fed dextrose was lower than at the troughs. Although the mRNA level of the hypoxia-inducible factor (HIF-1alpha) was higher at the trough than at the peak, its protein concentration in the nuclear fraction was not increased at the troughs compared with the peaks. In fact, the nuclear protein level of HIF-1alpha at the peak was significantly higher than in control samples, which is consistent with the presence of hypoxia at the peaks. Further analysis of the HIF-alpha degradation regulation revealed that prolyl 4-hydroxylase (P4ha1) and von Hippel-Lindau syndrome homolog (Vhl) were both up-regulated at the troughs compared with the peaks. The liver surface oxygen levels at the peaks were reduced compared with the control samples. The pO(2) levels fell abruptly when the vessels at the porta hepatis were clamped. When the clamp was removed, allowing reperfusion of the liver, pO(2) returned to baseline levels in the control, and at the troughs but not at the peaks. These results support the hypothesis that hypoxia occurs at the peaks of the BAL cycle and recovery from ischemia is impaired at the peaks.

Topics: Administration, Oral; Adrenomedullin; Animals; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Erythropoietin; Ethanol; Hypoxia; Liver; Liver Diseases, Alcoholic; Male; Oligonucleotide Array Sequence Analysis; Oxygen; Peptides; Rats; Rats, Wistar; Receptors, Adrenergic; Reperfusion Injury; RNA, Messenger

The placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K, identical to PFKFB3) is expressed in a variety of cells and tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, primary blood mononuclear cells and cancer cells. We observed previously that the enhancer region of the HP2K gene, which has been identified in the 5'-flanking region between -1265 and -1329, could respond to serum stimulation following the transfection of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. The HP2K enhancer region also contains two copies of the hypoxia-inducible factor-1 (HIF-1) binding motif (5'-ACGTG-3'). In this study we performed characterization of the HP2K gene expression in response to hypoxic conditions. Both electrophoretic mobility shift and co-transfection assays of the HP2K promoter-luciferase reporter with HIF-1 expression vectors indicated that HIF-1 binds to the hypoxia-responsive element (HRE) of HP2K, thereby upregulating its gene expression. In addition, we demonstrated using site-directed mutagenesis that a complete tandem repeat of the HIF-1 binding motif with a 4-bp interruption is required for full induction of HP2K expression (up to 22-fold) under hypoxic conditions, and that this response is much stronger than that of the erythropoietin (EPO) gene. These results suggest that the sequence 5'-ACGTGNNNNACGTG-3' in the HP2K enhancer is the authentic HRE consensus motif that mediates increased transcription, under hypoxic conditions, via HIF-1.

Topics: Amino Acid Motifs; Base Sequence; Cell Line; Cell Line, Tumor; Cell Nucleus; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation; Genetic Vectors; Glycolysis; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Luciferases; Molecular Sequence Data; Oligonucleotides; Phosphofructokinase-2; Placenta; Promoter Regions, Genetic; Protein Isoforms; Protein Structure, Tertiary; Time Factors; Tissue Distribution; Transcription Factors; Transcription, Genetic; Transfection; Up-Regulation

Topics: Adult; Circadian Rhythm; Erythropoietin; Humans; Hypercapnia; Hyperoxia; Hypoventilation; Hypoxia; Male; Middle Aged; Sleep Apnea, Obstructive; Syndrome

To study characteristics of iron deficiency anemia (IDA) in native population of high altitude territories.. 1300 women living in Tien-Shan areas were examined for serum and erythrocytic ferritins (SF and EF), general iron-binding capacity of the serum, transferrin saturation with iron, transferrin, soluble transferrin receptor (TR), erythropoietin (EP), Hb, erythrocytes, erythrocytic indices.. Regulation of erythropoiesis in women living at high altitude is specific, i.e. disagreement between complete depletion of iron (by SF and EF) and normal level of Hb, erythrocytes, EP and TR.. It is suggested that in population living at high altitudes (3000 m above the sea level and higher) long-term adaptation to hypoxia gave rise to an original mechanism of erythropoiesis regulation when all coming iron participates in hemoglobin synthesis without iron deposition. Under hypoxic hypoxia regulation of erythropoiesis is directed to prevention of tissue hypoxia.

Topics: Adaptation, Physiological; Altitude; Anemia, Iron-Deficiency; Erythrocyte Indices; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Ferritins; Humans; Hypoxia; Iron; Kyrgyzstan; Receptors, Transferrin; Transferrin

The aim of the present study was to evaluate whether the Epo isoforms in blood, induced by short-term and intermittent hypoxia, are different from those at normoxia at sea level and if this could be an impediment to the use of a direct Epo doping test based upon the electric charge of the Epo isoforms. Ten healthy subjects, 9 men and 1 woman, participated in the study. Median age was 22 years (range 20-32). Normobaric hypoxia was administered differently in 3 sub-groups; two groups with 12 h hypoxia and 12 h normoxia up to 10 days: IM 2000 and IM 2700 living in 16.2% and 14.9% O2, corresponding to 2000 and 2700 m above sea level, respectively, and training in normoxia. The third group, C 2700, lived in hypoxia, 14.9% O2 corresponding to 2700 m, continuously for 48 h. The mean serum Epo level increased from 10.9 IUL(-1) (range 8.8-12.5) to 23.5 IUL(-1) (15.6-29.1) after 2 days followed by 19.7 IUL(-1) (16.1-24.1) after 10 days exposure for intermittent hypoxia. The highest values 39.5 IUL(-1) (31.5-50) were obtained for the group exposed for continuous hypoxia for 48 h. The median electrophoretic mobility of the serum Epo isoforms was above the cut-off limit of 670 AMU, previously estimated for discrimination between recombinant and endogenous Epo, in all samples taken before and after exposure to hypoxia. The highest values, mean 730 mAMU (range 703-750) were obtained after 10 days of intermittent hypoxia.. If the method had been used as a doping test, no false positive results would have been registered for the 15 serum samples from the 10 individuals exposed for hypoxia. Thus, the results indicate that the basic principle for direct detection of recombinant Epo doping, based upon the change in electric charge on Epo, can be used also on individuals having lived in a hypoxic milieu.

Topics: Adult; Electrophoresis; Erythropoietin; Female; Humans; Hypoxia; Male; Protein Isoforms; Recombinant Proteins; Time Factors

In rats, in contrast with human subjects who develop megaloblastic anaemia due to vitamin B12 deficiency, haematological abnormalities with anaemia were not observed under normoxic conditions even though plasma vitamin B12 concentration was reduced to <15 % of a normal concentration by depleting dietary vitamin B12. To elucidate whether erythropoiesis was affected by vitamin B12 deficiency in rats, these vitamin B12-deficient rats were exposed to hypoxia (10.5 % O2) to stimulate erythropoiesis. In the vitamin B12-sufficient control rats, erythrocyte count was significantly (P<0.05) increased 1 week after starting the hypoxic exposure. However, the hypoxia-induced erythropoiesis was affected by vitamin B12 deficiency, and no significant increase in the erythrocyte count was observed even after 6-week exposure to hypoxia in the vitamin B12-deficient rats. In the vitamin B12-deficient rats in hypoxia, erythrocytes became abnormally enlarged, and haemoglobin concentration in peripheral blood was increased in proportion to the increase of mean corpuscular volume. However, the level of the increase in the haemoglobin concentration was significantly (P<0.05) lower in the vitamin B12-deficient rats compared with that in the -sufficient controls. In addition, in the vitamin B12-deficient rats, in contrast to the -sufficient rats, serum erythropoietin concentration was not normalized even after 6-week exposure to hypoxia. These results indicate that a megaloblastic anaemia-like symptom is induced when the vitamin B12-deficient rats are exposed to hypoxia.

Topics: Analysis of Variance; Anemia, Megaloblastic; Animals; Erythrocyte Count; Erythropoietin; Hypoxia; Male; Methylmalonic Acid; Models, Animal; Rats; Vitamin B 12; Vitamin B 12 Deficiency

Mice myocardia, in which plasma erythropoietin (EPO) concentrations were modified in response to different experimental conditions, were studied to evaluate contractility (dF/dt). CF1 mice were randomly separated into four main groups: group I, normocythaemic normoxic; group II-a, normocythaemic intermittently exposed to hypobaria for 72 h; group II-b, normocythaemic intermittently exposed to hypobaria for 3 weeks; group III, hypertransfused polycythaemic exposed to 72 h hypobaria; and group IV, hypertransfused polycythaemic maintained in normobaric air. Plasma EPO, contractile studies and binding assays were performed. The dF/dt was significantly higher in group II-a than in group I and group II-b; but in groups III and IV, the dF/dt was reduced. The toxic action of ouabain was reduced and delayed in its onset, accompanied by increased numbers of 3H-ouabain binding sites in group II-a. Contractility was positively correlated with plasma EPO (pEPO) in the different groups. Treating group I with recombinant human (rHu)-EPO enhanced contractility while treating group II-a with a monoclonal anti-EPO decreased the dF/dt. The inhibition of enzymatic pathway(s) known to participate in the cytokines signal transduction, decreased the basal dF/dt values on atria from group II-a and on group I atria treated with rHu-EPO. The results demonstrated: (1) a cardiac non-haematopoietic effect of EPO; (2) that mice in which the pEPO concentration increased showed improvement in contractility and in the therapeutic action of ouabain; and (3) it is possible that EPO may act as a cardioprotective agent by modulating the cardiac Na+-K+ pump.

Topics: Animals; Antibodies, Monoclonal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Erythropoietin; Female; Hypoxia; Mice; Myocardial Contraction; Ouabain; Recombinant Proteins; Sodium-Potassium-Exchanging ATPase

Current evidence suggests that a modulatory action on O(2)-dependent EPO secretion is exerted by the erythroid/precursor cell population in the erythropoietic organs through a negative feedback system. The hypothesis is based on studies of stimulated-EPO secretion performed in mice in whom the erythropoietic rates were either enhanced or depressed in the presence of normal plasma EPO half-lives. Since erythropoietic depression was elicited by cyclophosphamide administration, which could have altered EPO production directly, the aim of the present investigation was to estimate hypoxia-stimulated EPO secretion in a mouse model of functional depressed erythropoiesis induced by exposure to normobaric hyperoxia. Females CF#1 mice aged 70 d were divided into control (C) and experimental (E) groups. The former was maintained in plastic cages in a normal environment, while the latter was placed in an environment of 60% O(2)/40% N(2) in an 85-dm(3) atmospheric chamber with air flow of 1 L/min. Erythropoiesis was evaluated by either 24-h RBC-(59)Fe uptake or iron kinetics performed 3 h after IV injection of a tracer dose of (59)Fe. Both indexes of the red cell production rate were significantly depressed in E mice. Plasma disappearance of exogenous EPO in C mice, as well as in E mice exposed to hyperoxia for 4 d, was estimated by injecting (125)I-rHuEPO intravenously. Linear regression analysis indicated that neither the differences between the slopes of both curves nor the Y-intercepts were significant. Hypobaric hypoxemia was used as stimulus for EPO production. Plasma immuno-EPO titer after a 4-h exposure to hypobaric air was 73% higher in mice with hyperoxia-induced hypoerythropoiesis than in control mice with normal erythropoiesis. Data support the concept that the rate of erythropoiesis, perhaps through the number of the erythroid progenitor/precursor cell population, modulates O(2)-dependent EPO secretion.

Topics: Animals; Bone Marrow; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Hematocrit; Hyperoxia; Hypoxia; Iron Radioisotopes; Linear Models; Mice; Models, Animal; Regression Analysis; Spleen

Preconditioning phenomena provide evidence for adaptive responses to ischemia that have important implications for treatment/prevention of myocardial infarction. Hypoxia-inducible factor 1 (HIF-1) mediates adaptive transcriptional responses to hypoxia/ischemia.. Exposure of wild-type mice to intermittent hypoxia resulted in protection of isolated hearts against ischemia-reperfusion injury 24 hours later. Cardiac protection induced by intermittent hypoxia was lost in Hif1a+/- mice heterozygous for a knockout allele at the locus encoding HIF-1alpha. Erythropoietin (EPO) mRNA expression was induced in kidneys of wild-type mice subjected to intermittent hypoxia, resulting in increased plasma EPO levels. EPO mRNA expression was not induced in Hif1a+/- mice. EPO administration to rats increased functional recovery and decreased apoptosis in isolated hearts subjected to ischemia-reperfusion 24 hours later.. Hearts isolated from rodents subjected to intermittent hypoxia or EPO administration are protected against postischemic injury. Cardiac protection induced by intermittent hypoxia is critically dependent on Hif1a gene dosage. Our data suggest that additional studies to evaluate therapeutic applications of EPO administration are warranted.

Topics: Animals; Erythropoietin; Gene Expression; Heart; Heterozygote; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Kidney; Male; Mice; Mice, Mutant Strains; Myocardium; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Reperfusion Injury; RNA, Messenger; Time Factors; Transcription Factors

Cellular response to hypoxia is mainly controlled by hypoxia-inducible factor 1 (HIF-1). The HIF-1 target gene erythropoietin (EPO) has been described as neuroprotective. Thus, we hypothesize EPO to be an essential mediator of protection in hypoxic preconditioning.. We randomized Sv129 mice into groups for different pretreatments, different hypoxia-ischemia intervals, or different durations of ischemia. For hypoxic preconditioning, the animals were exposed to a hypoxic gas mixture (8% O2 and 92% N2) for 30, 60, 180, 300, or 360 minutes. At 0, 24, 48, 72, or 144 hours later, we performed middle cerebral artery occlusion and allowed reperfusion after 30, 45, 60, or 120 minutes, or occlusion was left to be permanent. We studied EPO gene expression in brain tissue with a real-time reverse transcriptase-polymerase chain reaction and measured HIF-1 DNA-binding activity with an electrophoretic mobility shift assay. To block endogenously produced EPO, we instilled soluble EPO receptor into the cerebral ventricle.. Hypoxic preconditioning for 180 or 300 minutes induced relative tolerance to transient focal cerebral ischemia, as evidenced by a reduction of infarct volumes to 75% or 54% of the control, respectively. Hypoxic pretreatment was effective only when applied 48 or 72 hours before middle cerebral artery occlusion. Sixty minutes after hypoxia, we found a marked activation of HIF-1 DNA-binding activity and a 7-fold induction of EPO transcription. Infusion of soluble EPO receptor significantly reduced the protective effect of hypoxic pretreatment by 40%.. Endogenously produced EPO is an essential mediator of ischemic preconditioning.

Topics: Animals; Brain; Cerebral Infarction; Disease Models, Animal; DNA; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Erythropoietin; Female; Hippocampus; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Infarction, Middle Cerebral Artery; Injections, Intraventricular; Ischemic Preconditioning; Male; Mice; Mice, Inbred Strains; Nuclear Proteins; Receptors, Erythropoietin; Reperfusion; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stroke; Time Factors; Transcription Factors

To investigate the influence of theophylline on erythropoiesis in chronic obstructive pulmonary disease (COPD) and explore the potential underlying mechanisms.. We evaluated the haematological parameters and erythropoietin (EPO) values in 38 COPD patients, 18 of which had been treated with theophylline (8 mg/kg daily) for at least 1 year, and the other 20 had never received this drug; 38 sex- and age-matched healthy volunteers served as controls. We further studied the development of BFU-E (bursts forming units of erythrocyte precursors) -derived colonies in semisolid methylcellulose cultures in blood samples from 7 patients randomly selected from both groups. In addition, we studied the effects of theophylline on the erythroid cell development by adding this agent to erythroid cell cultures from 6 healthy volunteers at various concentrations.. Haemoglobin values were found to be significantly lower in COPD patients treated with theophylline than in those untreated ( P<0.05). Both groups of patients exhibited significantly higher haemoglobin values than normal subjects ( P<0.01 and P<0.001 for treated and untreated patients, respectively). Serum EPO levels did not differ among the three studied groups. Unlike untreated patients and controls, the serum of the theophylline-treated patients produced a significant growth inhibition of erythroid bursts ( P<0.05); the in vitro use of theophylline showed a concentration-dependent inhibition ( P<0.001).. Our findings confirm the decrease of red cell production, which occurs following administration of theophylline, exclude the possibility of decreased EPO synthesis and suggest a direct inhibitory action of theophylline on erythropoiesis.

Topics: Aged; Bronchodilator Agents; Cells, Cultured; Erythropoiesis; Erythropoietin; Female; Humans; Hypoxia; Male; Pulmonary Disease, Chronic Obstructive; Theophylline

Therapeutic angiogenesis with gene encoding vascular endothelial growth factor (VEGF) is a new potential treatment in cardiovascular disease. However, unregulated VEGF-mediated angiogenesis has the potential to promote tumor growth, accelerate diabetic proliferative retinopathy, and promote rupture of atherosclerotic plaque. To be safe and effective, gene therapy with VEGF must be regulated. To limit the risk of pathological angiogenesis, we developed a hypoxia-inducible VEGF gene therapy system using the erythropoietin (Epo) enhancer and water-soluble lipopolymer (WSLP). pEpo-SV-VEGF or pSV-VEGF-Epo was constructed by insertion of the Epo enhancer upstream of the Simian Virus 40 (SV40) promoter or downstream of the poly(A) signal of pSV-VEGF. In vitro transfection showed that pEpo-SV-VEGF, not pSV-VEGF-Epo, induced the VEGF expression in hypoxic cells. In addition, the VEGF protein, which was produced from the Epo-SV-VEGF-transfected and hypoxia-incubated cells, was able to enhance the proliferation of the endothelial cells. Injection of the pEpo-SV-VEGF/WSLP complex showed that the expression of VEGF was induced in ischemic myocardium, compared to normal myo-cardium. Therefore, with the localized induction of VEGF and the low cytotoxicity of WSLP, the pEpo-SV-VEGF/WSLP system may be helpful to eventually treat ischemic heart disease.

Topics: Animals; Cell Division; Endothelial Growth Factors; Endothelium, Vascular; Enhancer Elements, Genetic; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Gene Expression; Genetic Engineering; Genetic Therapy; Humans; Hypoxia; Intercellular Signaling Peptides and Proteins; Liposomes; Luciferases; Lymphokines; Models, Animal; Myocardial Ischemia; Neovascularization, Physiologic; Polymers; Rabbits; Stem Cells; Transfection; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Apoptosis is a contributing cause of myocyte loss in ischemic heart disease. Recent work has shown that erythropoietin (EPO) offers protection against apoptosis in a wide variety of tissues. We demonstrate that the erythropoietin receptor (EPOR) is expressed in the neonatal rat ventricular myocyte (NRVM). Exposure of NRVMs to hypoxia, with recombinant human EPO, significantly decreased apoptosis as measured by TUNEL, flow cytometry, and caspase 3/7 like activity when compared to hypoxia treatment alone. EPO administered at the initiation of coronary artery occlusion in the rat significantly decreased apoptosis in the myocardial ischemic region. In the NRVM, EPO increased the activity of Akt. The anti-apoptotic effect of EPO was abrogated by co-treatment with LY294002, a specific blocker of phosphatidylinositol 3-kinase (PI3-K). Our study demonstrates that EPO inhibits apoptosis in the NRVM exposed to hypoxia, through an Akt-dependent pathway. EPO also inhibits apoptosis in the in vivo rat model of myocardial ischemia.

Topics: Animals; Apoptosis; Caspase 3; Caspase 7; Caspases; Erythropoietin; Flow Cytometry; Heart Ventricles; Hypoxia; Immunohistochemistry; In Situ Nick-End Labeling; Male; Myocardial Ischemia; Myocardium; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin

Necrotizing enterocolitis (NEC) is an important neonatal disease with a high mortality rate; erythropoietin (Epo) is a hematopoietic growth factor. Functional Epo receptors are in the fetal and postnatal small bowel and their ligands are available for binding. Excessive nitric oxide (NO) production by an isoform of NO synthase inducible by inflammatory stimuli leads to changes in vascular permeability and tissue injury. The aim of this study was to investigate NO formation in an experimental model of NEC and the possible role of NO in the protection Epo provides against NEC.. Twenty-four Wistar albino rat pups were divided into three groups: group 1 = control; group 2 = hypoxia-reoxygenation and saline; group 3 = hypoxia-reoxygenation and recombinant human EPO (rhEpo) pretreatment. rhEpo was given 750 U/kg/week by intraperitoneal injection 3 times a week for 2 weeks. On the 15th day, hypoxia was induced by placing the pups in a 100% CO(2) chamber for 5 min. After the hypoxia period the pups were reoxygenated for 10 min with 100% O(2) and returned to their mothers. All pups were killed 4 h after the hypoxia-reoxygenation period was over. The abdomen was opened and representative samples of injured areas were taken for histopathologic examination. Then nitrite levels were determined in the intestine by Griess Reagent.. On histopathological examination, injury scores in group-2 animals were found to be significantly higher than in group-3 animals (p = 0.001). Significantly increased intestinal nitrite levels were found in group-2 rats compared to the rats of groups 1 and 3 (p = 0.001 and p = 0.001, respectively). There was a positive correlation between the histological findings and the intestinal nitrite levels in group-2 and -3 animals (r = 0.94, p = 0.001; r = 0.99, p = 0.001, respectively).. The present study demonstrates that the Epo-pretreated group had decreased levels of NO and limited mucosal necrosis in intestinal tissue samples. We believe that these results deserve further experimental studies in order to elucidate the possible effector mechanisms involved in the inhibitory relationship between Epo, NO and NEC.

Topics: Animals; Enterocolitis, Necrotizing; Erythropoietin; Humans; Hypoxia; Intestinal Mucosa; Intestines; Nitrites; Rats; Rats, Wistar; Recombinant Proteins

Hypoxia-inducible factor 1 (HIF-1) is a phosphorylated protein and its phosphorylation is involved in HIF-1alpha subunit stabilization as well as in the regulation of HIF-1 transcriptional activity. In a variety of cell lines, the phosphorylation of HIF-1alpha is dependent on ERK or p38, two members of the mitogen-activated protein kinase (MAPK) superfamily. In addition, active MAPK could be inactivated through dephosphorylation by mitogen-activated protein kinase phosphatase-1 (MKP-1). MKP-1 has been identified as a hypoxia responsive gene, but its role in the response of cells to hypoxia is poorly understood. Here we found that hypoxia induces MKP-1 expression in human hepatoma cells HepG2 in a time-dependent manner. Inhibition of MKP-1 expression using siRNA technique could enhance HIF-1alpha phosphorylation, accompanied by an increase in transcriptionally active HIF-1 as well as a rise in the levels of HIF-1-induced erythropoietin expression.

Topics: Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; DNA-Binding Proteins; Down-Regulation; Dual Specificity Phosphatase 1; Enzyme Activation; Erythropoietin; Gene Expression Regulation, Neoplastic; Homeostasis; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Immediate-Early Proteins; Liver Neoplasms; Nuclear Proteins; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Sensitivity and Specificity; Transcription Factors; Transcriptional Activation

The source of the erythropoietin (EPO) that circulates in the fetus is unknown although it is known that EPO does not cross the placenta and that fetal kidneys, liver, and placenta express the EPO gene. This study tested to what extent in vivo EPO secretion by the fetal kidneys and placenta can be demonstrated under normoxic and hypoxic conditions.. Renal arterial and venous EPO concentrations were determined in eight late-gestation chronically catheterized fetal sheep made progressively anemic by exchange transfusion with saline solution over 5 to 8 days. In a separate additional series of experiments, umbilical arterial and venous EPO concentrations were determined in nine normoxic fetuses and in nine fetuses subjected to 12 hours of hypoxia induced by lowering maternal-inspired oxygen content. Organ secretion rates were calculated as the product of plasma flow rate and the arteriovenous concentration differences.. Renal vein plasma EPO concentration was higher than the arterial concentration in 36 of 40 paired samples (P<.0001) by 16.3%+/-2.7% (mean+/-SE). This difference was concentration independent over a range of 12 to 4100 mU/mL. Renal EPO secretion rates were variable and averaged 155+/-105 mU/min when hematocrit was 31.3%+/-1.6% (n=5) and 1124+/-300 mU/min post-exchange transfusion when hematocrit was 15.6%+/-0.8% (n=12). In contrast, umbilical venous and arterial EPO concentrations (range 9-35 mU/mL), although highly correlated (r=0.94), were not different during normoxia (Po(2)=21.6+/-0.5 mm Hg, n=9). Under hypoxic conditions (Po(2)=15.6+/-0.4 mm Hg, n=9), umbilical vein EPO concentration (range 151-1245 mU/mL) was higher than arterial concentration (range 140-951 mU/mL) in eight of nine paired samples by 13.6%+/-3.3% (P<.01). Under these conditions, estimated umbilical EPO secretion rate was 27,900+/-11,500 mU/min.. Under nonanemic, normoxic basal conditions, the kidneys secreted EPO into the fetal circulation, whereas secretion by the placenta was not demonstrated. In the phlebotomy-induced fetal anemia experiments, the kidney demonstrated marked, progressive increases in the rate of EPO production. Similarly, in the fetal hypoxemia experiments, the placenta demonstrated progressive increases--albeit an order of magnitude greater than the kidneys--in EPO production rate. As an extension of these findings, we speculate that the hypoproliferative neonatal anemia that invariably occurs in the early weeks after birth is in part the result of loss of EPO production by the placenta.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoiesis; Erythropoietin; Female; Fetal Blood; Hypoxia; Kidney; Oxygen; Oxygen Consumption; Placenta; Pregnancy; Pregnancy, Animal; Probability; Reference Values; Sheep, Domestic

Exposure to chronic hypoxia induces erythropoietin (EPO) production to facilitate oxygen delivery to hypoxic tissues. Previous studies from our laboratory found that ovariectomy (OVX) exacerbates the polycythemic response to hypoxia and treatment with 17beta-estradiol (E2-beta) inhibits this effect. We hypothesized that E2-beta decreases EPO gene expression during hypoxia. Because E2-beta can induce nitric oxide (NO) production and NO can attenuate EPO synthesis, we further hypothesized that E2-beta inhibition of EPO gene expression is mediated by NO. These hypotheses were tested in OVX catheterized rats treated with E2-beta (20 microg/day) or vehicle for 14 days and exposed to 8 or 12 h of hypoxia (12% O(2)) or normoxia. We found that E2-beta treatment significantly decreased EPO synthesis and gene expression during hypoxia. E2-beta treatment did not induce endothelial NO synthase (eNOS) expression in the kidney but potentiated hypoxia-induced increases in plasma nitrates. We conclude that E2-beta decreases hypoxic induction of EPO. However, this effect does not appear to be related to changes in renal eNOS expression.

Topics: Animals; Blood Gas Analysis; Erythropoietin; Estradiol; Female; Gene Expression Regulation; Hematocrit; Hypoxia; Immunohistochemistry; Isoenzymes; Kidney; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitrites; Organ Size; Ovariectomy; Rats; RNA, Messenger; Uterus

The epididymis consists of the interstitial tissue and the ductus epididymidis, an extremely tortuous duct, in which spermatozoa exported from the testis gain motility and fertilizing capacity. We found that the cultured mouse epididymis produces erythropoietin (Epo). The content of Epo mRNA in the epididymis from the adult mouse (8-week-old) amounts to 40% of that in the kidney. The epididymal Epo mRNA dramatically increased upon growth; its level increased 120-fold from the age of 3 weeks to 7 weeks when they complete sexual maturation, while the increase in the total RNA was 3-fold. Hypoxia induced a 5-fold increase in the epididymal Epo mRNA transiently, which is much lower than the induction in the kidney (28-fold). In situ hybridization technique elucidated that the site of Epo production was located in the interstitial space between ductus epididymidis. The epididymal Epo may have an unidentified function in the male reproductive organ.

Topics: Animals; Base Sequence; DNA Probes; Epididymis; Erythropoietin; Gene Expression Regulation, Developmental; Hypoxia; In Situ Hybridization; Kidney; Male; Mice; Mice, Inbred ICR; Polymerase Chain Reaction; RNA, Messenger; Testis

Erythropoietin (Epo), induced by hypoxia, controls the survival, proliferation, and differentiation of Epo receptor (EpoR)-bearing erythroid progenitors and plays a role in the protection of neurons from hypoxic damage. Hypoxia in malignant disease is associated with invasion, metastasis, resistance to therapy, and selection for cells with diminished apoptotic potential. The authors recently demonstrated the basal and hypoxia-stimulated expression of Epo and EpoR in human breast carcinoma cell lines and in breast carcinomas, suggesting a role for autocrine Epo signaling in the hypoxic adaptations of mammary neoplasms.. The authors characterized the expression of Epo and EpoR by immunohistochemistry in 184 invasive mammary carcinomas and 158 in situ mammary carcinomas and benign mammary epithelium. They analyzed the correlation of Epo and EpoR immunostaining with clinicopathologic tumor features and the patients' smoking history.. Benign mammary epithelial cells showed weak-to-moderate expression of Epo and EpoR. EpoR immunostaining was increased in carcinomas compared with benign epithelium both in nonsmokers and smokers, and Epo immunostaining was increased in carcinomas compared with benign epithelium in nonsmokers but not in smokers. Prominent Epo staining was seen in tumor cells adjacent to necrotic areas and at the infiltrating edge of tumors. EpoR staining, but not Epo staining, was significantly greater in tumors that showed high histologic grade, tumor necrosis, lymphovascular invasion, lymph node metastases, and loss of hormone receptor expression.. The current findings suggest that increased EpoR expression may play an important role in breast carcinogenesis. The induction of autocrine or paracrine Epo signaling may represent a novel mechanism by which hypoxia can promote breast carcinoma.

Topics: Breast Neoplasms; Carcinoma; Cell Transformation, Neoplastic; Erythropoietin; Female; Humans; Hypoxia; Immunohistochemistry; Receptors, Erythropoietin; Signal Transduction; Smoking

It has been hypothesized that O(2) sensing in type I cells of the carotid body and erythropoietin (EPO)-producing cells of the kidney involves protein components identical to the NADPH oxidase system responsible for the respiratory burst of phagocytes. In the present study, we evaluated O(2) sensing in mice with null mutant genotypes for two components of the phagocytic oxidase. Whole body plethysmography was used to study unanesthetized, unrestrained mice. When exposed to an acute hypoxic stimulus, gp91(phox)-null mutant and wild-type mice increased their minute ventilation by similar amounts. In contrast, p47(phox)-null mutant mice demonstrated increases in minute ventilation in response to hypoxia that exceeded that of their wild-type counterparts: 98.0 +/- 18.0 vs. 20.0 +/- 13.0% (n = 11, P = 0.003). In vitro recordings of carotid sinus nerve (CSN) activity demonstrated that resting (basal) neural activity was marginally elevated in p47(phox)-null mutant mice. With hypoxic challenge, mean CSN discharge was 1.5-fold greater in p47(phox)-null mutant than in wild-type mice: 109.61 +/- 13.29 vs. 72.54 +/- 7.65 impulses/s (n = 8 and 7, respectively, P = 0.026). Consequently, the hypoxia-evoked CSN discharge (stimulus-basal) was approximately 58% larger in p47(phox)-null mutant mice. Quantities of EPO mRNA in kidney were similar in gp91(phox)- and p47(phox)-null mutant mice and their respective wild-type controls exposed to hypobaric hypoxia for 72 h. These findings confirm the previous observation that absence of the gp91(phox) component of the phagocytic NADPH oxidase does not alter the O(2)-sensing mechanism of the carotid body. However, absence of the p47(phox) component significantly potentiates ventilatory and chemoreceptor responses to hypoxia. O(2) sensing in EPO-producing cells of the kidney appears to be independent of the gp91(phox) and p47(phox) components of the phagocytic NADPH oxidase.

Topics: Animals; Carotid Sinus; Chemoreceptor Cells; Erythropoietin; Gene Expression; Hypoxia; Kidney; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; NADPH Oxidase 2; NADPH Oxidases; Nervous System; Oxygen; Phagocytes; Phosphoproteins; Respiration; Rest

Anemia is a common clinical problem, and there is much interest in its role in promoting left ventricular hypertrophy through increasing cardiac workload. Normally, red blood cell production is adjusted through the regulation of erythropoietin (Epo) production by the kidney. One important cause of anemia is relative deficiency of Epo, which occurs in most types of renal disease. Clinically, this can be corrected by supplementation with recombinant Epo. Here we describe an oxygen-regulated gene therapy approach to treating homozygous erythropoietin-SV40 T antigen (Epo-TAg(h)) mice with relative erythropoietin deficiency. We used vectors in which murine Epo expression was directed by an Oxford Biomedica hypoxia response element (OBHRE) or a constitutive cytomegalovirus (CMV) promoter. Both corrected anemia, but CMV-Epo-treated mice acquired fatal polycythemia. In contrast, OBHRE-Epo corrected the hematocrit level in anemic mice to a normal physiologic level that stabilized without resulting in polycythemia. Importantly, the OBHRE-Epo vector had no significant effect on the hematocrit of control mice. Homozygous Epo-TAg(h) mice display cardiac hypertrophy, a common adaptive response in patients with chronic anemia. In the OBHRE-Epo-treated Epo-TAg(h) mice, we observed a significant reversal of cardiac hypertrophy. We conclude that the OBHRE promoter gives rise to physiologically regulated Epo secretion such that the hematocrit level is corrected to healthy in anemic Epo-TAg(h) mice. This establishes that a hypoxia regulatory mechanism similar to the natural mechanism can be achieved, and it makes EPO gene therapy more attractive and safer in clinical settings. We envisage that this control system will allow regulated delivery of therapeutic gene products in other ischemic settings.

Topics: Anemia; Animals; Antigens, Polyomavirus Transforming; Cell Line; Erythropoietin; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Hypoxia; Kidney; Lymphocyte Activation; Mice; Mice, Transgenic; Polymerase Chain Reaction; Simian virus 40; Spleen; Transfection

1. Acute moderate hypoxia modifies the catalytic activity and expression of certain isoenzymes of hepatic cytochrome P450 (P450). The aim of this study was to document whether hypoxia affects hepatic P450 directly or through the release of serum mediators. 2. Rabbits were subjected to a FiO(2) of 8% for 48 h, sacrificed, and serum and hepatocytes were isolated; hepatocytes from control and rabbits with hypoxia were incubated with serum from control and hypoxic rabbits for 4 and 24 h, and total P450 content, CYP1A1, 1A2 and 3A6 activities and expressions were assessed. Sera were fractionated by size exclusion chromatography and fractions tested for their ability to modify activity and amount of P450, and serum mediators were identified through neutralization experiments. 3. Total serum and fractions with proteins of 15-23 and 65-94 kDa of M(r) reduced P450 content and expression of CYP1A1, 1A2 and 3A6, as well as CYP1A1, 1A2 and 3A6 mRNA. Total serum and the fraction with 32-44 kDa proteins increased CYP3A6 activity and protein and mRNA. The serum mediators implicated in the decrease in activity and expression of CYP1A1, 1A2 and 3A6 were interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta) and IL-2. Erythropoietin (Epo) was partly responsible for the increase in P450 content and CYP3A6 expression. 4. In conclusion, acute moderate hypoxia diminishes the activity and expression of CYP1A1, 1A2 and CYP1A1, 1A2 mRNA, and increases CYP3A6 protein, activity and CYP3A6 mRNA. Several mechanisms contribute to these changes in P450, among them the release of cytokines acting as serum mediators.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Blood Proteins; Blotting, Northern; Blotting, Western; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Down-Regulation; Erythropoietin; Gene Expression Regulation, Enzymologic; Hepatocytes; Hypoxia; Interferon-gamma; Interleukin-1; Interleukin-2; Liver; Male; Molecular Weight; Rabbits; RNA, Messenger; Up-Regulation

The presence of increased numbers of nucleated red blood cells (NRBC) and increased levels of erythropoietin (EPO) in the circulation of neonates has been associated with states of relative hypoxia. The aim of this study is to assess the pattern of NRBC counts and EPO levels in a group of high-risk neonates under stress conditions and determine the short-term outcome for these babies by using these parameters.. There were 69 high-risk neonates; 14 intrauterine growth retarded (IUGR), 25 preterm infants, 18 term infants with asphyxia and 12 infants of diabetic mothers. Control groups included healthy, term infants delivered either vaginally (n = 18) or with cesarean section (n = 19). Three blood samples were obtained from each infant within 12 h (initial), 3 days and 7 days after birth to measure NRBC counts and EPO levels. Neonatal and short-term outcomes at 3 and 6 months of age were determined.. There was no significant difference among the groups with regard to the initial serum EPO concentrations. The initial NRBC counts were significantly lower in the control groups compared with the study groups (P = 0.002). While there was no significant difference between patients with good and poor outcome in terms of EPO concentrations of initial samples, a significant difference existed in terms of NRBC counts (P = 0.038).. Both serum EPO level and NRBC count provide limited clinical benefit in the detection of pathological conditions of the neonatal period, but NRBC count determination seems to be especially helpful in predicting short-term neurodevelopmental outcome.

Topics: Analysis of Variance; Erythroblasts; Erythrocyte Count; Erythropoietin; Female; Humans; Hypoxia; Infant, Low Birth Weight; Infant, Newborn; Infant, Premature; Male; Prospective Studies

The aim of this study was to further define the signal transduction pathways leading to hypoxia-inducible factor-1 (HIF-1) erythropoietin (EPO) gene expression.. Human hepatocellular carcinoma cells (Hep3B) were exposed to hypoxia (1% oxygen) and examined for mRNA expression, as well as gene transactivation with RT-PCR and luciferase reporter gene assays, respectively.. Treatment with LY294002 (a selective pharmacological inhibitor of phosphatidylinositol 3-kinase) significantly inhibited EPO protein and mRNA expression in Hep3B cells exposed to hypoxia for 24 hours, while treatment with PD098059 or SB203580 (selective pharmacological inhibitors of the MEK and p38 mitogen-activated protein kinase pathways, respectively) had no significant effects. The activity of AKT, a downstream target of PI3K, was increased by hypoxia and was also inhibited by LY294002. Genetic inhibition of AKT resulted in significant inhibition of NF-kappaB and HIF-1-mediated transactivation, as well as EPO gene expression, in response to hypoxia. Overexpression of constitutively active AKT resulted in increased NF-kappaB and HIF-1 transactivation. The selective inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), significantly blocked HIF-1 protein expression. Inhibition of NF-kappaB with a superrepressor dominant negative IkappaBalpha genetic construct also significantly blocked NF-kappaB and HIF-1 transactivation, as well as EPO gene expression.. We propose a key role for NF-kappaB in EPO gene regulation in response to hypoxia.

Topics: DNA-Binding Proteins; Erythropoietin; Gene Expression Regulation; Genes, Reporter; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; NF-kappa B; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

Obstructive sleep apnea (OSA) is associated with increased cardiovascular morbidity and mortality; however, some patients with OSA do not develop cardiovascular disease even in the presence of severe nocturnal oxygen desaturations. Vascular endothelial growth factor (VEGF) is a hypoxia-sensitive glycoprotein stimulating neoangiogenesis. We hypothesized that VEGF production is increased in OSA because of repetitive nocturnal hypoxia. Three different groups were investigated: 10 OSA patients with severe nighttime hypoxia (Group A), 10 OSA patients with moderate hypoxia (Group B), and 10 healthy volunteers (Group C). Serum levels of VEGF were measured by ELISA from peripheral venous blood samples obtained at 7 AM. Group A had significantly (p < 0.01) increased VEGF serum levels when compared with Group B and Group C (mean +/- SEM: 410 +/- 77 pg/ml versus 224 +/- 38 pg/ml and 245 +/- 61 pg/ml). The degree of nocturnal oxygen desaturation in OSA significantly correlated with the VEGF concentrations (r = 0.67, p < 0.01). In conclusion, serum levels of VEGF are elevated in severely hypoxic patients with OSA and are related to the degree of nocturnal oxygen desaturation. This might constitute an adaptive mechanism to counterbalance the emergence of OSA-related cardiovascular disease.

Topics: Adaptation, Physiological; Analysis of Variance; Case-Control Studies; DNA-Binding Proteins; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Forced Expiratory Volume; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Logistic Models; Lymphokines; Male; Nuclear Proteins; Obesity; Oximetry; Oxygen; Polysomnography; Recurrence; Severity of Illness Index; Sleep Apnea, Obstructive; Transcription Factors; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vital Capacity

It is well known that the anemia of chronic renal failure is associated with a blunted erythropoietin response. However, it is not clear why this response is blunted. Oxygen tension is an important regulator of erythropoietin production and release, but the effect of reduced renal mass on renal tissue oxygen tensions is currently unknown.. A computer-based simulation was used to determine how alterations in filtration fraction might impact on renal tissue oxygen tensions. In addition, direct measurements of oxygen tension with needle electrodes were employed, as well as conventional physiological measurements and ELISA measurements of plasma and tissue erythropoietin concentrations in rats subjected to 5/6th nephrectomy.. Remnant kidney rats had 39% and 52% decreases in tissue and plasma erythropoietin concentrations, respectively, that correlated with 73% increased oxygen tensions in both cortex and outer medulla in the remnant kidney (all P < 0.01). Estimations of filtration fraction were decreased by approximately 36% in the rats bearing remnant kidneys.. Higher oxygen tensions were observed in the remnant kidneys. We suggest that higher oxygen tensions are caused by a decrease in filtration fraction, and that these higher tissue oxygen tensions result in decreased renal erythropoietin production and anemia.

Topics: Anemia; Animals; Erythropoietin; Glomerular Filtration Rate; Hematocrit; Hypoxia; Kidney Cortex; Kidney Failure, Chronic; Kidney Medulla; Male; Nephrons; Oxygen; Partial Pressure; Rats; Rats, Sprague-Dawley; Renal Circulation

An erythropoietin (EPO)-deficient anaemia is recognized in Type 1 diabetic patients with early nephropathy and symptomatic autonomic neuropathy (DN). The aim of this study was to determine whether the EPO response to hypoxia was deficient in order to clarify the mechanisms involved in this process.. Five Type 1 diabetic patients DN (age 39 (28-48) years (mean (range))) with EPO-deficient anaemia (haemoglobin, Hb 10.6 (9.5-12.0) g/dl, EPO 5.0 (3.2-6.5) IU/l) and early diabetic nephropathy (persistent proteinuria 1161.6 (130-2835) mg/day, serum creatinine 97.6 (63-123) micromol/l)) were compared with nine normal subjects (age 31 (24-39) years, Hb 13.4 (11.8-15.7) g/dl, EPO 7.6 (5.6-10.3) IU/l) and four patients with non-diabetic advanced chronic renal failure RF (proteinuria 2157.5 (571-4578) mg/day, serum creatinine 490.2 (406-659) micromol/l, Hb 10.3 (9.0-11.3) g/dl, EPO 4.6 (2.9-8.5) IU/l). The subjects were exposed to 6 h of hypoxia (inspired oxygen 11.6-12.6%) by breathing a gas mixture via a hood. Hourly serum EPO levels were measured.. All groups showed a rise in EPO production after 2 h. The diabetic DN group achieved a similar maximal response to the normal subjects at 6 h (EPO 17.3 +/-5.4 vs. 17.8 +/-7.9 IU/l). The renal failure patients mounted an EPO response to hypoxia but at lower EPO levels.. Although the DN patients have inappropriately low EPO levels for the severity of their anaemia, they can mount an appropriate EPO response to moderate hypoxia. The mechanism underlying the EPO-deficient anaemia present in some diabetic patients remains unclear.

Topics: Adult; Anemia; Autonomic Nervous System Diseases; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Diabetic Neuropathies; Erythropoietin; Female; Humans; Hypoxia; Kidney Failure, Chronic; Male; Middle Aged; Proteinuria

Tolerance to cerebral ischemia is achieved by preconditioning sublethal stresses, such as ischemia or hypoxia, paradigms in which the decrease of O2 availability may constitute an early signal inducing tolerance. In accordance with this concept, this study shows that hypoxia induces tolerance against focal permanent ischemia in adult mice. Normobaric hypoxia (8% O2 of 1-hour, 3-hour, or 6-hour duration), performed 24 hours before ischemia, reduces infarct volume by approximately 30% when compared with controls. To elucidate the mechanisms underlying this neuroprotection, the authors investigated the effects of preconditioning on cerebral expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and its target genes, erythropoietin and vascular endothelial growth factor (VEGF). Hypoxia, whatever its duration (1 hour, 3 hours, 6 hours), rapidly increases the nuclear content of HIF-1alpha as well as the mRNA levels of erythropoietin and VEGF. Furthermore, erythropoietin and VEGF are upregulated at the protein level 24 hours after 6 hours of hypoxia. The authors' findings show that (1) hypoxia elicits a delayed, short-lasting (<72 hours) tolerance to focal permanent ischemia in the adult mouse brain; (2) HIF-1 target genes could contribute to the establishment of tolerance; and (3) this model might be a useful paradigm to further study the mechanisms of ischemic tolerance, to identify new therapeutic targets for stroke.

Topics: Animals; Brain; Brain Ischemia; DNA-Binding Proteins; Endothelial Growth Factors; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning; Lymphokines; Male; Mice; Nuclear Proteins; Oxygen; Time Factors; Transcription Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

We measured blood erythropoietin (EPO) concentration, arterial O(2) saturation (Sa(O(2))), and urine PO(2) in 48 subjects (32 men and 16 women) at sea level and after 6 and 24 h at simulated altitudes of 1,780, 2,085, 2,454, and 2,800 m. Renal blood flow (Doppler) and Hb were determined at sea level and after 6 h at each altitude (n = 24) to calculate renal O(2) delivery. EPO increased significantly after 6 h at all altitudes and continued to increase after 24 h at 2,454 and 2,800 m, although not at 1,780 or 2,085 m. The increase in EPO varied markedly among individuals, ranging from -41 to 400% after 24 h at 2,800 m. Similar to EPO, urine PO(2) decreased after 6 h at all altitudes and returned to baseline by 24 h at the two lowest altitudes but remained decreased at the two highest altitudes. Urine PO(2) was closely related to EPO via a curvilinear relationship (r(2) = 0.99), although also with prominent individual variability. Renal blood flow remained unchanged at all altitudes. Sa(O(2)) decreased slightly after 6 h at the lowest altitudes but decreased more prominently at the highest altitudes. There were only modest, albeit statistically significant, relationships between EPO and Sa(O(2)) (r = 0.41, P < 0.05) and no significant relationship with renal O(2) delivery. These data suggest that 1) the altitude-induced increase in EPO is "dose" dependent: altitudes > or =2,100-2,500 m appear to be a threshold for stimulating sustained EPO release in most subjects; 2) short-term acclimatization may restore renal tissue oxygenation and restrain the rise in EPO at the lowest altitudes; and 3) there is marked individual variability in the erythropoietic response to altitude that is only partially explained by "upstream" physiological factors such as those reflecting O(2) delivery to EPO-producing tissues.

Topics: Acute Disease; Adult; Altitude; Arteries; Atmospheric Pressure; Erythropoietin; Female; Humans; Hypoxia; Male; Oxygen; Renal Circulation

Erythropoietin (Epo) is upregulated by hypoxia and provides protection against apoptosis of erythroid progenitors in bone marrow and brain neurons. Here we show in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of Epo, fibroblast growth factor 2 and vascular endothelial growth factor via hypoxia-inducible factor-1alpha (HIF-1alpha) stabilization. Hypoxic preconditioning protects retinal morphology and function against light-induced apoptosis by interfering with caspase-1 activation, a downstream event in the intracellular death cascade. In contrast, induction of activator protein-1, an early event in the light-stressed retina, is not affected by hypoxia. The Epo receptor required for Epo signaling localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied Epo that crosses the blood retina barrier and prevents apoptosis even when given therapeutically after light insult. Application of Epo may, through the inhibition of apoptosis, be beneficial for the treatment of different forms of retinal disease.

Topics: Animals; DNA-Binding Proteins; Electroretinography; Endothelial Growth Factors; Erythropoietin; Fibroblast Growth Factor 2; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Light; Lymphokines; Mice; Mice, Inbred BALB C; Nuclear Proteins; Retinal Degeneration; Transcription Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Despite equivocal findings about the benefit of altitude training, current theory dictates that the best approach is to spend several weeks living at > or =2500 m but training near sea level. This paper summarizes six studies in which we used simulated altitude (normobaric hypoxia) to examine: (i) the assumption that moderate hypoxia compromises training intensity (two studies); and (ii) the nature of physiological adaptations to sleeping in moderate hypoxia (four studies). When submaximal exercise was >55% of sea level maximum oxygen uptake (VO2max), 1800 m simulated altitude significantly increased heart rate, blood lactate and perceived exertion of skiers. In addition, cyclists self-selected lower workloads during high-intensity exercise in hypoxia (2100 m) than in normoxia. Consequently, our findings partially confirm the rationale for 'living high, training low'. In the remaining four studies, serum erythropoietin increased 80% in the early stages of hypoxic exposure, but the reticulocyte response did not significantly exceed that of control subjects. There was no significant increase in haemoglobin mass (Hb(mass)) and VO2max tended to decrease. Performance in exercise tasks lasting approximately 4 min showed a non-significant trend toward improvement (1.0+/-0.4% vs. 0.1+/-0.4% for a control group; P=0.13 for group x time interaction). We conclude that sleeping in moderate hypoxia (2650-3000 m) for up to 23 days may offer practical benefit to elite athletes, but that any effect is not likely due to increased Hb(mass) or VO2max.

Topics: Adaptation, Physiological; Adult; Altitude; Erythrocyte Count; Erythrocyte Volume; Erythropoietin; Exercise; Exercise Tolerance; Female; Heart Rate; Hemodynamics; Humans; Hypoxia; Male; Oxygen; Sleep; Sports

The aim of this study was to assess the erythropoietic response to hypoxaemia in patients with diffuse idiopathic pulmonary fibrosis (DIPF), and to speculate on the underlying mechanisms. Patients on an established chronic respiratory failure due to DIPF or chronic obstructive pulmonary disease (COPD) were studied. The erythropoietic response to hypoxaemia in both conditions was assessed. We studied 18 patients with DIPF and 29 patients with COPD in respiratory failure in a stable stage, free from acute infection and congestive heart failure. Blood gases, erythrocytic parameters, as well the serum levels of iron, ferritin and erythropoietin were determined. All the DIPF patients studied, apart from two, had normal or subnormal haematocrit values. The patients with COPD had an inconsistant response to hypoxaemia; 12 had normal or subnormal haematocrit values and the remaining 17 were erythraemic. The mean value of erythropoietin (EPO) in both DIPF and COPD patients was significantly higher than normal. In conclusion, patients with DIPF exhibit a lack of erythropoietic response to hypoxaemia, despite the augmented erythropoietin levels. This may reflect a defective bone marrow erythropoietic response in DIPF patients. It is suggested that the pathophysiology of DIPF underlies this mechanism.

Topics: Adult; Aged; Aged, 80 and over; Blood Gas Analysis; Erythropoiesis; Erythropoietin; Female; Ferritins; Hematocrit; Humans; Hypoxia; Iron; Lung Diseases, Obstructive; Male; Middle Aged; Pulmonary Fibrosis; Reference Values; Regression Analysis

To better understand how humans adapt to hypoxia, the levels of hemoglobin (Hb), serum erythropoietin (Epo), and vascular endothelial growth factor (VEGF) were measured in 106 patients with severe obstructive sleep apnea-hypopnea syndrome. The results indicated that temporal hypoxic stimulation increases Hb. Furthermore, a minor increase in Epo and a substantial increase in VEGF were found. The induction in patients with severe sleep apnea was greater than that reported in other types of hypoxia. (Blood. 2001;98:1255-1257)

Topics: Case-Control Studies; Endothelial Growth Factors; Erythropoietin; Hemoglobins; Humans; Hypoxia; Lymphokines; Sleep Apnea Syndromes; Sleep Apnea, Obstructive; Syndrome; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Acclimatization to moderate high altitude accompanied by training at low altitude (living high-training low) has been shown to improve sea level endurance performance in accomplished, but not elite, runners. Whether elite athletes, who may be closer to the maximal structural and functional adaptive capacity of the respiratory (i.e., oxygen transport from environment to mitochondria) system, may achieve similar performance gains is unclear. To answer this question, we studied 14 elite men and 8 elite women before and after 27 days of living at 2,500 m while performing high-intensity training at 1,250 m. The altitude sojourn began 1 wk after the USA Track and Field National Championships, when the athletes were close to their season's fitness peak. Sea level 3,000-m time trial performance was significantly improved by 1.1% (95% confidence limits 0.3-1.9%). One-third of the athletes achieved personal best times for the distance after the altitude training camp. The improvement in running performance was accompanied by a 3% improvement in maximal oxygen uptake (72.1 +/- 1.5 to 74.4 +/- 1.5 ml x kg(-1) x min(-1)). Circulating erythropoietin levels were near double initial sea level values 20 h after ascent (8.5 +/- 0.5 to 16.2 +/- 1.0 IU/ml). Soluble transferrin receptor levels were significantly elevated on the 19th day at altitude, confirming a stimulation of erythropoiesis (2.1 +/- 0.7 to 2.5 +/- 0.6 microg/ml). Hb concentration measured at sea level increased 1 g/dl over the course of the camp (13.3 +/- 0.2 to 14.3 +/- 0.2 g/dl). We conclude that 4 wk of acclimatization to moderate altitude, accompanied by high-intensity training at low altitude, improves sea level endurance performance even in elite runners. Both the mechanism and magnitude of the effect appear similar to that observed in less accomplished runners, even for athletes who may have achieved near maximal oxygen transport capacity for humans.

Topics: Acclimatization; Adult; Altitude; Erythropoietin; Female; Hematocrit; Hemoglobins; Humans; Hypoxia; Male; Oxygen Consumption; Physical Endurance; Receptors, Transferrin; Running; Transferrin

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. Although HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of its alpha subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF-1 activation in vivo, we determined the spatial and temporal distribution of HIF-1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohistochemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF-1alpha is present in mice kept under normoxic conditions and is further increased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF-1alpha expression varies among different organs. In liver and kidney, HIF-1alpha reaches maximal levels after 1 h and gradually decreases to baseline levels after 4 h of continuous hypoxia. In the brain, however, HIF-1alpha is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF-1beta is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF-1alpha. Overall, HIF-1alpha expression in normoxic mice suggests that HIF-1 has an important role in tissue homeostasis.

Topics: Animals; Brain; Cell Nucleus; Cytoplasm; DNA-Binding Proteins; Dose-Response Relationship, Drug; Erythropoietin; Female; Gene Expression Regulation; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoblotting; Immunohistochemistry; Kidney; Liver; Mice; Mice, Inbred C57BL; Nuclear Proteins; Oxygen; RNA, Messenger; Time Factors; Tissue Distribution; Transcription Factors

This study examined exercise-induced hypoxaemia (EIH) and plasma volume contraction as modulators of serum erythropoietin (Epo) production. Five athletes cycled for 3 min at supra-maximal power outputs, at each of two different elevations (1,000 m and 2,100 m). Five subjects were exposed to normobaric hypoxia (F(I)O(2)=0.159), seven subjects underwent plasmapheresis to reduce plasma volume and eight subjects were time controls for Epo levels. Oxyhaemoglobin saturation was significantly reduced during exercise and during normobaric hypoxia. The time period of haemoglobin oxygen saturation <91% was 24+/-29 s (mean+/-S.D., n=5) for exercise at 1000 m, 136+/-77 s (mean+/-S.D., n=5) for exercise at 2100 m and 178+/-255 s (mean+/-S.D., n=5) with resting hypoxic exposure. However, significantly increased serum Epo levels were observed only following exercise (24+/-3%; mean+/-S.D., n=5 at 1,000 m and 36+/-5%; mean+/-S.D., n=5 at 2,100 m). Volume contraction also resulted in increased serum Epo (35+/-6%; mean+/-S.D., n=7) in spite of a significant rise in haematocrit of 2.2%. Despite similar degrees of arterial desaturation, only the hypoxaemia induced by exercise was associated with an increase in serum Epo. This finding indicates that other factors, in addition to hypoxaemia, are important in modulating the production of Epo in response to exercise. Volume depletion in the absence of exercise resulted in increases in Epo levels that were comparable with those observed in response to exercise. The paradoxical responses of the increased haematocrit and the increase in Epo in subjects undergoing plasmapheresis suggests that plasma volume may also modulate the production of Epo.

Topics: Adult; Case-Control Studies; Erythropoietin; Exercise; Exercise Test; Female; Hematocrit; Humans; Hypoxia; Male; Plasma Volume; Plasmapheresis; Regression Analysis; Statistics, Nonparametric

This study aimed to investigate the role of endogenous nitric oxide (NO) in erythropoietin (EPO) gene expression in mice in vivo. For this purpose EPO mRNA was semiquantitated by ribonuclease protection assay in livers and kidneys of three groups of mice: wild-type (wt), endothelial NO-synthase (NOS) knockout mice (eNOS-/-), and wt treated with the NOS inhibitor N(G)-nitro-L-arginine methyl ester (50 mg x kg(-1) x day(-1)) for 4 days (wt+L-NAME). EPO gene expression was stimulated by normobaric hypoxia (8% O2) or by 0.1% carbon monoxide (CO) inhalation for 4 h each, or by intraperitoneal injection of 60 mg/kg cobaltous chloride (CoCl2) for 6 h. Renal EPO mRNA in wt increased 12-, 40-, and 13-fold over normoxic levels in response to hypoxia, CO and CoCl2 respectively. EPO mRNA was detectable in the livers only after CO exposure. Renal and hepatic EPO gene expression in wt+L-NAME appeared moderately increased relative to wt with a maximal 2.5-fold enhancement after CO exposure. EPO mRNA levels in eNOS-/- mirrored those of wt+L-NAME, but the effects were less prominent. Our data suggest that endogenous NO attenuates EPO gene expression in mice. This effect is dependent on the rate of EPO gene induction.

Topics: Animals; DNA-Binding Proteins; Enzyme Inhibitors; Erythropoietin; Gene Expression Regulation, Enzymologic; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nuclear Proteins; RNA, Messenger; Transcription Factors; Transcriptional Activation

Erythropoietin (Epo), the major hormone controlling the hypoxia-induced increase in the number of erythrocytes, has also a functional role in the brain. However, few data exist as to the cellular source of brain-derived Epo as well as to the molecular mechanisms that control Epo expression in the central nervous system. Using patch-clamp and RT-PCR methods, we provide direct evidence that, besides astrocytes, neurons are a source of Epo in the brain. Both the astrocytic and neuronal expression of Epo mRNA are induced not only by hypoxia, but also by desferrioxamine (DFX) and cobalt chloride (CoCl(2)), two agents known to mimic the hypoxic induction of Epo in hepatoma cells. This induction is blocked by cycloheximide suggesting that de novo protein synthesis is required. Furthermore, the addition of H(2)O(2) decreases the hypoxia-induced Epo mRNA levels. These data indicate that, following hypoxia, a common oxygen sensing and signaling pathway leads to increased Epo gene expression in both nervous and hepatoma cells; this pathway would be dependent on the redox-state of the brain. Furthermore, we show that the in vivo administration of CoCl(2) and DFX to mice induces an increased Epo mRNA level in the neocortex. As Epo protects the brain against ischemia, our in vivo experiments suggest that the use of molecules such as CoCl(2) or DFX, that provoke an increased Epo gene expression in the brain, could be useful in the development of potential therapeutic strategies for the treatment of hypoxic or ischemic brain injury.

Topics: Animals; Astrocytes; Brain; Cells, Cultured; Erythropoietin; Hypoxia; Male; Mice; Mice, Inbred Strains; Neurons; Oxidation-Reduction; Oxygen; Polymerase Chain Reaction; RNA, Messenger; Time Factors

In order to investigate whether vascular endothelial growth factor (VEGF) and inflammatory pathways are activated during acute hypobaric hypoxia in subjects who are susceptible to high-altitude pulmonary oedema (HAPE-S), seven HAPE-S and five control subjects were exposed to simulated altitude corresponding to 4000 m in a hypobaric chamber for 1 day. Peripheral venous blood was taken at 450 m (Zürich level) and at 4000 m, and levels of erythropoietin (EPO), VEGF, interleukin-6 (IL-6) and the acute-phase proteins complement C3 (C3), alpha1-antitrypsin (alpha1AT), transferrin (Tf) and C-reactive protein (CRP) were measured. Peripheral arterial oxygen saturation (SaO2) was recorded. Chest radiography was performed before and immediately after the experiment. EPO increased during altitude exposure, correlating with SaO2, in both groups (r = -0.86, P < 0.001). Venous serum VEGF did not show any elevation despite a marked decrease in SaO2 in the HAPE-S subjects [mean (SD) HAPE-S: 69.6 (9.1)%; controls: 78.7 (5.2)%]. C3 and alpha1AT levels increased in HAPE-S during hypobaric hypoxia [from 0.94 (0.11) g/l to 1.07 (0.13) g/l, and from 1.16 (0.08) g/l to 1.49 (0.27) g/l, respectively; P < 0.05], but remained within the clinical reference ranges. No significant elevations of IL-6, Tf or CRP were observed in either group. The post-exposure chest radiography revealed no signs of oedema. We conclude that VEGF is not up-regulated in HAPE-S and thus does not seem to increase critically pulmonary vascular permeability during the 1st day at high altitude. Furthermore, our data provide evidence against a clinically relevant inflammation in the initial phase of exposure to hypoxia in HAPE-S, although C3 and alpha1AT are mildly induced.

Topics: Acute-Phase Proteins; Acute-Phase Reaction; Adult; Altitude; Atmospheric Pressure; Disease Susceptibility; Endothelial Growth Factors; Erythropoietin; Humans; Hypoxia; Lymphokines; Male; Middle Aged; Pulmonary Edema; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Previously, we showed that erythropoietin (Epo) is produced in the mouse uterus, where Epo is indispensable for estrogen (E(2))-dependent angiogenesis. Expression of uterine Epo mRNA is stimulated by E(2) and hypoxia. The hypoxic induction requires the presence of E(2). In the present study, we examined other female reproductive organs in the mouse with respect to Epo mRNA expression and its stimuli (E(2) and hypoxia)-induced changes. Although Epo mRNA expression was seen in the ovary and oviduct, the E(2)-induced stimulation of Epo mRNA was found only in the oviduct. The E(2)-induced stimulation in the oviduct was transient and rapidly downregulated. Epo mRNA expression in the oviduct was hypoxia inducible, in both the presence and the absence of E(2). E(2)-dependent production of Epo and its mRNA expression were also found by use of cultured oviducts. The E(2) action is probably mediated through the E(2) receptor, and de novo protein synthesis is not required for E(2) induction of Epo mRNA. In the oviduct, the ampulla and isthmus regions produce Epo.

Topics: Animals; Cycloheximide; Dactinomycin; Erythropoietin; Estradiol; Estrogen Antagonists; Fallopian Tubes; Female; Fulvestrant; Gene Expression; Hypoxia; Mice; Mice, Inbred ICR; Organ Culture Techniques; Ovary; Oxygen; Protein Synthesis Inhibitors; RNA, Messenger

This study aimed to determine whether brief hypoxic stimuli in a hypobaric chamber are able to elicit erythropoietin (EPO) secretion, and to effectively stimulate erythropoiesis in the short term. In two different experiments, a set of haematological, biochemical, haemorheological, aerobic performance, and medical tests were performed in two groups of healthy subjects. In the first experiment, the mean plasma concentration of EPO ([EPO]) increased from 8.7 to 13.5 mU.ml-1 (55.2%; P < 0.01) after 90 min of acute exposure at 540 hPa, and continued to rise until a peak was attained 3 h after the termination of hypoxia. In the second experiment, in which subjects were exposed to a simulated altitude of up to 5500 m (504 hPa) for 90 min, three times a week for 3 weeks, all haematological indicators of red cell mass increased significantly, reaching the highest mean values at the end of the programme or during the subsequent 2 weeks, including packed cell volume (from 42.5 to 45.1%; P < 0.01), red blood cell count (from 4.55 x 10(6) to 4.86 x 10(6).l-1; P < 0.01), reticulocytes (from 0.5 to 1.4%; P < 0.01), and haemoglobin concentration (from 14.3 to 16.2 g.dl-1; P < 0.01), without an increase in blood viscosity. Arterial blood oxygen saturation during hypoxia was improved (from 60% to 78%; P < 0.05). Our most relevant finding is the ability to effectively stimulate erythropoiesis through brief intermittent hypoxic stimuli (90 min), in a short period of time (3 weeks), leading to a lower arterial blood desaturation in hypoxia. The proposed mechanism for these haematological and functional adaptations is the repeated triggering effect of EPO production caused by the intermittent hypoxic stimuli.

Topics: Adaptation, Physiological; Adult; Altitude; Atmospheric Pressure; Blood Physiological Phenomena; Erythrocyte Count; Erythrocytes; Erythropoietin; Hematocrit; Hemoglobins; Humans; Hypoxia; Male; Middle Aged; Oxygen; Time Factors

N(G)-monomethyl-L-arginine (L-NMMA) has been reported to be elevated in uremic patients. Based on the hypothesis that the pathogenesis of the anemia of renal disease might be due to the perturbation of transcription factors of the erythropoietin (Epo) gene by L-NMMA, the present study was designed to investigate the effect of L-NMMA on Epo gene expression through the GATA transcription factor. L-NMMA caused decreased levels of NO, cyclic guanosine monophosphate (cGMP), and Epo protein in Hep3B cells. L-NAME (analogue of L-NMMA) also inhibited Epo production in anemic mice. Transfection of the Epo promoter-luciferase gene into Hep3B cells revealed that L-NMMA inhibited the Epo promoter activity. However, L-NMMA did not inhibit the Epo promoter activity when mutated Epo promoter (GATA to TATA) was transfected, and L-NMMA did not affect the enhancer activity. Electrophoretic mobility shift assays demonstrated the stimulation of GATA binding activity by L-NMMA. However, L-NMMA had no effect on the binding activity of hepatic nuclear factor-4, COUP-TF1, hypoxia-inducing factor-1, or NF-kappaB. Furthermore, cGMP inhibited the L-NMMA-induced GATA binding activity. L-NMMA also increased GATA-2 messenger RNA expression. These results demonstrate that L-NMMA suppresses Epo gene expression by up-regulation of the GATA transcription factor and support the hypothesis that L-NMMA is one of the candidate substances that underlie the pathogenesis of renal anemia. (Blood. 2000;96:1716-1722)

Topics: Animals; Base Sequence; Binding Sites; Cyclic GMP; DNA; DNA-Binding Proteins; Enzyme Inhibitors; Erythropoietin; GATA2 Transcription Factor; Gene Expression Regulation; Humans; Hypoxia; Luciferases; Mice; Molecular Sequence Data; Mutation; NG-Nitroarginine Methyl Ester; Nitric Oxide; omega-N-Methylarginine; Promoter Regions, Genetic; Protein Binding; Recombinant Fusion Proteins; Transcription Factors; Tumor Cells, Cultured

Erythropoiesis occurs in the presence of erythropoietin (EPO) without macrophages in vitro. In hematopoietic tissues, however, erythroid cells associate closely with stromal macrophages, forming erythroblastic islands via interactions with adhesion molecules. To elucidate the role of macrophages in erythropoiesis, we selectively abrogated stromal macrophages of splenic red pulp of phlebotomized mice by injection with dichloromethylene diphosphonate encapsulated in multilamellar liposomes (CL2MDP-liposome). In the spleen, no erythropoietic activity occurred until 5 days after the treatment. Colony assay revealed that the erythropoiesis was suppressed at the level of CFU-E. The splenic erythropoietic activity gradually developed from day 6 after the treatment, when F4/80+ macrophages began to appear in the red pulp. EPO mRNA was expressed in kidney but not in liver or spleen of phlebotomized mice injected with CL2MDP-liposome, and the serum EPO concentration in these mice was higher than that in phlebotomized mice. These findings suggest that abrogation of stromal macrophages by injection with CL2MDP-liposome impairs the splenic microenvironment for erythropoiesis induced by hypoxic stress, and this may be an excellent experimental model for further characterization of the in vivo role of splenic macrophages in erythropoiesis.

Topics: Anemia; Animals; Clodronic Acid; Colony-Forming Units Assay; Depression, Chemical; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Hemorrhage; Humans; Hypoxia; Kidney; Liposomes; Liver; Macrophages; Mice; Mice, Inbred BALB C; Phlebotomy; Recombinant Proteins; RNA, Messenger; Spleen; Stromal Cells

Both toxic exposure to cadmium and cancer therapy with cisplatin (CDDP) can induce anemia in patients owing to the insufficient production of erythropoietin (EPO). Therefore, the effects of cadmium chloride (Cd) and CDDP in the Hep3B human hepatoma cell line, which up-regulates EPO expression in response to hypoxia and cobalt (Co), were investigated. The induction of binding activity of the HIF-1 transcription factor and EPO mRNA expression and protein production were suppressed by Cd and CDDP in a dose-dependent manner with no apparent cell damage. Mercuric chloride also suppressed hypoxia- and Co-induced EPO production, mRNA expression, and HIF-1 binding in a manner similar to Cd and CDDP, whereas zinc chloride suppressed Co-induced EPO production, mRNA expression, and HIF-1 binding but did not affect hypoxia induction or that observed after simultaneous exposure to hypoxia and Co. In contrast, lead and tin salts had no effect on HIF-1 activation or EPO expression. These results indicate that Cd and CDDP have a strong and specific inhibitory effect on hypoxia- and Co-induced signaling and EPO induction in hepatic cells. It is likely that these agents cause anemia by directly impacting EPO production in the kidney. (Blood. 2000;96:3743-3747)

Topics: Cadmium; Cell Survival; Cisplatin; Cobalt; DNA-Binding Proteins; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Environmental Exposure; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Mercury; Metals, Heavy; Nuclear Proteins; Protein Binding; RNA, Messenger; Transcription Factors; Tumor Cells, Cultured; Zinc

Topics: Acute Disease; Biomarkers; Erythropoietin; Humans; Hypoxia; Leukemia, Myeloid; Neovascularization, Pathologic

The involvement of proximal kidney tubules in erythropoietin production and/or in its control has been a subject of controversy among researchers. The present study addresses the problem whether acute tubular impairment affects erythropoietin production and thus alters the basic hematological parameters. For this purpose rats with hypobaric hypoxia-stimulated (42.55 kPa) erythropoesis were studied and the rat model of gentamicin-induced (50 mg/kg daily for 15 days) tubular damage was employed. According to the study protocol 4 different groups of rats were used: control rats, rats exposed to hypobaric hypoxia, rats treated with gentamicin prior to hypoxic exposure and finally rats given a dose of 50 U human recombinant erythropoietin for 2 days following treatment with gentamicin. The evaluated hematological parameters included: hemoglobin and hematocrit levels, reticulocyte count and plasma creatinine concentration. Histological analysis of kidney sections was also used. INCSTAR (USA) immunoassay was employed to determine plasma erythropoietin level. Evidence (histological and laboratory-elevated plasma creatinine) of distinct tubular lesions was found in rats injected with gentamicin. Hemoglobin and hematocrit levels were decreased in rats treated with gentamicin but a rise in reticulocyte count was seen on day 3 after hypoxic exposure--39@1000 +/- 10 vs. 5@1000 +/- 1.3 on day 0 (p < 0.001). Reduced reticulocyte counts on day 0 were not seen only in rats injected with human recombinant erythropoietin--36.33@1000 (p < 0.01). Hypoxia-induced increase in plasma erythropoietin was inhibited in animals treated with gentamicin--15.3 mU/ml (on day 0). Both hypoxic exposure and erythropoietin administration were associated with a rise in plasma erythropoietin on day 0--40.5 +/- 4.4 mU/ml and 42.7 +/- 4.1 mU/ml respectively, which normalised on day 3. The results of the study support the hypothesis that the erythropoietin response in rats after stimulation is related to the functional condition of the proximal kidney tubules.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoiesis; Erythropoietin; Gentamicins; Hematologic Tests; Hypoxia; Kidney Tubular Necrosis, Acute; Kidney Tubules, Proximal; Male; Rats; Rats, Wistar; Recombinant Proteins

The most important stimulus for the enhanced synthesis of erythropoietin (Epo) is a lowered O2 tension in the tissue. However, the mechanism by which an impaired O2 supply is transduced into appropriate Epo production is still not fully understood. Recently, studies in human hepatoma cells (line HepG2) indicate that reactive O2 species are involved in the signal transduction from the cellular O2 sensor to the Epo gene. To clarify the role of reactive O2 species in the regulation of Epo synthesis in the kidney, the principal Epo-producing organ in vivo, we investigated the influence of potent pro- and antioxidants on Epo production in isolated perfused rat kidneys. Under normoxic conditions, the iron chelator desferrioxamine and the antioxidant vitamin A increased renal Epo production, mimicking hypoxic induction. In contrast, supplementation of the perfusion medium of hypoxically perfused kidneys with the prooxidant compounds H2O2 or pyrogallol caused a significant reduction of Epo synthesis. The inhibition of Epo formation by reactive O2 species could be completely antagonized by desferrioxamine and the hydroxyl radical-(OH*)-scavenger tetramethylthiourea. Vitamin A also antagonized the H2O2-dependent inhibition of hypoxically induced Epo synthesis. Interestingly, the addition of the antioxidant vitamin A to hypoxically perfused kidneys also induced Epo production significantly. Our data strongly support the idea that reactive O2 species, especially H2O2, are part of the signaling chain of the cellular O2-sensing mechanism regulating the renal synthesis of Epo.

Topics: Animals; Antioxidants; Chelating Agents; Deferoxamine; Erythropoietin; Free Radical Scavengers; Hydrogen Peroxide; Hypoxia; In Vitro Techniques; Kidney; Male; Oxidants; Pyrogallol; Rats; Rats, Sprague-Dawley; Reference Values; RNA, Messenger; Thiourea; Vitamin A

Topics: Erythropoietin; Hemoglobinometry; Hemoglobins; Humans; Hypoxia; Neoplasms; Oxygen

We designed our study to explore how the inhibition of prostaglandins (PGs) could affect erythropoiesis in bone marrow erythroblastic islands (EIs). To this end, we used hypoxic-stimulated rats-hypobaric hypoxia (42.55 kPa/6 h)-pretreated or not with indomethacin (4 mg/kg/3 days). Blood sampling was done at 0 h, 24 h, and 72 h after hypoxia. The study included estimations of the plasma erythropoietin (EPO) level (by radioimmunoassay), peripheral blood, number of EI from classes I to V per femur, rate of immature cell's differentiation into erythroblasts, and rate of repeated participation of macrophages in new EI reconstruction. Plasma EPO rose significantly (p < 0.01) in all hypoxic rats: 40.5+/-10.15 mU/ml and 46.75+/-16.28 mU/ml and at 0 h versus 13.83+/-6.82 mU/ml in controls. An increased rate of cell differentiation into erythroblasts in EIs (p < 0.01), an enhanced reconstruction in involuted EIs, and a reduced number of maturing EIs (p < 0.01) were observed in all hypoxic animals. However, in indomethacin-pretreated rats, the stimulation of bone marrow erythropoiesis was better expressed. Our results favor the concept that PG inhibition does not attenuate the erythropoietic response to hypoxia and support the hypothesis about the important role of EI macrophages as a local regulator of bone marrow erythropoiesis.

Topics: Animals; Bone Marrow; Cyclooxygenase Inhibitors; Erythroblasts; Erythropoiesis; Erythropoietin; Hypoxia; Indomethacin; Male; Rats; Rats, Wistar; Reticulocyte Count

The aim of this study was to determine if the hypoxaemic stimulus generated by intense exercise results in the physiological response of increased erythropoietin production. Twenty athletes exercised for 3 min at 109 +/- 2.8% (mean +/- s) maximal oxygen consumption. Estimated oxyhaemoglobin saturation was measured by reflective probe pulse oximetry (Nellcor N200) and was validated against arterial oxyhaemoglobin saturation by CO-oximetry in eight athletes. Serum erythropoietin concentrations-as measured using the INCSTAR Epo-Trac radioimmunoassay-increased significantly by 28 +/- 9% at 24 h post-exercise in 11 participants, who also had an arterial oxyhaemoglobin saturation < or = 91% (P < 0.05). Decreased ferritin levels and increased reticulocyte counts were observed at 96 h post-exercise. However, no significant changes in erythropoietin levels were observed in nine non-desaturating athletes and eight non-exercise controls. Good agreement was shown between arterial oxyhaemoglobin saturation and percent estimated oxyhaemoglobin saturation (limits of agreement = -3.9 to 3.7%). In conclusion, short supramaximal exercise can induce both hypoxaemia and increased erythropoietin levels in well-trained individuals. The decline of arterial hypoxaemia levels below 91% during exercise appears to be necessary for the exercise-induced elevation of serum erythropoietin levels. Furthermore, reflective probe pulse oximetry was found to be a valid predictor of percent arterial oxyhaemoglobin saturation during supramaximal exercise when percent estimated oxyhaemoglobin saturation > or = 86%.

Topics: Adult; Erythropoietin; Exercise; Exercise Test; Humans; Hypoxia; Lactic Acid; Male; Oximetry; Oxygen; Oxyhemoglobins

The glucocorticoid receptor (GR) coordinates a multitude of physiological responses in vivo. In vitro, glucocorticoids are required for sustained proliferation of erythroid progenitors (ebls). Here, we analyze the impact of the GR on erythropoiesis in vivo, using GR-deficient mice or mice expressing a GR defective for transactivation. In vitro, sustained proliferation of primary ebls requires an intact GR. In vivo, the GR is required for rapid expansion of ebls under stress situations like erythrolysis or hypoxia. A particular, GR-sensitive progenitor could be identified as being responsible for the stress response. Thus, GR-mediated regulation of ebl proliferation is essential for stress erythropoiesis in vivo.

Topics: Anemia; Animals; Cells, Cultured; Chickens; Culture Media, Serum-Free; Dimerization; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Hematopoiesis, Extramedullary; Hematopoietic Stem Cell Transplantation; Hemolysis; Hypoxia; Leukemia, Erythroblastic, Acute; Liver; Mice; Mice, Knockout; Radiation Chimera; Receptors, Glucocorticoid; Stem Cell Factor; Stress, Physiological; Transcriptional Activation

Topics: Animals; Erythrocytes; Erythropoietin; Heating; Heme; Hemin; Hypoxia; Kidney Cortex; Male; Metalloporphyrins; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Protoporphyrins; RNA, Messenger

Inflammation and hypoxia are frequently associated, but their interaction is poorly understood. In vitro studies have shown that hypoxia stimulates the genes of acute phase proteins (APP) and cytokines known to induce APP. We decided to determine kinetics and potential determinants of an acute phase response after cardiac arrest and to assess whether isolated moderate hypoxia can induce APP in humans in vivo.. Prospective, observational study in patients and human experiment.. Tertiary care university hospital.. 22 patients after primarily successful cardiopulmonary resuscitation (CPR) and 7 healthy volunteers.. None in patients; exposure of volunteers to simulated altitude (460 torr/6 h).. Following CPR, type-1 APP (C-reactive protein, alpha 1-acidglycoprotein, serum amyloid A) and type-2 APP (haptoglobin, alpha 1-antitrypsin) increased consistently within 1-2 days and the 'negative' APP transferrin was downregulated. This APP response occurred irrespective of the cause of arrest, the estimated time of anoxia, clinical course or patient outcome and was not different in patients with and without infectious complications. Exposure of healthy volunteers to less severe but more prolonged hypoxia did not induce APP, although a time dependent increase of serum erythropoietin (EPO) was measurable under these conditions, indicating the activation of oxygen dependent gene expression.. (i) A marked acute phase response occurs regularly after cardiac arrest, but within the complexity of this situation the severity of hypoxia is not a predominant determinant of this response. (ii) Despite in vitro evidence for similarities in the oxygen dependent regulation of APP and EPO production, the oxygen sensitivity of these proteins in vivo is different. (iii) Measurements of APP are not revealing regarding infectious complications in the early phase after CPR.

Topics: Acute-Phase Proteins; Acute-Phase Reaction; Adolescent; Adult; Aged; Cardiopulmonary Resuscitation; Erythropoietin; Female; Heart Arrest; Hemoglobins; Humans; Hypoxia; Inflammation; Kinetics; Male; Middle Aged; Prospective Studies

To examine whether perinatal hypoxia increases the risk of occurrence of hypoglycaemia--between first and second hour of life--in newborn of the diabetic mother.. The study material consisted of 151 newborns born to 58 pregestational and 93 gestational diabetes mothers. The occurrence of hypoglycaemia was examined in accordance with some perinatal hypoxia indicators such as: 1 and 5 minutes Apgar scores, umbilical arterial blood gas analysis and cord blood erythropoietin (EPO) level.. Newborns of the diabetic mothers in whom hypoglycaemia was recognised had lower 1 minutes Apgar scores, lower pH values, higher pCO2 values and higher EPO levels than those, in whom normoglycaemia was recognised.. Low 1 minutes Apgar scores and occurrence of even mild perinatal hypoxia are factors increasing the risk of hypoglycaemia in the group of newborns of the diabetic mothers in the time between first and second hour of life.

Topics: Adult; Blood Gas Analysis; Diabetes, Gestational; Erythropoietin; Female; Fetal Blood; Humans; Hypoglycemia; Hypoxia; Infant, Newborn; Mothers; Pregnancy; Risk Factors

We examined erythropoietin (EPO) gene expression and EPO production during hypoxia in two Sprague-Dawley rat strains with divergent polycythemic responses to hypoxia. Hilltop (H) rats develop severe polycythemia, severe hypoxemia, and pulmonary artery hypertension. The H rats often die from a syndrome indistinguishable from chronic mountain sickness (CMS) in humans. Madison (M) rats develop polycythemia and pulmonary artery hypertension that is modest and suffer no excess mortality. We tested the hypothesis that these rat strains have different stimulus-response characteristics governing EPO production. Rats of each strain were exposed to hypoxia (0.5 atm, 73 Torr inspired PO2), and renal tissue EPO mRNA and EPO levels, plasma EPO, ventilation, arterial and renal venous blood gases, and indexes of renal function were measured at fixed times during a 30-day hypoxic exposure. During extended hypoxic exposure, H rats had significantly elevated renal EPO mRNA, renal EPO, and plasma EPO levels compared with M rats. Ventilatory responses and indexes of renal function were similar in the strains during the hypoxic exposure. H rats had greater arterial hypoxemia from the onset of hypoxia and more severe renal tissue hypoxemia and greater polycythemia after 14 days of hypoxic exposure. When EPO responses were expressed as functions of renal venous PO2, the two strains appeared to lie on the same dose-response curves, but the responses of H rats were shifted along the curve toward more hypoxic values. We conclude that H rats have significantly greater polycythemia secondary to poorer renal tissue oxygenation, but the stimulus-response characteristics governing EPO gene expression and EPO production do not seem to differ between M and H rats. Finally, the regulation of EPO levels during hypoxia occurs primarily at the transcriptional level.

Topics: Altitude Sickness; Animals; Blood Gas Analysis; Blotting, Northern; Chronic Disease; Cobalt; Erythropoietin; Feedback; Gene Expression Regulation; Hypoxia; Kidney; Kidney Function Tests; Male; Polycythemia; Rats; Rats, Sprague-Dawley; Ribonucleases

When humans ascend to high altitude (ALT) their plasma volume (PV) and total blood volume (BV) decrease during the first few days. With continued residence over several weeks, the hypoxia-induced stimulation of erythropoietin increases red cell production which tends to restore BV. Because hypoxia also activates the beta-adrenergic system, which stimulates red blood cell production, we investigated the effect of adrenergic beta-receptor inhibition with propranolol on fluid volumes and the polycythemic response in 11 healthy unacclimatized men (21-33 years old exposed to an ALT of 4300 m (barometric pressure 460 Torr) for 3 weeks on Pikes Peak, Colorado. PV was determined by the Evans blue dye method (PVEB), BV by the carbon monoxide method (BVCO), red cell volume (RCV) was calculated from hematocrit (Hct) and BVCO, and serum erythropoietin concentration ([EPO]) and reticulocyte count, were also determined. All determinations were made at sea level and after 9-11 (ALT-10) and 19-20 (ALT-20) days at ALT. At sea level and ALT, six men received propranolol (pro, 240 mg x day[-1]), and five received a placebo (pla). Effective beta-blockade did not modify the mean (SE) maximal values of [EPO] [pla: 24.9 (3.5) vs pro: 24.5 (1.5) mU x ml(-1)] or reticulocyte count [pla: 2.7 (0.7) vs pro: 2.2 (0.5)%]; nor changes in PVEB [pla: -15.8 (3.8) vs pro: -19.9 (2.8)%], RCVCO [pla: +7.0 (6.7) vs pro: + 10.1 (6.1)%], or BVCO [pla: -7.3 (2.3) vs pro: -7.1 (3.9)%]. In the absence of weight loss, a redistribution of body water with no net loss is implied. Hence, activation of the beta-adrenergic system did not appear to affect the hypovolemic or polycythemic responses that occurred during 3 weeks at 4300 m ALT in these subjects.

Topics: Adrenergic beta-Antagonists; Adult; Altitude; Blood Volume; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Male; Plasma Volume; Polycythemia; Propranolol

Erythropoietin (EPO) is the primary regulator of mammalian erythropoiesis, providing a proliferative and differentiative signal to the early EPO-responsive erythroid progenitors, burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid, as well as to later EPO-responsive erythroid progenitors. EPO is secreted by the kidney in response to hypoxia and anemia. There is an extensive biological crossreactivity between human EPO and the EPOs of other mammals. Necas et al. have reported that this crossreactivity may not include the guinea pig (Cavia porcelllus). Because the specificity of the guinea pig's erythropoietic responses may be of biological significance, we compared guinea pig hypoxic serum with mouse (m) and human (h) recombinant (r) EPOs for their ability to induce erythroid progenitor proliferation and differentiation in semisolid cultures. Guinea pig bone marrow mononuclear cells (BMMCs) formed BFU-E colonies in response to guinea pig hypoxic serum, rhEPO, or rmEPO in a dose-dependent fashion. Neither human nor mouse BMMCs responded to guinea pig hypoxic serum; however, guinea pig hypoxic serum exerted no inhibitory effect on human or mouse in vitro erythroid differentiation in the presence of rhEPO or rmEPO. The intensity of the EPO band on Western blotting analysis of guinea pig hypoxic serum was significantly greater than in nonhypoxic serum. This suggests that guinea pig erythropoiesis is mediated by EPO and stimulated by hypoxia in a fashion similar to that observed in human and mouse erythropoiesis. Furthermore, guinea pig EPO did not stimulate human or mouse erythroid differentiation in vitro, whereas guinea pig erythroid progenitors could be stimulated by human or mouse EPO, suggesting structural differences in guinea pig EPO and EPO receptor (EPOR) compared with human or mouse EPO and EPOR. These differences probably evolved after the guinea pig's ancestors diverged from myomorph rodents. Further characterization of the guinea pig EPO and EPOR should facilitate our understanding of the interaction between EPO and EPOR.

Topics: Animals; Antibodies; Blotting, Western; Cells, Cultured; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Guinea Pigs; Humans; Hypoxia; Mammals; Mice; Phylogeny; Receptors, Erythropoietin; Recombinant Proteins; Species Specificity

Erythropoietin (Epo) synthesis is suppressed in normoxia and stimulated in hypoxia. To test the hypothesis that the cellular H2O2 level is important in the control of Epo synthesis, we have studied effects of modulators of H2O2 generation and degradation on Epo production in human hepatic cell cultures (hepatoma lines HepG2 and Hep3B). In addition, we measured the activities of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase) in cultures following hypoxia exposure or H2O2 treatment. The results show that the formation of immunoreactive Epo was stimulated in normoxic cultures by treatment with exogenous catalase thus mimicking the effect of hypoxia (24 h incubation periods). Epo production was also stimulated when scavengers of reactive O2 species (tetramethylthiourea, dihydrorhodamine) were added to the cells. On the other hand, stimulators of H2O2 generation (xanthine oxidase, glucose oxidase, NADH, NADPH) lowered Epo production in hypoxic cultures. Hypoxia exposure decreased superoxide dismutase activity and H2O2 treatment reduced catalase activity thus influencing the endogenous antioxidant defense system. These findings support the concept that reactive O2 species, primarily H2O2, act as messengers in the O2-dependent control of the hepatic production of Epo. Changes in the cellular activities of antioxidant enzymes appear to play only a minor role in this process.

Topics: Carcinoma, Hepatocellular; Catalase; Erythropoietin; Free Radical Scavengers; Glutathione Peroxidase; Humans; Hydrogen Peroxide; Hypoxia; Reactive Oxygen Species; Superoxide Dismutase; Tumor Cells, Cultured

Keeping in mind the renal origin of erythropoietin (EPO), we designed our study in order to estimate the role of kidney damage in the development of bone marrow erythropoiesis in erythroblastic islands (EI). The experiment was performed comparing intact rats with untreated and gentamicin-pretreated rats (50 mg/kg/15 days) exposed to hypobaric pressure (42.55 kPa/6 h) to stimulate hypoxia. Blood samples were taken following a 2-week period. The study included an estimation of plasma EPO levels by RIA, the number of peripheral blood parameters and bone marrow EI (classes I to V/femur), the rate of erythroid differentiation into erythroblasts, and the rate of repeated participation of macrophages in new EI reconstruction. Plasma EPO increased to 52.88 mU/ml (p < 0.01) and 23.45 mU/ml at 0 h immediately following hypobaric exposure in untreated and gentamicin-treated rats, respectively, as compared to 14.25 mU/ml in intact animals. Bone marrow recovery patterns were markedly expressed in untreated hypoxic animals throughout the observed period. The rate of erythroid differentiation into erythroblasts in EI was increased (p < 0.01) while the number of maturing EI decreased (p < 0.01); increased reconstruction was observed in involuted EI. Less pronounced stimulation of erythropoiesis was observed in hypoxic gentamicin-treated rats. The direct impact of the hypoxic stimulus on the erythropoietic bone marrow tissue was considered significant for the erythropoietic response via activation of macrophages. These data support the hypothesis that EI central macrophages play an important role as local regulators of bone marrow erythropoiesis.

Topics: Animals; Bone Marrow; Creatinine; Erythroblasts; Erythropoietin; Gentamicins; Hypoxia; Kidney Diseases; Kidney Tubules; Linear Models; Male; Rats; Rats, Wistar

Although a great deal of evidence supports the hypothesis that plasma erythropoietin (EPO) levels of mammals are related to the oxygen supply to the tissues relative to their oxygen needs, several observation millitate against its inherent simplicity. This study presents our results obtained from in vivo experiments that suggest that hypoxia-dependent EPO production can be altered by conditions which apparently do not modify the tissue oxygen supply/demand ratio. Hypoxia-dependent EPO production rate (EPO-PR), derived from plasma EPO titers and plasma EPO half-lives, were estimated in both transfused-polycythemic and normocythemic mouse models subjected to different treatments. From calculations of the O2 carrying capacity of blood and body O2 consumption, it was assumed that the tissue supply/demand ratios were similar in both experimental and control mice of the same model at the time of induction of EPO production. The following observations were worth noting: (1) EPO-PRs in transfused polycythemic mice whose erythropoietic rates were stimulated by intermittent exposure to hypobaria (0.5 atm, 18 hr/day x 3 weeks), phenylhydrazine administration (40 mg/kg at weekly intervals x 3 weeks) or repeated rh-EPO injections (1500 U/kg 3 times a week x 3 weeks) before transfusion were more than five times high than in comparabily polycythemic mice whose erythropoietic rates were not stimulated previously; and (2) EPO-PR in response to hypobaric hypoxia was 2.08 times normal in normocythemic mice with cyclophosphamide (100 mg/kg) induced depression of erythropoiesis, and 0.33 times normal in normocythemic mice with rh-EPO (400 U/kg x 2) induced enhancement of erythropoiesis. Although the results obtained in polycythemic mice are difficult to explain, those from normocythemic mice suggest the existence of a feedback mechanism between EPO-responsive cells and EPO-producing cells. Both demonstrate the existence of experimental conditions in which modulation of the hypoxia-dependent expression of the EPO gene appears to occur. This modulation would be dependent on factors other than oxygen.

Topics: Animals; Blood Transfusion; Erythropoiesis; Erythropoietin; Female; Half-Life; Hypoxia; Mice; Oxygen Consumption; Polycythemia; Recombinant Proteins

There is accumulating evidence from in vitro studies suggesting that the genes of endothelin-1, PDGF, and VEGF are, like the erythropoietin gene, regulated by oxygen tension and by divalent cations. Hypoxia-induced stimulation of, such as endothelin-1, PDGF or VEGF might be involved in the pathogenesis of acute or chronic renal failure, and in renal "inflammatory" diseases (glomerulonephritis, vasculitis, allograft rejection). Hypoxia (8% O2) for six hours caused a 55-fold/1.6-fold increase of renal erythropoietin/endothelin-1 gene expression, whereas endothelin-3, PDGF-A, PDGF-B, and VEGF gene expression was unchanged. Carbon monoxide (0.1%) treatment for six hours stimulated renal erythropoietin gene expression 140-fold; however, endothelin-1, endothelin-3, PDGF-A, PDGF-B, and VEGF gene expression was not affected. Finally, cobalt treatment (60 mg/kg CoCl2) increased only renal erythropoietin/PDGF-B gene expression 5-fold/1.65-fold. These findings suggest that hypoxia is a rather weak stimulus for renal endothelin-1 gene expression, and that renal PDGF and VEGF gene expression in vivo is not sensitive to tissue hypoxia, in contrast to cell culture experiments. The in vivo regulation of endothelin-1, PDGF, and VEGF differs substantially from that of erythropoietin, suggesting that the basic gene regulatory mechanisms may not be the same.

Topics: Animals; Endothelial Growth Factors; Endothelin-1; Endothelin-3; Erythropoietin; Gene Expression; Growth Substances; Hypoxia; Kidney; Kidney Diseases; Lymphokines; Male; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; RNA, Messenger; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

This study examined the expression of EPO, VEGF and VEGF receptor gene under conditions of reduced oxygen supply in primary cultures of rat hepatocytes, and compared it with the expression of these genes in hypoxic rat livers in vivo. To this end we exposed male Sprague-Dawley rats to hypoxia (10% and 8% O2), carbon monoxide (0.1% CO) or injected cobalt chloride (60 mg/kg CoCl2) subcutaneously. For the in vitro experiments we used primary cultures of rat hepatocytes which were kept at high (20% O2) and low (1% O2) oxygen tensions for three hours. The EPO mRNA was up-regulated by hypoxia in vitro and in vivo about 10-fold. The VEGF mRNA was up-regulated fivefold in the hepatocytes only, whereas the in vivo mRNA levels remained unchanged. The mRNA levels of flt-1 were up-regulated threefold by 8% O2 in livers, dependent on the strength of hypoxia (10% caused no changes in flt-1 gene expression) and on the kind of hypoxic stimulus (8% O2 was as effective as 0.1% CO and more effective than cobalt). The mRNA levels of flk-1/KDR and flt-4 remained unchanged in the liver. In vitro there were no changes in the mRNA levels of flt-1, flt-4 and flk-1/KDR. Consequently, the in vivo regulation of VEGF, which might be modulated by induction of flt-1 receptor gene expression, differs from the in vitro cell culture situation and might be different from the EPO regulation in vivo.

Topics: Animals; Base Sequence; Cell Hypoxia; DNA Primers; Endothelial Growth Factors; Erythropoietin; Gene Expression Regulation; Hypoxia; In Vitro Techniques; Liver; Lymphokines; Male; Molecular Sequence Data; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Receptor Protein-Tyrosine Kinases; Receptors, Cell Surface; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; RNA, Messenger; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-3; Vascular Endothelial Growth Factors

An important role in O2 sensing has been assigned to microsomal and membrane-bound b-type cytochromes which generate regulatory reactive O2 species (ROS). Recently, ROS have been shown to suppress the in vitro synthesis of erythropoietin (Epo). We investigated the potential of the antioxidant vitamins A, E and C to enhance renal and hepatic Epo production. Renal effects were studied in isolated serum-free perfused rat kidneys. In control experiments without antioxidant vitamins, Epo secretion amounted to 441 +/- 23 mU/g kidney (mean +/- SEM, N = 5) during the three hour period of hypoxic perfusion (arterial pO2 35 mm Hg). Epo secretion significantly increased to 674 +/- 92 mU/g kidney (N = 7) when vitamins A (0.5 microgram/ml), E (0.5 microgram/ml) and C (10 micrograms/ml) in combination were added to the perfusion medium. The effects of the single vitamins were studied in Epo-producing hepatoma cell cultures (lines HepG2 and Hep3B). Vitamin A induced a dose-dependent increase (half-maximal stimulation at 0.2 microgram/ml) in the production of immunoreactive Epo during 24 hours of incubation (such as 680 +/- 51 U Epo/g cell protein in HepG2 cultures with 3 micrograms/ml retinol acetate compared to 261 +/- 15 U/g in untreated controls; N = 4). In contrast, vitamin E (tested from 0.05 to 500 micrograms/ml) and vitamin C (tested from 2 to 200 micrograms/ml) did not increase Epo production in hepatoma cell cultures. Thus, while vitamins E and C may have the potential to protect cells from oxidative damage, vitamin A exerts a specific stimulation of Epo production. Preliminary evidence suggests that this effect of vitamin A involves increased mRNA levels of hypoxia-inducible factor 1 alpha (HIF-1 alpha).

Topics: Animals; Antioxidants; Ascorbic Acid; Cell Line; DNA-Binding Proteins; Erythropoietin; Gene Expression; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; In Vitro Techniques; Kidney; Liver; Male; Nuclear Proteins; Perfusion; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transcription Factors; Vitamin A; Vitamin E; Vitamins

This study sought to investigate the role of immediate early genes in the stimulation of erythropoietin (EPO) gene expression by hypoxia. To this end freshly isolated rat hepatocytes were exposed to either normoxia (20% oxygen) or to hypoxia (1% oxygen) and the mRNA levels of the early genes c-fos, c-jun, c-myc and EGR-1 were monitored together with EPO mRNA. Isolation of the cells from the livers strongly stimulated the expression of c-fos, c-jun, c-myc and of EGR-1, whilst EPO gene expression remained unchanged. Exposure of the isolated hepatocytes to hypoxia did not further change early gene expression when compared with cells kept under normoxic conditions. EPO mRNA increased time dependently with a delay of 1 h after onset of hypoxia. These findings suggest that even strong activation of early gene expression has no influence on EPO gene expression, whilst activation of EPO gene expression during hypoxia can happen without change of early gene expression. It appears, therefore, as if immediate early genes are not causally involved in the sequence of events by which hypoxia stimulates EPO gene expression.

Topics: Animals; Erythropoietin; Gene Expression; Genes, Immediate-Early; Hypoxia; In Vitro Techniques; Liver; Male; Oxygen; Rats; Rats, Sprague-Dawley; Ribonucleases; RNA, Messenger

Regulated expression of the erythropoietin (EPO) gene in the adult kidney plays a key role in the regulation of erythropoiesis. However, uncertainty exists regarding the type of kidney cells involved in EPO gene expression. We previously showed by light microscopy that the lacZ reporter gene is expressed and inducible by hypoxia/anemia in the proximal convoluted tubular (PCT) cells of the kidneys of transgenic mice carrying the 5'-lacZ construct, in which the lacZ gene was placed downstream of a 7.0-kb mouse EPO gene segment containing 6.5 kb of the 5'-flanking sequence. We, report here the light and transmission electron microscopic examination of lacZ expression in the kidneys of transgenic mice carrying the 5'-lacZ construct and two additional constructs carrying the 6.5-kb 5'-flanking sequence with the body of the gene alone, or along with the 1.2-kb 3'-flanking sequence. The electron microscopic analyses unequivocally demonstrated that lacZ under the regulatory control of the 6.5-kb 5'-flanking sequence with or without the body of the gene and the 1.2-kb 3'-flanking sequence was expressed predominantly in the proximal convoluted tubular cells of the kidney following hypoxia induction.

Topics: Anemia; Animals; Chimera; Erythropoietin; Gene Expression Regulation; Hypoxia; Kidney Tubules, Proximal; Lac Operon; Mice; Mice, Transgenic; Microscopy, Electron

Hypoxia-inducible factor 1 (HIF-1) binds to cis-acting hypoxia-response elements within the erythropoietin, vascular endothelial growth factor, and other genes to activate transcription in hypoxic cells. HIF-1 is a basic helix-loop-helix transcription factor composed of HIF-1alpha and HIF-1beta subunits. Here, we demonstrate that HIF-1alpha contains two transactivation domains located between amino acids 531 and 826. When expressed as GAL4 fusion proteins, the transcriptional activity of these domains increased in response to hypoxia. Fusion protein levels were unaffected by changes in cellular O2 tension. Two minimal transactivation domains were localized to amino acid residues 531-575 and 786-826. The transcriptional activation domains were separated by amino acid sequences that inhibited transactivation. Deletion analysis demonstrated that the gradual removal of inhibitory domain sequences (amino acids 576-785) was associated with progressively increased transcriptional activity of the fusion proteins, especially in cells cultured at 20% O2. Transcriptional activity of GAL4/HIF-1alpha fusion proteins was increased in cells exposed to 1% O2, cobalt chloride, or desferrioxamine, each of which also increased levels of endogenous HIF-1alpha protein but did not affect fusion protein levels. These results indicate that increased transcriptional activity mediated by HIF-1 in hypoxic cells results from both increased HIF-1alpha protein levels and increased activity of HIF-1alpha transactivation domains.

Topics: Animals; COS Cells; DNA-Binding Proteins; Endothelial Growth Factors; Erythropoietin; Genes, Reporter; Helix-Loop-Helix Motifs; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphokines; Nuclear Proteins; Oxygen; Peptide Fragments; Transcription Factors; Transcriptional Activation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

This study aimed to examine the influence of acute tissue hypoxygenation on the expression of immediate early genes in different rat tissues. To this end male Sprague-Dawley rats were exposed to 0.1% carbon monoxide for 0.5, 1 and 6 h or to 9% oxygen for 6 h and mRNA levels for c-jun, c-fos, c-myc and EGR-1 were assayed by RNase protection in hearts, kidneys, livers and lungs. We found that hypoxia increased c-jun mRNA levels between twofold (lung) and eightfold (liver) in all organs examined; c-fos mRNA increased between three-fold (lung) and 20-fold (heart); c-myc mRNA increased between twofold (lung) and sixfold (heart); and EGR-1 mRNA increased between twofold (lung) and sixfold (heart). Our findings suggest that acute tissue hypoxygenation is a general stimulus of the expression of immediate early genes in vivo. With regard to the sensitivity to hypoxia, organ differences appear to exist in that the lung is rather insensitive, whilst the heart is rather sensitive.

Topics: Animals; Carbon Monoxide; DNA-Binding Proteins; Early Growth Response Protein 1; Erythropoietin; Gene Expression Regulation; Genes, fos; Genes, Immediate-Early; Genes, jun; Hypoxia; Immediate-Early Proteins; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tissue Distribution; Transcription Factors

This in vivo study investigated whether adenosine (ADO) plays a role in oxygen-dependent production of erythropoietin (EPO). Exposure of rats to 0.075% carbon monoxide (CO) for four hours was used as a stimulus for EPO production. To inhibit potential effects of ADO, rats were treated with the non-specific ADO antagonist theophylline, the selective ADO A1 receptor blockers DPCPX and KW-3902, the selective ADO A2 receptor blocker DMPX, and AOPCP, an inhibitor of 5'-ectonucleotidase, an ADO generating enzyme that is expressed on the surface of EPO producing cells. To stimulate ADO receptor activity, animals were treated with the selective ADO A1 and A2 receptor agonists CHA and CGS 21680, the ADO reuptake inhibitors dipyridamole and soluflazine and the ADO desaminase inhibitor EHNA. At doses known to interfere with ADO signal transmission in vivo, none of these substances either influenced EPO serum levels in normoxic rats or affected the approximately 30-fold rise in EPO serum levels and the increase in renal EPO mRNA after exposure to carbon monoxide. Continuous administration of theophylline to normoxic rats for seven days did not alter hematocrit, hemoglobin or EPO serum levels. Taken together, these experiments do not support the hypothesis that ADO plays an important role in the regulation of EPO production.

Topics: Adenine; Adenosine; Adenosine Diphosphate; Animals; Antihypertensive Agents; Carbon Monoxide; Dipyridamole; Diuretics; Drug Synergism; Enzyme Inhibitors; Erythropoietin; Gene Expression; Hypoxia; Kidney; Male; Oxygen; Phenethylamines; Phosphodiesterase Inhibitors; Piperazines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P1; RNA, Messenger; Theophylline; Vasodilator Agents; Xanthines

This study aimed to investigate the influence of acute tissue hypoxygenation on the expression of NO synthase (NOS) genes in vivo. To this end, male Sprague-Dawley rats were exposed either to 9% oxygen or to 0.1% carbon monoxide for 6 h, and mRNA levels of NOS-I, -II, and -III in kidneys, livers, lungs, and left and right heart ventricles were assayed by ribonuclease protection. For comparison, mRNA levels of erythropoietin were also measured in these tissues. NOS-III mRNA was highly abundant in all organs investigated. NOS-II mRNA was detected in lungs and hearts but not in kidneys and livers. NOS-I mRNA was found in kidneys, lungs, and hearts but not in livers. NOS-III mRNA levels were upregulated by hypoxia in all tissues examined, with the least effect (1.2-fold) in the left ventricle and the greatest effect (2.6-fold) in the lung. NOS-II mRNA was substantially downregulated in the ventricles by both treatments but not changed in the lung. NOS-I mRNA was upregulated by carbon monoxide in kidneys and lungs and by 9% oxygen in the lung. These findings suggest that NOS-III and possibly also NOS-I gene expression behave like oxygen-regulated genes, whereas the general effect of tissue hypoxygenation on NOS-II gene expression is less clear. Because NOS-III is primarily expressed in endothelial cells, a general upregulation of NOS in these cells may be of relevance for the regulation and maintenance of blood flow through hypoxic tissues.

Topics: Actins; Animals; Carbon Monoxide; Erythropoietin; Gene Expression Regulation, Enzymologic; Hypoxia; Kidney; Liver; Lung; Male; Myocardium; Nitric Oxide Synthase; Organ Specificity; Oxygen; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transcription, Genetic

Proinflammatory cytokines play an important role in the pathogenesis of anemia in inflammatory diseases. Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) have been reported to inhibit the synthesis of erythropoietin (EPO) in vitro. To evaluate the in vivo significance of this observation, we have investigated effects of the administration of bacterial lipopolysaccharide (LPS) and IL-1 beta on renal EPO production in rats. Measurements by competitive reverse-transcription polymerase chain reaction showed that EPO mRNA levels were significantly reduced in the kidneys of normoxic rats 6 h after the injection of LPS (0.1 or 1 mg/kg). In addition, LPS and IL-1 beta (1 microgram/kg) inhibited the increase in EPO mRNA and plasma EPO levels when administered to rats before hypoxia exposure (8% O2 in the inspiratory gas). Evidence for an inflammatory reaction in the kidneys of LPS-treated rats was provided by measurements of greatly elevated renal TNF-alpha mRNA levels. Furthermore, kidneys isolated from LPS-created rats produced less immunoreactive EPO when perfused hypoxically in vitro for 2 h. Thus mediators of the immune response inhibit renal EPO gene expression in vivo, which is relevant with respect to the impaired synthesis of EPO in inflammatory diseases in humans.

Topics: Animals; DNA Primers; Erythropoietin; Escherichia coli; Glomerular Filtration Rate; Hypoxia; Interleukin-1; Kidney; Kinetics; Lipopolysaccharides; Male; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Renal Circulation; RNA, Messenger; Transcription, Genetic; Tumor Necrosis Factor-alpha; Vascular Resistance

Erythrocytosis arises from a variety of pathogenic mechanisms. We sequenced a 256-bp region 3' to the erythropoietin (Epo) gene which included a 24- to 50-bp minimal hypoxia-responsive element spanning HIF-1- and HNF-4-binding sites in 12 patients with erythrocytosis and 4 normal subjects. Four polymorphisms were found, none of which affected the HIF-1-binding site, although one polymorphism was present in the HNF-4 consensus region. The data indicate that none of these polymorphisms cause erythrocytosis.

Topics: Binding Sites; DNA-Binding Proteins; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Nuclear Proteins; Polycythemia; Transcription Factors

Topics: Anemia; Animals; Cell Transplantation; Cells, Cultured; Erythropoietin; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Humans; Hypoxia; Mice; Muscle Fibers, Skeletal; Oxygen; Recombinant Proteins; Transgenes

Current therapy for several forms of anemia involves a weekly regime of multiple subcutaneous injections of recombinant human erythropoietin (hEpo). In an effort to provide a physiologically regulated administration of erythropoietin, we are developing cell lines genetically engineered to release hEpo as a function of oxygen tension. C2C12 cells were transfected using a vector containing the hEpo cDNA driven by the hypoxia-responsive promoter to the murine phosphoglycerate kinase gene. In vitro, these cells showed a threefold increase in hEpo secretion as oxygen levels were shifted from 21% to 1.3% oxygen. To test in vivo response, C2C12-hEpo cells were encapsulated in a microporous membrane and implanted subcutaneously on the dorsal flank of DBA/2J mice. On average, serum hEpo levels in animals exposed to 7% oxygen were two-fold higher than values seen in their control counterparts kept at 21% oxygen. Similar studies employing rats confirmed that hEpo delivery is regulated as a function of oxygen tension. These results suggest the feasibility of developing an oxygen-regulated, encapsulated cell-based system for hEpo delivery.

Topics: Anemia; Animals; Cells, Cultured; DNA-Binding Proteins; Drug Compounding; Erythropoietin; Gene Expression Regulation; Genetic Therapy; Histocytochemistry; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Inbred DBA; Nuclear Proteins; Oxygen; Partial Pressure; Phosphoglycerate Kinase; Plasmids; Promoter Regions, Genetic; Rats; Rats, Inbred F344; Recombinant Proteins; Transcription Factors; Transgenes

This study aimed to investigate the influence of different forms of tissue hypoxia on the expression of the endothelin genes in kidneys and livers. Tissue hypoxia in rats was induced by five different manoeuvres, namely hypoxia (8% O2), functional anaemia (0.1% CO), haemorrhage (haematocrit, hct = 0.12), cobalt treatment (60 mg/kg) for 6 h each and renal artery stenosis (0.2-mm clips) for 2 days. Endothelin-1 (ET-1) mRNA levels in the kidneys were increased by 200% with renal artery stenosis, 70% by hypoxia, 50% by anaemia, 30% by CO, but were not changed by cobalt. ET-3 mRNA in the kidneys decreased during renal artery clipping and cobalt treatment and were not significantly changed under the other conditions. ET-2 mRNA was not detected in the kidneys and livers. The abundance of ET-1 in the livers of normoxic animals was about 15% of that found in the kidney. Hypoxia increased ET-1 mRNA by 200%, haemorrhage by 400%, whilst CO and cobalt did not change hepatic ET-1 gene expression. The abundance of ET-3 mRNA in the livers of normoxic animals was about 6% of that found in the kidneys. The expression of the ET-3 gene in the livers was decreased by CO, but was not changed by any of the other experimental conditions used. These findings suggest that hyoxaemia and tissue hypoxia are moderate stimuli for the expression of the ET-1 gene but not for the ET-3 gene in the kidney and more potent stimuli in the liver, whilst cobalt does not activate ET-1 gene expression in the kidneys nor the livers.

Topics: Anemia; Animals; Endothelins; Erythropoietin; Gene Expression; Hypoxia; Kidney; Liver; Male; Rats; Rats, Sprague-Dawley

A DNA response element, TR2RE-EPO (5'-TCTGACCTCTCGACCTAC-3') has been identified in the 3-minimal hypoxia-inducible enhancer of the human erythropoietin gene for the TR2 orphan receptor, an androgen-repressed transcription factor and a member of the steroid/thyroid hormone receptor superfamily. Electrophoretic mobility shift assay showed a specific binding with high affinity (Kd = 0.14 nM) between the TR2 orphan receptor and the TR2RE-EPO. Our data further indicated that this specific binding is not due to the homo-dimerization of the TR2 orphan receptor. In addition, reporter gene expression using chloramphenicol acetyltransferase assay demonstrated that the TR2 orphan receptor may suppress the expression of the chloramphenicol acetyltransferase activities via the TR2RE-EPO in the hypoxic/normoxic human hepatoma HepG2 cells. Finally, our in situ hybridization data also indicated that the TR2 orphan receptor and the erythropoietin transcripts can be co-expressed in mouse kidney and liver. Together, our data suggest that the human erythropoietin gene could represent the first human target gene regulated directly by the human TR2 orphan receptor.

Topics: Animals; Base Sequence; DNA Primers; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythropoietin; Gene Expression; Gene Expression Regulation; Humans; Hypoxia; In Situ Hybridization; Mice; Molecular Sequence Data; Nuclear Receptor Subfamily 2, Group C, Member 1; Receptors, Steroid; Receptors, Thyroid Hormone; Recombinant Proteins; Repressor Proteins; RNA, Messenger; Signal Transduction

Regular administration of recombinant erythropoietin (EPO) is frequently complicated by a rise in arterial blood pressure. We therefore asked if prolonged stimulation of endogenous EPO production has the same effect. To this end, male Sprague-Dawley rats were placed in a hypobaric chamber (390 mm Hg) for 24 days. The control (NL) group was placed in the chamber at normobaric condition. The animals were then removed from the chamber and monitored through day 108. Plasma EPO peaked within 24 hours and returned to baseline by day 7 and remained so thereafter. Hematocrit rose steadily during the hypoxic phase and declined steadily during the normobaric phase, reaching the baseline on day 45. This was accompanied by parallel changes in erythrocyte mass and blood volume. The rise in hematocrit during hypoxia was accompanied by a parallel rise in blood pressure which peaked on day 24. Despite the restoration of normal hematocrit, erythrocyte mass and blood volume following resumption of normoxia, blood pressure remained elevated throughout the observation period. To dissect the role of hypoxia from that of the associated rise in hematocrit, the experiments were repeated using a group of rats whose hematocrits were kept constant by repeated phlebotomies. These animals exhibited a sustained rise in blood pressure identical to that found in the original group. Thus, prolonged hypobaric hypoxia leads to a severe hematocrit-independent systemic hypertension (HTN) that persists long after the restoration of normoxia. Given the transient nature of the rise in its plasma concentration, endogenous EPO does not appear to play a role in the genesis of the observed systemic HTN. The authors believe that this animal model can be used for future studies of the mechanism, consequences and treatment of acquired HTN.

Topics: Air Pressure; Animals; Arginine Vasopressin; Atrial Natriuretic Factor; Blood Pressure; Blood Volume; Disease Models, Animal; Erythropoietin; Hematocrit; Hypertension; Hypoxia; Male; Rats; Rats, Sprague-Dawley; Time Factors

Hypoxia or anemia is the fundamental stimulus for erythropoietin (EPO) production. Recent in vitro studies suggest that EPO secretion in response to hypoxia is regulated by adenosine in the kidney. In order to examine the in vivo effect of adenosine on EPO production, we determined the effects of adenosine receptor agonists and antagonists on serum EPO concentration in normal and anemic rats. In normal rats, intravenous injection of adenosine agonists (NECA, CHA and CGS-21680) dose-dependently stimulated EPO production. Pretreatment with KW-3902, an adenosine A1 antagonist with modest A2b antagonistic action, or KF17837, an adenosine A2a antagonist, inhibited the NECA (0.1 mg/kg, i.v.)-stimulated EPO production. Anemic hypoxia, induced by 2% (v/w body weight) blood withdrawal, increased serum EPO concentration from 38 +/- 2 to 352 +/- 76 mU/ml, with the increased serum adenosine concentration in the renal vein. KF17837 (0.1 mg/kg, i.v.), but not KW-3902 (0.1 mg/kg, i.v.), inhibited the anemic hypoxia-induced increase in EPO production. The present findings support the notion that adenosine mediates the EPO production in response to hypoxia in the kidney.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Analysis of Variance; Anemia; Animals; Erythropoietin; Hematocrit; Hypoxia; Kinetics; Male; Nephrectomy; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Time Factors; Xanthines

Erythropoietin (EPO) is the main humoral stimulus of erythropoiesis. In adult mammals, the kidney releases EPO in response to hypoxic stress. Conflicting data have suggested either renal tubular or peritubular cell origins of EPO synthesis in vivo. In situ hybridization studies were performed to define further the kidney cell type(s) capable of increasing EPO gene expression during hypoxic stimulation. EPO gene expression was stimulated in mice exposed to acute hypobaric hypoxia. Kidneys from hypoxic and control normoxic mice were obtained. Six digoxigenin-labelled oligonucleotide probes complementary to EPO exon sequences were utilized for in situ hybridization for EPO messenger RNA. Positive hybridization signals were identified in some proximal tubular cells, confined to the inner third of the renal cortex of hypoxic mouse kidney.

Topics: Animals; Base Sequence; Digoxigenin; Erythropoietin; Gene Expression; Hypoxia; Kidney Tubules, Proximal; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Oligonucleotide Probes; RNA, Messenger

The aim of this study was first to investigate cardio-ventilatory and haematological responses induced by intermittent acclimation and second to study de-adaptation from high altitude observed after descent. To achieve these objectives nine subjects were submitted to intermittent acclimation in a low barometric chamber (8 h daily for 5 days, day 1 at 4500 m, day 5 at 8500 m) before an expedition to the Himalayas. Cardio-ventilatory changes were measured during a hypobaric poikilocapnic hypoxic test (4500 m, barometric pressure = 589 hPa) and haematological changes were studied at sea level. These measurements were performed before and after acclimation, after return to sea level, but also 1 and 2 months after the expedition. In addition, partial pressures of oxygen and carbon dioxide in arterial blood (PaO2, PaCO2) and arterial erythropoietin concentration [EPO] were measured at rest during the hypoxic test. Results suggested the pre-adaptation protocol was efficient since an increased PaO2 (+12%, P < 0.05), a smaller difference in alveolo-arterial PO2 ( -63%, P < 0.05) and a lower PaCO2 ( -11%, P < 0.05), subsequent to ventilatory changes, were observed after acclimation with a significant increase in reticulocytes and in sea level [EPO] (+44% and +62% respectively, P < 0.05). De-adaptation was characterized by a loss of these cardio-ventilatory changes 1 month after descent, whereas the haematological changes (increased red blood cells and packed cell volume, P < 0.05) persisted for 1 month before disappearing 2 months after descent. This study would also suggest that acute hypoxia performed after a sojourn at high altitude could induce significantly depressed EPO responses (P < 0.05).

Topics: Adaptation, Physiological; Adult; Altitude; Erythrocyte Count; Erythropoietin; Exercise; Heart; Heart Rate; Hematocrit; Hematologic Tests; Humans; Hypoxia; Male; Nepal; Respiration; Reticulocyte Count; Tibet

We examined the effect of H2O2 on the regulation of the human erythropoietin (Epo) gene through the GATA sequence in the Epo promoter in Hep3B cells. The addition of exogenous H2O2 in Hep3B cells inhibited hypoxia-induced Epo production of mRNA as assessed by competitive polymerase chain reaction (PCR) and protein by EIA. Likewise, menadione, which increased production of endogenous H2O2, inhibited hypoxia-induced Epo production in Hep3B cells. Furthermore, the expression of the human GATA (hGATA) transcription factor was increased dose-dependently by the addition of H2O2 or menadione in Hep3B cells as assessed by gel mobility shift assay. We concluded that H2O2 inhibits Epo gene expression, because it enhanced the expression of the hGATA transcription factor. Our findings support the hypothesis that GATA is a transcription factor for the Epo gene acting through the oxygen sensor.

Topics: Cells, Cultured; Erythropoietin; Gene Expression; Humans; Hydrogen Peroxide; Hypoxia; Promoter Regions, Genetic; Transcription Factors; Vitamin K

There is accumulating evidence from in vitro experiments that the gene expression of the vascular endothelial growth factor (VEGF) is, like that of the erythropoietin (EPO) gene, regulated by the oxygen tension and by divalent cations such as cobalt. Since the information about the regulation of VEGF gene expression in vivo is rather scarce, this study aimed to examine the influence of hypoxia and of cobalt on VEGF gene expression in different rat organs and to compare it with that on EPO gene expression. To this end male Sprague-Dawley rats were exposed to carbon monoxide (0.1% CO), hypoxia (8% O2 ) or to cobalt chloride (12 and 60 mg/kg s.c.) for 6 h. mRNA levels for VEGF- 188, -164, and -120 amino acid isoforms in lungs, hearts, kidneys and livers were semiquantitated by RNase protection. For these organs we found a rank order of VEGF mRNA abundance of lung >> heart > kidney = liver. EPO mRNA levels were semiquantitated in kidneys and livers. Hypoxia, CO and cobalt increased EPO mRNA levels 60-fold, 140-fold and 5-fold, respectively, in the kidneys, and 11-fold, 11-fold and 3-fold, respectively, in the livers. None of these manoeuvres caused significant changes of VEGF mRNA in lung, heart or kidneys. Only in the livers did hypoxia lead to a significant (50%) increase of VEGF mRNA. These findings suggest that, in contrast to the in vitro situation, the expression of the VEGF gene in normal rat tissues is rather insensitive to hypoxia. In consequence, the in vivo regulation of the VEGF and the EPO genes appear to differ substantially, suggesting that the regulation of the VEGF and EPO genes may not follow the same essential mechanisms in vivo.

Topics: Animals; Base Sequence; Carbon Monoxide; Cobalt; Endothelial Growth Factors; Erythropoietin; Gene Expression Regulation; Hypoxia; In Vitro Techniques; Lymphokines; Male; Organ Specificity; Rats; Rats, Sprague-Dawley; RNA, Messenger; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

This study investigated the human erythropoietin (EPO) response to short-term hypocapnic hypoxia, its relationship to a normoxic or hypoxic increase of the haemoglobin oxygen affinity, and its suppression by the addition of CO2 to the hypoxic gas. On separate days, eight healthy male subjects were exposed to 2 h each of hypocapnic hypoxia, normocapnic hypoxia, hypocapnic normoxia, and normal breathing of room air (control experiment). During the control experiment, serum-EPO showed significant variations (ANOVA P = 0.047) with a 15% increase in mean values. The serum-EPO measured in the other experiments were corrected for these spontaneous variations in each individual. At 2 h after ending hypocapnic hypoxia (10% O2 in nitrogen), mean serum-EPO increased by 28% [baseline 8.00 (SEM 0.84) U.l-1, post-hypoxia 10.24 (SEM 0.95) U.l-1, P = 0.005]. Normocapnic hypoxia was produced by the addition of CO2 (10% Co2 with 10% O2) to the hypoxic gas mixture. This elicited an increased ventilation, unaltered arterial pH and haemoglobin oxygen affinity, a lower degree of hypoxia than during hypocapnic hypoxia, and no significant changes in serum-EPO (ANOVA P > 0.05). Hypocapnic normoxia, produced by hyperventilation of room air, elicited a normoxic increase in the haemoglobin oxygen affinity without changing serum-EPO. Among the measured blood gas and acid-base parameters, only the partial pressures of oxygen in arterial blood during hypocapnic hypoxia were related to the peak values of serum-EPO (r = -0.81, P = 0.01). The present human EPO responses to hypoxia were lower than those which have previously been reported in rodents and humans. In contrast with the earlier rodent studies, it was found that human EPO production could not be triggered by short-term increases in pH and haemoglobin oxygen affinity per se, and the human EPO response to hypoxia could be suppressed by concomitant normocapnia without acidosis.

Topics: Adult; Carbon Dioxide; Erythropoietin; Hemoglobins; Humans; Hydrogen-Ion Concentration; Hypocapnia; Hypoxia; Male; Oxygen

The haematopoietic growth factor erythropoietin is the primary regulator of mammalian erythropoiesis and is produced by the kidney and the liver in an oxygen-dependent manner. We and others have recently demonstrated erythropoietin gene expression in the rodent brain. In this work, we show that cerebral erythropoietin gene expression is not restricted to rodents but occurs also in the primate brain. Erythropoietin mRNA was detected in biopsies from the human hippocampus, amygdala and temporal cortex and in various brain areas of the monkey Macaca mulatta. Exposure to a low level of oxygen led to elevated erythropoietin mRNA levels in the monkey brain, as did anaemia in the mouse brain. In addition, erythropoietin receptor mRNA was detected in all brain biopsies tested from man, monkey and mouse. Analysis of primary cerebral cells isolated from newborn mice revealed that astrocytes, but not microglia cells, expressed erythropoietin. When incubated at 1% oxygen, astrocytes showed >100-fold time-dependent erythropoietin mRNA accumulation, as measured with the quantitative reverse transcription-polymerase chain reaction. The specificity of hypoxic gene induction in these cells was confirmed by quantitative Northern blot analysis showing hypoxic up-regulation of mRNA encoding the vascular endothelial growth factor, but not of other genes. These findings demonstrate that erythropoietin and its receptor are expressed in the brain of primates as they are in rodents, and that, at least in mice, primary astrocytes are a source of cerebral erythropoietin expression which can be up-regulated by reduced oxygenation.

Topics: Adult; Animals; Brain; Endothelial Growth Factors; Erythropoietin; Female; Gene Expression; Humans; Hypoxia; Lymphokines; Macaca mulatta; Male; Mice; Oxygen; Receptors, Erythropoietin; RNA, Messenger; Tissue Distribution; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Tissue hypoxia is less effective in increasing erythropoietin plasma levels in animals with post-transfusion polycythaemia. Since more red blood cells are decomposed under this condition, the effects of exogenous haemin and of lysed or heat-damaged red blood cells on activation of the erythropoietin gene have been studied in mice rendered hypoxic. Total RNA was extracted from the kidney and the liver and subjected to northern blot analysis with a probe containing part of the murine erythropoietin gene. Blood plasma was collected and erythropoietin levels were determined by radioimmunoassay. Erythropoietin gene activation was suppressed by haemin and increased red blood cell haemolysis. Tin (Sn) protoporphyrin, a haeme analogue which cannot bind oxygen, did not share the effect of haemin. On the other hand, when injected with haemin, Sn-protoporphyrin potentiated the suppressive effect of haemin, probably through inhibition of haemin catabolism. We conclude that the intracellular haeme concentration inhibits the kidney oxygen sensor and that this inhibition, mediated by products red blood cell degradation, is a physiological safeguard mechanism against excessive polycythaemia and its deleterious effects upon blood circulation.

Topics: Animals; Blotting, Northern; Erythrocytes; Erythropoietin; Hemolysis; Hot Temperature; Hypoxia; Kidney; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Oxygen; RNA, Messenger

We have previously reported that nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) may be involved in the regulation of erythropoietin (Epo) production in response to hypoxia both in vivo and in vitro (20). In the present studies, we have used the isolated perfused rat kidney to assess the role of NO in oxygen sensing and Epo production. When arterial PO2 was reduced from 100 mmHg (normoxemic) to 30 mmHg (hypoxemic) in the perfusate of this system, perfusate levels of Epo were significantly increased. This hypoxia-induced increase in Epo production was significantly decreased by the addition of NG-nitro-L-arginine methyl ester (L-NAME; 1 mM) to the perfusates. Hypoxemic perfusion also produced a significant increase, and L-NAME significantly inhibited this increase, in intracellular cGMP levels in the kidney when compared with normoxemic perfused kidneys. Quantitative reverse transcription-polymerase chain reaction also revealed that hypoxemic perfusion produced significant increases in Epo mRNA levels in the kidney, which was blocked by L-NAME. Our findings further support an important role for the NO/cGMP system in hypoxic regulation of Epo production.

Topics: Animals; Arginine; Base Sequence; DNA, Complementary; Erythropoietin; Hypoxia; In Vitro Techniques; Kidney; Male; Molecular Probes; Molecular Sequence Data; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxygen; Partial Pressure; Perfusion; Polymerase Chain Reaction; Rats; Rats, Wistar; RNA, Messenger

This study investigates the effects of hypoxia and of cobalt on erythropoietin (EPO) gene expression in hepatocytes in vivo and in vitro in neonatal, juvenile, and adult rats. With the use of the ribonuclease protection assay to quantify RNA, both hypoxia (0.1% CO or 9% O2) and cobalt (60 mg/kg) elicit production of increased amounts of EPO mRNA in neonatal and juvenile rat liver in vivo. In vitro hepatocyte EPO gene expression could be reproducibly stimulated by hypoxia (3% O2) but not by cobaltous chloride (50-150 microM) within 2-20 h. Conversely, cobalt substantially attenuated the rise of EPO mRNA levels in response to hypoxia. This inhibitory effect of cobalt was mimicked by zinc but not by other metals. CO attenuated the rise of EPO mRNA levels in vitro in response to hypoxia; this inhibitory effect coincided with an inhibition of total RNA synthesis as determined by [3H]uridine incorporation. The lack of specific inhibitory effects of CO and of specific stimulatory effects of cobalt on hepatocyte EPO gene expression in vitro suggests that a specific heme oxygen sensor may be less important than in hepatoma cells.

Topics: Animals; Animals, Newborn; Carbon Monoxide; Cells, Cultured; Cobalt; Erythropoietin; Gene Expression; Hypoxia; In Vitro Techniques; Liver; Male; Oxygen; Rats; Rats, Sprague-Dawley; RNA, Messenger

We previously found that augmentation of polycythemia by exogenous human recombinant erythropoietin (EPO) failed to worsen the severity of hypoxic pulmonary hypertension in rats. We asked whether this unexpected finding was related to reductions in cardiac output, left ventricular end-diastolic pressure, pulmonary vascular resistance, or some combination of these factors. Four groups of Sprague-Dawley rats were studied over a 3-wk period: hypoxic (0.5 ATM) and normoxic animals each injected with EPO (500 U/kg sc thrice weekly) or saline (control animals). As observed previously, we found that pulmonary arterial (PA) pressures and right ventricular hypertrophy were not increased in EPO-treated rats despite significant increases in hematocrit and blood viscosity. Cardiac outputs, blood volumes, and left ventricular end-diastolic pressures were similar in EPO-treated and control rats. Acute PA pressure responses to acute normoxia in hypoxic rats and to acute hypoxia in normoxic rats were similar, suggesting no differences in vasoreactivity. However, lungs isolated from EPO-treated hypoxic rats had lower pulmonary vascular resistance than saline-treated hypoxic rats when perfused with blood from normocythemic donor rats. PA medial thickness and the percentage of muscularized small PAs were significantly lower in EPO-treated hypoxic rats. These results indicate that augmented polycythemia fails to worsen hypoxic pulmonary hypertension in rats because of a decrease in the severity of structural remodeling.

Topics: Adaptation, Physiological; Animals; Blood Pressure; Blood Vessels; Blood Viscosity; Chronic Disease; Erythropoietin; Hematocrit; Hemodynamics; Humans; Hypoxia; Male; Polycythemia; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Recombinant Proteins

The purpose of this study was to characterize the effects of two new adenosine A2 agonists, 2-(p-(2-carboxyethyl)phenethyl amino)-5'-N-ethylcarboxamidoadenosine (CGS-21680) and N6-(2(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl)-adenosine (DPMA), on erythropoietin (EPO) production in vivo and in vitro. Intravenous injections of CGS-21680 (100 to 500 nmol/kg mouse/day) and DPMA (50 to 500 nmol/kg mouse/day) for 4 days produced significant increases in serum levels of EPO in exhypoxic polycythemic mice. CGS-21680 (10(-7) to 10(-6) mol/L) and DPMA (10(-8) to 10(-5) mol/L) also produced significant increases in medium levels of EPO in a cloned EPO-producing Hep3B hepatocellular carcinoma cell line after 18 hours of incubation in 1% O2. Both compounds also increased cellular cAMP levels significantly in a dose-dependent manner after 1 hour of incubation. A2 receptor binding assays with tritiated CGS-21680 revealed a single type of adenosine receptor binding site on Hep3B cell membranes with a dissociation constant of 132.9 nmol/L and a binding capacity of 270.6 fmol/mg protein. The Ki competition binding values versus tritiated CGS-21680 were 217 nmol/L for CGS-21680 and 86.8 nmol/L for DPMA. These results indicate that adenosine A2 receptor activation amplifies EPO production in response to hypoxia, both in vivo and in vitro.

Topics: Adenosine; Animals; Binding, Competitive; Carcinoma, Hepatocellular; Cyclic AMP; Erythropoietin; Female; Humans; Hypoxia; Kinetics; Liver Neoplasms; Mice; Phenethylamines; Polycythemia; Purinergic P1 Receptor Agonists; Tumor Cells, Cultured

We measured nocturnal plasma erythropoietin concentration (EPO) in eight subjects with obstructive sleep apnea syndrome (OSAS) and eight healthy overweight subjects while they were undergoing nocturnal polysomnography. We also measured EPO (radioimmunoassay) after 120 min of exposure to 10.5% O2. The subjects with OSAS had a respiratory disturbance index (RDI) of 50.8 +/- 41.9 and a maximal O2 desaturation of 65 +/- 13.3%, and they spent 104.5 +/- 89.3 min out of a total sleep time of 356 +/- 54 min below 90% oxygen saturation (T90). Nocturnal EPO concentrations were normal and did not differ between the two groups. We found no correlation between the T90, T80, or RDI and either the mean or maximal EPO concentrations. After the exposure to 10.5% oxygen, during which oxygen saturations were between 80 and 85%, the healthy subjects and those with OSAS developed increases in EPO of 34 and 49%, respectively, 240 min after the initiation of exposure (p < 0.05). We conclude that the nocturnal hypoxemia occurring in these subjects with OSAS may not have been sufficiently severe to stimulate an increased EPO production. The lack of EPO response was not due to down-regulation of EPO production.

Topics: Adaptation, Physiological; Adult; Case-Control Studies; Down-Regulation; Erythropoietin; Female; Humans; Hypoxia; Male; Obesity; Polysomnography; Radioimmunoassay; Sleep Apnea Syndromes; Time Factors

Recent studies on the induction of erythropoietin gene expression by hypoxia have indicated that erythropoietin forms part of a widely operative system of gene regulation by oxygen. Similar responses to hypoxia, cobaltous ions and desferrioxamine have indicated that the action of these agents is closely connected with the mechanism of oxygen sensing. To consider further the mechanisms underlying these responses, the effect of iodonium compounds was tested on five genes which show oxygen-regulated expression; erythropoietin, vascular endothelial growth factor (VEGF), lactate dehydrogenase-A (LDH-A), glucose transporter-1 (GLUT-1) and placental growth factor (PLGF). In each case, the response to hypoxia was specifically inhibited by low doses of diphenylene iodonium (Ph1I+). This occurred irrespective of whether the hypoxic response was induction of gene expression (erythropoietin, vascular endothelial growth factor, lactate dehydrogenase-A, glucose transporter-1) or inhibition of gene expression (PLGF). In contrast, the induction of gene expression by cobaltous ions or desferrioxamine was not inhibited by Ph2I+. The differential action of Ph2I+ on the response to hypoxia versus the response to cobaltous ions or desferrioxamine must reflect a difference in the mechanism of action of these stimuli, which will require accommodation in any model of the oxygen-sensing mechanism. Based on the known properties of Ph2I+, the implication of these findings is that the mechanism of oxygen sensing most probably involves the operation of a flavoprotein oxidoreductase.

Topics: Base Sequence; Biphenyl Compounds; Cobalt; Deferoxamine; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythropoietin; Gene Expression; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Molecular Sequence Data; Nuclear Proteins; Oligodeoxyribonucleotides; Onium Compounds; Oxygen; Transcription Factors; Tumor Cells, Cultured

Hypoxia is the best physiological stress to disturb the erythropoietic steady state. The present study has been undertaken in the aim to analize the splenic erythropoietic proliferative response with different doses of recombinant human erythropoietin under hypoxic conditions along 18 days using the DNA synthesis assay. Normoxic splenic progenitors failed to show significative erythroid replication at 0 days. A clearly rh Epo response was noticed from 2 to 8 days of hypoxia. Splenic proliferation returned to basal pattern from 10 days to the end of the experience. The highest proliferative activity, 25 fold increase over control (p < 0.001), was found at 6 days from 62.5 to 250 mU/ml rh Epo. These results support suggestions that hypoxia induces a transiently erythroid splenic proliferative response changing its quantitative parameters in the Epo dose-response relationship during the physiological adaptation.

Topics: Animals; Cell Culture Techniques; Cell Division; DNA; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Female; Hypoxia; Mice; Mice, Inbred Strains; Spleen; Time Factors

Hypoxia during haemodialysis, mainly acetate, has been reported several times. In our study we have monitored oxygen status during 258 bicarbonate haemodialyses. A significant drop below 80 mmHg in mean oxygen tension occurred. Mean oxygen saturation reflected this drop but did not reach levels below 90%. The mean oxygen concentration was on the whole critical low, though slightly increasing during each haemodialysis session due to ultrafiltration. It is concluded that both hypoxia and hypoxaemia do occur during bicarbonate haemodialysis. To a group of patients generally having limited cardiac reserves, a poor oxygen status is a potentially serious complication to haemodialysis. Monitoring oxygen status is thus advisable.

Topics: Acetates; Adult; Aged; Bicarbonates; Carbon Dioxide; Cardiac Output; Dialysis Solutions; Erythropoietin; Female; Hemoglobins; Humans; Hypoxia; Male; Middle Aged; Oxygen; Oxygen Consumption; Renal Dialysis; Ultrafiltration

Effects of thyroid hormones on the production of erythropoietin (Epo) were investigated in isolated perfused rat kidneys and in the human hepatoma cell line, HepG2. Epo protein was measured by radioimmunoassay. L-triiodothyronine and L-thyroxine stimulated hypoxia-induced Epo formation both in the kidney and in HepG2 cells in a dose-dependent fashion. Quantitation of Epo mRNA by competitive polymerase chain reaction (PCR) showed that hypoxic HepG2 cells had three-fold higher Epo messenger RNA levels when treated with thyroid hormones for 3 hours. Measurements of oxygen consumption revealed that this effect was not due to an increase in the degree of hypoxia. Thus, apart from the known direct effect on erythroid precursors, thyroid hormones appear to stimulate erythropoiesis by a noncalorigenic increase in Epo production.

Topics: alpha-Fetoproteins; Animals; Carcinoma, Hepatocellular; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Erythropoietin; Humans; Hypoxia; Kidney; Liver Neoplasms; Male; Oxygen Consumption; Polymerase Chain Reaction; Radioimmunoassay; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thyroxine; Triiodothyronine; Tumor Cells, Cultured

Fetal hemoglobin (HbF) synthesis in children with congenital cyanotic heart disease was compared that in normal children. Children with hypoxemia had higher levels of hemoglobin, total HbF, and HbF synthesis. In these children there was also an inverse correlation between HbF synthesis and oxygen content, as well as between HbF synthesis and hemoglobin concentration. Thus hypoxemia increases HbF synthesis.

Topics: Child; Child, Preschool; Cyanosis; Erythropoietin; Fetal Hemoglobin; Heart Defects, Congenital; Humans; Hypoxia; Infant

Vascular endothelial growth factor (VEGF) expression is highly stimulated by hypoxia, both in vitro and in vivo. Recent findings suggest that the VEGF gene utilizes an oxygen sensing mechanism similar to the one used by the erythropoietin gene. The genomic sequences that control the VEGF response to hypoxia are, however, largely unknown. In utilizing transient transfection assays in HeLa cells we determined that hypoxia/cobalt responsive enhancer elements are present at the 5' and 3' flanking regions of the human VEGF gene. The 3' enhancer is contained in a 160 bp fragment located about 60 bp downstream of the polyadenylation site. It contains a sequence stretch of about 12 bp which are highly homologous to sequences in the erythropoietin hypoxia-responsive enhancer. The 5' flanking enhancer is contained in a 100 bp fragment located about 800 bp upstream of the start site. This fragment does not contain significant homologies with the erythropoietin enhancer. Thus, it appears that the response to hypoxia of the VEGF gene is controlled by two regulatory elements; one which may be related to the erythropoietin enhancer and a second, which appears to be a completely unrelated sequence.

Topics: Base Sequence; Cell Hypoxia; Cobalt; DNA; Endothelial Growth Factors; Enhancer Elements, Genetic; Erythropoietin; Genes, Regulator; HeLa Cells; Humans; Hypoxia; Lymphokines; Molecular Sequence Data; Neovascularization, Pathologic; Sequence Homology, Nucleic Acid; Transfection; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Studies were performed to reexamine the response of erythropoietin (Epo) production to acute hypoxic stimuli in patients with end-stage renal disease (ESRD). In the absence of acute bleeding or hypoxia, the serum Epo level in ESRD was similar to that of normal subjects despite severe anemia. In 11 dialysis patients with acute bleeding, the decrease in the Hb level from 8.9 to 5.8 g/dL provoked a significant increase in serum Epo up to 52.2 times the normal value. The increase in serum Epo was associated with a significant increase in corrected reticulocyte. Systemic hypoxemia (PaO2 < 65 mm Hg) in 8 dialysis patients provoked a significant elevation in the serum Epo level up to 24.6 times the normal level. There was an inverse relationship between serum Epo and arterial PaO2 (r = -0.715). The serum Epo level in these patients declined to or near the normal value after recovery from acute hypoxic stress. These data suggest that the ability of the Epo production is well preserved in ESRD, indicating that acute hypoxic stimuli provoke a significant increase in serum Epo.

Topics: Acute Disease; Adult; Aged; Anemia, Iron-Deficiency; Erythropoietin; Female; Hemoglobins; Hemorrhage; Humans; Hypoxia; Kidney Failure, Chronic; Male; Middle Aged; Oxygen; Partial Pressure; Renal Dialysis

The present study was undertaken to assess the effect of prazosin, a selective postsynaptic alpha 1-adrenergic receptor blocking agent, on normoxic and hypoxic mice, in order to evaluate experimentally its use in the treatment of the excessive erythrocytosis that characterizes chronic mountain sickness. The drug, injected intraperitoneally to adult mice at a dose of 400 micrograms/kg per day, induced a significant depression of the rate or erythropoiesis, as measured by red blood cell 59iron uptake, with a decrease in the hematocrit from the 3rd day. The drug also inhibited the oxygen-dependent secretion of erythropoietin (estimated by the plasma immunoreactive hormone concentration) in hypoxemic mice when injected between 0 and 2 h after initiation of the hypoxic stimulation. When injected daily into mice exposed to intermittent hypobaric hypoxia, prazosin limited the degree of polycythemia or induced a sustained decrease in the hematocrit when polycythemia was already present due to previous exposure. It is postulated that the drug, by reducing the peripheral vascular resistance seen during hypoxia, could increase renal blood flow, thus improving the renal oxygen supply and partially restoring the imbalance between gas supply and demand, which drives erythropoietin formation.

Topics: Adrenergic alpha-1 Receptor Antagonists; Animals; Erythropoiesis; Erythropoietin; Female; Hypoxia; Male; Mice; Polycythemia; Prazosin

In an attempt to assess the erythropoietin (Epo) production site(s) in rat kidney, Epo response to hypoxia and renal histopathological changes were studied in rats administered with graded doses of gentamicin. Male Sprague-Dawley rats of 9 to 11 weeks old were used. Following a 14-day subcutaneous administration (67.5 or 33.8 mg kg-1 day-1) of gentamicin, a nephrotoxic aminoglycoside, selective proximal tubular lesions were produced. These gentamicin-administered rats were compared with normal rats with respect to Epo response to hypoxia. Two different kinds of hypoxic load, either 0.35 atm hypobaric hypoxia (PIO2 = 46 torr) or acute anaemia (Ht: 29.3 +/- 0.2% and [Hb]: 9.7 +/- 0.3 g dl-1) by withdrawing of blood corresponding to 1-2% of body weight was used. During the hypoxic period of up to 48 h, the peak renal venous plasma Epo titres of 3.1 +/- 0.6 and 4.3 +/- 0.6 U ml-1 was observed at the 6th h in normal hypobaric hypoxic and anaemic rats, compared with the prehypoxic value of 0.7 +/- 0.1 U ml-1. The Epo titres then declined gradually. In the rats which were administered gentamicin, Epo response pattern was the same as that observed in the normal rats, but the peak value decreased significantly to 0.8 +/- 0.3 and 1.1 +/- 0.4 U ml-1 in hypoxic and anaemic rats (P < 0.05). Histological examination revealed the selective damage to renal proximal convoluted tubules. The Epo response was reduced by the tissue damages, and restoration of the gentamicin-induced tissue injury was accompanied with restored Epo response to hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Anemia; Animals; Atmospheric Pressure; Blood Volume; Erythropoietin; Gentamicins; Hypoxia; Kidney Tubules, Proximal; Male; Plasma Volume; Rats; Rats, Sprague-Dawley

Erythropoietin (Epo), a 30.4-kD glycoprotein, is the principal regulator of erythropoiesis. To evaluate the concept that in vivo gene transfer might be used as an alternative to recombinant human Epo (rhEpo) in applications requiring a 1- to 3-week stimulation of erythropoiesis, the replication-deficient recombinant adenovirus AdMLP.Epo was constructed by deleting the majority of E1 from adenovirus type 5, and replacing E1 with an expression cassette containing the adenovirus type 5 major late promoter (MLP) and the human Epo gene, including the 3' cis-acting hypoxia response element. In vitro studies showed that infection of the human hepatocyte cell line Hep3B with AdMLP.Epo resulted in a 15-fold increase in Epo production in 24 hours that was enhanced to 116-fold in the presence of a hypoxic stimulus. One-time in vivo administration of AdMLP.Epo (7 x 10(9) plaque-forming units/kg) to the peritoneum of cotton rats caused a marked increase in red blood cell production, with a 2.6-fold increase in bone marrow erythroid precursors by day 4, and sevenfold increase in reticulocyte count by day 7. The hematocrit increased gradually, with a maximum of 64% +/- 4% at day 14 (compared with an untreated baseline of 46% +/- 2%), and a level of 55% +/- 1% at day 24. Furthermore, one-time subcutaneous administration of AdMLP.Epo caused an increase in hematocrit that peaked at 14 days (57% +/- 2%) and was still elevated at day 42. Hematocrit level in animals receiving subcutaneous administration of AdMLP.Epo sustained a long-term increase compared with animals receiving intraperitoneal administration. In the context of these observations, gene therapy with a single administration of an adenovirus vector containing the human EPO gene may provide a means of significantly augmenting the circulating red blood cell mass over the 1- to 3-week period necessary for many clinical applications.

Topics: Adenoviridae; Animals; Base Sequence; Erythropoiesis; Erythropoietin; Genetic Therapy; Genetic Vectors; Humans; Hypoxia; Molecular Sequence Data; Promoter Regions, Genetic; Sigmodontinae

The addition of exogenous H2O2 inhibited hypoxia-induced erythropoietin (Epo) production in the human hepatoma cell line HepG2. Likewise, elevation of endogenous H2O2 levels by the addition of menadione or the catalase inhibitor, aminotriazole, dose-dependently lowered Epo production. The inhibitory effect of exogenous H2O2 on Epo formation could be completely overcome by co-incubation with catalase. When GSH levels in HepG2 cells were lowered, Epo production was more susceptible to H2O2-induced inhibition, indicating that H2O2 might affect thiol groups in regulatory proteins. Endogenous production of H2O2 in HepG2 cells was dependent on the pericellular O2 tension, being lowest under conditions of hypoxia. Our results support the hypothesis that an H2O2-generating haem protein might be part of the O2 sensor that controls Epo production. High H2O2 levels under conditions of normoxia suppress, whereas lower levels in hypoxic cells allow epo gene expression.

Topics: Amitrole; Base Sequence; Carcinoma, Hepatocellular; Catalase; DNA Primers; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression Regulation; Glutathione; Humans; Hydrogen Peroxide; Hypoxia; Liver Neoplasms; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Vitamin K

Erythropoietin (EPO), triiodothyronine, thyroxine, thyroid-stimulating hormone (TSH), hemoglobin (Hb), reticulocytes, packed cell volume, and plasma volume changes were studied in 29 male Austrian mountain rescue soldiers before and several days after an ascent from 744 to 2,315 m in the Alps. EPO concentrations showed a remarkable increase 48 h after the ascent (P < 0.01). No significant changes were found in triiodothyronine, thyroxine, and TSH. Reticulocytes increased only slightly. Hb decreased during the week (P < 0.01). Packed cell volume did not change, whereas plasma volume showed a slight decrease for 48 h after the ascent but then increased constantly (P < 0.01). It seems that thyroid hormones, TSH, and Hb play only a minor role in the regulation of EPO production and release under mild hypobaric-hypoxic conditions (2,315 m).

Topics: Adult; Altitude; Blood Cell Count; Erythropoietin; Hemoglobins; Humans; Hypoxia; Male; Middle Aged; Plasma Volume; Reticulocyte Count; Thyroid Hormones; Thyrotropin; Thyroxine; Triiodothyronine

We have obtained transgenic mice in which an erythropoietin-SV40 virus T antigen fusion gene is homologously recombined into the native Epo locus. This gene is expressed in a tissue-specific manner closely resembling that of the native Epo gene. Immunohistochemical detection of SV40 T antigen has been used to characterize the hepatic cell populations expressing the transgene. In mice stimulated by anaemia or hypobaric hypoxia, SV40 T antigen was demonstrated in two liver cell populations: a subset of hepatocytes and a nonparenchymal cell type. Immunohistochemical and ultrastructural characterization of these cells by light and electron microscopy showed the nonparenchymal cell type to be the Ito cells, which lie in a persinusoidal position within the space of Disse. We therefore conclude that Ito cells are the nonhepatocytic source of liver Epo production. These cells show many similarities to the Epo-producing fibroblastoid interstitial cells of the kidney.

Topics: Anemia; Animals; Antigens, Polyomavirus Transforming; Erythropoietin; Gene Expression; Hypoxia; Immunohistochemistry; Liver; Mice; Mice, Transgenic; Microscopy, Immunoelectron; Organ Specificity; Phenotype; Recombinant Fusion Proteins; RNA, Messenger

Results on the localization of erythropoietin (Epo) synthesis in renal cells have been contradictory, implicating either interstitial or tubular cells. We fused the lacZ gene to a 7-kb DNA fragment of the mouse Epo gene encompassing a portion of the first intron, the first exon, and a 6-kb sequence of the 5'-flanking region. Transgenic mice carrying this construct show a low level of specific expression of the lacZ gene in proximal convoluted tubule (PCT) cells. Without hypoxia, no significant expression was detected in the liver. Hypoxia induced a large degree of lacZ expression, mainly in kidney PCT cells and to a lesser degree in liver. However, anemia induced lacZ expression in both kidney and liver. These findings indicate that, under these conditions, Epo is expressed in tubular cells, specifically PCT cells.

Topics: Anemia; Animals; beta-Galactosidase; Erythropoietin; Exons; Gene Expression; Genes, Reporter; Hypoxia; Introns; Kidney Tubules, Proximal; Liver; Mice; Mice, Transgenic; Promoter Regions, Genetic; Recombinant Fusion Proteins

The role of the submandibular salivary gland (SG) in the renal and extrarenal erythropoietin (Epo) response to hypoxia was evaluated in adult male mice with chronic renal failure from partial nephrectomy. A partial nephrectomy model for chronic renal failure was used in an attempt to evaluate erythropoiesis and Epo production in mice whose renal source of Epo may be compromised and thus more dependent on extrarenal sources. Mice with two-thirds of total renal mass excised developed a three-fold increase in serum creatinine concentration, polyuria, and polydipsia but not anemia. They responded to the combined challenge of hypobaric hypoxia (17 hours, 0.5 atm) and anemia from phenylhydrazine treatment (60 mg/kg intraperitoneally [i.p.]) with a consistent increase in serum Epo. This response was not affected by either acute or chronic bilateral submandibularectomy. However, bilateral submandibularectomy in mice with chronic renal failure was associated with a reduction in serum creatinine (p < 0.01). The possibility that reduction of renal mass might increase extrarenal secretion of Epo was examined in mice with chronic renal failure by removing the kidney remnant and exposing them to a severe hypoxic challenge. While acute bilateral submandibularectomy did not influence the extrarenal Epo response to severe hypoxia, mice with partial nephrectomies had a greater Epo response to hypoxia than control mice with a recent bilateral nephrectomy. In conclusion, the submandibular salivary glands do not appear to be an extrarenal source of Epo, nor do they appear to contribute to the enhanced extrarenal Epo response of mice with chronic renal failure.

Topics: Animals; Creatinine; Erythropoietin; Hypoxia; Injections, Intraperitoneal; Kidney Failure, Chronic; Male; Mice; Mice, Inbred Strains; Nephrectomy; Phenylhydrazines; Submandibular Gland

The mean erythropoietin concentration in the cord blood of neonates whose mothers were smokers was greater than in neonates whose mothers were not smokers. There was a significant positive correlation between erythropoietin and hemoglobin concentrations. The findings suggest that approximately one of five fetuses who are exposed to tobacco smoke are in a state of chronic hypoxia.

Topics: Arginine Vasopressin; Case-Control Studies; Erythropoietin; Female; Fetal Blood; Fetal Diseases; Hemoglobins; Humans; Hypoxia; Infant, Newborn; Pregnancy; Smoking

In order to investigate positive and negative regulatory elements of the erythropoietin (Epo) gene, synthetic oligonucleotides were designed to control Epo transcription by means of an antigen strategy. We devised a new method for detecting regulatory elements of genes that have a weak promoter. Synthetic oligonucleotides were incubated with Hep3B cells in the presence or absence of CoCl2 or hypoxia. To exclude the effect of translational regulation, Epo mRNA concentration was determined by competitive polymerase chain reaction. The addition of antisense oligonucleotide for CACCC elements decreased the production of Epo mRNA in a dose-dependent fashion when cells were stimulated by CoCl2 or hypoxia. In contrast, the addition of antisense oligonucleotide for the GATA element caused a dose-dependent stimulation of Epo mRNA production either in the presence or absence of CoCl2 or hypoxia. Triple helix formation was revealed by electrophoresis. CACCC elements were demonstrated to be positive regulatory elements of the Epo gene, whereas the GATA element was a negative regulatory element. Furthermore, by gel mobility shift assays, we demonstrated evidence for the presence of factors in Hep3B cell nuclear extract that specifically bind to CACCC or GATA elements. Based on these observations, we presented the possibility that triple helix formation could serve as a novel means for transcriptional regulation of the gene.

Topics: Base Sequence; Binding Sites; Cell Line; Cobalt; DNA Primers; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; In Vitro Techniques; Molecular Sequence Data; Nuclear Proteins; Nucleic Acid Conformation; Oligonucleotides; Promoter Regions, Genetic; RNA, Messenger

Altitude hypoxia induces an increase in erythropoiesis. Some of the factors involved in the control of altitude polycythemia were studied. Ten subjects (4 women, 6 men) were exposed for 3 wk to extreme altitude (6,542 m). Blood was withdrawn in normoxia (N) and after 1 wk (H1), 2 wk (H2), or 3 wk (H3) at 6,542 m for the measurement of serum erythropoietin (EPO), blood hemoglobin (Hb), hematocrit (Hct), intraerythrocyte folate (Fol), and plasma ferritin (Fer) concentrations. Renal blood flow (RBF) and absolute proximal reabsorption rate (APR) were measured by the p-aminohippuric acid and lithium clearance, respectively, in N and H2 conditions. O2 supply to the kidneys was calculated using RBF and arterial O2 content (CaO2). After an initial sharp increase in EPO, it decreased at H2 and H3. Hct and Hb increased from N to H1 and H2 and then unexpectedly decreased from H2 to H3. Mean corpuscular Hb content (MCHC = Hb/Hct) was lower in all H than in N conditions. Increase in EPO at H1 varied from 3- to 134-fold among individuals. Women showed a smaller increase in Hct and Hb and a greater decrease in MCHC. Two women showed a large increase in EPO without increase in Hb. Fol was not modified by altitude hypoxia. Fer showed a marked decrease in H1 and H3 compared with N. Hb was positively related to Fer in hypoxia. Iron intake in food was markedly decreased during the 2-wk ascent to 6,542 m. EPO was inversely related to CaO2 and positively related to APR.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acute Disease; Adult; Altitude; Altitude Sickness; Erythropoiesis; Erythropoietin; Female; Hormones; Humans; Hypoxia; Kidney; Male; Severity of Illness Index; Sex Characteristics; Time Factors

To examine the endogenous erythropoietin response in critically ill children with acute anemia or acute hypoxemia.. A prospective case study of critically ill acutely anemic, and acutely hypoxemic pediatric patients compared with control groups of critically ill nonanemic and nonhypoxemic patients and with a hemoglobin and age-matched, chronically anemic patient group.. Multidisciplinary, tertiary, pediatric intensive care unit (ICU).. Critically ill patients admitted to the pediatric ICU during an 11-month period between February 1992 and March 1993 with acute anemia (n = 21), acute hypoxemia (n = 18), or neither anemia nor hypoxemia (n = 10). Outpatients with chronic anemia (n = 21) and no acute illness were also studied as a comparison group.. None.. Ages were equivalent among the groups and averaged 57.4 +/- 47.2 months (range 1 to 144). Acutely hypoxemic and critically ill control patients had normal hemoglobin levels. Acutely anemic patients had a hemoglobin level equivalent to chronically anemic outpatients, but lower (p < .001) hemoglobin levels than acutely hypoxemic and critically ill control patients. The serum erythropoietin concentrations in the acutely anemic group were significantly lower than erythropoietin values in the chronically anemic group (39.3 +/- 62.2 vs. 861 +/- 758 mU/mL, p < .001) and similar to erythropoietin concentrations in the critically ill control (13.5 +/- 10.5 mU/mL) and acutely hypoxemic (5.2 +/- 3.3 mU/mL) patient groups. Only ten of 49 critically ill patients had an erythropoietin concentration above normal, compared with 20 of 21 chronically anemic patients, whose erythropoietin concentrations were above normal.. The erythropoietin response to known physiologic stimuli is blunted in critically ill children. This blunted erythropoietin response may result in increased transfusion requirements.

Topics: Acute Disease; Analysis of Variance; Anemia; Blood Transfusion; Case-Control Studies; Child; Child, Preschool; Chronic Disease; Critical Illness; Erythropoietin; Hemoglobins; Humans; Hypoxia; Infant; Infant, Newborn; Intensive Care Units, Pediatric; Matched-Pair Analysis; Outpatients; Prospective Studies; Severity of Illness Index

Topics: Animals; Cytochrome b Group; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Oxygen; Photosystem II Protein Complex; Signal Transduction

Topics: Animals; Erythropoietin; Female; Hypoxia; Kidney Tubules, Proximal; Mercuric Chloride; Necrosis; Potassium Dichromate; Rats; Rats, Wistar; Reference Values; Urine

Topics: Animals; Erythropoietin; Humans; Hypoxia; Oxygen

The ability to adapt successfully to periods of relative hypoxia is crucial to the survival of all higher life forms. Several genes have previously been identified which are up-regulated in response to hypoxia; these include the genes encoding erythropoietin (Epo), platelet-derived growth factor B chain, endothelin, interleukin-1 alpha, ornithine decarboxylase, and vascular endothelial growth factor (VEGF). However, the molecular mechanisms by which hypoxia is sensed remain enigmatic. In addition, it is unknown whether the genes mentioned share a common oxygen-sensing signal transduction pathway. In this report we demonstrate multiple similarities between the oxygen-sensing mechanisms regulating the expression of VEGF and Epo. The expression of both mRNAs is significantly up-regulated by hypoxia and cobalt chloride (CoCl2), and the half-life of both mRNAs is markedly prolonged by cycloheximide. In addition, hypoxic induction of both Epo and VEGF is inhibited by carbon monoxide. As part of our investigation into the signal transduction pathway responsible for the hypoxia and cobalt induction of these genes, we discovered that the expression of members of the jun and fos protooncogene families is also up-regulated early after exposure to either of these stimuli. These findings provide support for the hypothesis that the mechanism(s) by which hypoxia is sensed at a molecular level may be highly conserved and tightly regulated.

Topics: Animals; Cobalt; Cycloheximide; Endothelial Growth Factors; Erythropoietin; Gene Expression Regulation; Genes, fos; Genes, jun; Heme; Humans; Hypoxia; In Vitro Techniques; Lymphokines; Oxygen; Rats; RNA, Messenger; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The human hepatoma cell line, Hep 3B, produces biologically active erythropoietin (Epo) in response to normal physiologic stimuli and thus provides a model system for the study of Epo regulation. The addition of phorbol 12-myristate 13-acetate (PMA) to Hep 3B cells subsequently grown under hypoxic conditions resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 95 +/- 1% with half-maximal inhibition at 8 ng/mL. By Northern blot analysis, Epo mRNA levels were correspondingly decreased after treatment with PMA. Direct measurement of both membrane and cytosolic protein kinase C activity in Hep 3B cells following treatment with PMA demonstrated a biphasic response as a function of time. Membrane-associated protein kinase C activity initially increased but subsequently decreased to baseline levels by 12 hours. The PMA-induced inhibition of hypoxia-induced Epo production was shown to occur as early as 3 hours after PMA addition, suggesting that the initial activation, rather than the subsequent decrease in protein kinase C activity, is of primary importance. The relative specificity of the PMA-induced inhibition of Epo production was demonstrated by 1) the finding that overall protein and RNA synthesis were not similarly decreased as measured by 3H-leucine and 3H-uridine pulse labeling studies and 2) the observation that the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, failed to have any effect on hypoxia-induced Epo production. In addition, the synthetic analog of diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore, A23187, inhibited hypoxia-induced Epo production up to 85 +/- 3% and 82 +/- 4%, respectively, in a dose-dependent manner. Taken together, these findings suggest that hypoxia-induced Epo production may be negatively regulated by activators of a protein kinase C-mediated pathway.

Topics: Blotting, Northern; Calcimycin; Carcinoma, Hepatocellular; Diglycerides; DNA; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Leucine; Liver Neoplasms; Protein Kinase C; Radioimmunoassay; RNA; Tetradecanoylphorbol Acetate; Tritium; Tumor Cells, Cultured; Uridine

The low plasma erythropoietic (Epo) activity which is non-adequate to manifestation of anemia, and the lack of correlation between Epo activity and the degree of anemia and hypoxia were found in children with sepsis. The lowest Epo activity was determined in plasma of patients after repeatedly blood transfusion and in emaciated children. The non-specific Epo activity inhibitor was determined in acute period of sepsis in majority of patients. We suppose that the low Epo activity was due to the violation of Epo synthesis regulation mechanism or was connected with the presence of non-specific inhibitors. These results suggest recombinant Epo for the treatment of anemia in children with sepsis.

Topics: Anemia, Hypochromic; Animals; Bacterial Infections; Blood Transfusion; Cells, Cultured; Erythropoietin; Hemoglobins; Humans; Hypoxia; Infant; Mice; Mice, Inbred BALB C; Reference Values

1. Fetal sheep at 120 days gestation were fitted with upper and lower body arterial and venous catheters in addition to a flow sensor and occluder placed around the aorta below the renal arteries. 2. After 7 days of recovery, the occluder was partially inflated to reduce aortic blood flow to 70% of control. Blood flow reduction was maintained at this level for the remainder of the experiment. 3. Blood samples were taken after 60 min of blood flow reduction and again after 3 or more days of blood flow reduction. 4. There was no change in upper body arterial or venous blood pressure. Lower body arterial blood pressure decreased, as expected. Arterial PO2 decreased while packed cell volume and haemoglobin concentration increased. There was no change in plasma erythropoietin concentrations or plasma renin activity. 5. While both red cell mass and haemoglobin mass increased during the period of the study, the rate of increase was no different from the rate of blood volume increase.

Topics: Animals; Blood Flow Velocity; Blood Pressure; Blood Volume; Erythrocytes; Erythropoietin; Female; Fetal Blood; Fetal Hypoxia; Heart Rate; Hematocrit; Hemoglobins; Hypoxia; Pregnancy; Sheep

The present studies were undertaken to assess the effects of 5'-N-ethylcarboxamideadenosine (NECA), an adenosine analogue, on erythropoietin (Epo) production. NECA (0.05 and 0.1 mumol/kg i.v.) produced significant increases in serum Epo levels (368.8 +/- 56.1 and 384.6 +/- 45.9 mU/ml, respectively) in exhypoxic polycythemic mice after a four hour exposure to hypoxia when compared with hypoxia controls (133.2 +/- 18.2 mU/ml). The hypoxic kidney Epo levels were 46.4 +/- 13.4 mU/kg kidney which were significantly higher than normoxic kidney Ep levels (< 1.24 mU/kg kidney). Theophylline (20 mg/kg i.p.), an adenosine receptor antagonist, significantly inhibited the stimulatory effects of NECA on serum Epo levels. In vitro cultures of an Epo producing hepatocellular carcinoma (Hep3B) cell line with NECA (> or = 10(-6) M) for 20 hours under hypoxic conditions (1% O2) produced significant increases in medium levels of Epo when compared with hypoxia controls. Hepatocellular carcinoma cells treated with NECA at a concentration range of 10(-7) M to 5 x 10(-5) M for one hour in a hypoxic atmosphere also had significantly higher cAMP levels than that of hypoxia controls. Scatchard analyses of [3H]NECA binding to membrane preparations of hepatocellular carcinoma cells showed low affinity binding sites with a dissociation-constant (Kd) of 0.44 microM and a binding capacity of 863 fmol/mg protein. These findings suggest that the increase in Epo production in response to NECA under hypoxic conditions can be attributed, at least in part, to stimulation of adenosine A2 receptors which is coupled to adenylyl cyclase activation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Erythropoietin; Female; Hypoxia; Liver Neoplasms; Mice; Mice, Inbred Strains; Osmolar Concentration; Polycythemia; Theophylline; Tumor Cells, Cultured

Erythropoietin (Epo) production in response to hypoxia or cobalt is primarily mediated by activation of transcription of the Epo gene. Recently an hypoxia responsive enhancer was identified in the 3' flanking region of the mouse and human Epo genes. Using functional analysis in Hep 3B cells we define here the minimal enhancer element as a 29-bp segment starting at the Apa1 site in the 3' flanking region of the human Epo gene. Mutagenesis studies of the minimal element identified three different areas that are necessary for full enhancer activity. Electrophoretic mobility shift assays show the presence of hypoxia- and/or cobalt-inducible nuclear DNA-binding proteins that bind to one of the active sites of the enhancer. Induction of hypoxia-binding activity was abolished by Anisomycin, a potent protein synthesis inhibitor, suggesting that de novo protein synthesis is necessary for the activation process. Further characterization of DNA-binding proteins by use of UV light crosslinking identified a protein of molecular weight of approximately 120-Kd that was present only in hypoxic extracts. This protein was found to be present in hypoxic nuclear extracts from both Epo-producing and non-Epo-producing cells, suggesting that it may be involved in a more generalized mechanism of cellular response to hypoxia.

Topics: Base Sequence; Cell Line; Deoxycholic Acid; DNA Mutational Analysis; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; In Vitro Techniques; Molecular Sequence Data; Molecular Weight; Nuclear Proteins; Protein Binding

Since in juvenile rats the liver is the predominant site of erythropoietin (EPO) gene expression, we have used primary cultures of juvenile rat hepatocytes to establish and in vitro system for investigation of oxygen-dependent EPO formation. When isolated hepatocytes were incubated at reduced oxygen tensions for 18-48 h, we found increased secretion of EPO protein and elevated levels of EPO mRNA, as determined by RNas protection. This increase was maximal at 3% O2, where EPO mRNA levels after 18 h were approximately 15-fold higher than at 20% O2. The increase in EPO mRNA at low oxygen tensions was specific insofar as [3H]uridine incorporation, as a measure of total RNA synthesis, was reduced by approximately 50% at 3% O2, and it appeared to involve gene transcription since it was abolished in the presence of actinomycin D (35 microM). Significant increases in EPO mRNA were also observed in cells kept at 20% oxygen in the presence of cobalt chloride (50 microM) and nickel chloride (400 microM), but EPO mRNA levels achieved under these conditions were less than 7% of those in cells incubated at 3% oxygen. No increase in EPO mRNA levels was observed in cultures incubated at 20% O2 in the presence of cyclic dibutyryl-AMP (10 microM-3 mM), cyclic 8-bromoGMP (10 microM-1 mM), cyclohexyladenosine (1 microM), 5'-N-ethylcarboxamidoadenosine (1 microM) and phorbol 12-myristate 13-acetate (3 nM). In the presence of 10% carbon monoxide, used to block haem proteins in their oxy conformation, EPO mRNA levels in hepatocytes incubated at low oxygen tensions were reduced to 63%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Survival; Cells, Cultured; Centrifugation; Erythropoietin; Gene Expression Regulation; Hypoxia; Liver; Male; Oxygen; Protein Biosynthesis; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Second Messenger Systems

At present, nearly all infants with birth weights of < 1 kg receive blood transfusions for treatment of clinical signs of tissue hypoxia resulting from anemia of prematurity. In contrast to the successful use of recombinant human erythropoietin (rhEp) in adults, treatment of anemic neonates with rhEp to stimulate red cell production and reduce the need for transfusions that pose serious infectious and immunologic risk has not been effective. The present study investigates the pharmacodynamics (PD) of endogenous erythropoietin (Ep) in sheep fetuses to determine possible causes for the poor rhEp response in early development. The dynamic relationship between plasma Ep and plasma iron resulting from spontaneous hypoxemic episodes is investigated by PD system analysis. The erythropoietic effect of Ep is measured in terms of the mobilization of plasma iron needed in the production on new erythrocytes. A hysteresis minimization approach is employed to determine the intrinsic PD dose-response relationship (transduction) of Ep. The dose-response relationship shows a well-defined threshold level that has to be exceeded before Ep begins to show a significant effect on plasma iron. It is postulated that the threshold mechanism may serve a useful purpose during early development by reducing the risk of the fetus developing a pathological degree of polycythemia and hyperviscosity in the relatively hypoxemic fetal environment. At the same time, the threshold serves the purpose of providing a needed response to more severe pathologic hypoxemic episodes. The occurrence of anemia during subsequent postnatal life when PaO2 levels increase markedly may be the inevitable, but unfortunate corollary of a continuation of this mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Dose-Response Relationship, Drug; Erythropoietin; Fetus; Hypoxia; Iron; Sheep

1. To clarify the relationship between nocturnal oxygen desaturation and erythropoietin production in patients with chronic obstructive pulmonary disease, we determined arterial oxygen saturation and serum immunoreactive erythropoietin levels over 24 h in eight patients with chronic obstructive pulmonary disease and in nine normal subjects. 2. In the normal subjects, there was a significant circadian variation in serum erythropoietin levels with the highest mean deviation from the geometric mean at 22.00 hours and the nadir at 05.00 hours. 3. The three patients with chronic obstructive pulmonary disease with the most marked nocturnal desaturation (lowest arterial oxygen saturation < 57%) and most marked daytime hypoxaemia (daytime arterial partial pressure of oxygen < 6 kPa) had raised nocturnal serum erythropoietin levels. In two of these patients, the serum erythropoietin level was raised throughout the 24 h and erythrocyte mass was also raised. In the other patient, the serum erythropoietin level was not raised in five daytime samples and erythrocyte mass was normal. 4. The other five patients with chronic obstructive pulmonary disease with less severe nocturnal hypoxaemia (lowest arterial oxygen saturation range 78-86%) had serum erythropoietin levels (range 14-36 m-i.u./ml) which were indistinguishable from normal (range 12-44 m-i.u./ml) and showed circadian changes which were not significantly different (P = 0.35) from those in the normal subjects. 5. Thus, mild nocturnal oxygen desaturation is not associated with elevation of serum erythropoietin levels, whereas daytime hypoxaemia with associated severe nocturnal desaturation is associated with increased serum erythropoietin levels both by day and by night.

Topics: Aged; Chronic Disease; Circadian Rhythm; Erythropoietin; Female; Humans; Hypoxia; Lung Diseases, Obstructive; Male; Oxygen; Sleep

In two rat strains (H and M) with differing susceptibilities to chronic hypoxia we examined the role of polycythemia in the differing hypoxic pulmonary hemodynamic responses. We hypothesized that augmentation of hematocrit (Hct) during hypoxia in the resistant M strain would render cardiopulmonary responses similar to those obtained in the susceptible H strain. Administration of human recombinant erythropoietin (EPO) in doses of 100, 250 and 500 U.kg-1 s.c. thrice weekly for three weeks raised Hct similarly in both strains indicating that normoxic rats had similar sensitivities to EPO. In rats exposed to hypobaric hypoxia (0.5 atm) for 21 days, EPO (500 U.kg-1 thrice weekly) significantly increased Hct and whole blood viscosity as expected. Surprisingly, right ventricular (RV) to body weight (BW) ratio as an index of right ventricular hypertrophy (RVH) and RV peak systolic pressure did not increase in EPO-injected rats of either strain compared to hypoxic controls. Among hypoxic animals, Hct correlated highly with viscosity but not with RV/BW. We conclude, contrary to our hypothesis, that polycythemia does not appear to be responsible for the strain difference in RVH and pulmonary hypertension.

Topics: Animals; Erythropoietin; Hematocrit; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Polycythemia; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Species Specificity

The major stimulus for erythropoietin production is tissue hypoxia. We sought to investigate the relationship of beta-sympathomimetic administration for tocolysis and fetal serum erythropoietin.. Umbilical cord blood was obtained from infants whose mothers received intravenous beta-sympathomimetic tocolysis and who were delivered at < or = 34 weeks' gestation. Serum erythropoietin was measured by radioimmunoassay. On the basis of the presumed 2- to 4-hour half-life of fetal erythropoietin, the infants were divided into two groups. In group 1 (n = 16) beta-sympathomimetic therapy was discontinued < 24 hours before delivery; in group 2 (n = 11) it was discontinued > or = 24 hours before delivery.. Group 1 fetuses had significantly higher erythropoietin levels than did group 2 fetuses (37.3 vs 13.9 mU/ml, p = 0.02). The duration of beta-sympathomimetic tocolysis and the maximum infusion rate were not different. The two groups did not differ in gestational age, birth weight, route of delivery, presence of labor, or duration of first or second stage of labor.. We speculate that intravenous beta-sympathomimetic tocolytic therapy stimulates fetal erythropoietin production by decreasing fetal oxygenation as a result of the reversible fetal metabolic effects of the tocolysis. These data suggest that beta-sympathomimetic tocolysis should be undertaken cautiously if fetal compromise is suspected, fetal well-being should be assessed carefully if tocolysis is undertaken, and treatment should be discontinued promptly if a clear benefit is not realized.

Topics: Erythropoietin; Female; Fetal Blood; Fetal Diseases; Fetus; Humans; Hypoxia; Infusions, Intravenous; Pregnancy; Sympathomimetics; Tocolytic Agents

Information on morphological and functional effects of anemia in kidney is scarce, although this organ plays a major role in erythropoietin production, which is strongly stimulated in anemia. We undertook a morphological study of kidneys of anemic rats. Anemia was induced by X-irradiation and subsequent injection of a hemolytic drug. The most striking effects of anemia on renal morphology were damages in the proximal tubule and a volume increase of the peritubular space. These effects were evident only in the cortical labyrinth. Morphometry showed that the enlargement of the peritubular space reflected an increase of the volumes of both capillaries and interstitium. The structural changes in the cortical interstitium were associated with increased activity of the ecto-5'-nucleotidase in the fibroblasts. We suggest that hypoxia accounts for most of the observed alterations. The hypoxic proximal tubule might release the nucleotide AMP, which would be hydrolyzed to adenosine by the ecto-5'-nucleotidase in the interstitium. Adenosine has been reported to trigger the synthesis of erythropoietin and the growth of blood capillaries.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Monophosphate; Anemia; Animals; Erythropoietin; Fibroblasts; Fluorescent Antibody Technique; Hematocrit; Hypoxia; Kidney Cortex; Kidney Medulla; Male; Microscopy, Electron; Organ Size; Rats; Rats, Wistar

We have used in situ hybridization to determine the localization and distribution of cells expressing the erythropoietin (EPO) gene in kidneys of rats exposed to reduced oxygen tensions to characterize the control of renal EPO formation during hypoxic hypoxia. Animals were subjected to severe hypoxia (7.5% O2) for 4, 8 and 32 hours to assess changes related to the duration of hypoxic exposure, and additionally to 9% and 11.5% O2 for eight hours to define changes related to the degree of hypoxia. The number of cells containing EPO mRNA were counted on tissue sections and compared to tissue concentrations of EPO mRNA and to the serum hormone concentrations. In situ hybridization revealed expression of the EPO gene exclusively in peritubular cells that were predominantly located in the cortical labyrinth under all conditions tested. After four hours of severe hypoxia (7.5% O2) approximately 170-fold more cells were found to contain EPO mRNA than under normoxic conditions. The number of EPO producing cells did not change significantly between four and eight hours exposure to 7.5% O2, but the amount of EPO mRNA per kidney increased approximately threefold. Further continuation of hypoxia resulted in down-regulation of renal EPO mRNA levels, which was mainly due to a reduction in the number of cells containing EPO mRNA. Comparison of graded degrees of hypoxia applied for eight hours showed an inverse exponential relationship between oxygen tension and the number of EPO producing cells. This recruitment of cells expressing the EPO gene occurred along a gradient extending from the corticomedullary border to the subcapsular tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Anemia; Animals; Erythropoietin; Gene Expression; Hypoxia; In Situ Hybridization; Kidney; Male; Rats; RNA, Messenger; Time Factors; Tissue Distribution

The 5'-flanking region of the human erythropoietin (Epo) gene contains a 0.14-kb sequence that is conserved in the Epo gene from mouse and located within a promoter that is activated under hypoxic conditions such as anemia. Using a fragment containing this sequence in DNA mobility shift assays, we found that specific DNA-binding proteins were induced in mouse kidney nuclei under anemic hypoxia. Using synthetic double-stranded oligonucleotides that contain this sequence, the essential binding site was defined to be the -40 to -20 region upstream of the transcription initiation site in the human Epo gene. By DNA affinity chromatography using a column with the immobilized 5'-flanking sequence, two inducible binding proteins with apparent molecular masses of 55 and 45 kDa were identified in the nuclei of mouse kidney and liver under anemic hypoxia. These binding proteins were also induced during cobalt exposure.

Topics: Anemia; Animals; Base Sequence; Cell Line; Chromatography, Affinity; Cobalt; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Gene Expression; Humans; Hypoxia; Kidney; Male; Mice; Mice, Inbred BALB C; Molecular Sequence Data

The role of the submandibular salivary gland in erythropoiesis in the male mouse (MRC TO strain) was evaluated by subjecting mice without submandibular salivary glands (SX) and control (C) sham-operated mice to a variety of stimuli intended to stress the erythropoietic system. In SX mice, after removal of the submandibular glands at age 4 weeks and observation for 8 weeks, mean hematocrit was the same as in C mice, but mean body weight was less. Bilateral removal of the submandibular glands at age 6 weeks neither affected the rate of fall and subsequent recovery of hematocrit which followed treatment with phenylhydrazine (80 mg/kg intraperitoneally [i.p.] 9 days after operation) nor altered the rate of increase in hematocrit or change in body weight which occurred during hypobaric hypoxia (0.5 atm, > 23 hours/day) for 23 days. Mean (SEM) estimates of serum immunoreactive erythropoietin after 17 hours' continuous hypobaric (0.5 atm) exposure were not significantly different between SX [186 (30) mU/mL, n = 7] and C mice [232 (17) mU/mL, n = 7]. In mice given bilateral nephrectomies at age 6 weeks--2 weeks after SX or C surgery--and then both treated with phenylhydrazine (60 mg/kg i.p.) and exposed for 17 hours to hypobaric (0.5 atm) hypoxia, mean estimates of serum immunoreactive erythropoietin were 22.6 (10.6) mU/mL and 22.3 (5.4) mU/mL in SX (n = 5) and C (n = 5) mice. Results of the study do not support the premise that the submandibular salivary glands either contribute to the erythropoietic response or are a source of extrarenal erythropoietin.

Topics: Animals; Atmospheric Pressure; Body Weight; Erythropoiesis; Erythropoietin; Hematocrit; Hypoxia; Male; Mice; Phenylhydrazines; Submandibular Gland

The present study was performed to evaluate the effects of chronic inspiratory hypoxia and its combination with physical exercise on plasma erythropoietin concentration ([EPO]). Eight natives from the Bolivian Plateau were investigated at 3,600 m above sea level at rest as well as during and up to 48 h after exhaustive exercise (EE) and 60 min of submaximal (60%) cycle ergometer exercise (SE). Ten sea-level subjects were used as a control group for resting values. The mean resting plasma [EPO] of the high-altitude group (19.5 +/- 0.7 mU/ml) did not differ from that of the sea-level group (18.1 +/- 0.4 mU/ml) but was higher than would be expected from the relationship between [EPO] and hematocrit at sea level. Five hours after both types of exercise, [EPO] decreased by 2.1 +/- 0.8 (EE, P < 0.01) and 1.6 +/- 0.8 mU/ml (SE, P < 0.05); 48 h after SE, [EPO] increased by 2.6 +/- 0.9 mU/ml (P < 0.05). It is concluded that 1) high-altitude natives need relatively high [EPO] to maintain their high hematocrit and 2) exercise at low basal arterial PO2 does not directly increase plasma [EPO] in high-altitude residents but seems to exert suppressive effects.

Topics: Acclimatization; Adult; Altitude; Erythropoietin; Exercise; Hematocrit; Humans; Hypoxia; Male; Oxygen; Plasma Volume

Topics: Animals; Erythropoietin; Feedback; Hematocrit; Hypoxia; In Vitro Techniques; Kidney; Male; Oxygen; Perfusion; Rats; Rats, Sprague-Dawley

A relationship between erythropoietin (EPO) and erythroid colony-stimulating activity (ECSA) in mouse plasma was examined in fetal mouse liver cell (FMLC) cultures using a monoclonal antibody (MoAb) R2 raised against recombinant human EPO. Most of the ECSA in plasma from normal, anemic, and hypoxic mice was neutralized by MoAb. This neutralization could be reversed by addition of excess of anemic plasma or by preincubation of MoAb with goat anti-mouse IgG antibody. Most of the plasma ECSA was bound to an immunoadsorbent column containing the immobilized MoAb, and the retained ECSA was completely neutralized by MoAb. The plasma ECSA and standard EPO showed parallel dose-response curves and additive effect on CFU-E stimulation. Based on these findings, we conclude that mouse plasma ECSA detected by CFU-E assay using FMLCs is mainly due to EPO.

Topics: Anemia; Animals; Antibodies, Monoclonal; Chromatography, Affinity; Colony-Forming Units Assay; Dose-Response Relationship, Immunologic; Erythroid Precursor Cells; Erythropoietin; Humans; Hypoxia; Male; Mice; Mice, Inbred ICR; Neutralization Tests; Recombinant Proteins

This study reports the effects of cyclic adenosine 3'-5' monophosphate (cAMP) and hypoxia on erythropoietin biosynthesis in an erythropoietin-producing hepatocellular carcinoma cell line (Hep3B). Erythropoietin levels in the medium and cell extracts of low-density Hep3B cells after 20-hour incubation under hypoxic conditions (1% O2) were 25.33 +/- 1.50 mU/ml/10(7) cells and 3.60 +/- 0.50 mU/10(7) cells, respectively. These levels were significantly higher than in the respective normoxic controls (medium, 2.51 +/- 0.31 mU/ml/10(7) cells; cell extracts, undetectable [less than 0.31 mU/10(7) cells]). Cobalt also produced a significant increase in medium and cell erythropoietin levels. However, hypoxia and cobalt alone failed to produce an increase in cAMP accumulation in the cell cultures. Erythropoietin levels in the medium and cell extracts from cells exposed to 8-bromo cAMP (1 x 10(-4) mol/L) and forskolin (4 x 10(-6) mol/L) in a hypoxic atmosphere were significantly (p less than 0.05) higher than in the respective hypoxic controls. In addition, forskolin produced a significant (p less than 0.05) increase in cAMP accumulation (180 +/- 11.5 pmol/10(6) cells) under hypoxic conditions compared with the hypoxic controls (cAMP, 2.27 +/- 0.33 pmol/10(6) cells). These results suggest that cAMP elevation is not required in vitro in Hep3B cells for the increase in medium and cell levels of erythropoietin after hypoxia, but may be involved indirectly in erythropoietin biosynthesis, secretion, or both in vivo through some synergistic action with hypoxia.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Carcinoma, Hepatocellular; Cobalt; Colforsin; Cyclic AMP; Erythropoietin; Humans; Hypoxia; Liver Neoplasms; Tumor Cells, Cultured

Erythropoietin (EPO) is mainly produced in the kidneys and is regulated by blood oxygen availability. Studies with isolated perfused kidneys have established that an oxygen-sensing system exists intrarenally but the mechanisms involved are poorly understood. Using a quantitative RNase protection assay, we have demonstrated oxygen-dependent EPO mRNA production in isolated perfused rat kidneys, with EPO mRNA levels increasing 30-fold when perfusate pO2 was reduced from 474 to 25 mm Hg. To determine if the high amplitude changes in EPO mRNA levels in response to hypoxia are mediated by cyclic AMP, four agents, which activate the cyclic AMP system in different ways, were administered to isolated kidneys perfused over a range of perfusate pO2. Salbutamol and N6-ethyl carboxamidoadenosine, which activate adenylate cyclase, dibutyryl cyclic AMP (a cyclic AMP analogue) and forskolin did not augment EPO mRNA production, and no significant differences in the regression of log (EPO mRNA) on perfusate pO2, were found between experimental groups exposed to each of these compounds and controls. We conclude that the rapid increase in EPO mRNA levels in response to hypoxia is not mediated or substantially modulated by a cyclic AMP-dependent mechanism.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Albuterol; Animals; Bucladesine; Colforsin; Cyclic AMP; Erythropoietin; Hypoxia; In Vitro Techniques; Kidney; Male; Oxygen; Perfusion; Rats; Rats, Inbred Strains; RNA, Messenger

Hypoxia causes an increased production of erythropoietin (EPO), but the time course of the EPO response in humans has not been well characterized. This study examines the relationship between the duration of normobaric hypoxic exposure and plasma EPO levels in healthy human subjects. Six volunteers breathed a gas mixture of 10.5% O2-89.5% N2 continuously for 5, 60, and 120 or intermittently for 240 min. O2 saturations were maintained between 75 and 85% during the exposure. Arterial pH was 7.467 +/- 0.019, PO2 37.05 +/- 2.43 Torr, and PCO2 36.69 +/- 2.05 Torr. O2 half-saturation pressures of hemoglobin were normal for all subjects. Plasma EPO was measured every 30 min for 360 min by radioimmunoassay. No increase in EPO was seen after the 5- and 60-min exposures. However, a 50% increase was seen 240 min after the initiation of the 120-min hypoxic exposure (P less than 0.01). Intermittent exposure resulted in an increase of EPO by 52% 360 min after the onset of exposure (P less than 0.05). Therefore, exposing humans continuously to an inspiratory O2 fraction of 0.105 for 120 min or intermittently for 240 min provides a sufficient stimulus to increase production of EPO.

Topics: Adult; Altitude; Erythropoietin; Humans; Hypoxia; Kinetics; Male; Polycythemia; Sleep Apnea Syndromes

Erythropoietin (EPO) mRNA levels were measured by ribonuclease (RNase) protection in organs from unstimulated rats and from animals after normobaric hypoxia or hemorrhagic anemia. Both liver and kidney responded to stimulation with large increases in EPO mRNA, but the response characteristic to graded stimulation was different. The liver responded poorly to mild normobaric hypoxia, accounting for only 2 +/- 1% of total EPO mRNA at 11% O2, but hepatic EPO mRNA levels increased steeply with more severe hypoxia so that at 7.5% O2 the liver contributed to 33 +/- 7% of the total. After hemorrhagic anemia, the liver also responded more strongly to more severe stimulation, but at all points it accounted for a significant proportion of total EPO mRNA, contributing 18 +/- 6% after removal of 2.5 ml (hematocrit 37.2 +/- 1.3%), increasing to 37 +/- 14% after venesection of 10.5 ml (hematocrit 15.8 +/- 0.8%). Studies of EPO mRNA in other organs confirmed that EPO production outside the liver and kidney were quantitatively insignificant in stimulated animals. However, the hypoxia-induced increases in EPO mRNA in brain, testis, and spleen suggest the existence of an oxygen-sensing mechanism at other sites.

Topics: Acute Disease; Anemia; Animals; Erythropoietin; Feedback; Hemorrhage; Hypoxia; Kidney; Liver; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors; Tissue Distribution

We studied erythropoietin (EPO) metabolism, regulation, and pharmacokinetics in rats with nephrotic syndrome. Sprague-Dawley rats were randomized into nephrotic (puromycin-induced) and pair-fed control groups. Animals were studied at baseline and after induction of anemia or exposure to hypobaric conditions (32 cmHg). The nephrotic group showed a reduced hematocrit (P < 0.05), a significant urinary EPO excretion, and an inappropriately low plasma EPO. Induction of anemia and exposure to hypoxia resulted in a less pronounced elevation of plasma EPO in the nephrotic group than in the control group (P < 0.05). The blunted plasma EPO response to hypoxia in nephrotic animals was associated with a marked rise in urinary EPO excretion. Pharmacokinetic studies following intravenous injection of recombinant EPO, 100 U/kg, revealed a shorter plasma half-life (t1/2) (P < 0.05), larger apparent volume of distribution (P < 0.05), and greater clearance (P < 0.02) in the nephrotic group than in the controls. Estimated endogenous EPO production rate in nephrotic rats with severe anemia was significantly lower (P < 0.05) than that of equally anemic controls. Thus puromycin-induced nephrotic syndrome is associated with marked urinary loss of EPO, relatively depressed plasma EPO response to anemia and hypoxia, as well as reduced plasma t1/2, increased volume of distribution, and clearance of exogenous EPO.

Topics: Anemia; Animals; Blood Cell Count; Erythropoietin; Hematocrit; Hypoxia; Male; Nephrosis; Puromycin; Rats; Rats, Sprague-Dawley

Chronic mountain sickness, which affects permanent residents of high altitudes, is the outcome of a progressive loss of ventilatory rate which naturally occurs with age and resulting in excessive hypoxemia and polycythemia. A theoretical model predicts the progressive failure of homeostatic control of the hemoglobin concentration when the values increase above those found at sea level. This is confirmed by lack of feedback mechanism between high altitude erythrocytosis and serum erythropoietin. The results of epidemiological studies are in agreement with the physiological findings. In a male population living at 4,300 m, an increase with age of the prevalences of excessive erythrocytosis (Hb > 213 g/l), blood oxygen saturation < 83%, headaches and a high score of symptoms of chronic mountain sickness has been found. The studies suggest the possibility that in addition to an accentuated hypoxemia, the excessive erythrocytosis may also result from an overreaction of the bone marrow to a fixed level of hypoxemia in ageing individuals.

Topics: Adult; Aged; Aging; Altitude Sickness; Chronic Disease; Erythropoietin; Homeostasis; Humans; Hypoventilation; Hypoxia; Male; Middle Aged; Models, Theoretical; Peru; Polycythemia; Prevalence

A polysomnographic study was undertaken in nine patients with unexplained polycythaemia and nine age- and sex-matched controls. Circulating plasma levels of immunoreactive erythropoietin (IrEPO) were analysed before and after sleep. Seven out of nine polycythaemia patients were found to have sleep-disordered breathing and fulfilled the criteria for the sleep apnoea syndrome. Erythrocyte volume fraction was by definition higher among polycythaemia patients, and showed a weak positive correlation with minimum oxygen saturation during sleep (P less than 0.05). However, plasma IrEPO did not differ between the two groups or between morning and evening samples within the respective groups. In a separate study, IrEPO was repeatedly analysed during sleep in a group of six patients with severe obstructive sleep apnoea and six matched controls. No correlation with severity of sleep-disordered breathing was found. None of these patients had polycythaemia, and there was no obvious diurnal variation in IrEPO levels. A nocturnal sleep study may be warranted in patients with unexplained polycythaemia. Obstructive sleep apnoea does not appear to be related to increased IrEPO levels, although polycythaemia has been reported as a relatively common finding in this disease.

Topics: Circadian Rhythm; Erythropoietin; Humans; Hypoxia; Middle Aged; Monitoring, Physiologic; Polycythemia; Sleep Apnea Syndromes

We measured arterial oxygen saturation (SaO2) and serum erythropoietin (EPO) levels in patients with obstructive sleep apnea syndrome (OSAS) and in healthy subjects. SaO2 profile was analyzed as the cumulative distribution of SaO2 over time. OSAS patients spent 25.5 +/- 30.9% (SD) of the time at SaO2 of less than 88% vs. 0.64 +/- 1.6% in healthy subjects (P less than 0.05) and had 59 +/- 25 desaturations (greater than 4%) per hour vs. less than 5 per hour in healthy subjects (P less than 0.05). EPO measurements (radioimmunoassay) were made in blood samples withdrawn every 1 or 2 h over a 24-h period. The mean EPO concentration was higher (P less than 0.05) for OSAS patients (45 +/- 33 mU/ml) than for normal subjects (17 +/- 8 mU/ml). There was a significant variability in EPO values over the 24-h period. To analyze the EPO pattern over 24 h, EPO time series were fit to a general cosine function. Data from normal subjects conformed to a cosine function with an amplitude of 3.5 +/- 2.1 (CV of 60%) and an acrophase of 1,000 +/- 184 min after 0800 (CV of 18%), indicating a zenith about 1 A.M. and a nadir around 1 P.M. Data from OSAS subjects fit a similar cosine function with an amplitude of 19.9 +/- 41.0 (CV of 206%) and acrophase of 582 +/- 408 min (CV of 70%), indicating a greater variability in the magnitude and the timing of peak serum EPO levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Aged; Circadian Rhythm; Erythropoietin; Humans; Hypoxia; Male; Middle Aged; Oxygen; Sleep Apnea Syndromes

Hilltop (H) and Madison (M) strains of Sprague-Dawley rats exhibit strikingly different susceptibilities to the effects of chronic altitude exposure. The H rats develop greater polycythemia, hypoxemia, and pulmonary hypertension. We studied ventilation, pulmonary gas exchange, tissue oxygenation, and hematologic adaptations in the two rat strains during a 50-day exposure to a simulated altitude (HA) of 5,500 m (18,000 ft). There were no strain differences among the variables we studied under sea level (SL) conditions. Within the first 14 days of hypoxic exposure, the only significant strain differences were that erythropoietin (EPO) rose much higher and erythroid activity was greater in the H rats, even though arterial Po2 and PCo2 (Pao2 and PaCo2, respectively), renal venous PO2 (Prvo2), and ventilation (VE) were equivalent in the two strains during this time. By day 14 at HA, the H rats had significantly higher erythroid activity, hematocrit (Hct), and EPO levels, significantly lower PaO2 and PrvO2, but equivalent VE and PaCO2. These changes persisted for the remainder of the exposure, except that the Hct continued to rise and the increase was greater in H rats. Despite the greater O2-carrying capacity of H rats in the later stages of hypoxic exposure, PaO2 and PrvO2 were significantly lower in H rats. There were no strain differences at either SL or HA in ventilatory responses to hypercapnia or hypoxia, in blood O2 affinity or 2,3-diphosphoglycerate, in extrarenal production of EPO, or in EPO clearance. We conclude that early in the hypoxic exposure the H rats produce more EPO at apparently equivalent levels of hypoxia, and this is the first step in the pathogenesis of the maladaptation to HA manifest by H rats. We find no consistent evidence that differences in VE contribute to the variable susceptibility to hypoxia in the two rat strains.

Topics: Altitude Sickness; Animals; Disease Models, Animal; Erythropoietin; Hematopoiesis; Hypoxia; Kidney; Male; Oxygen; Rats; Rats, Inbred Strains; Respiration; Species Specificity

The mechanisms which control the production of erythropoietin (Epo) remain enigmatic. Recent data suggest that the half-time of Epo messenger RNA (mRNA) is increased by hypoxia in Hep 3B cells, a human hepatoma line. The post-transcriptional regulation of other rapidly degraded mRNAs is mediated by sequence-specific mRNA binding proteins. In order to determine if Epo mRNA specific binding proteins exist, we probed cytosolic lysates from Hep 3B cells and mouse tissues with radiolabeled Epo RNA. A cytosolic protein that binds specifically to Epo RNA was identified in the Epo-producing, hepatoblastoma Hep 3B cell line by gel mobility shift assay. This protein was identified in both normoxic and hypoxic cells and bound specifically to a 120-base fragment of the 3'-untranslated region (3'-UTR) of Epo mRNA. Binding was completed with unlabeled Epo RNA, but not with granulocyte-macrophage colony-stimulating factor RNA. Ultraviolet light cross-linked Epo RNA-protein complexes migrated as two bands of 70 and 135-140 kD on sodium dodecyl sulfate-polyacrylamide gels. Binding activity was markedly increased in brain and spleen lysates from mice subjected to 24 h of hypoxia. Therefore, the post-transcriptional regulation of Epo expression in response to hypoxia may in part be due to the interaction of Epo RNA with its specific binding protein.

Topics: Animals; Binding, Competitive; Carcinoma, Hepatocellular; Carrier Proteins; Cytosol; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; Mice; Oxygen; Restriction Mapping; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; RNA, Messenger; Tumor Cells, Cultured; Up-Regulation

The present studies were undertaken to assess the direct effects of N6-cyclohexyladenosine (CHA), a stable adenosine analogue, on erythropoietin (Ep) secretion in hepatocellular carcinoma cells (Hep 3B). Ep levels in the medium of low density Hep 3B cells treated with CHA in concentrations of 10(-5) and 5 x 10(-5) M for 20 h under hypoxic conditions (1% O2) were significantly higher than that of hypoxic controls. In addition, CHA at the same concentrations produced significant increases in adenosine 3',5'-cyclic monophosphate (cAMP) levels in Hep 3B cells after 1-h incubation under hypoxic conditions when compared with hypoxic controls. Dibutyryl cAMP (10(-5), 10(-4) M) also caused significant increases in Ep secretion when compared with control hypoxic cells. On the other hand, 8-phenyltheophylline, an adenosine receptor antagonist, significantly inhibited the stimulatory effects of CHA on both Ep secretion and cAMP accumulation in the Hep 3B cell cultures in response to hypoxia. These data suggest that Ep secretion may be regulated by adenosine receptor-coupled activation of adenylyl cyclase and the generation of cAMP.

Topics: Adenosine; Anaerobiosis; Bucladesine; Carcinoma, Hepatocellular; Cell Line; Cell Survival; Cyclic AMP; Erythropoietin; Humans; Hypoxia; Kinetics; Liver Neoplasms; Theophylline

This study was carried out to investigate the influence of different exercise regimens on serum immunoreactive erythropoietin concentration (EPO). The same untrained male subjects performed bouts of maximal and submaximal exercise (60 min at 60% of maximal performance) under normoxia (n = 10) and normobaric hypoxia (PIO2 92 mmHg, n = 9). Five of them were exposed to hypoxia for 90 min under resting conditions (RTH). [EPO] was unchanged up to five hours after maximal (MEN) and submaximal (SEN) exercise under normoxia. After RTH, [EPO] increased after 3 hours by 5.0 mU/ml (p less than 0.01). Submaximal exercise under hypoxia (SEH) led to a similar increase in [EPO] (after 3 hours: + 5.5 mU/ml), which remained elevated the following days (after 24 h: + 6.1 mU/ml, 48 h: + 5.3 mU/ml; ANOVA p less than 0.001). Maximal exercise under hypoxia (MEH) had no significant effect. The results indicate that exercise has no immediate effect on serum [EPO], whereas the higher EPO level one and two days after SEH could result from the occurring hemodilution as is indicated by a slight negative correlation between [EPO] and Hct (r = 0.59, p less than 0.001). The number of reticulocytes increased after all hypoxic experiments and after MEN without any correlation to [EPO].

Topics: Adult; Erythropoietin; Exercise; Exercise Test; Heart Rate; Hemoglobins; Humans; Hypoxia; Male; Oxygen Consumption; Reticulocytes

Eight patients with obstructive sleep apnoea and a normal haemoglobin concentration underwent nocturnal studies during which oxyhaemoglobin saturation was recorded continuously with an ear oximeter and serum erythropoietin concentration was measured hourly by means of a radioimmunoassay. Serum erythropoietin concentrations remained within the normal range throughout the study despite falls in oxyhaemaglobin saturation in individuals to 33-78%. There was no relation between the degree of nocturnal hypoxaemia and serum erythropoietin concentrations. The brief cyclical episodes of hypoxaemia typical of obstructive sleep apnoea may not be a sufficient stimulus for erythropoietin secretion.

Topics: Adult; Aged; Erythropoietin; Humans; Hypoxia; Male; Middle Aged; Oxyhemoglobins; Sleep Apnea Syndromes

Erythropoietin gene expression is greatly stimulated under conditions of hypoxia. The activation of the erythropoietin gene appears regulated primarily at the level of gene transcription. To study cis-acting elements involved in the response to hypoxia a mini-gene was constructed by an internal deletion from exon II to V of the human erythropoietin gene and used in transient transfection assays in the erythropoietin producing Hep 3B cell line. It was initially found that hypoxia responsiveness was present in an erythropoietin fragment containing 400 base pairs (bp) of 5'-flanking and 600 bp of 3'-flanking regions. Deletion analysis showed no significant effect on the response to hypoxia when highly conserved regions of 5'-flanking sequence, exon and intron I, and exon V were removed from the mini-gene construct. However, removal of a fragment containing the 3' end of the gene and 3'-flanking sequences completely eliminated hypoxia responsiveness. Reinsertion of the above fragment upstream of the 5' end of the mini-gene restored the response to hypoxia. Further analysis using hybrid erythropoietin-chloramphenicol-acetyltransferase constructs allowed the localization of enhancer-like element(s) in the 3'-flanking region, approximately 120 bp downstream of the polyadenylation site of the human erythropoietin gene. Activation by these sequences were position- and orientation-independent and stimulated 15-fold transcription of the erythropoietin gene in response to hypoxia.

Topics: Cells, Cultured; Chloramphenicol O-Acetyltransferase; DNA; Enhancer Elements, Genetic; Erythropoietin; HeLa Cells; Humans; Hypoxia; Oxygen; Plasmids; Restriction Mapping; Transcription, Genetic; Transfection

We used RNAase protection assays to measure low levels of erythropoietin messenger RNA (EPO mRNA) in the organs of unstimulated rats, and to compare basal and stimulated levels of EPO mRNA in the kidneys and extrarenal organs of rats rendered uremic by subtotal nephrectomy, with pair-fed controls. Using this sensitive assay, EPO mRNA was measured in the kidneys of unstimulated control animals and was detectable, at lower levels, in the liver and lung. After exposure to hypoxia, there was a 150-fold increase in renal EPO mRNA. Hepatic EPO mRNA was also greatly increased and accounted for 39 +/- 10% of the total. The small quantity of EPO mRNA in lung did not increase, but EPO mRNA became detectable in spleen. Animals subjected to subtotal nephrectomy became uremic and anemic (hematocrit 0.32 +/- 0.04 vs. 0.43 +/- 0.04 in controls, P = 0.002), but serum EPO concentrations were not significantly increased (32 +/- 9 vs. 24 +/- 6 mU/ml, P = 0.14). However, after hypoxic exposure, uremic animals increased serum EPO concentrations greatly, although the response was less than in controls (349 +/- 82 vs. 1009 +/- 238 mU/ml, P = 0.002). After hypoxia, extrarenal EPO mRNA levels in uremic animals were similar to controls. In particular, the large hepatic potential for EPO mRNA synthesis was unchanged but accounted for a greater proportion (84 +/- 5%) of the total EPO mRNA. The renal EPO mRNA content was reduced, as expected, after subtotal nephrectomy, but increased 50-fold after hypoxia. In this model of chronic renal failure, despite anemia, a large potential for EPO synthesis exists in liver and remnant kidney.

Topics: Amino Acid Sequence; Animals; Erythropoietin; Hypoxia; Male; Molecular Sequence Data; Rats; Rats, Inbred Strains; Reference Values; RNA, Messenger; Tissue Distribution; Uremia

Synthesis of erythropoietin, the primary humoral regulator of erythropoiesis, in liver and kidney is inducible by anemia or hypoxia. Analysis of human erythropoietin gene expression in transgenic mice revealed that sequences located 6-14 kilobases 5' to the gene direct expression to the kidney, whereas sequences within the immediate 3'-flanking region control hepatocyte-specific expression. Human erythropoietin transcription initiation sites were differentially utilized in liver and kidney. Inducible transgene expression was precisely targeted to peritubular interstitial cells in the renal cortex that synthesize endogenous mouse erythropoietin. These studies demonstrate that multiple erythropoietin gene regulatory elements control cell-type-specific expression and inducibility by a fundamental physiologic stimulus, hypoxia.

Topics: Anemia; Animals; Base Sequence; Blotting, Northern; Erythropoietin; Gene Expression Regulation; Genes; Humans; Hypoxia; Kidney; Liver; Mice; Mice, Transgenic; Molecular Sequence Data; Nucleic Acid Hybridization; Regulatory Sequences, Nucleic Acid; Restriction Mapping; RNA, Messenger; Transcription, Genetic

Immunohistochemically detectable erythropoietin-like substance(Epo) in granular convoluted tubule(GCT) cells of submandibular glands (SMG's) was examined in mice in which hemolytic anemia had been induced by phenylhydrazine (ph), and in mice subjected to hypoxia, nephrectomy, or testosterone (TP) injections. Staining for Epo was negative in GCT cells of SMG's in normal mice, while positive staining occurred in GCT cells of the anemic mice and mice subjected to hypoxia or nephrectomy. A positive Epo reaction was also revealed at the luminal borders of distal tubules, and in cells of proximal and distal tubules in the kidney, and in some hepatic and spleen cells, of mice that had received combination regimens producing anemia and hypoxia, or had been nephrectomized. Increased staining of Epo was found in GCT cells of SMG's, and in proximal and distal kidney tubules of mice given the combination treatment plus TP injections. The detection of Epo in GCT cells suggests these extrarenal cells to be sites of accumulation or biosynthesis of the protein under certain specific conditions such as hemolytic anemia and hypoxia.

Topics: Anemia, Hemolytic; Animals; Erythropoietin; Female; Hypoxia; Immunoenzyme Techniques; Male; Mice; Nephrectomy; Submandibular Gland; Testosterone

Quantification of 32P in bands after gel electrophoresis was performed using the flat-bed scintillation counter (Betaplate). The most convenient system involved placing fragments of dried gel between two glass fiber sheets, each previously sealed in a thin plastic bag with liquid scintillant. Good pulse-height spectra and counting efficiencies were obtained with low cross talk and background. The method has been used to quantify mRNA in RNA antisense-protection assays that were linear over a wide range (1-20000 cpm). Cross talk and background could be reduced further by an alternative technique utilizing plastic trays with shallow wells in which a solid scintillant had been melted. Fragments were immersed in the molten scintillant (90 degrees C), which was allowed to solidify, by cooling, before counting.

Topics: Animals; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Hypoxia; Kidney; Phosphorus Radioisotopes; Rats; RNA, Messenger; Scintillation Counting

Consecutive determinations of erythropoietin in serum (s-Epo) were made in ten patients with chronic hypoxia starting domiciliary long-term oxygen therapy (LTO). After 24 h of supplementary oxygen treatment there was a fall in the median s-Epo level from 11.3 to 4.4 IU.l-1 (p less than 0.01). The initial decrease in s-Epo in conjunction with oxygen treatment was not sustained after one and three months of LTO. S-Epo levels above the reference range (3.3-13.5 IU.l-1) were found in three patients before and during LTO and in another two patients during LTO. Markedly elevated s-Epo levels were found in two patients with hypoxia and hypercapnia at the time of blood sampling. A significant negative relationship was found between the arterial oxygen tension and the log value for the s-Epo level (r = 0.40, p less than 0.005). The s-Epo levels were found to be normal in half of the measurements in patients using oxygen less than 15 h daily, a fact that indicates that s-Epo measurements are probably not suitable as indicators of compliance with LTO.

Topics: Aged; Aged, 80 and over; Chronic Disease; Erythropoietin; Home Care Services; Humans; Hypoxia; Long-Term Care; Male; Middle Aged; Oxygen Inhalation Therapy; Patient Compliance

Because the human hepatoma cell line Hep3B produces erythropoietin (Epo) in a regulated fashion, it can be used to investigate the cis-acting regulatory elements of the Epo gene. Comparison of primate and mouse sequences shows strong homology not only in the coding sequence but also within the 5' flanking region, the first intron, and the 3' flanking region. These portions of the Epo gene were inserted 5' and 3' to a reporter gene, human growth hormone (GH). 5A is a 1,192-base pair (bp) HindIII-Xbal fragment that extends from 378 bp 5' to the cap site through the first intron. To obviate the problem of false initiation of translation from the Epo ATG start codon, this site was changed to TAG by site-directed mutagenesis. 3A is a 255-bp Accl-BglII fragment that extends 67 bp upstream from the Epo termination codon and covers most of the 3' noncoding region of homology. The plasmid DNAs were transfected by electroporation into Hep3B cells with RSVCAT as an internal standard to correct for transfection efficiency. One aliquot of cells was exposed to 50 mumol/L CoCl2 or to 1% O2. At the end of the incubations, GH and Epo were measured in the cell media and the cell pellet was assayed for CAT. Production of GH was stimulated 1.7-fold by cobalt or hypoxia. Furthermore, addition of 3A to the GH gene, irrespective of orientation, stimulated GH production 2.6-fold with CoCl2 and 2.3-fold with hypoxia. Stable cell lines were produced by cotransfection of the above constructions, along with the selectable marker pSV-Neo. In two clones, exposure to hypoxia resulted in much more marked (16-fold) induction of GH. Stimulus of both GH and Epo production by hypoxia was partially abrogated by carbon monoxide. These results demonstrate the presence of promoter and enhancer elements within the human Epo gene that are appropriately responsive to hypoxia and cobalt.

Topics: Animals; Cell Line; Chlorocebus aethiops; Cobalt; Erythropoietin; Gene Expression Regulation; Humans; Hypoxia; In Vitro Techniques; Regulatory Sequences, Nucleic Acid; Transcription, Genetic; Transfection

Reduced oxygen tension is regarded as the primary physiologic signal for the production of erythropoietin (EPO). There is little information available about early changes of EPO production in man due to severe hypoxia. The purpose of the present study was to examine the time course of EPO in serum of patients with acute cardiogenic pulmonary edema (ACPE). In 29 patients (seventy-five +/- six years, mean age +/- SEM) who were hospitalized within two hours after onset of symptoms of ACPE, serum EPO concentrations were monitored for up to seventy-two hours. At the moment of admission all patients showed significantly increased EPO concentrations of 121 +/- 64 mU/mL (mean +/- SEM) compared with a healthy population (15-35 mU/mL). Twenty-three patients who recovered within thirty minutes (group A) exhibited a quick return of their EPO serum levels to normal. The remaining 6 patients (group B) had a protracted clinical course and their EPO concentration showed a further increase up to the end of the observation period. The comparative monitoring of concentrations of alpha-1-proteinase inhibitor, antithrombin III, C-reactive protein, fibronectin, hapotoglobin, and transerrin in serum and plasma revealed no significant changes. Thus a major contribution of fluid shifts into or from the intravascular compartment to the observed changes in EPO concentration seems to be unlikely. The data suggest that the production and release of EPO in the kidneys due to altered oxygen delivery is a fast-responding mechanism.

Topics: Acute Disease; Aged; Aged, 80 and over; Blood Proteins; Cardiovascular Diseases; Erythropoietin; Female; Humans; Hypoxia; Kidney; Male; Pulmonary Edema

The renal glycoprotein hormone erythropoietin (Epo) interacts with erythrocytic progenitors to stimulate their proliferation and differentiation in the bone marrow. The renal O2-sensing mechanism in the control of the synthesis of Epo is still poorly understood. Therefore, the capacity of isolated rat kidneys to produce Epo during hypoxic and anemic perfusion was studied. The kidneys were perfused at a constant perfusion pressure of 100 mm Hg with a substrate-enriched Krebs-Henseleit solution containing 60 g/liter BSA and freshly drawn human erythrocytes. Epo was measured by RIA. When the kidneys were perfused at an arterial pO2 of 720 or 150 mm Hg (hematocrit, 5%), Epo production was very low (0.1-0.2 U/g kidney within 3 h of perfusion). When the arterial pO2 was lowered to 35 or 20 mm Hg, Epo production increased to 0.4 and 0.9 U/g kidney, respectively. The release of Epo during hypoxic perfusion (pO2 35 and 20 mm Hg) was little affected by changes in the hematocrit, i.e. the O2-carrying capacity of the perfusion medium over a wide range (0-40%). These results indicate that the production of Epo in the isolated perfused kidney depends on the availability of O2 and can be modulated by changes in the arterial pO2.

Topics: Anemia; Animals; Erythropoietin; Hematocrit; Hypoxia; In Vitro Techniques; Kidney; Male; Perfusion; Radioimmunoassay; Rats; Rats, Inbred Strains

Human erythropoietin gene expression in liver and kidney is inducible by anemia or hypoxia. DNase I-hypersensitive sites were identified 3' to the human erythropoietin gene in liver nuclei. A 256-base-pair region of 3' flanking sequence was shown by DNase I protection and electrophoretic mobility-shift assays to bind four or more different nuclear factors, at least two of which are induced by anemia in both liver and kidney, and the region functioned as a hypoxia-inducible enhancer in transient expression assays. These results provide insight into the molecular basis for the regulation of gene expression by a fundamental physiologic stimulus, hypoxia.

Topics: Animals; Base Sequence; Cell Nucleus; Chromosome Mapping; Deoxyribonuclease I; DNA-Binding Proteins; Enhancer Elements, Genetic; Erythropoietin; Gene Expression Regulation; Genes; Humans; Hypoxia; Mice; Mice, Transgenic; Molecular Sequence Data; Nuclear Proteins; Regulatory Sequences, Nucleic Acid; Restriction Mapping

Several previous studies have shown that hypoxia increases erythropoiesis and decreases thrombocytopoiesis in mice. It has been postulated that the thrombocytopenia is caused by stem cell competition between the erythrocytic and megakaryocytic cell lines. In the present work, we compared the effects of severe hypoxia (5.5-6.0% O2) in both male and female C3H and BALB/c mice by measuring their abilities to produce red blood cells and platelets. All mice had significant increases in packed cell volumes and marked decreases in platelet production after hypoxia; however, there were significant differences in the degree of stimulation in the two mouse strains. After 14 days of hypoxia, the percentage of 35S incorporation into platelets, total circulating platelet counts and total circulating platelet masses were lower in C3H mice than in BALB/c mice, but platelet sizes were larger. Also, hypoxia caused greater changes in male mice than in female mice, with male C3H mice showing the greatest increase in packed cell volumes and the lowest platelet counts of all mice tested. The least responses were observed in female BALB/c mice. BALB/c mice had higher P50 (right-shifted O2 dissociation curves) and lower erythrocyte 2,3-diphosphoglycerate values than C3H mice, indicating a lower hemoglobin O2 affinity for BALB/c mice. The results indicate that the effects of hypoxia are not direct upon platelet production, but that the thrombocytopenia is a result of stimulation of erythropoiesis. These data support the stem cell competition hypothesis and illustrate that the degree of the inverse relationship between red blood cells and platelet production of hypoxic mice is dependent, to a large degree, upon the sex and strain of mice that are used.

Topics: Animals; Blood Platelets; Erythropoiesis; Erythropoietin; Female; Hematopoiesis; Hypoxia; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Platelet Count; Sex Factors; Species Specificity

Erythropoietin (EPO) producing cells were examined by the immunofluorescent antibody technique, and the alterations in the Ia antigen expression in glomeruli by hypoxic stimuli. Antisera were obtained from rabbits immunized with pure human EPO. Positive fluorescence was localized in glomerular mesangial regions of the hypoxic rat kidney. Although hypoxia showed neither increase in the number of glomerular cells nor hypertrophy of the mesangial matrix, it induced an increase of the Ia antigen expressions in glomeruli. The peak of the increase occurred 2 hr after hypoxia and then decreased until 24 hr later. The increase of Ia antigen after 2 hr of hypoxic stimuli was 176.5 +/- 7.0% of normal controls, and ranged from 151 to 209%. In conclusion, Ia positive cells in glomeruli may have some relationship to the stimulation of EPO production.

Topics: Animals; Erythropoietin; Histocompatibility Antigens Class II; Hypoxia; Immunohistochemistry; Kidney; Kidney Glomerulus; Male; Rats; Rats, Inbred Strains

Erythropoietin (Epo) gene transcription is stimulated in Hep3B cells under hypoxic conditions. We have prepared transcriptionally active nuclear extracts from normal and hypoxia-induced Hep3B cells and shown that the hypoxic extracts produce a consistent increase in the level of Epo transcription in vitro, relative to control Hep3B cells. Hypoxic treated HeLa cells failed to express the endogenous Epo gene in vivo, and extracts prepared from them did not show increased Epo transcription in vitro. The Epo transcript which is induced in vitro is initiated at the same site as Epo RNA synthesized in intact Hep3B cells and in human kidney adenocarcinoma cells. This system will facilitate the purification and analysis of factors and sequences required for Epo gene transcription in response to changes in tissue oxygen tension.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Cell Line; Erythropoietin; Genes; Humans; Hypoxia; Liver Neoplasms; Restriction Mapping; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic

A noninvasive, inexpensive method of excluding significant sleep-associated hypoxemia would be desirable for patients being investigated and treated for obstructive sleep apnea (OSA). Sixty-eight such patients provided specimens before and after sleep studies for estimation of urinary uric acid:creatinine ratio (UA:Cr), serum erythropoietin (EPO), and blood 2,3-diphosphoglycerate (2,3-DPG). Mean (SD) morning 2,3-DPG was higher in 26 patients with overnight hypoxemia than in 42 normoxemic patients (2.54 [0.46] versus 2.24 [0.44] mmol/L; p = 0.01). Neither overnight change nor absolute values of serum EPO or urinary UA:Cr were significantly different between hypoxemic and normoxemic groups. There was a diurnal variation in serum EPO in normoxemic patients (P.M. EPO = 14.8 [7.1] mU/ml; A.M. EPO = 10.7 [7.1] mU/ml; p less than 0.05) but not in hypoxemic patients. Eighteen hypoxemic patients were restudied after using nasal continuous positive airway pressure (nCPAP) for at least 4 wk. Seven normoxemic patients not using nCPAP were restudied after a similar time. There were no significant differences between pretreatment and posttreatment nights in absolute values or percentage overnight change of blood 2,3-DPG or serum EPO in either group. In the hypoxemic (nCPAP) group, overnight change in urinary UA:Cr was lower on the second night (p = 0.04); there was no significant change in the control group. We conclude that although urinary UA:Cr, serum EPO, and 2,3-DPG may be physiologically related to hypoxemia, none of these measures can be used to predict accurately the presence of moderate nocturnal hypoxemia in patients with OSA or in monitoring the effect of their therapy.

Topics: 2,3-Diphosphoglycerate; Circadian Rhythm; Creatinine; Diphosphoglyceric Acids; Erythropoietin; Female; Humans; Hypoxia; Male; Middle Aged; Positive-Pressure Respiration; Sleep Apnea Syndromes; Uric Acid

Some differences between erythropoietin biogenesis under the action of natural stimuli and that resulting from testosterone administration are commented. Evidences are presented suggesting that androgens, apart from amplifying erythropoietin production, might exert some action on the stroma as specific erythrocytic tissue. This action could be anabolic or trophic and not erythropoietin dependent, at least to some extent, its effect being applied on the quantitative erythropoietic homeostasis.

Topics: Animals; Bone Marrow; Carbon Monoxide; Erythropoiesis; Erythropoietin; Female; Hypoxia; Mice; Mice, Inbred C3H; Stimulation, Chemical; Testosterone; Whole-Body Irradiation

Topics: 2,3-Diphosphoglycerate; Acid-Base Equilibrium; Animals; Diphosphoglyceric Acids; Erythropoietin; Hemoglobins; Hypoxia; Kidney; Rabbits; Uremia

Erythropoietin (EPO) production in response to hypoxic hypoxia is known to be attenuated by simultaneous hypercapnia. This study aimed to investigate whether this inhibitory effect of hypercapnia is 1) a direct effect of carbon dioxide or mediated by changes in pH or bicarbonate, 2) affects also carbon monoxide hypoxia, and 3) influences either the synthesis and release of EPO or the mechanisms by which hypoxia triggers an increase in EPO production rate. We found that EPO formation in mice exposed to normobaric hypoxia (8% O2) or to carbon monoxide (0.1%) was reduced by 30 and 42% when animals were simultaneously exposed to hypercapnia (7% CO2), by 35 and 38% when subjected to metabolic acidosis (NH4Cl), and unchanged when subjected to metabolic alkalosis (NaHCO3). In animals exposed to brief hypoxia (15 min) and subsequent normoxia (2 h), metabolic acidosis did not affect EPO levels when initiated after the hypoxic period. The results indicate that acidosis inhibits hypoxia-induced triggering of EPO formation independently of PCO2 and HCO3 levels. Because this inhibitory effect is also present during carbon monoxide hypoxia, it appears not solely due to potentiated hyperpnea. Alternatively, it may result from a facilitated intrarenal oxygen release or a direct effect at the EPO production sites.

Topics: Acid-Base Equilibrium; Acidosis; Acidosis, Respiratory; Animals; Carbon Monoxide; Erythropoietin; Hydrogen-Ion Concentration; Hypoxia; Male; Mice

We examined the relationship between the duration of hypoxic exposure and serum erythropoietin (EPO) production. Adult male Long-Evans rats were exposed to hypobaric hypoxia (HH = 0.5 atm) for a period of 15 min to 20 days. Serum for EPO by radioimmunoassay was collected immediately, 1 or 2 h after HH exposure. A significant rise in EPO levels occurred 1 h after a 30-min HH exposure that was not sustained 2 h after termination. One hour of HH exposure resulted in increased EPO levels 1 h after termination of exposure and further increased levels 2 h after termination of exposure. With prolonged exposure, serum levels remained elevated at 6 and 20 days, despite the development of polycythemia. We concluded that the hypoxic stimulus for elevation of serum EPO could be as short as 30 min and that EPO levels remained elevated after chronic HH. The experimental data were consistent with a mathematical model in which stimulated EPO production was proportional to the time of HH stimulus.

Topics: Animals; Erythropoietin; Hypoxia; Male; Models, Biological; Rats; Rats, Inbred Strains; Time Factors

Serum erythropoietin (EPO) levels in response to hypoxia are known to decline before an increase in blood oxygen carrying capacity. To define the possible mechanisms underlying this phenomenon, we have investigated 1) how renal EPO mRNA content and EPO production rate underlying the early kinetics of serum EPO levels change under different degrees of normobaric hypoxia, and 2) if a feedback inhibition of either EPO formation or EPO survival in the circulation exists by the hormone itself. We found that serum immunoreactive EPO levels in rats peaked after 12-h exposure to 7.5 or 9% oxygen (2,949 +/- 600 and 756 +/- 108 mU/ml, respectively, mean +/- SE) and declined to 29 and 64% of peak levels, respectively, after 36 h of hypoxia. EPO levels in response to 11.5% oxygen showed no consistent change between 12 (122 +/- 21 mU/ml, mean +/- SE) and 36 h (182 +/- 35 mU/ml) of hypoxia. The decline in EPO levels under severe hypoxia (7.5% O2) was paralleled by a marked reduction in renal EPO mRNA content, indicating that it was primarily a result of diminished hormone production. The observed reductions in serum EPO after 36 h corresponded to preceding declines of calculated EPO production rates from 163- to 62-fold (7.5% O2) and 36- to 25-fold (9% O2) basal values. Application of 50 IU recombinant human EPO to rats 12 h, 6 h, or immediately before hypoxic exposure to mimic the early increase in EPO levels did not affect endogenous EPO formation during a subsequent hypoxic exposure of 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Biomechanical Phenomena; Erythropoietin; Feedback; Hypoxia; Kidney; Kinetics; Male; Rats; Rats, Inbred Strains; RNA, Messenger

Patients with solid tumors are often anemic even before they undergo cytotoxic therapy. Since the cause of the anemia of cancer is unknown, we examined the possible role of erythropoietin. Using a sensitive radioimmunoassay, we determined serum immunoreactive erythropoietin levels in 81 anemic patients with solid tumors. For any given degree of anemia, the serum concentration of immunoreactive erythropoietin was lower in this group of patients than in a group of control patients with iron-deficiency anemia (P = 0.0001). Furthermore, the expected inverse linear relation between serum levels of immunoreactive erythropoietin and of hemoglobin was absent in the group with cancer. The erythropoietin response was further decreased in patients receiving chemotherapy; it was not influenced by the presence or absence of cisplatin in the treatment regimen. The inability of the patients with cancer to produce erythropoietin was not absolute; if they had hypoxemia, adequate erythropoietin production was restored. We conclude that erythropoietin levels are inappropriately low in anemia associated with cancer, and that erythropoietin deficiency may contribute to the development of this form of anemia. Treatment of the anemia of cancer with erythropoietin may be of value.

Topics: Anemia; Anemia, Hypochromic; Antineoplastic Agents; Erythropoietin; Female; Hemoglobins; Humans; Hypoxia; Male; Neoplasms; Radioimmunoassay

Because cobalt administration is known to elicit erythropoietin response, it is a reasonable hypothesis that cobalt would also stimulate the O2-sensing process in the peripheral chemoreceptors. We tested this hypothesis by measuring the effects of cobalt chloride on carotid chemosensory fibers in pentobarbital-anesthetized cats that were paralyzed and artificially ventilated. Responses of carotid chemoreceptor afferents to graded doses of cobalt given by intra-arterial injections (0.08-2.10 mumols) were measured at constant blood gases. Responses of the same chemoreceptor afferents to hypoxia, before and after a saturation dose of cobalt, were measured. In two experiments carotid body tissue PO2 was also simultaneously measured. The chemosensory fibers showed prolonged excitation after a brief period of inhibition subsequent to cobalt administration. The stimulatory effect showed a dose-dependent saturation response. Cobalt augmented rather than blocked carotid chemoreceptor response to hypoxia. The effect of cobalt was not mediated by tissue PO2. These results are consistent with the hypothesis that cobalt stimulates the O2-sensing mechanism, although a direct effect of cobalt on the excitability of the chemosensory terminal remains a possibility.

Topics: Animals; Calcium; Carotid Body; Cats; Chemoreceptor Cells; Cobalt; Electrophysiology; Erythropoietin; Hypoxia; Oxygen

Topics: Animals; Erythropoietin; Humans; Hypoxia; Male; Oxygen; Rats; Rats, Inbred Strains; Sleep Apnea Syndromes

Short-lasting hypobaric hypoxia was used in male white rats as a stimulus of erythropoietin synthesis (0.42 atm.). Plasma erythropoietin activity was determined by a biologic method on highly sensitive to exogenous erythropoietin recipients-posthypoxic polycytemic mice. Erythropoietin activity was estimated by measuring the percentage of incorporated 59Fe in newly formed erythrocytes of mice under the influence of the examined plasma, using automatic scintillation gamma-counter. Hypoxia induced an increase of plasma erythropoietin activity with 54% in comparison with control animals. Administration of actinomycin D in a dose of 0.4 microgram/g of body mass before exposure of the animals to hypoxia diminished production of erythropoietin with 58% in comparison with that of rats, exposed to pure hypoxia. A suggestion could be made that the formation of erythropoietin requires DNA dependent RNA-synthesis.

Topics: Animals; Dactinomycin; Erythropoietin; Hyperglycemia; Hypoxia; Male; Rats; Rats, Inbred Strains; RNA

Erythropoietin producing cells were identified in the murine hypoxic kidney by in situ hybridization. The positive cells were peritubular cells, most likely endothelial cells of the cortex and outer medulla. Glomerular and tubular cells were not labelled. In three patients with renal adenocarcinomas associated with polycythemia, a strong Epo message was observed on Northern blot analysis. Using in situ hybridization, a strong labelling was observed in all cases on the tumor cells which are of tubular origin.

Topics: Animals; Carcinoma, Renal Cell; Erythropoietin; Gene Expression Regulation; Genes; Humans; Hypoxia; Kidney Cortex; Kidney Neoplasms; Mice; Neoplasm Proteins; Nucleic Acid Hybridization; Polycythemia; Transcription, Genetic

To determine the maternal cardiovascular responses to long-term hypoxemia, we studied three groups of animals: 1) pregnant ewes (n = 20) at 110-115 days gestation subjected to hypoxia for up to 28 days; 2) pregnant ewes (n = 4) that served as normoxic controls; and 3) nonpregnant ewes (n = 6) subjected to hypoxemia for up to 28 days. We measured mean arterial pressure, heart rate, uterine blood flow, and uterine vascular resistance continuously for 1 h/day while the ewe was exposed to an inspired O2 fraction of 12-13% for at least 17 days. Arterial PO2, O2 saturation, hemoglobin, arteriovenous O2 difference, and uterine O2 uptake were measured daily while blood volume and erythropoietin concentration were measured weekly. In the pregnant hypoxic group arterial PO2 decreased from a control value of 101.5 +/- 5.1 to 59.2 +/- 5.1 Torr within a few minutes, where it remained throughout the study. The hemoglobin concentration increased from 8.9 +/- 0.5 to 10.0 +/- 0.5 g/dl within 24 h where it remained, whereas erythropoietin concentration increased from 16.6 +/- 2.1 to 39.1 +/- 7.8 mU/ml at 24 h but then returned to near-control levels. Arterial glucose concentration, mean arterial pressure, and cardiac output decreased slightly but insignificantly. In contrast, body weight, heart rate, blood volume, uterine blood flow, uterine O2 flow, uteroplacental O2 uptake, and the concentrations of catecholamines and cortisol remained relatively constant. Thus both pregnant and nonpregnant sheep experience relatively minor cardiovascular and hematologic responses in response to long-term hypoxemia of moderate severity.

Topics: Animals; Blood Pressure; Blood Volume; Carbon Dioxide; Cardiac Output; Erythropoietin; Female; Hematocrit; Hemodynamics; Hemoglobins; Hypoxia; Oxygen; Oxygen Consumption; Oxyhemoglobins; Partial Pressure; Pregnancy; Pregnancy Complications; Reference Values; Sheep; Uterus; Vascular Resistance

The effect of acute or short-term hypoxia on fetal cardiovascular hemodynamics has been well known; however, little is known about the effect of long-term hypoxemia. To determine the fetal hemodynamic responses to this stress we studied two groups of animals: 1) pregnant ewes (n = 20) at 110-115 days of gestation subjected to hypoxia for up to 28 days and 2) pregnant ewes (n = 4) that served as normoxic controls. We chronically catheterized the fetal brachiocephalic artery and vein. Five to 6 days after surgery, control measurements were made of mean arterial blood pressure, heart rate, arterial PO2, O2 saturation, hemoglobin, hematocrit, blood volume, and the concentrations of erythropoietin, cortisol, epinephrine, and norepinephrine. The next day the ewes were placed in a chamber with an inspired O2 fraction of 12-13%. Within a few minutes fetal arterial PO2 decreased from control value of 29.7 +/- 2.1 to 19.1 +/- 2.1 Torr, where it remained. Hemoglobin increased from 10.0 +/- 1.0 to 12.9 +/- 1.9 g/dl by day 7, where it remained. This was associated with an increase of erythropoietin from 22.8 +/- 2.2 to 144 +/- 37 mU/ml within 24 h, but by day 7 it had returned to levels slightly above normal. Epinephrine also increased moderately and remained elevated throughout the study. However, values of mean arterial pressure and heart rate did not differ from controls. Perhaps surprisingly, these fetuses were able to compensate so that at term their body weights were normal, 3.77 +/- 0.2 kg.

Topics: Animals; Blood Pressure; Blood Volume; Carbon Dioxide; Erythropoietin; Female; Fetal Blood; Fetus; Glucose; Heart Rate, Fetal; Hemodynamics; Hydrogen-Ion Concentration; Hypoxia; Maternal-Fetal Exchange; Oxygen; Partial Pressure; Pregnancy; Pregnancy Complications; Sheep

Topics: Carcinoma, Hepatocellular; Erythropoietin; Gene Expression Regulation; Genes, Regulator; Hemeproteins; Humans; Hypoxia; Liver Neoplasms; Metals; Oxygen; Tumor Cells, Cultured

Topics: Anemia; Animals; Erythropoietin; Female; Fetus; Hypoxia; Kidney; Mice; Mice, Inbred BALB C; Pregnancy; RNA, Messenger

Topics: Adolescent; Adult; Aged; Anemia; Erythropoietin; Exercise Test; Female; Humans; Hypoxia; Kidney Failure, Chronic; Male; Middle Aged; Oxygen; Recombinant Proteins; Renal Dialysis; Spirometry

Topics: Adult; Anemia; Erythropoietin; Exercise Test; Female; Hemoglobinometry; Humans; Hypoxia; Kidney Failure, Chronic; Male; Oxygen; Recombinant Proteins; Renal Dialysis

Temporal changes of plasma erythropoietin (Epo) in mice exposed to hypobaric hypoxia were studied by a fetal mouse liver cell culture method. Since a colony formation inhibitory activity was found in the mouse plasma, thirteen pretreatment procedures for bioassay were compared and the procedure of shaking with chloroform followed by dialysis was concluded to be the best. When normal mice (P50 = 40.4 +/- 2.2 Torr) were exposed to hypoxia of 350 Torr, the plasma Epo level was elevated, with peak at the 2nd to 3rd day, and afterwards declined gradually. On the contrary, cyanated mice (P50 = 30.1 +/- 1.5 Torr) showed much less of the Epo response when exposed to 350 Torr. Under 200 Torr hypoxia, both mice exhibited a similar and remarkable extent of the response. These results suggest that the renal Epo-producing tissue or its oxygen-sensing system is less hypoxic in cyanated mice than in normal mice under 350 Torr, and that the physiologically optimal oxygen affinity of blood is variable depending on hypoxic degrees. The fact that the inhibitory activity showed an inverse temporal change to that of Epo, suggested a possible important role of this activity in the regulation of erythropoiesis under hypoxia.

Topics: Administration, Oral; Animals; Atmospheric Pressure; Chloroform; Cyanates; Erythropoietin; Hypoxia; Mice; Mice, Inbred ICR; Oxygen; Time Factors

The plasma erythropoietin activity in diabetes patients was studied using methods for biological testing on posthypoxic polycythemic mice, based on the incorporation of 59Fe in the newly-formed red blood cells of the animals. The data are read on an automatic scintillation gamma-counter. Considerably higher erythropoietin activity of the plasma obtained from diabetics (p less than 0.01) compared with clinically health individuals has been obtained, as well as a statistically significant difference in the percentage of 59F incorporated in the newly-formed erythrocytes of the animals treated with that plasma, compared with the animals treated with saline. A dependence has been found between the chronic hyperglycaemia and the plasma erythropoietin level.

Topics: Animals; Blood Glucose; Blood Pressure; Diabetes Mellitus, Type 2; Erythropoietin; Female; Hemoglobins; Humans; Hypoxia; Iron; Iron Radioisotopes; Lipoproteins; Male; Mice; Middle Aged; Polycythemia

Our present study was undertaken to determine the serum erythropoietin concentration (radioimmunoassay), hematocrit, red cell mass, and body weight of mice exposed to hypoxia in a hypobaric chamber (0.42 atm, 22 h/day) for 14 days and during the 10 posthypoxic days at ambient pressure to clarify the correlation of the red cell mass and erythropoietin production during hypoxia. The mean serum erythropoietin titer was 326.23 +/- 77.04 mU/ml after 2 days, reached the highest level after 3 days (452.2 +/- 114.5 mU/ml), then gradually declined to a level of 36.5 +/- 11.4 mU/ml after 14 days of hypoxia, and was undetectable during the 10-day posthypoxic period. The hematocrit values were significantly increased from 41.09 +/- 0.50% at day 0 to 51.65 +/- 1.08% after 3 days and to 72.20 +/- 1.53% after 14 days of hypoxia. The red cell mass (calculated from initial body weight) increased from 3.24 +/- 0.1 ml/100 g at day 0 to 7.32 +/- 0.46 ml/100 g after 14 days of hypoxia and declined to 6.66 +/- 0.53 ml/100 g at the end of the 10-day posthypoxic period. The mice lost weight while they were in the hypobaric chamber and showed a significant increase in body weight during the 10-day posthypoxic period. These studies support the concept that chronic intermittent hypoxia causes an early increase, followed by a rapid decline, in erythropoietin production, which is correlated with the gradual increase in red cell mass.

Topics: Animals; Atmospheric Pressure; Body Weight; Erythrocyte Volume; Erythropoietin; Female; Hematocrit; Hypoxia; Mice; Polycythemia

Regulation of renal erythropoietin (EPO) production is based on an intrarenal oxygen sensor. Whereas the sensitivity of this oxygen sensor to variations in renal oxygen supply is well established, the influence of changes in renal oxygen consumption has not yet been elucidated. Diuretic drugs, which inhibit active sodium reabsorption, reduce tubular oxygen consumption. We therefore investigated the effects of acetazolamide, furosemide, hydrochlorothiazide, and amiloride, known to preferentially inhibit sodium reabsorption at different segments of the nephron, on hypoxia-induced EPO formation in mice. Those drugs that are considered to act mainly in the loop of Henle, distal tubule, and collecting duct (furosemide, hydrochlorothiazide, and amiloride) did not impair EPO formation. Acetazolamide on the other hand, which is thought to act predominantly at the proximal tubular site, significantly reduced EPO formation in response to normobaric hypoxia (8 and 14% O2) and functional anemia (0.1% carbon monoxide). This inhibitory effect of acetazolamide was dose dependent and correlated with the natriuresis induced. It appeared not to depend on the metabolic acidosis induced by the drug, since the simultaneous administration of sodium bicarbonate, which restored standard bicarbonate levels to normal, did not diminish the inhibitory effect of acetazolamide on EPO production. In conclusion the data suggest that the regulation of EPO production is likely to be related to proximal tubular function.

Topics: Animals; Diuretics; Erythropoietin; Humans; Hypoxia; Kidney Tubules, Proximal; Kinetics; Male; Mice; Mice, Inbred ICR; Natriuresis; Urine

Previous studies of the erythropoietic response to hypoxia in high-altitude natives suggest that the hematocrit and hemoglobin values in Himalayan natives (Sherpas) are lower than expected for the altitude, perhaps because of a genetic adaptation. However, differences in sampling techniques and experimental methods make comparisons difficult. Our studies were carried out to compare the erythropoietic response with the same altitude in age-matched natives of the Himalayas and Andes by the same experimental techniques. Healthy male subjects were selected in Ollagüe, Chile (n = 29, 27.3 +/- 5.9 yr) and in Khunde, Nepal (n = 30, 24.7 +/- 3.8 yr). Both of these villages are located at 3,700 m above sea level. Hematologic measurements confirmed lower hematocrit values in Nepal (48.4 +/- 4.5%) than in Chile (52.2 +/- 4.6%) (P less than 0.003). When subjects were matched for hematocrit, erythropoietin concentrations in Chile were higher than in Nepal (P less than 0.01). Detailed measurements of blood O2 affinity in Nepal showed no differences in shape or position of the O2 equilibrium curve between Sherpas and Western sojourners. Our results indicate that although Quechua Indians have higher hematocrits than Sherpas living at the same altitude, nevertheless they may be functionally anemic.

Topics: Adult; Altitude; Chile; Erythrocyte Indices; Erythrocytes; Erythropoietin; Hematocrit; Humans; Hypoxia; Indians, South American; Male; Nepal; Osmotic Fragility

This study was carried out to investigate the early changes in erythropoietin (EPO) formation in humans in response to hypoxia. Six volunteers were exposed to simulated altitudes of 3,000 and 4,000 m in a decompression chamber for 5.5 h. EPO was measured by radioimmunoassay in serum samples withdrawn every 30 min during altitude exposure and also in two subjects after termination of hypoxia (4,000 m). EPO levels during hypoxia were significantly elevated after 114 and 84 min (3,000 and 4,000 m), rising thereafter continuously for the period investigated. Mean values increased from 16.0 to 22.5 mU/ml (3,000 m) and from 16.7 to 28.0 mU/ml (4,000 m). This rise in EPO levels corresponds to 1.8-fold (3,000 m) and 3.0-fold (4,000 m) increases in the calculated production rate of the hormone. After termination of hypoxia, EPO levels continued to rise for approximately 1.5 h and after 3 h declined exponentially with an average half-life time of 5.2 h.

Topics: Adult; Atmospheric Pressure; Erythropoietin; Humans; Hypoxia; Male; Radioimmunoassay

A mathematical model has been developed which describes the regulation of erythropoiesis in mice and rats. The main model assumptions are: (1) Regulation is mediated by erythropoietin (EPO). (2) The production of EPO depends exponentially on the tissue oxygen pressure (e.g. in the renal production sites). (3) There are sigmoidal dose-response curves relating the EPO concentration in the plasma to the mitotic activity of CFU-E and proliferative erythropoietic precursors. For maximum stimulation two to four additional mitoses may occur, while for an absent stimulus three to five mitoses may be omitted. (4) The normal precursor transit time of three to four days may be shortened by more than 50% during maximum stimulation. (5) The erythrocytes have a normal lifespan of 42-56 days, which may be reduced to 15-20 days under erythropoietic stimulation. Among these assumptions, the dose-response relationships between EPO and the mitotic activity of CFU-E and the proliferative erythropoietic precursors are the most important hypotheses of the model. This is the first of a series of three papers and gives a description of the mathematical formalism and the parameters used. In the subsequent papers computer simulations on erythropoietic stimulation and suppression are presented.

Topics: Animals; Colony-Forming Units Assay; Erythrocytes; Erythropoiesis; Erythropoietin; Feedback; Hematopoietic Stem Cells; Hypoxia; Mathematics; Mice; Mitosis; Models, Theoretical; Rats; Reticulocytes

A mathematical model of erythropoietic cell production and its regulation process has been proposed in a preceding paper. It is primarily based on the assumption that the number of cell divisions taking place in the CFU-E and erythropoietic precursor stages is regulated depending on the oxygen supply of the tissue. Quantitative dose-response relationships for in vivo erythropoiesis are suggested. Here, we demonstrate that this model adequately reproduces data obtained in situations of stimulated erythropoiesis in mice and rats. In detail, this implies a quantitative description of the following processes: (1) Changes in tissue oxygen tension (Pto2) following removal of red cells (bleeding, haemolytic anaemia) or increase in plasma volume (dilution anaemia) or decrease in atmospheric oxygen pressure (hypoxia). (2) Pto2 dependent erythropoietin (EPO) production. (3) Dose-response of EPO on erythropoietic amplification (up to two to four additional mitoses). (4) The changes of the marrow transit time. Model simulations are compared with experimental data for changes of erythropoiesis during hypoxia, EPO-injection, and different forms of anaemia. A satisfactory agreement suggests that the model adequately describes and correlates different direct and indirect ways to stimulate erythropoiesis. It quantifies the role and relative contribution of the haematocrit, haemoglobin concentration, atmospheric oxygen pressure, tissue oxygen pressure, and plasma volume as triggers in erythropoietic stimulation under various conditions. Furthermore, the model may allow to optimize the scheme of EPO-administration and to find the maximum increase of erythropoiesis for a given amount of erythropoietin.

Topics: Anemia; Animals; Blood Volume; Erythropoiesis; Erythropoietin; Hematocrit; Hematopoietic Stem Cells; Hemorrhage; Hypoxia; Mice; Phenylhydrazines; Rats; Reticulocytes

Determination of plasma erythropoietin activity was performed on highly sensitive to exogenous erythropoietin recipients-posthypoxic polycytemic mice. After submitting male white rats to a two-hour hypobaric hypoxia (0.42 atm.) we obtained an increase in the plasma erythropoietic activity with 367% in comparison with control animals. Amanitin effect inhibited hypoxic stimulated plasma erythropoietin activity in male rabbits of the strain Chinchilla, which was statistically significant (p less than 0.01), but 4 hours after its application erythropoietin level was lowered with 68% in rabbits with a preceding hypoxia and intraperitoneal administration of Amanitin in comparison with nontreated hypoxic animals. The results were similar 48 hours after amanitin application as well. The results suggest that intraperitoneal administration of Amanita faloides before the onset of hypoxia diminishes erythropoietin production.

Topics: Amantadine; Animals; Erythropoietin; Hypoxia; Male; Mice; Rats; Rats, Inbred Strains; RNA Polymerase II

Erythropoietin, a plasma glycoprotein produced primarily by the kidney, is a growth and differentiation factor for erythroid progenitor cells. Production of renal erythropoietin is regulated by modulation of mRNA levels in response to changes in tissue oxygenation. Exposure to cobalt, a nonphysiologic stimulus for erythropoietin production, also acts by inducing mRNA accumulation. To determine whether variations in erythropoietin mRNA levels result from enhanced transcription of the erythropoietin gene, in vitro transcription reactions were performed using isolated rat kidney cell nuclei. Quantitation of specific nuclear RNAs labeled during in vitro transcription revealed active erythropoietin gene transcription in kidney nuclei from anemic-hypoxic and cobalt-treated animals while erythropoietin transcriptional activity was undetectable in normal kidney nuclei. Time course studies showed that stimulation of transcription begins between two and four hours following cobalt treatment and parallels the kinetics of mRNA and plasma erythropoietin accumulation. These results indicate that tissue hypoxia and cobalt exposure specifically enhance erythropoietin gene expression. This increase in erythropoietin production is regulated at least in part at the level of gene transcription.

Topics: Animals; Cobalt; Erythropoietin; Gene Expression Regulation; Hypoxia; Kidney; Male; Rats; Rats, Inbred Strains; RNA, Messenger; Transcription, Genetic

The main regulatory hormone for the control of erythropoiesis is erythropoietin (EPO). This glycoprotein has a molecular weight of 34,000 daltons, about 40% of which is represented by carbohydrates. EPO leads to an enhanced mitosis and differentiation of erythroid precursors (colony-forming unit erythroid) in the bone marrow by binding to specific receptors. The major stimulus for EPO formation in the kidney is hypoxia. The tubular parts that are intimately involved in this O2-sensing mechanism are the proximal tubular cells as can be inferred from the use of site-specific transport inhibitors. The minimal time necessary for a hypoxic signal to induce EPO formation was found to be 30 min, including wash out of oxygen stores, activation of the EPO gene and equilibration of the EPO distribution space. About 1.5 h after the onset of hypoxia, EPO mRNA was found to accumulate in the kidney, thus pointing toward the possibility of an oxygen-regulated transcription or oxygen-dependent regulation of the stability of EPO mRNA. Possibly, a decrease in the intracellular calcium concentration is involved in this signalling process.

Topics: Animals; Diuretics; Erythropoietin; Humans; Hypoxia; Kidney; Mice; Rats; RNA, Messenger; Time Factors

Animals subjected to hypoxia become hypocapnic and after some hours show an increase in circulating erythropoietin. The steps involved in the increased production of erythropoietin in response to hypoxia are not fully understood, although it has been postulated that changes in coincident variables such as acid-base balance may contribute to the mechanism of increased erythropoietin production. A rabbit model has been used to determine the physiological changes which occur in short-term hypobaric hypoxia. After 1 h, no changes were found in pCO2, pH, P50, base excess, standard bicarbonate or serum erythropoietic activity (SEA). After 3 h the pCO2, pH, base excess and standard bicarbonate had decreased while the P50 and SEA had increased. After 6 h, although the pCO2 was still significantly reduced, the pH, base excess and standard bicarbonate had returned to the initial levels and maximal SEA values. 20-fold greater than the pre-hypoxia values were found. Overall the data are consistent with the view that the magnitude of the erythropoietic response to hypoxia is modified by changes in acid-base balance.

Topics: Animals; Carbon Dioxide; Erythropoiesis; Erythropoietin; Hypoxia; Partial Pressure; Rabbits; Reference Values

The relationship of serum erythropoietin (Ep) levels to hematocrit and glomerular (GFR) filtration rate was evaluated in patients with chronic renal disease. The Ep level was measured by radioimmunoassay in 119 blood samples from 48 patients obtained over a period of up to 5 years. Hematocrit values correlated significantly with the GFR, but serum Ep levels did not change with a decline in the GFR. Significant anemia was noted only when the GFR fell below 20 ml/min/1.73 m2. Episodes of spontaneous acute hypoxic stress were observed in six patients with chronic renal failure. Serum Ep levels obtained during these episodes (mean +/- SEM: 273 +/- 76 mU/ml) were tenfold higher than Ep levels during stable steady-state chronic renal failure (26 +/- 6 mU/ml), even though Ep levels were inappropriately low for the degree of anemia in the stable state. Our findings suggest that the tissue oxygenation-Ep-hematocrit feedback mechanism operates at a lower set point in patients with chronic renal failure in comparison with normal subjects.

Topics: Adolescent; Adult; Child; Child, Preschool; Erythropoietin; Glomerular Filtration Rate; Hematocrit; Humans; Hypoxia; Kidney Failure, Chronic; Kidney Function Tests; Renal Dialysis

The ability of non-thyroidal stimuli to affect the binding affinity and capacity of solubilized nuclear receptors for thyroid hormones was studied in a normal homeostatic system (erythropoiesis) and a pathobiologic one (lung-ozone interaction). No significant effects on affinity were found, as Kd control values for receptors derived from rat bone marrow averaged 57 (+/- 28) pM while experimental (hypoxic) values averaged 89 (+/- 55) pM. Kd control values in rat lung were found to average 142 (+/- 22) pM while average values derived from experimental protocols with ozone and methimazole were 267 (+/- 44) pM and 161 (+/- 35) pM respectively. Finally, Kd control values for receptors derived from cultured MEL cells averaged 19 (+/- 2.6) pM while experimental values during exposure to DMSO or IGF1 were 23 (+/- 3.6) pM and 26 (+/- 11) pM respectively. In contrast, binding capacity (expressed as fmoles of hormone bound per unit protein of solubilized receptor) was markedly perturbed in several tissues by various agents: ozone effects on lung were shown by an average control value of 3.3 (+/- 0.4) as opposed to an experimental average of 28 (+/- 1.9); and hypoxia effects on erythroid tissue were displayed by an average control value of 0.7 (+/- 0.07) as opposed to the experimental figure of 1.8 (+/- 0.03). In cultured MEL cells, binding capacity was seen to be increased from control values of 388 (+/- 15) sites/cell to 1243 (+/- 142) sites/cell after DMSO exposure and 2002 (+/- 10) sites/cell after IGF1 exposure. Parallel experiments done with receptors derived from rat liver yielded values similar to those reported by other investigators and were unaffected by the experimental agents. These data are consistent with the hypothesis that some thyroid target tissues may vary their response to homeostatic concentrations of hormone by modulating their production of receptors as a consequence of exposure to non-thyroidal stimuli.

Topics: Animals; Cell Line; Erythropoietin; Hypoxia; Leukemia, Erythroblastic, Acute; Lung; Male; Ozone; Rats; Rats, Inbred Strains; Receptors, Thyroid Hormone

The role of the kidney tubules in the renal formation of erythropoietin is incompletely understood. Therefore, the capability to produce erythropoietin in response to hypoxia was studied in rats with tubular lesions. Nephron damage was induced in two different ways. First, rats were treated with the nephrotoxic aminoglycoside gentamicin (67.5 mg/kg and day) for 14 days. The animals were then subjected to simulated altitude (6,800 m) for 6 h. The resulting plasma erythropoietin concentration was significantly lower (0.5 IU/ml) than in saline treated control rats exposed to hypoxia (1.0 IU/ml). Second, unilateral hydronephrosis was induced by ureteral ligation. The contralateral kidney was removed immediately before the animals were exposed to simulated altitude for 6 h. The plasma erythropoietin concentration in the ureter-ligated rats did not increase above the value (0.3 IU/ml) in hypoxia exposed anephric rats. These results indicate that the production of erythropoietin is reduced following tubular injury. Tubule cells may directly produce the hormone or interfere with the O2-sensing mechanisms controlling its synthesis. The latter hypothesis would seem to be supported by our failure to demonstrate in vitro erythropoietin production by the two established kidney tubule cell lines, LLC-PK1 and PK-15.

Topics: Altitude; Animals; Cell Line; Erythropoietin; Gentamicins; Hydronephrosis; Hypoxia; Kidney Tubular Necrosis, Acute; Kidney Tubules; Male; Rats; Rats, Inbred Strains

Dynamic muscular exercises intensified the erythropoiesis within one week and the erythrocytosis within two weeks in rats. An increase in the marrow erythroid cells proliferative activity was the basis of this reaction. The dynamics of this index coincide with the blood reticulocyte concentration dynamics. The serum erythropoietin activity was found to increase considerably. These findings suggest that erythropoietin can increase the erythroid cells proliferative activity.

Topics: Adaptation, Physiological; Animals; Blood Volume; Erythropoiesis; Erythropoietin; Female; Hypoxia; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Muscles; Polycythemia; Rats; Swimming; Time Factors

A comparative study of renin and erythropoietin content in the blood serum of rats with "endocrine" kidneys and changes in their activity following the action of specific erythropoietic stimulus (4-hour hypoxia) has been conducted. The presence of "endocrine" kidneys increased renin and erythropoietin activity in such animals. Acute hypoxia produced further increase in erythropoietin titre in the blood serum, with renin remaining at the same level. Possible differences in the mechanisms of renin and erythropoietin biogenesis in the kidneys are considered.

Topics: Acute Disease; Air Pressure; Animals; Aorta, Abdominal; Erythropoietin; Hypoxia; Kidney; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Rats; Rats, Inbred Strains; Renin; Time Factors

Erythropoietin (Epo)-producing cells were identified in the murine hypoxic kidney by in situ hybridization. Profound anemia was induced in order to greatly increase Epo production. This resulted in high levels of Epo mRNA in the kidney. 35S-labeled DNA fragments of the murine Epo gene were used as probes for in situ hybridization. Control experiments conducted in parallel included kidneys of nonanemic mice, RNase-treated hypoxic kidney sections, and 35S-labeled non-Epo-related DNA. The Epo probe gave a specific hybridization signal in the hypoxic kidney in the cortex and to a lesser extent in the outer medulla. Glomerular and tubular cells were not labeled. All positive cells were identified as peritubular cells. Using immunofluorescence, we showed that cells with the same topography contained Factor VIII-related antigen. These data demonstrated that peritubular cells, most likely endothelial cells, constitute the major site of Epo production in the murine hypoxic kidney.

Topics: Animals; Erythropoietin; Hypoxia; Kidney; Mice; Nucleic Acid Hybridization; RNA, Messenger

Indomethacin elevates the plasma erythropoietin titer of hypoxic anephric rats and probably does so by increasing extrarenal erythropoietin production. Because indomethacin is a potent cyclooxygenase inhibitor, we postulated that it alters extrarenal erythropoietin production via its effects on tissue prostaglandin levels. Studies were, therefore, performed to determine the effects of indomethacin on hepatic prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) titers. Indomethacin significantly decreased the PGF2 alpha level but its effects on the PGE2 level were, in most experiments, insignificant. We next studied the effects of infusing PGF2 alpha and PGE2 on the plasma erythropoietin levels of hypoxic rats. PGF2 alpha significantly reduced the plasma erythropoietin titer of anephric rats and neutralized the effect of indomethacin when both substances were administered. PGE2 infusion, on the other hand, did not significantly affect the plasma erythropoietin level of anephric rats. The data support the conclusion that PGF2 alpha is a potent inhibitor of extrarenal erythropoietin production, and that indomethacin enhances the rate of extrarenal erythropoietin production by reducing the PGF2 alpha titer in the liver.

Topics: Animals; Drug Interactions; Erythropoietin; Hypoxia; Indomethacin; Kidney; Liver; Male; Nephrectomy; Prostaglandins E; Prostaglandins F; Radioimmunoassay; Rats; Rats, Inbred Strains

Topics: Animals; Carbon Monoxide Poisoning; Erythropoiesis; Erythropoietin; Female; Hypoxia; Mice

We hypothesized that children with cyanotic congenital heart disease and moderate hypoxemia, as a result of erythrocytosis, and adequate iron stores would have low serum erythropoietin titers, low tissue oxygen delivery, and normal red cell 2,3-diphosphoglycerate (DPG) concentrations. We assessed hemoglobin levels, aortic oxygen saturation, iron stores, red cell 2,3-DPG, oxygen consumption, and systemic O2 transport in 19 hypoxemic patients, aged 3 months to 8 years. Low erythropoietin titers (less than 30 mU/dl) were found in 14 patients. Patients with high erythropoietin titers had lower Pao2 (36 +/- 7 vs 49 +/- 7 mm Hg, p less than 0.01), lower aortic saturation (68 +/- 12 vs 81 +/- 9%, p less than 0.01), and higher red cell 2,3-DPG (2.47 +/- 0.34 vs 3.23 +/- 0.73 mumol/ml, p less than 0.01). Aortic oxygen saturation higher than 80% was associated with a low erythropoietin titer and a hemoglobin level below that associated with hyperviscosity. The relationship between aortic oxygen saturation and hemoglobin concentration was strong (r = 0.77). These data suggest that for children less than 8 years of age, adequate compensation for moderate hypoxemia can occur with moderate increases in hemoglobin levels.

Topics: 2,3-Diphosphoglycerate; Child; Child, Preschool; Cyanosis; Diphosphoglyceric Acids; Erythropoietin; Female; Heart Defects, Congenital; Hemoglobins; Humans; Hypoxia; Infant; Iron; Male; Oxygen; Oxygen Consumption; Polycythemia

The effects of adenosine (ADE) and ADE agonists on erythropoietin (Ep) production were determined using percent (%) 59Fe incorporation in red cells of exhypoxic polycythemic mice. The hemisulfate salt of ADE produced a significant increase in % 59Fe incorporation in response to hypoxia in concentrations of 400 to 1600 nmol/kg/day (i.v.). 5'-N-ethyl-carboxamideadenosine (NECA), a selective A2 receptor agonist, increased radioiron incorporation in a dose-dependent manner (10-100 nmol/kg/day, i.v.). In contrast, N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, did not affect radioiron incorporation in concentrations up to 1600 nmol/kg/day (i.v.). Albuterol, a beta 2-adrenergic agonist, enhanced % 59Fe incorporation in polycythemic mice and low doses of CHA (50 and 100 nmol/kg/day), which were not effective alone on % 59Fe incorporation in polycythemic mice exposed to hypoxia, inhibited the enhancement in radioiron induced by albuterol (25 and 100 micrograms/kg/day, i.p.) plus hypoxia. Theophylline (20 and 80 mg/kg/day, i.p.), a well-known antagonist of ADE receptors, blocked the ADE and NECA enhancement in radioiron incorporation at a dose of theophylline alone which produced only a slight enhancement of % 59Fe incorporation. These results suggest that ADE may both inhibit through A1 receptor activation and increase via A2 receptor stimulation the production of Ep.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Albuterol; Animals; Erythrocytes; Erythropoietin; Female; Hypoxia; Iron Radioisotopes; Mice; Mice, Inbred ICR; Polycythemia; Receptors, Purinergic; Theophylline

Production of immuno and biologically active erythropoietin was documented to occur in the human hepatoblastoma cell line HepG-2. The expression of the erythropoietin gene was further verified by Northern blot analysis using a single stranded RNA probe. In vitro studies showed that erythropoietin production by these cells was not stimulated by hypoxia or cobalt chloride, but was related to the proliferative activity of the cells in culture. In addition it was found that the secretion of erythropoietin was almost completely abrogated by tunicamycin, an inhibitor of N-linked glycosylation. This effect of tunicamycin was also observed in a permanently transfected cell line that secretes erythropoietin in large quantities.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cobalt; Cricetinae; Erythropoietin; Glycosylation; Humans; Hypoxia; Liver Neoplasms; Protein Processing, Post-Translational; RNA, Messenger; Transfection; Tumor Cells, Cultured; Tunicamycin

In a survey the correlations between hypoxia, acidosis and nephrology are presented. In chronic oxygen deficiency the individual aspects concern the uncertainties of erythropoietin and the carotid-sinusoidal natriuresis stimulated by chemoreceptors. The effects of acute ischaemic hypoxic reactions are described with regard to the acute renal failure. The regulatory renal function of acidification and its disturbance in chronic renal insufficiency as well as the renotubular acidosis are discussed. Finally the authors enter the influence on the renal function during positive pressure respiration (e.g. for the purpose of the normalisation of the pulmonary gas exchange in acute respiratory insufficiency) as well as on reactions of the haemodialysis (bicarbonate and acetate dialysis) to the blood gas and acid-base metabolism, taking into consideration the pulmonary function.

Topics: Acid-Base Equilibrium; Acidosis, Renal Tubular; Acidosis, Respiratory; Acute Kidney Injury; Erythropoietin; Glomerular Filtration Rate; Humans; Hypercapnia; Hypoxia; Kidney; Kidney Tubules; Natriuresis; Positive-Pressure Respiration; Renal Dialysis

Serum immunoreactive erythropoietin (siEp) levels were measured in 35 full-term infants aged 0-13 weeks, 31 of whom had congenital heart disease. The infants displayed a wide range in arterial oxygen tension (PaO2) and oxygen saturation (SaO2). During the first days of life siEp varied widely with a range from less than 3 to more than 10,000 mIU/ml. The wide variation is consistent with findings in cord blood at term. The siEp levels did not correlate significantly with haemoglobin, haematocrit, PaO2, SaO2, or arterial oxygen content in the total sample, nor when the cohort was split up into different age groups. Cyanotic infants aged 2-13 weeks had significantly higher siEp concentrations than normal adults (p less than 0.001) and than children with cyanotic congenital heart disease, aged 4 months-10 years (p less than 0.001). The raised siEp levels in cyanotic children aged 2-13 weeks found in this study and the normal levels found in their older counterparts (4 months-10 years) (reported elsewhere) are consistent with the pattern observed in man and animals exposed to prolonged hypobaric hypoxia, in which after an initial rise in erythropoietin concentrations the levels fall to normal while increased erythropoiesis is sustained.

Topics: Cross-Sectional Studies; Erythropoietin; Heart Defects, Congenital; Hemoglobins; Humans; Hypoxia; Infant; Infant, Newborn; Male; Oxygen; Radioimmunoassay

Rat erythropoietin (Ep) cross-reacts in the radioimmunoassay (RIA) for human Ep developed in this laboratory. Immunoreactive Ep was measured in serum and tissues of male rats in response to short-term hypoxia (0.43 atm for 24 h). In the unstimulated rat all tissues examined had low levels of Ep, with the exception of the submaxillary or salivary gland (SG). Exposure to hypoxia for 24 h resulted in significant increases in kidney and serum levels of Ep, with no apparent change in SG content. Sialectomy immediately prior to exposure reduced renal Ep production and serum levels significantly after 4 h of exposure. Nephrectomy (N) confirmed previous results by others: Ep production after exposure to hypoxia was reduced but not abolished. The effect of N plus sialectomy was identical to that of N alone, thus excluding the SG as a source of extrarenal Ep in nephrectomized rats. The long-term effect of SG ablation to the same constant stimulus was a steady decline of the Ep response during the first week after surgery, both in renal production and serum levels. Thereafter, from one to six weeks the serum levels remained constant, being higher than in the unstimulated rat but significantly lower than in intact hypoxic animals. No cross-reactivity in the RIA was found with renin, renin substrate, nerve and epidermal growth factor, or somatomedins. If this Ep-like substance in the SG were the source of extrarenal Ep, it should have been possible to document an increase in serum concentration before an increase could be measured in renal content. It appears, however, that the presence of the SG is necessary for renal tissue to be able to synthesize Ep during hypoxia.

Topics: Animals; Erythropoietin; Hypoxia; Immunologic Techniques; Kidney; Male; Rats; Salivary Glands; Tissue Distribution

In order to determine the influence of calcium on erythropoietin release in response to hypoxia, the effects of the calcium entry blocker verapamil on erythropoietin production were investigated. Hypoxia was induced in male Sprague-Dawley rats by placing them in a hypobaric chamber at 0.42 atmospheres for 6 and 12 hr. Serum levels of erythropoietin were measured by the exhypoxic polycythemic mouse bioassay and a sensitive radioimmunoassay for erythropoietin. Verapamil (5, 10 and 20 mg/kg i.p.) produced significant increases in mean serum erythropoietin levels after 6 and 12 hr of hypoxia, which was significantly higher than those of saline-injected hypoxic control rats. Mean arterial blood pressure values in rats injected with 5 and 10 mg/kg of verapamil were not significantly different from the preinjection controls when measured at ambient pressure over a 12-hr period. A dosage of 20 mg/kg of verapamil i.p. in rats produced a significant decrease in mean arterial pressure between 5 and 30 min after injection but was not significantly different from the vehicle controls between 30 min and 12 hr postinjection. In conclusion, the calcium entry blocker verapamil enhanced erythropoietin production in response to hypoxia. Thus, it is postulated that calcium plays a significant role in erythropoietin production and/or release.

Topics: Animals; Biological Assay; Blood Pressure; Calcium Channel Blockers; Erythropoietin; Female; Hypoxia; Male; Mice; Polycythemia; Rats; Rats, Inbred Strains; Time Factors; Verapamil

The erythropoiesis stimulating factor(s) (ESF) in plasma from 20-day-old WLO-mice have previously been studied by a cell culture assay, and also by means of gel filtration chromatography and affinity chromatography. It was concluded that the high levels of ESF found in the neonatal mouse plasma probably consisted of erythropoietin (Ep) alone. The objective of the present investigation was to obtain further information of whether this high ESF found in vitro is Ep alone, or Ep in combination with other factors. To accomplish this plasma from 20-day-old WLO mice and a standard Ep were studied in vivo by the exhypoxic polycythaemic mice assay for Ep, with and without preincubation with rabbit anti-Ep serum (AS). Aliquots of some samples were also studied in vitro by the exhypoxic polycythaemic mice assay for Ep, with and without pre- in both assays (P less than 0.001). However, incubation with AS significantly reduced (P less than 0.001) but did not totally block either the in vivo or the in vitro activity of the plasma (P less than 0.005). This also was the case regarding the in vivo activity of the standard Ep (P less than 0.001), while the in vitro activity of this Ep preparation was totally blocked by incubation with AS (P greater than 0.3). These results indicate that a considerable part of the high erythropoietic stimulatory activity found in plasma from 20-day-old mice, with both assays, is Ep. This supports the previous in vitro studies. However, the present results also support the conclusion that part of the activity is due to non-Ep stimulatory factors.

Topics: Age Factors; Anemia; Animals; Erythropoiesis; Erythropoietin; Female; Hypoxia; Male; Mice; Mice, Inbred Strains; Polycythemia; Sheep; Stimulation, Chemical

Plasma erythropoietin titers (Ep) were compared in rats subjected to different kinds of systemic hypoxia. Ep increased exponentially, when hypoxia was induced by exposure to simulated altitude (from less than 0.025 IU Ep/ml plasma at sea-level up to 3.65 IU Ep/ml at 7000 m). An acute increase in the O2 affinity of blood augmented Ep production in normal rats but not in rats exposed to hypobaria. Ep also rose exponentially when isovolemic anemia was induced (e.g., 0.5 IU Ep/ml plasma were found at 7 g Hb/dl blood). When the same reduction in blood O2 carrying capacity was produced by CO-inhalation (0.1% CO in air), Ep increased to 5.57 IU/ml plasma. This very high value was considered to be partly due to the very high O2 affinity of carboxyhemoglobin. These results indicate that a left shift in the hemoglobin-O2 dissociation curve produces a stimulation of Ep production, particularly, when the O2-carrying capacity of blood is lowered.

Topics: Anemia; Animals; Atmospheric Pressure; Carbon Monoxide Poisoning; Erythropoietin; Hemoglobins; Hypoxia; Male; Oxygen; Rats; Rats, Inbred Strains

Although erythropoietin (Epo) is produced primarily by the kidneys in response to hypoxia, the precise cell type(s) and mechanisms by which these cells regulate production are poorly understood. In the experiments we report, the kinetics of renal Epo production in response to acute hypoxia and the intrarenal localization of cellular Epo synthesis were studied at the level of Epo mRNA. Erythropoietin mRNA expression was determined by Northern blot analysis of rat kidney RNAs using a probe derived from the mouse Epo gene. Renal Epo mRNA content increased as early as 1 hour after initiation of hypoxia and continued to accumulate during 4 hours of stimulation. Discontinuation of the hypoxic stimulus resulted in rapid decay of mRNA levels. Kidney and plasma Epo levels measured by radioimmunoassay paralleled, with respective lag times, the changes in renal Epo mRNA content, suggesting that Epo production in response to acute hypoxia represents de novo synthesis and is regulated by changes in Epo mRNA. Northern blot analysis of RNAs extracted from separated glomerular and tubular tissue fractions revealed Epo mRNA in the tubular fraction, whereas glomerular tissue did not contain Epo mRNA. Thus, the site of cellular Epo synthesis is located in the renal tubule or its interstitium and not in the glomerular tuft.

Topics: Animals; Collodion; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Hypoxia; Kidney; Kidney Glomerulus; Kidney Tubules; Kinetics; Male; Rats; Rats, Inbred Strains; RNA, Messenger; Time Factors; Tissue Distribution; Tissue Extracts

The normal response to anemic or hypoxic hypoxia is synthesis and release of erythropoietin in accord with the concept that erythropoietin production is controlled by a renal oxygen sensor. In this study, erythropoietin production, as predicted, was abrogated in patients with renal impairment (55 cases), but normal in nonuremic individuals. Specifically, patients with rheumatoid arthritis (34 cases), sickle cell anemia (25 cases), aregenerative anemia (27 cases), and aplastic anemia (13 cases) had erythropoietin titers overlapping with those observed in simple anemia (61 cases) at corresponding hematocrits. The response of polycythemic laboratory animals to hypoxia is more difficult to fit within the concept of an oxygen sensor responsive both to anemic and hypoxic hypoxia. If the polycythemia was induced by hypertransfusion, erythropoietin production in response to hypoxia was, as predicted, less than that observed in normal animals. If, however the polycythemia was induced by previous exposure to hypoxia, the animals responded to hypoxia as though they were not polycythemic. An explanation for this challenging observation may provide a clue as to the operation of the oxygen sensor.

Topics: Anemia; Animals; Arthritis, Rheumatoid; Erythropoietin; Feedback; Humans; Hypoxia; Kidney; Kidney Diseases; Mice; Oxygen; Polycythemia

Erythropoiesis was stimulated in 2- to 5-day-old normal neonatal rats nursed by phlebotomized mothers, and in 12-day-old hypertransfused neonatal suckled for 4 days by a twice-bled mother. Erythropoietic stimulation was evidenced as increases in hemoglobin levels and numbers of peripheral reticulocytes in the 2- to 5-day-olds, and by a higher 48-hour RBC-59Fe incorporation in the hypertransfused than in control 12-day-olds. The reticulocyte maturation curve was shifted to the left in 14-day-old transfused rats suckled by an anemic mother, in 14-day-old normal rats suckled by an anemic mother, and in 12-day-old transfused neonates fed cow milk to which erythropoietin (Ep) had been added. The oral administration of cow milk containing Ep to 10-day-old normal neonates induced a reticulocytosis. One-day-old rats suckled by mothers bled 72 h prior to delivery exhibited significant amounts of Ep in their plasma as compared to nondetectable levels in 1-day-old control rats of normal mothers. Decreases in relative percentages of nucleated erythrocytes were noted in spleens of hypertransfused 12-day-old neonates nursed by anemic mothers, in spleens of hypertransfused 12-day-old injected with Ep, and in spleens of 14-day-old normal rats suckled by an anemic mother. Nucleated erythrocyte numbers were reduced in both spleen and marrow of 12-day-old anemic neonates. The data suggest that: Ep is transmitted to neonatal rats via maternal milk, and in the process of gastrointestinal tract absorption, escapes inactivation, thereby stimulating erythropoiesis in these animals; Ep acts on already differentiated erythroid cells by influencing their rate of maturation.

Topics: Anemia; Animals; Animals, Newborn; Bone Marrow Cells; Erythropoiesis; Erythropoietin; Hypoxia; Milk; Polycythemia; Rats; Reticulocytes; Spleen

Topics: Animals; DNA; Erythropoietin; Hypoxia; Kidney; Mice; Mice, Inbred Strains; RNA, Messenger

The erythropoietic response, measured as 59Fe uptake by RBC, and the erythropoietin content of kidney tissue after exposure to hypobaric hypoxia were determined in both posthypoxic (PH) and hypertransfused (HT) rats. Plasma Ep titers were also measured in both PH and HT nephrectomized rats treated similarly. PH and HT rats with similar degrees of polycythemia greatly differed in their responses to hypoxia, with radioiron uptake by RBC about six times higher in the former than in the latter. Kidney Ep titers were significantly enhanced after exposure to hypoxia in either normal or polycythemic rats. However, kidney Ep titers in PH rats were significantly higher than the values found in normal or HT rats. Plasma Ep titers in both PH and HT nephrectomized rats exposed to hypoxia were similar and significantly lower than the plasma values of normal or nephrectomized, nonpolycythemic rats treated similarly. These results suggest that exposure to hypoxia induces sensitization of the renal, but not extrarenal, mechanism involved in Ep production.

Topics: Animals; Blood Transfusion; Erythrocyte Indices; Erythropoietin; Hypoxia; Kidney; Male; Nephrectomy; Polycythemia; Rats; Rats, Inbred Strains

Immunoreactive erythropoietin (Ep) was measured in normoxic and hypoxic (0.5 atm; 18 hours) fetal rats from day 14 to day 21 of gestation and in neonatal rats from birth to weaning, and was compared to the adult rat. Amniotic fluid (AF) Ep was approximately 100 mU/mL on day 14 and 15, and decreased to 20 mU/mL on day 20, with no difference between the hypoxic and normoxic mothers. Only on day 21 did the Ep in the AF increase slightly in the hypoxic group, while the Ep in the control group continued to fall to 15 mU/mL on day 21, the last day of pregnancy. Before day 17 of gestation the rat fetus appears to have hypoxia-independent, extrahepatic Ep available which is followed by hepatic and renal Ep production, both of which become sensitive to maternal hypoxia during the last days of pregnancy. In the neonatal rat plasma and tissue, Ep levels varied greatly during the first three weeks of life regardless of whether the animals were hypoxic or not. With the exception of the first and ninth days of life, circulating Ep levels were higher than adult levels in normal newborn rats. Neonatal rats responded to hypoxia with increasing Ep levels, and the response increased with age such that during the third week of life the plasma Ep levels were significantly higher than in adult hypoxic rats. No sex difference in male and female response to hypoxia could be documented until sexual maturity (day 42). In the normoxic neonatal rat more Ep originated from the liver than the kidneys until day 10, while under hypoxic conditions the switch occurred as early as two days after birth.

Topics: Age Factors; Animals; Animals, Newborn; Erythropoietin; Fetus; Hypoxia; Kidney; Liver; Milk; Rats

An in vitro technique was used to determine the unidirectional Fe3+ uptake rates in mouse duodenal fragments. In animals in which erythropoiesis had been stimulated by hypoxia, Fe3+ uptake by the duodenal fragments was enhanced due to an increase in Vappmax, However, when erythropoiesis was increased by injections of erythropoietin, intestinal Fe3+ uptake rates were unaffected. Mice subjected to sub-total nephrectomy showed an increased Vappmax for Fe3+ after exposure to hypoxia, despite the absence of an erythropoietic response. When the bone marrow was obliterated by treatment with 89Sr, a small increase in Vappmax for Fe3+ was found: permeability studies and morphometric analyses demonstrated no apparent irradiation damage to the duodenal mucosa of these animals. Exposure of 89Sr-treated mice to hypoxia caused a further significant increase in Vappmax. These results indicate that the increase in intestinal mucosal iron uptake which follows hypoxia is not mediated by erythropoietin or other factors associated with increased erythropoiesis.

Topics: Animals; Bone Marrow; Duodenum; Erythropoiesis; Erythropoietin; Ferric Compounds; Hypoxia; Intestinal Absorption; Intestinal Mucosa; Male; Mice; Splenectomy

The effects of interferon (IF) on erythropoietin (Ep) action and production were studied in mice. In comparison to control animals, Ep action in exhypoxic, polycythemic mice was significantly decreased (p less than 0.05) following two low dose injections of IF (2.9-3.5 X 10(4) units). In addition, renal Ep production in normal intact mice was also significantly decreased (p less than 0.01) following a single injection of IF (5.4-6.3 X 10(4) units) and hypoxic exposure.

Topics: Animals; Erythropoiesis; Erythropoietin; Hypoxia; Interferons; Kidney; Mice

A compensated hemolytic state was induced in rats by injection of phenylhydrazine (PHZ) over a 6-week period. The liver and kidney were perfused to determine the levels and time of appearance of a hepatic erythropoietic factor (HEF) which induces the production of hepatic erythropoietin (Ep) and its antagonist, a renal inhibitory factor (RIF). Erythropoietin assays on the perfusates have been previously reported. The amount of HEF in perfusates recovered from the livers of the PHZ-treated rats was significantly higher during the 4th to 5th weeks of treatment, coinciding with the time of increased liver production of Ep. During the 6th week of PHZ treatment, the titer of RIF in perfusates recovered from the kidney was markedly increased and the HEF titer was decreased to near the control level, suggesting inhibitory action of RIF on HEF synthesis and/or effect. These findings indicate that an HEF/RIF control mechanism regulates hepatic Ep production in this compensated hemolytic condition.

Topics: Animals; Blood Proteins; Erythropoietin; Female; Hemolysis; Hepatocyte Growth Factor; Hypoxia; Kidney; Liver; Male; Mice; Perfusion; Phenylhydrazines; Rats; Time Factors

The site of erythropoietin (Ep) production and/or storage in the rat liver was determined. A guinea pig anti-Ep was produced against purified rat Ep (64,096 +/- 4j064 IU/mg). This antibody was found to be highly specific using rocket immunoelectrophoresis, Ouchterlony gel diffusion methods, and immunoprecipitin reactions as well as Ep neutralization tests (capable of completely neutralizing up to 2,000 IU Ep/mg). This anti-Ep was labeled with either fluorescein for light microscopic study or ferritin for electron microscopy. Kupffer cells showed varying degrees of labeling after hepatectomy alone or hepatectomy combined with nephrectomy and/or hypoxia. Greatest labeling was seen in Kupffer cells of rats that were nephrectomized 48 hr posthepatectomy and kept at ambient pressure. No labeling of hepatocytes or vascular and bile duct endothelium was noted.

Topics: Animals; Antibodies; Erythropoietin; Female; Ferritins; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Antibody Technique; Guinea Pigs; Hypoxia; Kupffer Cells; Liver Regeneration; Macrophages; Male; Rats; Rats, Inbred Strains; Thiocyanates

The erythropoietic response, measured as RBC-59Fe uptake, in response to either 24-h exposure to hypoxia or administration of dexamethasone, isoproterenol, testosterone, or erythropoietin, was determined in both posthypoxic (PH) and hypertransfused (HT) polycythemic mice. Highly significant differences between PH and HT mice exposed to hypoxia or injected with dexamethasone, isoproterenol, or testosterone were observed, isotope incorporation being always higher in PH than in HT mice. On the other hand, the response to erythropoietin did not show a significant difference between PH and HT mice. These results suggest that PH mice have been preconditioned by exposure to hypoxia in a way that makes them more sensitive to at least some kinds of erythropoietic stimuli. Since these stimuli have been shown by others to increase erythropoietin production, the results support our hypothesis that hypoxia induces sensitization of the erythropoietin-producing organ(s).

Topics: Animals; Blood Transfusion; Dexamethasone; Erythrocyte Volume; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Hypoxia; Iron; Iron Radioisotopes; Isoproterenol; Mice; Polycythemia; Testosterone

On the basis that fetal levels of plasma erythropoietin (Ep) may reflect fetal oxygenation the primary purpose of the present study was to assess the relation between Ep measured in cord plasma at delivery and the intrapartum fetal heart rate (FHR) record. A scoring system for interpreting FHR recordings blindly was prospectively utilized in 41 selected human pregnancies during the 4 h immediately preceding birth. The correlation of the overall mean FHR score for each individual patient with cord plasma Ep was significant such that the highest Ep levels were observed in those infants with the most abnormal FHR scores. Furthermore, when the birthweights of the infants were adjusted for gestational age, sex, and birth order, birthweight centile was negatively correlated with cord plasma Ep. When both FHR score and birthweight were simultaneously correlated with cord plasma Ep using multiple regression, the combined effect of these two factors improved the association of either alone with both contributing approximately equally.

Topics: Birth Weight; Delivery, Obstetric; Erythropoietin; Female; Fetal Blood; Fetal Heart; Fetal Monitoring; Heart Rate; Humans; Hypoxia; Pregnancy; Prospective Studies

Effects of an acute increase in blood O2 affinity on erythropoietin production were studied in normoxic and hypoxic male rats. Blood O2 affinity was increased by exchange-transfusion with blood from sodium cyanate treated rats. P50 was lowered to 27.6 torr (pH 7.4, PCO2 = 40 torr, 37 degrees C) in the recipients compared to 41.8 torr in control rats exchange transfused with normal blood. Hypoxia was induced by exposure to simulated high altitude (4750 or 7000 m) for 16 h. Erythropoietin was determined by in vivo bioassay. In rats with normal blood O2 affinity, plasma erythropoietin was undetectable at 300 m, 0.26 +/- 0.10 U/ml at 4750 m (mean +/- SEM; n = 7), and 3.52 +/- 0.58 U/ml at 7000 m (n = 10). Plasma erythropoietin titers were significantly enhanced in rats with high blood O2 affinity at 300 m (0.05 +/- 0.01 U/ml; n = 4) and moderately increased at 4750 m (0.57 +/- 0.12 U/ml; n = 7), but unchanged at 7000 m (3.88 +/- 0.74 U/ml; n = 10). These results indicate that a high blood O2 affinity reduces the O2 delivery to the cells controlling erythropoietin production in normoxia and moderate hypoxia. However, this is offset at severe hypoxia most likely by an improved O2 loading to the blood.

Topics: Altitude; Animals; Cyanates; Erythropoietin; Exchange Transfusion, Whole Blood; Hypoxia; Male; Oxygen; Rats; Rats, Inbred Strains; Reference Values

Severe erythrocytosis is associated with increased whole blood viscosity and impaired blood flow. Since a reduced blood flow will cause tissue hypoxia and since tissue hypoxia is associated with increased synthesis of erythropoietin, erythrocytosis per se should cause an increase in the rate of red cell production. This, however, does not occur and severe erythrocytosis in patients with polycythemia vera does not lead to increased synthesis of erythropoietin. We propose here that the reason for this discrepancy is that decreased blood flow to the kidneys, the site of erythropoietin synthesis, does not cause renal tissue hypoxia. The oxygen tension in the kidneys is to a great extent determined by the consumption of oxygen used for sodium reabsorption and since sodium reabsorption is roughly proportional to glomerular filtration, a decreased flow of blood should be matched by a decreased oxygen consumption leaving the tissue tension of oxygen unchanged. Consequently, the location of an oxygen sensor in the kidneys controlling erythropoietin production appears to be most fortuitous since it prevents the development of a vicious circle, with erythrocytosis causing more erythrocytosis.

Topics: Blood Viscosity; Erythropoietin; Glomerular Filtration Rate; Hematocrit; Humans; Hypoxia; Kidney; Oxygen Consumption; Polycythemia; Polycythemia Vera; Radioimmunoassay; Regional Blood Flow; Sodium

The relationship between plasma erythropoietin levels and kidney erythropoietin content was studied in rats subjected to hypoxia for various periods. Plasma and kidney erythropoietin followed a parallel course with detectable levels observed as early as after 1 hour of hypoxia. However, the kidney erythropoietin content reached a maximum at 6 hours, and its plasma erythropoietin content reached its maximum at 24 hours. Despite continuous hypoxia, the erythropoietin in both kidney and plasma decreased after reaching their maximal values, and leveled off after 72 hours. This parallel decrease in plasma and kidney erythropoietin suggests that this decrease in plasma erythropoietin observed after prolonged hypoxia is secondary to a decrease in erythropoietin production rather than to an increase in peripheral consumption. To identify the cell(s) involved in erythropoietin production, kidney cells from anemic-hypoxic animals were separated into their glomerular and tubular components utilizing a successive sieving procedure. Measurement of erythropoietin by bioassay revealed four to five times as much erythropoietin in the tubular fraction as in the glomerular fraction. Radioimmunoassay of these fractions revealed erythropoietin content similar to that by bioassay, ruling out the possible presence of biologically nonactive material. Renin activity, on the other hand, was about equally distributed between both of these fractions. Although our studies do not rule out participation of glomerular or juxtaglomerular cells in erythropoietin production, they suggest that a tubular origin of the bulk of renal erythropoietin is more likely.

Topics: Animals; Biological Assay; Cell Separation; Erythropoietin; Hypoxia; Kidney; Kidney Glomerulus; Kidney Tubules; Male; Mice; Radioimmunoassay; Rats; Rats, Inbred Strains; Renin; Time Factors; Tissue Extracts

Splenic CFU-E in mice were determined during stimulation and suppression of erythropoiesis by means of the methylcellulose culture system originally used for bone marrow CFU-E. It was found that under steady-state conditions the number of CFU-E in the spleen comprises less than 20% of the CFU-E in the total bone marrow mass. The number of CFU-E decreased with suppression and increased after stimulation of erythropoiesis. The response to exogenous Ep stimulation on day 10 posthypoxia was more pronounced in the spleen than in the bone marrow (the number of CFU-E was 1.6 times greater in the spleen than in the total bone marrow mass) indicating the importance of the spleen in stimulated erythropoiesis in mice.

Topics: Animals; Blood Transfusion; Bone Marrow Cells; Colony-Forming Units Assay; Cyclophosphamide; Depression, Chemical; Erythrocyte Transfusion; Erythropoiesis; Erythropoietin; Female; Hypoxia; Mice; Mice, Inbred CBA; Spleen

Exposure to hypoxia results in an increase in the plasma erythropoietin (Ep) content to a peak level in 10-12 h. If hypoxia is discontinued before maximum plasma Ep concentrations are reached, then the plasma Ep level continues to rise before it declines toward normal. To clarify this phenomenon, we determined the Ep level in the plasma and kidneys at various times after rats that had been exposed to 0.42 atm returned to ambient pressure. The plasma Ep level 2 h after hypoxia was 4-5 times as high as it was immediately after hypoxia and then gradually declined. The Ep content of the kidneys rose for only 1 h after which it rapidly fell to an undetectable level. The most plausible interpretation of this data is that renal Ep production decelerates rapidly within 1 h after return to ambient pressure, but plasma Ep levels continue to rise because of the release of preformed Ep into the plasma. Experiments were also performed to determine whether an increased plasma Ep level inhibits the production or secretion of Ep. Three different types of experiments were performed in which the plasma Ep level was increased by intravenously injecting a large amount of Ep prior to, during, or after exposure to hypoxia and determining its effect on the plasma Ep activity from 15 min to 4 h after injection. In all experiments the plasma and renal Ep levels rose comparably in Ep-treated and control rats during exposure to hypoxia. The data do not support the hypothesis that a rise in the plasma Ep level inhibits Ep production or secretion into the plasma.

Topics: Animals; Erythropoietin; Hypoxia; Kidney; Kinetics; Male; Rats; Rats, Inbred Strains; Time Factors

Topics: Anemia; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Infant, Newborn; Infant, Premature, Diseases

PG synthesis inhibitors were used to study the role of PG in modulating Ep effects on erythroid progenitors. The stimulating effect of exogenous Ep on both 59Fe incorporation into RBC and CFU-E number in polycythaemic mice was inhibited with indomethacin and aspirin treatment during the posthypoxic period. The inhibitory effect of indomethacin on CFU-E derived colonies was demonstrated only when cultured bone marrow and spleen cells were obtained from mice stimulated by prolonged exposure to hypoxia. The results implicate the role of PG in the Ep dependent regulation of the erythroid progenitor cell compartment size and Ep responsiveness in vivo.

Topics: Animals; Aspirin; Bone Marrow; Bone Marrow Cells; Colony-Forming Units Assay; Erythropoiesis; Erythropoietin; Female; Hypoxia; Indomethacin; Mice; Mice, Inbred CBA; Polycythemia; Prostaglandin Antagonists; Spleen

The development of a secondary erythrocytosis is usually considered a compensatory effort to counteract tissue hypoxia. However, the associated increase in viscosity tends to decrease blood flow and in theory should augment rather than relieve tissue hypoxia. Clinical observations have supported this concern and phlebotomies have been used to treat cardiopulmonary patients with high hematocrits and to prepare acclimatized mountain climbers for strenuous exercises. Direct measurements of tissue tension in rats and mice have shown that a moderate increase in hematocrit does increase the tissue tension of oxygen, probably due to a concomital increase in blood volume, and only severe increases in hematocrit are detrimental. In contrast, it was found that erythropoietin production in mice and man is decreased at even the most extreme hematocrits, suggesting that the tissue tension in the kidneys is not affected by high hematocrits and sluggish blood flow. This lack of renal hypoxia at high blood viscosities appears to serve an important purpose by preventing a vicious circle in which hypoxia will cause erythrocytosis leading to more hypoxia and more erythrocytosis and so on. However, well maintained secondary erythrocytosis cannot always be considered optimal for overall oxygen transport and has to be evaluated clinically for its potential benefit or harm.

Topics: Animals; Blood Viscosity; Erythropoietin; Hematocrit; Humans; Hypoxia; Kidney; Oxygen; Partial Pressure; Polycythemia; Rats; Regional Blood Flow

Hypoxia stimulates erythropoietin (Ep) production and subsequent stimulation of erythropoiesis. Chronically applied hypoxia results in a variety of physiological alterations including polycythemia, hemoglobinemia, and vascular and metabolic changes. As red cell mass increases to such an extent to allow for adequate tissue oxygenation, plasma Ep levels drop and the rate of erythropoiesis is lowered. In the present work, the precise contribution of the kidney and the liver to the Ep response to acutely administered hypoxia (6-24 hr/0.4 atm) was determined using an in situ tandem perfusion of the liver and kidneys. The results indicate that hepatic and renal Ep production during different intervals of acute hypoxia is not uniform. This alteration may be partially explained by the reported reduction in splanchnic blood flow and a possible redistribution of hepatic microcirculation, which may cause the liver to become more hypoxic than the kidney under the same ambient pO2. The role of the liver becomes more pronounced as the time of acute hypoxic exposure increases, whereas the Ep content of renal perfusates is not drastically altered. At certain intervals during hypoxia, the liver displays a greater potential to elaborate Ep.

Topics: Animals; Erythropoietin; Hypoxia; Kidney; Kinetics; Liver; Macrophages; Male; Rats

Changes in erythropoiesis with age were studied by examining the hematocrit increase in response to hypoxia in aged mice and by assessing the change in erythropoiesis following the injection of erythropoietin in young and old polycythemic mice. The increase in hematocrit after exposure to hypoxia was more variable and generally lower in old mice than in young mice. When erythropoietin was injected into polycythemic animals, the increase in differentiated erythroid cells and 59Fe incorporation into erythroid marrow and peripheral blood cells was significantly lower in old mice than in young mice. In contrast to differentiated erythroid cells, there was less evidence of a reduced response to stimulation of the more primitive erythroid progenitor cells of aged animals. The early undifferentiated erythroid progenitor, burst-forming units, did not decrease when either young or aged mice were made polycythemic, and no change following erythropoietin injection was noted. Polycythemia suppressed the late-differentiated erythroid progenitor, erythroid colony-forming units, to a greater extent in aged animals, but when erythropoietin was injected, the percent increase over the subsequent 24 hours was identical to that in young mice. These observations indicate a reduced erythropoietic capacity with age, the abnormality being most obvious in the more mature erythroid precursors.

Topics: Aging; Animals; Erythropoiesis; Erythropoietin; Female; Hematocrit; Hematopoietic Stem Cells; Hypoxia; Mice; Mice, Inbred C57BL; Polycythemia

Removal of 15% of blood volume in the mouse increases erythropoiesis by a factor of 2.2 when measured 12 h after bleeding. Exposure of normal mice to 40% reduced barometric pressure for the same period of time increases erythropoiesis only by a factor of 1.6. The response to hypoxia takes place in the presence of a 40% reduction of oxygen consumption and tissue-venous PO2, changes which are concomitant with a 5-fold increase in plasma erythropoietin activity. The larger response in anemic animals on the other hand occurs without any detectable change in these parameters. These results cast serious doubts about the interpretation of the quantitative homeostatic control of erythropoiesis based solely on the action of erythropoietin.

Topics: Anemia; Animals; Erythropoiesis; Erythropoietin; Female; Homeostasis; Hypoxia; Mice; Mice, Inbred C3H; Oxygen; Oxygen Consumption

The macromolecular polymer, methyl cellulose (MC), is a known hepatosplenomegalic agent which promotes a state of experimental hypersplenism in rats. This is characterized by massive splenomegaly, pancytopenia of the blood elements, medullary hypoplasia, and marked gross and histologic alteration of the liver, kidney, adrenals, and lungs. Massive splenomegaly results from storage of this inert material by splenic macrophages. In the present study, chronic MC administration in rats augmented the hepatic Ep response to hypoxia but did not appreciably affect renal production of Ep. Splenectomy resulted in a decrease in the extrarenal Ep response to hypoxia indicating a possible role of the massively enlarged spleen of these MC-treated rats in extrarenal Ep production. The augmentation of extrarenal Ep elaboration may be attributed to a stimulatory effect of MC on the hepatic and splenic macrophages.

Topics: Animals; Erythropoietin; Female; Hematocrit; Hypoxia; Kidney; Liver; Methylcellulose; Rats; Spleen

Polyacrylamide gel electrophoresis was used to reveal in the rabbit bone marrow cells 5 main fractions of histone proteins which are present in other eukaryotic cells. The degree of acetylation and phosphorylation increased after erythropoietin injection or exposure to hypoxia. Phosphorylation was enhanced in every fraction. Stimulation of acetylation was noted in histones rich in arginine. Erythropoietin had no effect on the synthesis of marrow histones in the early period after a hypoxic stimulus.

Topics: Acetylation; Animals; Bone Marrow; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Female; Histones; Hypoxia; Mice; Phosphorus Radioisotopes; Phosphorylation; Rabbits

Topics: Acidosis; Animals; Blood Gas Analysis; Electric Stimulation; Erythropoietin; Hemoglobins; Hypercapnia; Hypoxia; Iron; Kidney; Male; Oxygen Consumption; Protein Binding; Rabbits

An increase in hemoglobin concentration characterizes the normal compensatory response to chronic tissue hypoxia. We observed no such increase in 42 chronically hypoxic patients with cystic fibrosis, in whom the mean concentration was 12.6 gm/dl; one third of the patients were anemic. Compared with patients with cyanotic heart disease, patients with cystic fibrosis did not have a compensatory increase in P50 or 2,3-diphosphoglycerate. Despite anemia, erythropoietin levels in patients with cystic fibrosis were not significantly different from normal control values. The growth of colony-forming units-erythroid in patients with cystic fibrosis was similar to that in control subjects, and there was no inhibition of growth with the addition of autologous serum. Erythropoietin sensitivity, determined by measuring the CFUe dose response curve, was normal in both patients and controls. Results of iron studies were consistent with iron deficiency in the majority of patients. Impaired absorption of iron was observed in six of 13 iron-deficient patients with cystic fibrosis. An inverse correlation between erythrocyte sedimentation rate and peak serum iron was obtained during the iron absorption study. Eight patients who underwent a therapeutic trial of iron demonstrated a 1.8 gm/dl rise in hemoglobin concentration. Two patients with previously documented iron malabsorption responded to parenteral iron therapy after failure to respond to oral supplementation. These studies demonstrate that patients with cystic fibrosis not only have an impaired erythroid response to hypoxia, but are frequently anemic. Their inadequate erythroid response to hypoxia results in part from disturbances in erythropoietin regulation and iron availability.

Topics: 2,3-Diphosphoglycerate; Adolescent; Adult; Biological Availability; Blood Sedimentation; Child; Child, Preschool; Cystic Fibrosis; Diphosphoglyceric Acids; Erythropoietin; Heart Defects, Congenital; Hematopoietic Stem Cells; Hemoglobins; Humans; Hypoxia; In Vitro Techniques; Intestinal Absorption; Iron; Oxygen Consumption

The purpose of the present study was to analyze the effect of two different beta blocking agents, metipranolol (Trimepranol) and dl-propranolol (Inderal), upon erythropoietin production, erythropoietin responsive cell compartment and erythrocyte production. The effect on erythropoietin production: in rats metipranolol or dl-propranolol was injected daily in a single dose of 10 mg/kg b.wt. On the 5th day, the rats were exposed to an atmospheric pressure of 353.7 torr (47.1 kPa, 6000 m) to stimulate erythropoietin production. The onset of hypoxic exposure was started 1, 4, 12 or 24 hr after the last injection of beta blocking drugs. After the termination of hypoxia, erythropoietin plasma levels were determined in polycythemic mice. The rats treated with metipranolol 1 and 4 hr before hypoxia produced significantly less erythropoietin in response to hypoxia than saline-treated control animals. The effect on erythropoietin responsive cell compartment: polycythemic mice were treated with metipranolol or dl-propranolol in doses of 10 to 40 mg/kg b.wt. and with an erythropoietin standard. Radioiron incorporation into red blood cells in control mice and in mice pretreated with beta blocking agents was not significantly different. The effect on erythrocyte production: in rats treated daily with metipranolol in a single dose of 10 mg/kg b.wt., for a period of 10 days, the rate of erythropoiesis measured by the incorporation of 59Fe into red blood cells was decreased, dl-propranolol (10 mg/kg b.wt.) did not reduce the rate of erythropoiesis. Our results demonstrate that the changes in erythropoiesis after administration of metipranolol may be caused by reduction of erythropoietin production with a consequent reduction of the erythrocyte production rate.

Topics: Adrenergic beta-Antagonists; Animals; Atmospheric Pressure; Erythropoiesis; Erythropoietin; Female; Hypoxia; Male; Metipranolol; Mice; Mice, Inbred ICR; Propanolamines; Propranolol; Rats; Rats, Inbred Strains

Topics: Animals; Erythrocyte Membrane; Erythropoiesis; Erythropoietin; Hematocrit; Humans; Hypoxia; Propranolol; Rats; Receptors, Adrenergic, beta; Ribose

Topics: Alprostadil; Anemia; Animals; Dinoprostone; Epoprostenol; Erythropoietin; Humans; Hypoxia; Kidney; Kidney Failure, Chronic; Kidney Glomerulus; Kidney Neoplasms; Liver; Molecular Weight; Neoplasms; Parathyroid Hormone; Phospholipases; Polycythemia; Prostaglandins; Prostaglandins E; Tissue Distribution

We investigated 28 cystic fibrosis (CF) patients to determine why hypoxia from their obstructive pulmonary disease does not produce polycythemia. Oxygen saturation was lower and erythropoietin levels were higher in CF patients than in 25 age-comparable reference subjects (90.8% and 47 mimu vs. 94.7% and 29 mimu, p less than 0.01). Hematocrit and red blood cell (RBC) indices were not different between groups. Serum vitamin and iron levels, ferrokinetics, RBC volume, and RBC survival were studied in 10 of the 28 CF patients. Total iron-binding capacity and vitamin E levels were low, and serum iron, ferritin, vitamin B12, and folate levels were normal in these patients. Red blood cell survival was minimally decreased in six patients although there was no other evidence for hemolysis. Ferrokinetics (59Fe) indicated a reduction in total erythropoiesis in only two patients. Plasma volume was high-normal in five and above normal in four CF patients; RBC mass was increased appropriately for each patient's degree of hypoxia, when compared to healthy individuals living at different altitudes. These results suggest that CF patients are able to compensate for hypoxia by increasing RBC mass; however, an expanded plasma volume prevents a detectable rise in hematocrit.

Topics: 2,3-Diphosphoglycerate; Adolescent; Adult; Child; Chromium Radioisotopes; Cystic Fibrosis; Diphosphoglyceric Acids; Erythrocyte Aging; Erythrocyte Volume; Erythropoiesis; Erythropoietin; Hematocrit; Humans; Hypoxia; Iron; Iron Radioisotopes; Kinetics; Oxygen; Plasma Volume

The erythropoietic response, measured as RBC-59Fe uptake after 24 h-exposure to hypoxia, were determined in posthypoxic, "acute" and "chronic" hypertransfused, and posthypoxic-hypertransfused mice. Radioiron uptake by erythrocytes was about 25 times greater in post-hypoxic than in both types of hypertransfused mice. Posthypoxic-hypertransfused mice showed an erythropoietic response to hypoxia which was not significantly different from that of posthypoxic mice and about 20 times greater than those of "acute" and "chronic" hypertransfused mice. When hypertransfused mice were exposed to hypoxia before transfusions, a linear increase in 59Fe uptake values in response to hypoxia was observed with exposures between 6 and 30 h. No further increase was observed for exposures of 46 to 216 h. These results suggest a sensitization of the erythropoietin-producing organ(s) by hypoxia.

Topics: Animals; Blood Transfusion; Erythropoiesis; Erythropoietin; Female; Hypoxia; Iron; Mice; Mice, Inbred Strains; Polycythemia; Time Factors; Transfusion Reaction

An established human testis germ cell line (1411-H) was found to produce significant amounts of erythropoietin (Ep), the primary regulator of erythropoiesis. Media conditioned by the 1411-H cells stimulated erythropoiesis both in vivo and in vitro. This activity was neutralized by anti-Ep. This continuous cell line provides a unique model for the study of the mechanisms of control of Ep biogenesis.

Topics: Cell Line; Cell Survival; Erythrocytes; Erythropoietin; Humans; Hypoxia; In Vitro Techniques; Iron Radioisotopes; Male; Testis

Topics: Animals; Cobalt; Erythropoiesis; Erythropoietin; Hypoxia; Kidney; Male; Rats; Rats, Inbred Strains

Topics: Animals; Blood Transfusion; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Hypoxia; Iron Radioisotopes; Mice; Mice, Inbred Strains; Polycythemia

In order to test the hypothesis that the early cessation of erythropoietin (Ep) production during hypobaric hypoxia is induced by lowered food intake, we have compared the plasma Ep titer of rats after exposure to continuous hypoxia (42.6 kPa = 7000 m altitude) for 4 days with that in fed or fasted rats after exposure to discontinuous hypoxia. We found that plasma Ep was rather low after 4 days of continuous hypoxia. However, the Ep titer significantly rose again, when rats were maintained normoxic for 18 h and then exposed to repeated hypoxia for 6 h. Because this was also found in rats which were deprived of food during the normoxic interval and the second hypoxic period, we conclude that the fall of the Ep titer during continuous hypoxia is not primarily due to reduced food intake. In addition, our findings show that fasting per se lowers the Ep-response to hypoxia in normal rats but not exhypoxic rats.

Topics: Animals; Body Weight; Eating; Erythropoietin; Fasting; Hypoxia; Male; Rats; Rats, Inbred Strains

An attempt to evaluate the role of the liver in extrarenal erythropoietin production was made by measuring the content of erythropoietin in homogenates and perfusates from hypoxic rat livers. Extracts from livers from nephric or anephric animals rendered both anemic and hypoxic showed no detectable erythropoietin despite the fact that both plasma and kidney extracts contained large amounts of erythropoietin. This lack of measurable erythropoietin in the liver is not caused by degradation of erythropoietin during the extraction procedure because exogenously added rat erythropoietin was recovered to the same extent from livers or kidney homogenates. More likely, however, it is caused by the fact that extrarenal erythropoietin production accounts for only one-fifth of total erythropoietin production and that the liver mass is about six times that of both kidneys. Consequently, the erythropoietin content of 1 g of liver should be about one-thirtieth of that of 1 g of kidney, an amount that is below the limit of detection of the assay. On the other hand, the 2-h in situ perfusates of livers from similarly stimulated animals contained significant amounts of secreted erythropoietin. It is concluded that the liver participates actively in extrarenal erythropoietin production in the adult rat. However, the small amount expected to be present in tissue homogenates cannot be detected with our current bioassay.

Topics: Anemia; Animals; Biological Assay; Erythropoietin; Hypoxia; Kidney; Liver; Male; Mice; Rats; Rats, Inbred Strains

The four existing animal models of cyclic hematopoiesis are briefly described. The unusual erythropoietin (Ep) responses of the W/Wv mouse, the Sl/Sld mouse, and cyclic hematopoietic dog are reviewed. The facts reviewed indicate that the bone marrow itself is capable of influencing regulatory events of hematopoiesis.

Topics: Anemia; Animals; Bone Marrow; Bone Marrow Cells; Disease Models, Animal; Dog Diseases; Dogs; Erythropoiesis; Erythropoietin; Hematopoiesis; Humans; Hypoxia; Lithium; Mice; Mice, Inbred Strains; Mink; Neutropenia; Periodicity; Prednisolone; Strontium Radioisotopes

We investigated whether erythropoietin (Ep) can be extracted from renal glomeruli of hypoxic rats. Hypoxia was induced in a hypobaric chamber at 0.42 atmospheres for usually 6 h. Glomeruli were isolated with a sieving technique from kidneys that were flushed free of blood. Ep activities in glomeruli homogenates were determined in the fetal mouse liver cell assay and in the hypoxia exposed mouse assay. We found in vitro erythropoietic activity of glomerular extracts, which increased about 25-fold during hypoxia. Incubation of the extracts with anti-Ep serum abolished the activity. Its binding characteristics on Wheat germ lectin. Ricinus communis lectin and DEAE-cellulose suggested that glomeruli contained Ep precursor(s) that lacked terminal sugar residues. Such an incomplete carbohydrate portion seems also compatible with the finding that glomerular extracts did not stimulate erythropoiesis in hypoxia exposed mice.

Topics: Animals; Erythropoietin; Female; Fetus; Humans; Hypoxia; Kidney Glomerulus; Liver; Male; Mice; Mice, Inbred Strains; Molecular Weight; Pregnancy; Rats; Rats, Inbred Strains; Receptors, Mitogen; Time Factors

Topics: Animals; Blood Glucose; Erythropoietin; Female; Fetal Diseases; Glucose; Hyperglycemia; Hypoxia; Infusions, Intra-Arterial; Insulin; Oxygen; Pregnancy; Sheep

The effects of infusing subpressor doses of angiotensin II into hypoxic and anemic rats on plasma Ep levels were determined. The effect was greatest when 5 micrograms of angiotensin II per hour was infused into rats made hypoxic 18 hr after nephrectomy. Infusion of larger amounts of angiotensin II had a lesser effect on extrarenal Ep production than did infusion of 5 micrograms/hr. Infusion of angiotensin II into rats nephrectomized 1 hr prior to exposure to hypoxia affected extrarenal Ep production to a lesser degree than the infusion into rats nephrectomized 18 hr prior to hypoxia. In contrast, administration of carbon tetrachloride per os stimulated extrarenal Ep production only when nephrectomy was performed just prior to exposure to hypoxia. Administration of both CCl4 and angiotensin II to hypoxic anephric rats elevated the plasma Ep level to approximately 1.0 IRP U/ml.

Topics: Anemia; Angiotensin II; Angiotensin III; Angiotensins; Animals; Carbon Tetrachloride; Erythropoietin; Female; Hypoxia; Infusions, Parenteral; Nephrectomy; Rats; Rats, Inbred Strains; Time Factors

Plasma erythropoietin levels during continuous exposure to hypobaric hypoxia in mice with marrow aplasia induced by whole body X-irradiation of 5-fluorouracil injection were higher than in control mice similarly exposed. There findings give support to the hypothesis that a relationship exists between erythropoietin production rate and erythroid responsiveness to the hormone.

Topics: Animals; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Fluorouracil; Hypoxia; Iron; Mice

Plasma titers of erythropoietin (Ep) are known to increase initially during hypoxia and to return then towards prehypoxia values. To find out if this pattern of plasma Ep might be related to changes in the production of the hormone, I have compared plasma with kidney Ep titers in hypoxic rats. Rats were exposed to hypoxia in a hypobaric chamber at 0.42 atm for various time intervals for up to 4 days. Kidney Ep titers were assayed in extracts from kidneys that had been flushed free of blood in situ. It was found that kidneys of normal rats do not store significant amounts of Ep. Kidney Ep titers increased transiently during hypoxia. They reached maximum values after 6h and then declined to almost undetectable levels at continued hypoxia. In the plasma, maximum values were found after 12-18h of hypoxia. Additional studies were done on the effects of discontinuous hypoxia. It was found that, even after 3 days of previous hypoxia exposure, plasma and kidney Ep titers increased again in rats when these were maintained intermittently in normoxia for 18 h. It is concluded that the rise and fall in plasma Ep titers during hypoxia reflect similar changes in kidney Ep production.

Topics: Animals; Erythropoietin; Hypoxia; Kidney; Male; Rats; Rats, Inbred Strains; Time Factors

Experiments were performed to determine the erythropoietin (Ep) content of homogenates of kidneys and livers of male and female rats of various ages. In all studies, homogenates were adjusted to a concentration of 4 g of tissue per 12 ml of phosphate-buffered-saline, and the stimulus to Ep production consisted of exposure to 0.42 atmosphere for 4 h. The concentration of Ep in kidneys of male rats was about three times that found in those of females and was contained predominantly in the cortical portion of the kidneys. Ep was not detectable in kidneys of rats younger than 3 weeks of age, and reached a maximum concentration after 4 weeks of age. The Ep content of the liver was barely detectable regardless of the age of the rat or its plasma Ep titer; and did not increase significantly by administering angiotensin II or CCl4 (substances which increase extrarenal Ep production).

Topics: Aging; Animals; Erythropoietin; Female; Hypoxia; Kidney; Kidney Cortex; Kidney Medulla; Liver; Male; Rats; Rats, Inbred Strains; Sex Factors

Erythropoietin production in response to hypoxic-hypoxia is markedly reduced in the newborn when compared to the adult rat. This response improves steadily with age and reaches adult values at about 4 wk. When animals of the same age are stimulated with anemic-hypoxia, considerably higher levels of erythropoietin are found. The erythropoietin level is proportional to the degree of anemia and independent of the age of the animal. Extraction of erythropoietin from tissue homogenates revealed a parallelism between the plasma and kidney erythropoietin content, while no erythropoietin could be extracted from liver tissue at any age. The lack of response to hypoxia in the newborn appears to be related to the high hemoglobin oxygen affinity during the neonatal period, which facilitates oxygen loading. Newborn rats have a very low intraerythrocytic concentration of 2-3 DPG and a marked shift to the left in the oxygen hemoglobin dissociation curve that slowly increases to adult values at 4 wk of age. The response to anemia on the other hand, appears to be normal and not affected by age or by hemoglobin oxygen affinity. These studies suggest that the newborn rat, when properly stimulated, is able to produce normal amounts of erythropoietin, most likely renal in origin.

Topics: Anemia; Animals; Animals, Newborn; Diphosphoglyceric Acids; Erythrocytes; Erythropoietin; Hypoxia; Kidney; Liver; Phenylhydrazines; Rats

Topics: Animals; Charcoal; Colloids; Erythrocytes; Erythropoiesis; Erythropoietin; Hypoxia; Mice; Monocytes; Polycythemia; Rats; Time Factors

Topics: Animals; Cell Compartmentation; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Hypoxia; Iron Radioisotopes; Isoproterenol; Mice

Topics: Acetylation; Animals; Bone Marrow; Erythropoietin; Female; Histones; Hypoxia; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Oxidative Phosphorylation; Rabbits; RNA

The purpose of this study was twofold: (1) to define the relationship between erythropoietin (Ep) production and some of the coincident variables which control oxygen delivery in hypoxic and hypoxic-hypercarbic rats (2) to define the mechanism whereby hypercarbia suppresses Ep production in hypoxemic rats. Rats were exposed to O2 concentrations ranging from 5% to 9% O2 for either 3 or 16 h. Arterial whole blood pH, pCO2, pO2, O2 saturation, p50 and Ep levels were measured in each rat. There was a highly significant inverse correlation between both the arterial pO2 and O2 saturation and the Ep level. Ep levels were not increased above normal if the pO2 was greater than 50 mm Hg or the O2 saturation was greater than 80%. The addition of 5% CO2 to all inhaled gas mixtures was associated with a 10 mm Hg increment in the pO2 together with a marked reduction in plasma Ep levels. Of the measured variables only the pO2 and O2 saturation showed a consistent correlation with the Ep levels when rats exposed to hypoxia were compared with rats exposed to hypoxia + CO2.

Topics: Alkalosis, Respiratory; Animals; Carbon Dioxide; Erythropoietin; Female; Hydrogen-Ion Concentration; Hypercapnia; Hypoxia; Iron; Oxygen; Rats

Evidence is presented for the existence of a factor in renal venous blood, evoked by subtotal hepatectomy (hepx), which inhibits the production of Ep in nephrectomized (nephrx) rats exposed to hypoxia. Significant inhibitory activity is not observed in blood obtained from sites other than the renal vein. This inhibitory effect is believed to be due to the action of a specific renal inhibitory factor (RIF) which reduces the extrarenal (hepatic) Ep response to hypoxia indirectly, by decreasing the production or effectiveness of an antagonistic liver principle, the hepatic erythropoietic factor (HEF). The HEF has previously been shown to augment hepatic Ep production following hypoxia in renally-deficient animals. The RIF has no anti-Ep action and its activity is not influenced by the accumulation of metabolic wastes. A mechanism for a renal-hepatic antagonism in the Ep response to hypoxia is hypothesized.

Topics: Animals; Biological Products; Erythropoietin; Hepatectomy; Hypoxia; Kidney; Ligation; Liver; Male; Nephrectomy; Rats; Ureter

A mathematical model for the control of erythropoiesis has been developed based on the balance between oxygen supply and demand at a renal oxygen detector which in turn controls erythropoietin release and red cell production. Tissue oxygen tension is regulated by adjustments of hemoglobin levels resulting from the output of a renal-bone marrow controller. Special consideration given to the determinants of tissue oxygenation included evaluation of the influence of blood flow, capillary diffusion, oxygen uptake, and oxygen-hemoglobin affinity. A theoretical analysis of the overall control system is presented including: a) dynamic and steady-state responses, b) sensitivity analysis to determine the relative importance of parameters and their influence on model behavior, c) properties of the model as a proportional controller, d) analysis of steady-state errors, and e) effectiveness of feedback regulation. Computer simulations of altitude hypoxia, descent from altitude, red cell infusion, and hemolytic anemia demonstrate the validity of the model for general human application.

Topics: Anemia, Hemolytic; Biological Transport; Bone Marrow; Cell Differentiation; Computers; Erythrocyte Aging; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Humans; Hypoxia; Kidney; Models, Biological; Oxygen; Polycythemia; Pregnancy

Porphobilinogen is the substrate of two enzymes: porphobilinogen deaminase and porphobilinogen-oxygenase. The first one transforms it into the metabolic precursors of heme and the second diverts it from this metabolic pathway by oxidizing porphobilinogen to 5-oxopyrrolinones. Rat blood is devoid of porphobilinogen-oxygenase under normal conditions while it carries porphobilinogen-deaminase activity. When the rats were submitted to hypoxia (pO2 = 0.42 atm) for 18 days, the activity of porphobilinogen-oxygenase appeared at the tenth day of hypoxia and reached the maximum at the 14-16th day. It decreased to a half after 2 days (half-life of the enzyme) and disappeared after 4 days of return to normal oxygen pressure. Porphobilinogen-deaminase activity increased after the first day of hypoxia, reached a maximum at the 14-16th day and did not decrease to normal values until the 15th day after return to normal oxygen pressure. The activities of both porphobilinogen-oxygenase and porphobilinogen-deaminase were induced by administration of erythropoietin. When rats were made anaemic with phenylhydrazine, porphobilinogen-oxygenase activity also appeared in the blood cells. Although the reticulocyte concentration was higher when compared to that obtained under hypoxia, the activities of the oxygenase obtained under both conditions were comparable. Porphobilinogen-deaminase activity was always closely related to the reticulocyte content. The appearance of porphobilinogenase-oxygenase under the described erythropoietic conditions was due to a de novo induction of the enzyme, as shown by its inhibition with actinomycin D and cycloheximide. Porphobilinogen-oxygenase as well as porphobilinogen-deaminase were present in the rat bone marrow under normal conditions. Their activities increased in phenylhydrazine treated rats. The properties and kinetics of porphobilinogen-oxygenase from the rat blood and bone marrow were determined and found it differ in several aspects.

Topics: Ammonia-Lyases; Animals; Bone Marrow; Cycloheximide; Dactinomycin; Erythropoiesis; Erythropoietin; Female; Half-Life; Hydroxymethylbilane Synthase; Hypoxia; Kinetics; Mixed Function Oxygenases; Phenylhydrazines; Porphobilinogen; Rats

The effect of T3 replacement and glucose supplementation on erythropoietin production was investigated in fasted hypoxic rats. It was found that 48 hr of fasting significantly reduced the circulating levels of thyroid hormones and the production of renal and extrarenal erythropoietin in response to hypoxia. These effects of fasting were completely abolished when the animals had free access to 25% glucose solution as drinking water, despite their lack of protein intake. Replacement doses of T3 (0.5 micrograms/100 gm per day) restored erythropoietin production in the fasted animals but also increased the response of the fed controls. To avoid the effect of endogenous T3, the experiments were repeated in thyroidectomized rats. Erythropoietin production in athyroid rats was found to be markedly decreased, with values equivalent to those found in normal fasted animals, and were not affected by fasting or glucose supplementation. Replacement doses of T3 increased erythropoietin production in all three groups, but the fasted animals needed five times as much T3 to obtain a response similar to that observed in the fed group. Glucose supplementation enhanced the effect of T3 in the fasted animals but did not completely restore it. These results indicate that caloric deprivation is primarily responsible for the decreased erythropoietin production induced by fasting and that this effect is probably mediated by both a decreased level of T3 and a decreased responsiveness to it.

Topics: Acid-Base Equilibrium; Animals; Biological Assay; Dietary Carbohydrates; Erythropoietin; Fasting; Glucose; Hypoxia; Kinetics; Male; Mice; Rats; Rats, Inbred Strains; Triiodothyronine

Mice from newborn until adult age were exposed to hypobaric hypoxia of 0.5 atm for 6 h. In all age groups the animals responded with increased plasma erythropoietin (Ep) levels. A greater response was elicited from adult mice compared to newborn animals. No sex difference in response occurred. Twelve hours after the hypoxic exposure no plasma Ep activity was detectable. Though high plasma Ep levels were present in non-hypoxic mice 9 and 20 days old, hypoxia further increased the Ep activity. It is concluded that hypoxia stimulates Ep production during hepatic as well as during erythropoiesis. Though erythroid target cells may be maximally stimulated by Ep in the young mouse (9-20 days old) a reverse capacity for Ep production exists. Increased capacity for Ep production may develop towards adult age.

Topics: Aging; Animals; Atmospheric Pressure; Erythropoietin; Female; Hypoxia; Kinetics; Male; Mice

Topics: Animals; Blood Transfusion; Bone Marrow; Dogs; Erythropoietin; Female; Food Deprivation; Guinea Pigs; Hematopoietic Stem Cells; Humans; Hypoxia; Male; Methods; Mice; Polycythemia; Radioimmunoassay; Rats

Normal and plethoric bone marrow cells were grown in plasma clot diffusion chambers (PCDC) implanted into the peritoneum of normal mice or mice submitted to 7 her of hypoxia (23,000 ft) daily, on a single day or on 2 consecutive days at different times after implantation of the PCDC's. Daily discontinuous hypoxia (DDH) produced more 6-day bursts than other treatments. Hypoxia on days 1 and 2 after implantation was nearly as effective as DDH on day-6 bursts. Later bouts of hypoxia or a singly hypoxic exposure on day 1 or 2 was less effective. Erythropoietin (Ep) levels were measured by bioassay on both diffusion chamber (DC) contents and serum. Serum Ep levels peaked at 160 mU/ml after a 7-hr hypoxic exposure while the DC content Ep levels were in the nondetectable range (less than 50 mU/ml). The data implies that either higher than normal Ep levels or a companion molecules (s) produced by hypoxia are required for 1-2 days early in the culture period of force an increasing number of BFU-d-e down the erythrocytic pathway and thus increase red cell production at times of need in vivo.

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cell Division; Cells, Cultured; Clone Cells; Environment, Controlled; Erythrocytes; Erythropoietin; Hypoxia; Male; Mice; Time Factors

Previous attempts to extract erythropoietin from the kidneys have not been uniformly successful and have yielded only small amounts. Attempts were therefore made to extract erythropoietin from renal extracts prepared from the supernatant obtained after homogenizing kidneys in various liquids and centrifuging at 2300 x g. Detectable amounts of erythropoietin were recovered from kidneys of nonhypoxic male but not female rats. After exposure to hypoxia, the amounts increased to levels greater than 6.0 U/kidney from males after 8 hr of hypoxia. Erythropoietin became detectable in extracts from kidneys obtained from females after 2 hr of hypoxia and rose to levels of 0.54 U/kidney after 4 hr of hypoxia. The amount of plasma trapped in the kidneys at the time of extraction was not sufficient to account for a significant amount of this erythropoietin, and erythropoietin was not detected in extracts made from livers or spleens. Severalfold larger titers of erythropoietin were detected in renal extracts, by a simple extraction procedure, than was the case in previous attempts. The reasons for this difference are discussed.

Topics: Animals; Biological Assay; Erythropoietin; Female; Hypoxia; Kidney; Male; Radioimmunoassay; Rats; Time Factors; Tissue Distribution

In vitro and in vivo studies showed that methylation of homologous DNA in nuclear homogenates of rat bone marrow is controlled by cyclic nucleotides and some hormones. The cyclic nucleotides and their dibutyryl analogs inhibited homologous methylation of DNA in the presence and absence of phosphodiesterase inhibitors. In the presence of Ca2+ the inhibiting effect of the nucleotides was more pronounced. Prostaglandins inhibited DNA methylation in a weaker degree while insulin and erythropoietin had a stronger effect in comparison with the nucleotides. Histamine stimulated DNA methylation, whereas acute hypoxic hypoxia caused a reduction in the rate of DNA methylation.

Topics: Animals; Bone Marrow; Bucladesine; Calcium; Cell Nucleus; Cyclic AMP; Cyclic GMP; Dibutyryl Cyclic GMP; DNA; DNA (Cytosine-5-)-Methyltransferases; Erythropoietin; Hypoxia; Insulin; Kinetics; Methylation; Methyltransferases; Rats

Double partial hepatectomy (hepx) evokes an elevation in serum erythropoietin (Ep) levels in anephric hypoxic animals when compared to non-hypoxic or sham hepx controls. But this Ep response is significantly lower than that found in singly hepx, anephric hypoxic rats. Double hepx also induces numerous cytological changes in the liver. Extravascular accumulation of fat, fibrous scarring, localized necroses, and multiple abscesses, as well as decreased vascularity, occur following the second hepx. A humoral factor was detected in the serum of these animals that is capable of inducing hepatic Ep production when injected into normal rats 18 hours before nephrectomy and hypoxia. This factor, termed hepatopoietin (Hp), was previously demonstrated in the venous serum of singly hepx rats. The serum from animals subjected to double partial hepx is not as potent in inducing Ep production as the serum from singly hepx animals. The discrepancies noted between the single and double hepx groups is attributed to the necrotic cytological changes described above.

Topics: Animals; Blood Proteins; Erythropoietin; Hepatectomy; Hepatocyte Growth Factor; Hypoxia; Kupffer Cells; Liver; Nephrectomy; Rats

The effect of prostaglandin synthetase inhibitors, aspirin and indomethacin, salt overload and salt depletion, as well as renomedullary dissection on serum Ep level in rats exposed to acute hypoxia was studied. Male rats were given aspirin or saline for seven days prior to the exposure to hypoxia. Other group of animals was fed salt free diet for the same period of time. Indomethacin was given on two consecutive days and renomedullary dissection was performed three weeks before exposure to hypoxia. Ep level was indirectly determined by measuring the 48 h 59Fe incorporation into RBC of mice with posthypoxic polycythemia. Hypoxia induced Ep production was diminished after blocking the PG synthesis using PG synthesis inhibitors. Salt overload, procedure known to decrease PG synthesis, had the same effect. Further on, dissection of the renal medulla, the main source of renal PG, decreased Ep production. The results indicate that PG are part of the mechanism which controls Ep production.

Topics: Acute Disease; Animals; Aspirin; Cyclooxygenase Inhibitors; Dissection; Erythropoietin; Hypoxia; Indomethacin; Kidney Medulla; Male; Organic Chemicals; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred Strains; Sodium Chloride

The erythrocytes of rats treated with 1, 25-dihydroxycholecalciferol or 1, 25-dihydroxycholecalciferol glycoside showed decreased levels of 2, 3-diphosphoglycerate. The same result has been obtained in vitro, indicating a direct effect of the sterol on the red cell. The glycoside is less active than the free sterol in vivo and more active in vitro. The decreased levels of diphosphoglycerate induced tissue hypoxia as shown by a higher plasma lactate/pyruvate ratio and a three fold increase in plasma erythropoietin concentration.

Topics: Animals; Calcitriol; Dihydroxycholecalciferols; Diphosphoglyceric Acids; Erythrocytes; Erythropoietin; Female; Hydroxycholecalciferols; Hypoxia; Lactates; Male; Oxygen; Pyruvates; Rats

Evidence is presented implicating the renin-angiotensin system in the regulation of plasma erythropoietin concentration. Male and female rats were exposed to hypoxia at 0.43 atm for 8 hr. In male rats, individual erythropoietin values showed a positive correlation with renin, renin substrate, and angiotensin I after 8 hr of hypoxia. In female rats, renin was not elevated during hypoxia. However, after renin was injected subcutaneously, plasma renin values became similar to those in male rats and erythropoietin doubled. Individual erythropoietin values of the combined groups showed a positive correlation with plasma renin. A single oral dose of SQ 14225, an angiotensin I-converting enzyme inhibitor, reduced erythropoietin to undetectable levels in renin-injected female rats. Angiotensin II, in subpressor amounts, prevented the suppression of erythropoietin by SQ 14225. SQ 14225 also reduced renin substrate concentration at ambient pressure and during hypoxic exposure. The correlation coefficient between renin substrate and erythropoietin in rats given SQ 14225 to lower substrate or angiotensin II to increase substrate was 0.85; p < 0.0005. The increase in renin substrate that occurred during hypoxia in female rats given angiotensin II correlated positively with erythropoietin, r = 0.86; p < 0.0005.

Topics: Angiotensin I; Angiotensin II; Animals; Captopril; Erythropoietin; Female; Hypoxia; Male; Rats; Renin

During chronic hypoxia, the number of splenic erythroid progenitor cells in mice, particularly CFU-E, increased dramatically but transiently. Since all three classes of erythroid progenitors in the femoral bone marrow were suppressed, a large part of this increase might be attributed to migration of CFU-E and/or their progenitors from the medullary cavity. The changes in CFU-E were preceded 48-72 hours earlier by an increase in serum erythropoietin (Ep) titers which, in turn, had been preceded by a rapid and marked "shift-to-the-right" in the hemoglobin oxygen dissociation curve. During hypoxia, the mice lost a considerable fraction of their body weight. Computer simulations, using a mathematical model of erythropoietic regulation, suggest that this weight loss, either indirectly by reducing the need for red cells in a smaller-than-control animal or by directly altering the sensitivity of the Ep-producing mechanism, is the major cause of the falling Ep titers despite continuation of the hypoxic stress. Because of high endogenous 59Fe incorporation levels, it was not possible to confirm the thesis that animals with an expanded Erythropoietin Responsive Cell (ERC) compartment would be more sensitive to exogenous erythropoietin than are mice with a normal or reduced ERC population.

Topics: Animals; Bone Marrow Cells; Chronic Disease; Colony-Forming Units Assay; Computers; Disease Models, Animal; Erythrocyte Aging; Erythropoiesis; Erythropoietin; Female; Hematopoietic Stem Cells; Hypoxia; Mice; Mice, Inbred C3H; Spleen

Erythropoietin (Ep) in large amounts was detected in extracts of renal tissue from hypoxic rats. These extractions were performed by homogenization of kidney tissue in phosphate buffered saline, centrifugation at 3,000 g and collection of the supernate. Male kidneys contained more Ep than did females and the major portion of Ep is located in the renal cortex. Comparison of intrarenal and plasma Ep levels at various times following initiation and cessation of hypoxia appears to be a useful method for studying the kinetics of erythropoietin production and release, and also for studying feedback mechanisms that influence these functions.

Topics: Animals; Erythropoietin; Feedback; Female; Hypoxia; Kidney; Kinetics; Male; Rats; Rats, Inbred Strains; Subcellular Fractions

If 'sensitivity' is defined as the ratio of haem synthesis in erythropoietin (Ep)-stimulated cultures to that in cultures without added Ep, then haematopoietic tissue of ex-hypoxic mice is less sensitive to Ep in vitro than similar tissue from normal donors. This appears to be due primarily to the loss of erythropoietin responsive cells from the marrow and spleen. In addition, saline extracts of mature erythrocytes inhibited erythropoiesis in normal bone marrow cultures in a manner directly related to the 'haematocrits' used during their preparation. These data support recent suggestions that the preference of erythrocytotic mice over normal animals for Ep assay probably can be attributed to their low baseline level of erythropoiesis rather than an increased sensitivity to Ep.

Topics: Animals; Bone Marrow; Cell Extracts; Cells, Cultured; Erythrocytes; Erythropoietin; Female; Heme; Hypoxia; Mice; Mice, Inbred C3H; Polycythemia; Spleen

Topics: Animals; Erythropoiesis; Erythropoietin; Female; Glucagon; Hypoxia; Iron; Male; Mice; Polycythemia; Protamines; Rats; Time Factors

Plasma erythropoietin (Ep) activity was detectable in fetal rats near term and in rats less than 2 old. Ep activity was not found 12--48 h after birth. A hypoxic period (0.5 atmospheres for 6 h) increased the plasma Ep level in fetal and newborn rats, and in pregnant animals. After long-term hypoxia (0.5 atmospheres for 8 days) the plasma Ep level was neither increased in fetal rats nor in their mothers, while a compensatory increase in the hematocrit level occurred. It is concluded that hypoxia stimulates Ep production during hepatic erythropoiesis. During long-term hypoxia compensatory mechanisms for tissue oxygenation occur.

Topics: Animals; Animals, Newborn; Erythropoietin; Female; Fetus; Hematocrit; Hypoxia; Male; Pregnancy; Rats

The present studies were done to determine whether preventing the respiratory alkalosis, which is known to occur with acute "hypoxic" stimuli, would lead to alterations in plasma concentrations of erythropoietin (Ep). Rats were subjected to two acute stresses, hypoxia and blood loss, separately and in combination, with and without the added stress of hypercarbia. Hypercarbia in all experimental groups was associated with a decrease in plasma concentrations of Ep. This reduction in plasma Ep with hypercarbia could not be fully explained by the higher arterial pO2S or p50S of the hypercarbic rats. Hypercarbia may have indirectly suppressed Ep production by increasing blood flow to the site of Ep production. Alternatively, the cell of origin of Ep could be sensitive to changes in pH and/or PCO2. It was further demonstrated that neither the onset nor the degree of reticulocytosis could be predicted by the plasma Ep concentrations. It is likely that the removal of red blood cells led to a decrease in marrow transit time with the early emergence of reticulocytes after acute blood loss.

Topics: Anemia; Animals; Carbon Dioxide; Erythropoietin; Female; Hypoxia; Oxygen; Rats; Respiration

Topics: Albuterol; Animals; Dogs; Erythropoietin; Female; Hypoxia; In Vitro Techniques; Indomethacin; Kidney; Perfusion; Pressure; Receptors, Adrenergic, beta

Topics: Anestrus; Animals; Castration; Erythropoietin; Estradiol; Estrus; Female; Hematocrit; Hemoglobins; Hypoxia; Iron; Male; Nephrectomy; Pregnancy; Rats; Testosterone

Topics: Animals; Erythropoietin; Hypoxia; Kidney; Kupffer Cells; Male; Nephrectomy; Rats

Hemin was found to enhance the growth of murine erythroid colonies in culture. In the presence of 100 mU/ml erythropoietin (EPO), the addition of hemin (0.05-0.2 mM) resulted in the growth of twice as many colonies as were obtained with EPO alone. Hemin also significantly increased erythroid colony formation in culture in the absence of added EPO. Hemoglobin synthesis as measured by the incorporation of 59Fe into cyclohexanone extractable heme was augmented in culture by hemin. Neither delta-aminolevulinic acid, a hemin precursor, nor FeCl3 increased colony number.

Topics: Animals; Bone Marrow Cells; Cell Division; Cells, Cultured; Clone Cells; Erythropoiesis; Erythropoietin; Female; Heme; Hemin; Hemoglobins; Hypoxia; Iron Radioisotopes; Mice

Liver regeneration after partial hepatectomy is associated with an increase in extrarenal Ep production; however, this surgical procedure is time-consuming and difficult to standardize. Carbon tetrachloride (CCl4) ingestion in rats is an easy and reproducible way to induce liver damage and regeneration. We therefore studied the effects of CCl4 on extrarenal Ep production in rats. Just as is the case following partial hepatectomy, extrarenal Ep production in response to hypoxia was reduced immediately after ingestion of CCl4. Thereafter it rose to supranormal levels which peaked 3 to 4 days after CCl4 ingestion (at this time Ep titers of nephrectomized, CCl4-fed rats rose to greater than 1.0 U/ml of plasma after exposure to 0.42 atmosphere for 7 hr). Extrarenal Ep production then declined, but was still supranormal 7 days after CCl4. Carbon tetrachloride did not significantly affect extrarenal Ep production in rats nephrectomized 18 hr prior to initiation of hypoxia even if they received injections of renin prior to being made hypoxic, nor did it affect Ep production in response to hypoxia in nonnephrectomized rats under the conditions used in this study.

Topics: Animals; Carbon Tetrachloride Poisoning; Erythropoietin; Female; Hypoxia; Kidney; Liver; Liver Regeneration; Nephrectomy; Rats; Renin

Radioiron uptake by erythrocytes, metabolic rate, erythropoietin formation during hypoxia and erythroid responsiveness to exogenous erythropoietin were determined in both starved and water deprived rats. The feed intake showed a marked and progressive reduction during water deprivation. The metabolic rates of rats deprived of either food of water declined progressively showing a 40% reduction 5d after water deprivation or starvation began. At this time, the 24 h red blood cells 59Fe incorporation was 85% lower in both starved and dehydrated rats than in normal rats. Plasma erythropoietin levels in response to hypoxia were approximately 50% decreased in both starved and dehydrated rats. Both polycythaemic starved and polycythaemic water deprived rats injected with human urinary erythropoietin showed a 75% decrease in 59Fe incorporation into erythrocytes when compared to control rats. It is suggested that depression of erythropoiesis during water deprivation in the rat depends on a reduced sensitivity to erythropoietin, possibly associated with decreased production of the hormone. Since water deprived rats drastically reduce feed intake it is suggested that secondary starvation is the principal cause of the decreased erythropoiesis induced in the rat by water deprivation.

Topics: Animals; Dehydration; Energy Metabolism; Erythropoiesis; Erythropoietin; Female; Hypoxia; Male; Polycythemia; Rats; Starvation; Water Deprivation

Topics: Animals; Aspirin; Erythropoietin; Hydronephrosis; Hypoxia; Kidney; Male; Prostaglandins; Rats

Hepatic cells in rats were evaluated after subtotal hepatectomy using scintillation scanning with technetium sulfur colloid (TSC), autoradiography, and microstereology techniques. The ability of the liver to accumulate TSC increased during the course of the regeneration as did the labeling of Kupffer and parenchymal cells with tritiated thymidine (3H-tdR). Kupffer to parenchymal cell number ratios and Kupffer cell relative areas were also elevated, attaining peak values at 72 hours post-hepatectomy. This period corresponds to the time of peak erythropoietin (Ep) production in rats with regenerating livers after nephrectomy and exposure to hypoxia. These findings suggest that the Kupffer cell may function as a cellular site of Ep formation.

Topics: Animals; Autoradiography; Cell Count; Erythropoietin; Hepatectomy; Hypoxia; Kupffer Cells; Liver; Liver Regeneration; Male; Mononuclear Phagocyte System; Nephrectomy; Rats; Technetium; Thymidine

A simplified modification of the fetal mouse liver cell (FMLC) assay has been used to investigate the relationship between in vivo and in vitro detectable Erythroid Regulatory Factors (ERF) in the serum of dogs exposed to hypoxia. The results suggest that the FMLC assay is a useful technique for following changes in serum ERF. However, the data obtained cannot be directly equated with the in vivo erythrocythemic mouse assay unless a correction factor is applied. This is necessary because the in vitro assay, in general, detects 50% more ERF than does the standard mouse assay.

Topics: Animals; Biological Assay; Dogs; Erythropoietin; Female; Fetus; Hypoxia; Liver; Mice

A radioimmunoassay for erythropoietin has been developed using 125I labeled pure human erythropoietin and an anti-erythropoietin antiserum produced in a rabbit immunized with human erythropoietin. Two techniques are presented for labeling erythropoietin, both resulting in an immunologically reactive labeled reactant. One method involves the use of lactoperoxidase and the other a reagent known as IODO-GEN. The second International Reference Preparation of human erythropoietin is used as a standard and a double antibody scheme is used for the separation of the free and antibody bound labeled hormone. The radioimmunoassay is sensitive to an absolute amount of erythropoietin equivalent to 0.4 milliunits. Bioassays for erythropoietin require approximately 100 times this amount. The use of pure erythropoietin as the labeled reactant has removed certain discrepancies seen in previous attempts to develop radioimmunoassays for this hormone, e.g., sera from patients without kidneys do not give the high values previously seen. Sera from anemic individuals not only give rise to high radioimmunoassay values but also show a parallel relationship with the erythropoietin standard when halving dilutions are analyzed. Desialated erythropoietin is also reactive with the same parallelism. Bleeding of a normal individual increases the serum erythropoietin level and transfusion decreases it. Erythropoietin from a variety of laboratory animals is also reactive in the radioimmunoassay, with very high values being observed in hypoxic animals.

Topics: Anemia; Animals; Antibody Specificity; Erythropoietin; Humans; Hypoxia; Iodine Radioisotopes; Rabbits; Radioimmunoassay

Topics: Animals; Atmospheric Pressure; Carbon; Erythropoietin; Hypoxia; Kidney; Liver; Male; Mononuclear Phagocyte System; Phagocytosis; Rats; Spleen

Topics: Acute Disease; Anesthesia; Animals; Erythropoietin; Female; Hemoglobins; Hydrogen-Ion Concentration; Hypercapnia; Hypoxia; Lactates; Male; Mice; Oxygen; Oxygen Consumption; Phosphates; Rabbits; Time Factors

To assess the adaptive value of the right-shift of the oxyhemoglobin dissociation curve (decreased affinity for oxygen) observed in humans upon altitude exposure, the short-term physiologic responses to altitude-induced hypoxia were evaluated in two subjects with a high oxygen affinity hemoglobin (Hb Andrew-Minneapolis) and in two of their normal siblings. In striking contrast to normal subjects, at moderately high altitude (3,100 m) the high affinity subjects manifested: (a) lesser increments in resting heart rate; (b) minimal increases in plasma and urinary erythropoietin; (c) no decrement in maximal oxygen consumption; and (d) no thrombocytopenia. There was no difference between subject pairs in 2,3-diphosphoglycerate response to altitude exposure. These results tend to contradict the belief that a decrease in hemoglobin oxygen affinity is of adaptive value to humans at moderate altitudes. Rather, they support the hypothesis that, despite disadvantages at low altitude, a left-shifted oxyhemoglobin dissociation curve may confer a degree of preadaptation to altitude.

Topics: Adaptation, Physiological; Adolescent; Altitude; Blood Cell Count; Blood Platelets; Child; Diphosphoglyceric Acids; Erythropoiesis; Erythropoietin; Female; Heart Rate; Hemoglobins; Hemoglobins, Abnormal; Humans; Hydrogen-Ion Concentration; Hypoxia; Male; Oxygen; Oxyhemoglobins

Topics: Animals; Carotid Body; Erythropoietin; Hypoxia; Liver; Rabbits; Rats; Spleen

Topics: Aging; Animals; Animals, Newborn; Erythropoietin; Hypoxia; Kidney; Liver; Rats

Our hypothesis is that PGs released within the kidney play a role in the modulation of kidney production of Ep. PGs release probably at medullary and/or cortical sites following erythropoietic stimuli such as hypoxic hypoxia, anemic hypoxia, and ischemic hypoxia induced by renal artery constriction increase kidney production of Ep. PGs which are released probably activate a renal cortical adenylate cyclase thereby enhancing the production of intracellular cAMP. This initiates the cascade of events resulting in the production and/or secretion of Ep by the kidney. The endoperoxide analogs and PGE2 have been found to produce a dose-related and Ep-dependent increase in radioiron incorporation into newly formed red blood cells of exhypoxic polycythemic mice. Indomethacin, a potent PG cyclo-oxygenase inhibitior, attenuates Ep production and the appearance of PGE in the renal venous effluent of animals exposed to hypoxic hypoxia and renal artery constriction. Arachidonic acid (C20:4) and PGE2 infusion into the posthypoxic isolated perfused dog kidney produced a significant elevation in Ep titers in the perfusate. The increase in Ep production caused by arachidonate is blocked by indomethacin. It has been previously reported that PGs of the E series stimulate cAMP formation in several tissues. We have found that not only are renal cortical cAMP levels significantly elevated in rats following exposure to hypobaric hypoxia but that dibutyryl cAMP administration produces an increase in hematocrit and circulating red cell mass in normal mice. Our data thus far strongly support the hypothesis that the renal PGs and the cyclic nucleotides are intimately involved in the pharmacologic and/or pathophysiologic control of Ep production. Further work is necessary to determine whether the PGs and cyclic nucleotides are involved in the day-to-day control of Ep production by the mammalian kidney.

Topics: Animals; Blood Pressure; Cyclic AMP; Dogs; Erythrocytes; Erythropoiesis; Erythropoietin; Hypoxia; Indomethacin; Iron; Kidney; Mice; Prostaglandins; Prostaglandins E; Prostaglandins F

In CBA mice the absolute and relative (per 10(6) spleen cells) number of antibody-forming cells (AFC) in the spleen was cut by half on the 1st, 4th, and 7th days after acute hypoxia (12 hours, 6700 m), and on the 1st and 4th days after cessation of chronic hypoxia (16 days, 16 hours, 6700 m). The number of AFC in the spleen returned to the normal level on the 7th day after cessation of chronic hypoxia. Single or double erythropoietin injections caused approximately a 1.15--2-fold decrease in spleen AFC number in posthypoxic mice in comparison with control animals.

Topics: Animals; Antibody Formation; Antibody-Producing Cells; Erythropoietin; Hypoxia; Immunization; Mice; Mice, Inbred CBA; Spleen; Time Factors

Topics: Animals; Erythropoietin; Hypoxia; Kidney; Liver; Male; Nephrectomy; Rats; Spleen; Time Factors

In order to determine if there were any relationships between thrombopoietin, erythropoietin and erythrogenin, supernatant fluids from culture lines of foetal mouse liver, foetal and adult bovine kidney, and adult rabbit kidney cells have been assayed for their content of the presumptive humoral regulators of haematopoiesis. Hormone production varied markedly from one culture line to another confirming other studies that the optimal culture conditions for the generation of haematologically-active hormones have not been delineated. The lack of any discernable relationship between the production of thrombopoietin and erythropoietin, thrombopoiet in these and erythrogenin or erythropoietin and erythrogenin suggests that materials represent three distinct entities.

Topics: Animals; Cattle; Cell Line; Cells, Cultured; Erythropoietin; Female; Glycoproteins; Hypoxia; Kidney; Liver; Male; Mice; Mice, Inbred C3H; Rabbits; Thrombopoietin

The production of erythropoietin by rats fed a protein-deficient diet is markedly decreased. This defect can be rapidly reversed by feeding protein. In the reported experiments we investigated the effect of protein deficiency on the extra-renal production of erythropoietin. The results indicate that the production of erythropoietin in nephrectomized animals is insensitive to protein depletion.

Topics: Animals; Dietary Proteins; Erythropoietin; Female; Hemoglobins; Hypoxia; Kidney; Nephrectomy; Protein Deficiency; Rats

So far the question has not been elucidated whether latent ESF-deficiency exists in patients with chronic renal disease during initial development of renal anemia. Therefore, ESF was determined in urine and serum of non anemic patients being exposed to acute hypoxia: 8 healthy volunteers and 8 patients who had a Ccreat below 48 ml/min were studied while in a hypobaric chamber for 4 subsequent periods of 10 h each (simulated maximal altitude 4000 m INA = 462 mm Hg). We also determined plasma iron turnover and reticulocyte counts. 10 h after the study began, the patients showed a significantly higher ESF-level than the normal volunteers. In the course of 48 h collection periods of urine under hypoxic conditions the mean ESF excretion in patients corresponded to 9.9 and in healthy persons to 7.3 Units. With regard to plasma iron turnover and reticulocyte count an increase was shown, but no significant differences existed between the two groups. A latent ESF-deficiency as the initial cause of renal anemia does not exist.

Topics: Adult; Chronic Disease; Deficiency Diseases; Erythrocyte Count; Erythropoietin; Female; Hematocrit; Hemoglobins; Humans; Hypoxia; Iron; Kidney Diseases; Male; Middle Aged

Hypophysectomized or sham-operated male rats were exposed to hypoxia (0.42--0.40 or 0.37--0.35 atm for 6, 12, or 24 hr) applied 2 wk to 7 mo after surgery. Erythropoietin (Ep) levels in rat serum were evaluated on the basis of the exhypoxic polycythemic mouse assay. Ep activity evoked by hypoxia was significantly lower in hypophysectomized rats than in sham-operated controls. Progressive increase of the EP response to hypoxia correlated with extension of the time interval between hypophysectomy and hypoxia from 2 wk to 2--4 mo apparently mediated by the simultaneous inverse decline of red cell mass (RCM) values, i.e., of the "relative plethora" induced by a low O2 demand associated with relatively high RCM values. However, after 3--7 mo hypoxic Ep activity was still lower than in sham-operated controls. In these ablated animals the relative plethora became negligible or absent; accordingly, the Ep response apparently had reached plateau levels. These studies indicate that hypophysis (hypophyseal and target hormones, with the exception of estrogens) modulates Ep production under hypoxic conditions, possibly via a permissive enhancement of renal Ep activity.

Topics: Animals; Erythrocytes; Erythropoietin; Female; Hypophysectomy; Hypoxia; Male; Mice; Pituitary Gland; Rats; Time Factors

Studies were done of cell production by marrow in diffusion chambers implanted in the peritoneal cavity of rabbits subjected to various stimuli to hematopoiesis. In chambers in neutropenic hosts and in hosts injected with endotoxin, animals presumed to have an increased stimulus to granulopoiesis, there was increased production of granulocytes but there was also increased production of red cells. Although red cell production was decreased in chambers in polycythemic hosts, granulocyte production was not different from that in controls. Stimulation of erythropoiesis by erythropoietin injections or by exposure to hypoxia increased red cell production by marrow in the implanted diffusion chambers without diminishing granulopoiesis. Only in chambers in hosts made anemic by bleeding was there an increase in red cell production accompanied by a decrease in granulocyte production. In these anemic hosts induction of neutropenia led to an increase in granulopoiesis without any depression of erythropoiesis.

Topics: Anemia; Animals; Bone Marrow Cells; Cells, Cultured; Endotoxins; Erythrocytes; Erythropoietin; Hematopoiesis; Hypoxia; Leukocytes; Male; Mechlorethamine; Neutropenia; Polycythemia; Rabbits

Topics: Acute Kidney Injury; Adolescent; Adult; Animals; Blood Proteins; Erythropoiesis; Erythropoietin; Female; Humans; Hypoxia; Male; Mice; Middle Aged; Polycythemia; Transfusion Reaction

Topics: Anemia; Animals; Chronic Disease; Dose-Response Relationship, Drug; Erythropoietin; Hypoxia; Kidney; Lead; Lead Poisoning; Male; Rats

In 22 healthy newborns and in 31 newborns with transposition of the major arterial vessels (TMAV) erythropoietin and erythropoiesis inhibitor was studied on a model of polycythemic mice. Erythropoietin was not detected, whereas erythropoiesis inhibitor was revealed in the plasma and urine of 5--7-day-old healthy newborns. The newborns with TMAV displayed a rise of erythropoietin level up to the 14th day and its reduction during the period of from 14th to the 56th days. Erythropoiesis inhibitor was also found in the urine concentrates of the newborn with TMAV during the period of 3--4 weeks. The role of erythropoiesis inhibitor as a physiological regulator appearing in the blood of healthy and hypoxic newborns to normalize the erythropoiesis in the course of the initial weeks after birth is discussed.

Topics: Erythropoietin; Humans; Hypoxia; Infant; Infant, Newborn; Transposition of Great Vessels

The addition of carbon dioxide during a short bout (6 to 24 hr) of hypoxia suppresses the production of Ep. The effects of carbon dioxide on Ep production and erythropoiesis were determined in hypoxic rats over a 6-day period. Carbon dioxide suppressed both renal and extrarenal Ep production induced by hypoxia. However, rats exposed to a hypoxic environment with 5% CO2 became as polycythemic as rats exposed to the same hypoxia without CO2. This may result from the following. (1) A factor other than Ep is involved in stimulating erythropoiesis in these animals. (2) The large amount of Ep produced during hypoxia is in excess, and only a small amount is needed to compensate for the hypoxia by producing increased numbers of RBCs. (3) A significant degree of hemoconcentration occured, resulting in the increased number of RBCs observed.

Topics: Animals; Biological Assay; Carbon Dioxide; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Hematocrit; Hypoxia; Rats; Reticulocytes

The ability of arachidonic acid (AA), the bisenoic prostaglandin precursor to stimulate erythropoiesis and erythropoietin (Ep) production in exhypoxic polycythemic mice and the programmed isolated perfused canine kidney was investigated. Arachidonate in the lowest dose tested (50 microgram/kg i.p.) maximally stimulated erythropoiesis when administered to exhypoxic polycythemic mice. Kidneys from dogs made hypoxic for 4 hr at 0.42 atm pressure were perfused (2--3 ml/g/min, 37 degrees C) in a closed circuit system for 5 hr with blood from nonhypoxic donors. AA infusion (80 microgram/min) caused a significant (P less than .05) increase in erythropoietic activity of the perfusate as measured by the percentage of 48-hr 59Fe incorporation into red blood cells of exhypoxic polycythemic mice per milliliter of perfusate from an initial value of 1.66 +/- 0.50% to 6.05 +/- 0.96% over the 1st hr of infusion whereas vehicle controls showed no change. To determine whether this increase in Ep production was dependent on biosynthesis of renal prostaglandins and their intermediates, the ability of indomethacin to block AA-induced Ep production was studied. When kidney donors were twice pretreated with indomethacin 12 hr and immediately before their hypoxic exposure, no increase in Ep titers were seen during AA infusion. These results support the hypothesis that endogenously synthesized prostaglandins, their intermediates and/or other products of AA metabolism, such as prostacyclin and prostaglandins play an important role in the control Ep production.

Topics: Animals; Arachidonic Acids; Dogs; Erythropoiesis; Erythropoietin; Female; Hypoxia; In Vitro Techniques; Kidney; Mice; Polycythemia; Prostaglandins

Topics: Amino Acids; Animals; Cystine; Erythropoietin; Female; Hypoxia; Methionine; Protein Deficiency; Rats

The question of a latent erythropoietin (ESF)-deficiency was studied in incipient renal anemia using a double stimulation technique for ESF. After a 4-week stimulation of ESF production with oral administration of fluoxymesterone (flu) intermittent hypoxic ESF stimulation was performed corresponding to a maximum altitude of 4000 m in 7 patients with chronic renal disease without or with incipient renal anemia (mean hematocrit 40%) and in 11 normal subjects (mean hematocrit 46%). Double stimulation in patients was compared with hypoxic stimulation alone and both were compared with controls. After flu alone only ESF excretion was increased in patients and in normal subjects. After flu plus 10 h of hypoxia serum ESF--titers were higher in healthy subjects than in the patients. The mean ESF titer after double stimulation was 81 mU/ml in patients and 115 mU/ml in normal persons. Forty-eight hour ESF excretion was 11 U and 43 U respectively. Compared to hypoxic stimulation alone double stimulation increased serum ESF titers in patients by 5% versus 80% in controls. Correspondingly, ESF excretion was enhanced by 19% and 49%, respectively. Finally, renal ESF clearance was increased by 42% versus 200%. After hypoxia alone non-anemic patients had higher serum ESF titers than healthy controls excluding a latent ESF deficiency in incipient renal anemia. It is concluded that decreased ESF production after double stimulation in patients was due to a nephrotoxic effect of flu followed by a decreased excretory and ESF-producing function of the damaged kidneys.

Topics: Adult; Anemia; Chronic Disease; Erythropoiesis; Erythropoietin; Female; Fluoxymesterone; Hematocrit; Humans; Hypoxia; Kidney; Kidney Diseases; Male; Reticulocytes

Topics: Animals; Blood Coagulation; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Erythrocytes; Erythropoietin; Granulocytes; Hypoxia; Male; Rats; Time Factors

Topics: Adult; Aldosterone; Erythropoietin; Female; Hematocrit; Humans; Hypoxia; Kidney; Kidney Transplantation; Male; Middle Aged; Renin

A case study is presented of a 55-year-old man who had clear cell renal carcinoma with pulmonary metastases and erythrocytosis. The increase in red blood cell mass was associated with an elevation in erythropoietic stimulatory activity in serum, pleural fluid, and tumor-cyst fluid as determined by the exhypoxic polycythemic mouse assay. It is postulated that the increased erythropoietic stimulatory activity represents autonomous tumor secretion of erythropoietin or an erythropoietin-like material. Electron microscopic studies confirmed the proximal tubular origin of this tumor.

Topics: Adenocarcinoma; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Kidney Neoplasms; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Polycythemia; Renal Veins; Vascular Diseases

The insensitivity of Tfm mutant mice to androgens is due to a greatly diminished ability to bind 5alpha-dihydrotestosterone to androgen-binding receptors in the cytoplasm of target tissues. Testosterone stimulates hemopoiesis through several mechanisms. The Tfm mutant mouse was used in an attempt to delineate hemopoietic effects of androgens that are mediated by androgen-binding receptors from those that require other cellular mechanisms. Mice of the Tfm mutant and related (androgen-binding) genotypes were treated with multiple doses of 5alpha-dihydrotestosterone (5alpha-DHT). The Tfm mutant was not stimulated to incorporate 59Fe, suggesting that androgen-binding receptors mediate this hemopoietic action of 5alpha-DHT. In contrast, the mutation did not block the ability of a single dose of 5alpha-DHT to increase the absolute number of committed granulocyte/macrophage precursors (CFU-C) in the femurs of mutant mice. The latter finding suggests that androgen-binding receptors are not involved in the mechanisms that lead to amplification of the CFU-C population by 5alpha-DHT. Tfm mutant mice respond to nonandrogenic stimuli of erythropoiesis. These findings emphasize the specificity of the diminished ability of the Tfm mutant mouse to respond erythropoietically to androgen. Accordingly, the Tfm mutant mouse appears to be a useful analytical tool for studying mechanisms underlying the hematopoietic effects of testosterone and related steroids.

Topics: Animals; Dihydrotestosterone; Erythropoiesis; Erythropoietin; Female; Hematopoiesis; Hypoxia; Iron Radioisotopes; Male; Mice; Mice, Inbred Strains; Mutation; Receptors, Androgen; Receptors, Steroid

Serum from patients with chronic renal failure (CRF serum) contains a substance inhibitory to erythropoiesis in vitro. This paper explores the mechanism of the inhibition. Four experiments were performed to evaluate the effects of CRF serum on erythropoietin (EP). In the first 2 experiments, the effect of exposure of EP solutions to CRF serum was evaluated using the plethoric mouse EP assay system and a tissue culture system containing normal dog marrow cells. In the third study, dog marrow cells were preincubated with CRF serum before being stimulated with EP. Finally, EP-dose response curves were constructed in the dog marrow tissue culture system and analyzed using an enzyme kinetic model. The results show no evidence of inhibition or inactivation of EP by CRF serum, although in vitro heme synthesis is clearly depressed in the presence of CRF serum. We conclude that CRF serum inhibits erythropoiesis by directly, although reversibly, impairing the ability of erythroblasts to synthesize heme.

Topics: Aged; Animals; Bone Marrow Cells; Dogs; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Heme; Humans; Hypoxia; Kidney Failure, Chronic; Mice; Middle Aged; Time Factors

The number of erythroid burst-(BFU-E) and colony-forming units (CFU-E), as well as of myeloid-macrophage colony-forming units (CFU-C), has been evaluated in tibial marrow and spleen of ex-hypoxic polycythaemic mice, at sequential time intervals after the end of hypoxia. In both marrow and spleen, the kinetics of the CFU-E pool is characterized by a sharp fall from above normal to lower than normal values. BFU-E and CFU-C however rise from below normal to higher than normal levels. These results have been correlated with both the erythropoietin (Ep) and the erythropoietic activity curves. It is apparent that Ep levels largely control both the differentiation and the amplification of the CFU-E pool and it is suggested that Ep may act as a 'survival factor' at the CFU-E level and/or increase the flow of cells from BFU-E to CFU-E. The difference in response between CFU-E and BFU-E favours a clearcut distinction between these populations, whereas the similarity between the BFU-E and CFU-C response suggest a close relationship between these two cell populations. It is also of interest that the murine spleen functions as a large reservoir of erythroid microenvironment for hypoxia-induced stress erythropoiesis.

Topics: Animals; Bone Marrow Cells; Erythropoiesis; Erythropoietin; Female; Hematopoietic Stem Cells; Hypoxia; Kinetics; Mice; Polycythemia; Spleen; Time Factors

Topics: Anemia; Erythropoiesis; Erythropoietin; Fetal Hemoglobin; Humans; Hypoxia; Infant, Newborn; Infant, Premature, Diseases

The regenerating liver produces erythropoietin in response to hypoxia. The amounts of erythropoietin produced in animals subjected to hepatectomy are significantly higher than those observed in sham-operated animals. Hepatic erythropoietin production appears to be dependent upon the stage of regeneration with the highest levels being produced during the period of greatest proliferation and increase in liver mass.

Topics: Animals; Biological Assay; Erythropoietin; Hypoxia; Liver; Liver Regeneration; Male; Nephrectomy; Rats

1. Erythropoietin has been measured in the serum of hypoxic rats that have been exposed to 6 hr of hypoxia, by use of a biological assay. 2. The effects of section of splanchnic, renal and sinus nerves on the concentration of Erythropoietin present in the serum ofthe hypoxic animals has been investigated. 3. Serum levels of Erythropoietin were reduced in the hypoxic animals immediately after cutting the splanchnic nerves, but unaltered one week after the operation. 4. The concentration of Erythropoietin in the serum of hypoxic rats was increased by the secretion of the renal nerves. Section of the sinus nerves abolished the effect. 5. Elevated levels of Erythropoietin were present in the sera of hypoxic rats immediately after section of the sinus nerves. The effect was still obtained when the animals were exposed to hypoxia 1 week after cutting the nerves, though in attenuated form. 6. The rise in the concentration of the hormone after chronic section of the sinus nerves was estimated as as 261% (fiducial limits 167-513, P = 0-05), by parallel line bio-assay. 7. The concentration of Erythropoietin in the serum of hypoxic rats was progressively elevated as the arterial PO2 of the animals was reduced. Section of the sinus nerves did not alter the secretion of Erythropoietin in these experiments.

Topics: Animals; Autonomic Nervous System; Erythropoietin; Female; Hypoxia; Kidney; Mice; Oxygen; Respiration; Splanchnic Nerves

Erythropoietin production was studied in 30-day-old and adult rats previously injected with pharmacological doses of isoproterenol. The animals were stimulated either by hypobaric hypoxia or by a combination of hypoxia and acute anemia. The erythropoietic activity of the plasma was measured by 59Fe incorporation in total circulating red blood cells of mice made polycythemic by chronic hypoxia. The results were converted to units of erythropoietin (IRP). A significant reduction of erythropoietin production was observed only in young rats. Our interpretation of the findings is that isoproterenol induced the differentiation of the salivary glands in the young animals hence reducing hormone production at the extrarenal (submandibular) level.

Topics: Age Factors; Anemia; Animals; Erythropoiesis; Erythropoietin; Hypoxia; Injections, Intraperitoneal; Isoproterenol; Male; Rats

Erythropoietin (Ep) is produced mainly by the liver and spleen during fetal and neonatal periods and by the kidney during adolescent and adult life. The liver is also an important extrarenal producer of Ep in the hypoxic, anephric adult animal. Subtotal hepatectomy results in a substantial elevation in serum Ep levels at 30-72 hours after hepatectomy in rats subsequently nephrectomized and rendered hypoxic. Ep production is related to the mass of regenerating liver with peak Ep production occurring during times of greatest tissue proliferation. Regenerative and erythropoietic responses to hepatectomy decline with advancing age. Rats undergoing repeated hepatectomies do not recover full liver mass but the initial rate of regeneration increases following each successive hepatectomy. Ep levels decline in anephric hypoxic rats undergoing multiple hepatectomies when compared to sham-operated controls.

Topics: Age Factors; Animals; Erythropoietin; Hepatectomy; Hypoxia; Liver Regeneration; Male; Nephrectomy; Rats

Studies were performed to determine the effects of partial hepatectomy on extra-renal erythropoietin production. Rats were either partially hepatectomized or sham operated. At intervals of from 5 min to 7 days afterward, both kidneys were removed from cohorts of the above two groups of rats and the animals were then exposed to hypoxia for 7.5 hr. Immediately afterward, their plasma was collected and its erythropoietin titer was assayed. Rats which were partially hepatectomized 2-4 days prior to nephrectomy and hypoxia had significantly higher plasma erythropoietin levels than did sham-operated controls, whereas rats hepatectomized 5 min, 1 day, or 7 days prior to nephrectomy and hypoxia did not. These data are consistent with the conclusion that extrarenal erythropoietin production is enhanced in association with rapid regeneration of hepatic cells.

Topics: Animals; Erythropoietin; Female; Hepatectomy; Hypoxia; Liver; Nephrectomy; Rats

Hypoxemia (45-minute) influence in vivo on erythropoietic activity of the kidney, liver, spleen, and sternum was studied by normoxemic perfusion of the isolated organs. The erythropoietic activity proved to increase after 6-hour perfusion of the liver; this confirmed the participation of this organ in the extrarenal secretion of the erythropoietic factor.

Topics: Animals; Dogs; Erythropoietin; Hypoxia; Kidney; Liver; Perfusion; Spleen; Sternum

The plasma erythropoietic activity of anephric mice and anephric mice without submandibular glands was compared. The erythropoietin production was stimulated by anemia (phenylhydrazine) and hypobaric hypoxia (0.4 atm). The activity was measured by the 59Fe incorporation in total circulating red blood cells in mice rendered polycythemic by hypoxia. The results were also calculated in units (IRP) of erythropoietin. The plasma erythropoietic activity of anephric mice was neutralized by antiserum against normal erythropoietin. Extrarenal erythropoietin production was significantly reduced in mice deprived of submandibular glands. These data indicate that this phenomenon is not restricted to the rat and support the concept that the submandibular glands are important extrarenal sources of erythropoietin.

Topics: Anemia; Animals; Erythropoietin; Hypoxia; Male; Mice; Phenylhydrazines; Species Specificity; Submandibular Gland

The effect of the androgen fluoxymesterone (Flu) upon the hypoxic stimulus of erythropoietin (ESF) production was studied in 8 male and 3 female normal human subjects. After 4 weeks Flu pretreatment (40 mg/day/m2 body surface in men and 10 mg in women) hypoxic stimulation corresponding a maximum of 4000 m altitude was employed. After Flu pretreatment serum ESF titers increased by 80%, ESF excretion by 490%, and ESF clearance by 200% compared to hypoxic stimulation alone. The increase in serum ESF titers after Flu plus hypoxic stimulation employing only 25% of the Flu dose in females was higher than in males. ESF excretion, however, was lower in females than in males. Whereas Flu causes a marked increase in ESF production, ESF clearance is even more enhanced. As in the case of other serum proteins not only glomerular filtration, but also tubular processes may be involved in the excretion of ESF. Since results are available after a short term experiment of 24 h the double stimulation (androgen + hypoxia) may be recommended for clinical trials in men testing the ESF stimulating effect of androgens and anabolic hormones.

Topics: Adult; Erythropoiesis; Erythropoietin; Female; Fluoxymesterone; Humans; Hypoxia; Male; Middle Aged; Stimulation, Chemical

Erythropoiesis inhibiting factor (EIF) has been described for the first time in the serum of normal human subjects. EIF seems to be a physiological factor regulating erythropoiesis and the antagonist of erythropoietin.

Topics: Animals; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Iron; Male; Mice; Polycythemia

Topics: Animals; Atropine; Atropine Derivatives; Cholinergic Fibers; Dogs; Erythropoietin; Female; Hypoxia; Receptors, Adrenergic; Receptors, Cholinergic

The effects of protein deprivation on erythropoietin (Ep) production were studied. The posthypoxia plasma Ep levels of rats fed a protein-free diet for only 1 day prior to being exposed to 0.5 atmosphere for 7 hr were significantly lower than those of comparably hypoxic rats maintained on a normal diet. The postthypoxia plasma Ep levels were even lower in rats kept on protein-depleted diets for longer periods of time. Rats fed diets with 0.5%, 5% or 12% protein content for 6 days had lower posthypoxia plasma Ep levels than those fed a normal diet (20% protein content). When a single protein meal was force-fed to protein-deprived rats 0-4 hr after initiating the exposure to hypoxia, the posthypoxia plasma Ep levels were significantly higher than in protein-deprived rats that were fed water or dextrose. The posthypoxia plasma Ep titers of protein-deprived rats fed protein 4-8 hr prior to exposure to hypoxia did not differ significantly from those of protein-deprived rats. Although the posthypoxia plasma Ep levels of protein-deprived rats fed a hemolysate containing 0.8 g of hemoglobin 4 hr after beginning hypoxia were greater than those of protein-deprived rats fed only water, the rate of oxygen consumption in these two groups did not differ. We conclude that, in addition to its response to the availability of oxygen, Ep production is infl,enced by the availability of amino acids.

Topics: Animals; Dietary Proteins; Erythropoiesis; Erythropoietin; Female; Hypoxia; Oxygen Consumption; Protein Deficiency; Rats; Time Factors

The ability of either beta-adrenergic blockade or bilateral renal denervation to alter erythropoietin (ESF) production in rabbits exposed to hypobaric hypoxia was studied. ESF elaboration during 5 h of exposure to hypoxia was not affected by beta-blockade, but was markedly reduced by prior surgical denervation of both kidneys. After 18 h of hypoxia plasma ESF levels in renal denervated rabbits did not differ significantly from those of sham-operated controls. Previous studies have shown that ESF production during this more prolonged exposure to hypoxia was significantly inhibited by some beta-adrenergic blocking agents. Combined renal denervation and beta-blockade were more effective than renal denervation alone in attenuating ESF production during 5 h of exposure to hypoxia. However, ESF elaboration during 18 h of hypoxia was significantly greater in animals with combined denervation and beta-blockade than in control rabbits. These results suggest the existence of two distinct mechanisms for ESF production in rabbits exposed to hypobaric hypoxia.

Topics: Adrenergic beta-Antagonists; Denervation; Erythropoietin; Hypoxia; Kidney; Practolol

Renal erythropoietic factor (REF) serves as a substrate for a cyclic nucleotide-dependent protein kinase. The phosphorylation of REF is associated with increased erythropoietic activity. Conversely, REF isolated from hypoxic rats is a poor phosphate acceptor. These findings and the presence of a positive correlation between erythropoietin levels and urinary excretion of adenosine 3',5' monophosphate (cyclic-AMP) in anemic individuals suggest that the cyclic-AMP-protein kinase system plays an important role in the activation of REF.

Topics: Anemia; Animals; Cyclic AMP; Erythropoietin; Humans; Hypoxia; Kidney; Phosphates; Protein Kinases; Rats

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cells, Cultured; Cyclic AMP; Cyclic GMP; Erythropoietin; Female; Humans; Hypoxia; Mice; Mice, Inbred Strains; Rabbits; Spleen; Time Factors

The functioning canine renal allograft produces plasma renin activity (PRA) and erythropoietin (ESF) activity and can maintain normal blood pressure and normal erythropoiesis. Moreover, in response to various provocative stimuli it can: (i) increase plasma renin activity in response to low sodium intake; (ii) suppress PRA in response to high sodium intake; (iii) produce increased serum erythropoietin in response to hypoxia. The granulation activity of the juxtaglomerular apparatus correlates best with the degree of graft rejection and with the PRA in groups manipulated by changing sodium balance. This is not the case with hypoxia. Thus, the juxtaglomerular apparatus, even in the presence of vascular changes seen with the severe degree of rejection in renal allografts, can respond to stimuli that can regulate renin release. Renin production by the transplanted kidney can be dissociated from ESF secretion. Blood pressure changes in the present model were not directly associated with increased PRA or juxtaglomerular apparatus activity. In such conditions hypertension can exist in the presence of suppressed PRA and without hypergranulation of the apparatus. The majority of correlations of this study thus establish a close association of the degree of juxtaglomerular index activity with PRA levels, rather than ESF.

Topics: Animals; Azathioprine; Blood Pressure; Cobalt; Diet; Dogs; Erythropoietin; Female; Graft Rejection; Hypoxia; Juxtaglomerular Apparatus; Kidney Transplantation; Male; Renin; Sodium Chloride; Testosterone; Transplantation, Homologous

Thymectomized, irradiated and syngeneic bone marrow-repopulated (TIR) mice, after hemopoietic recovery, were more effective in producing erythropoietin than normal controls in response to hypobaric hypoxia. Mice which were only thmectomized responded to hypoxia with more erythropoietin production than controls but less than TIR animals. It is suggested that lymphoid elements of thymic origin may produce inhibitors of the erythropoietic system. This may play a role in the anemia of lymphomas and certain other clinical syndromes.

Topics: Animals; Atmospheric Pressure; Bone Marrow Cells; Bone Marrow Transplantation; Erythropoietin; Female; Hypoxia; Iron Radioisotopes; Kidney; Mice; Mice, Inbred C57BL; Thymus Gland; Transplantation, Homologous

Topics: Animals; Animals, Newborn; Blood Transfusion; Erythropoiesis; Erythropoietin; Female; Hypoxia; Male; Rats