endothelin-1 and Anemia--Sickle-Cell

Increased endothelin-1 (ET-1) levels in patients with sickle cell disease (SCD) and transgenic mouse models of SCD contribute to disordered hematological, vascular, and inflammatory responses. Mineralocorticoid receptor (MR) activation by aldosterone, a critical component of the Renin-Angiotensin-Aldosterone-System, modulates inflammation and vascular reactivity, partly through increased ET-1 expression. However, the role of MR in SCD remains unclear. We hypothesized that MR blockade in transgenic SCD mice would reduce ET-1 levels, improve hematological parameters, and reduce inflammation. Berkeley SCD (BERK) mice, a model of severe SCD, were randomized to either sickle standard chow or chow containing the MR antagonist (MRA), eplerenone (156 mg/Kg), for 14 days. We found that MRA treatment reduced ET-1 plasma levels (p = .04), improved red cell density gradient profile (D

Topics: Aldosterone; Anemia, Sickle Cell; Animals; Disease Models, Animal; Endothelial Cells; Endothelin-1; Humans; Inflammation; Mice; Mice, Transgenic; Mineralocorticoid Receptor Antagonists; Receptors, Mineralocorticoid; Vascular Diseases

Renal involvement is common in sickle cell disease (SCD). Early demonstration of renal injury and commencement of appropriate treatment will increase survival and quality of life in these patients. We investigated renal manifestations in our pediatric and adult SCD patients and evaluated the role of cystatin C, Beta2 microglobulin (B2M), retinol-binding protein (RBP), N-acetyl-beta-D-glucosaminidase (NAG), and endothelin-1 (ET-1) to indicate renal damage.. The study involved 45 pediatric and 10 adult patients with SCD and 20 healthy children and 10 healthy adults as a control. All the patients were questioned for possible renal manifestations. 24-hour urine samples were collected and glomerular filtration rates (GFRs) were calculated by using creatinine (GFR(creatinine)), Schwartz formula (GFR(Schwartz)), and cystatin C (GFR(cystatin C)). Blood and urine samples were collected and serum cystatin C, urine B2M, RBP, NAG, and ET-1 levels were measured.. Nocturnal enuresis and proteinuria were the most common renal manifestations in SCD patients. When the groups were compared in terms of GFR, GFR(creatinine) and GFR(Schwartz) levels were higher in group 1 and 2 patients than in control 1 and 2 patients (P < .05). Cystatin C, B2M, RBP, NAG, and ET-1 values were normal in both the patient and the control groups. However, B2M/creatinine levels were higher than 160 μg/mg creatinine levels in 10 patients.. Serum cystatin C, urine NAG, RBP, and ET-1 levels were found to be insufficient for the evaluation of SCD nephropathy. Increased B2M/creatinie levels can be valuable in estimating possible glomerular and tubular damage in SCD.

Topics: Acetylglucosaminidase; Adolescent; Adult; Anemia, Sickle Cell; beta 2-Microglobulin; Child; Child, Preschool; Creatinine; Cystatin C; Endothelin-1; Female; Humans; Infant; Kidney Diseases; Male; Middle Aged; Retinol-Binding Proteins, Cellular

Topics: Anemia, Sickle Cell; Bosentan; Endothelial Cells; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Erythrocyte Deformability; Erythrocytes; Humans; Polymerization; Receptors, Endothelin; Sulfonamides; Thrombospondins

Priapism is a urologic emergency characterized by an uncontrolled, persistent and painful erection in the absence of sexual stimulation, which can lead to penile fibrosis and impotence. It is highly frequent in sickle cell disease (SCD) associated with hemolytic episodes. Our aim was to investigate molecules that may participate in the regulation of vascular tone. Eighty eight individuals with SCD were included, of whom thirty-seven reported a history of priapism. Priapism was found to be associated with alterations in laboratory biomarkers, as well as lower levels of HbF. Patients with sickle cell anemia using hydroxyurea and those who received blood products seemed to be less affected by priapism. Multivariate analysis suggested that low HbF and NOm were independently associated with priapism. The frequency of polymorphisms in genes NOS3 and EDN1 was not statistically significant between the studied groups, and the presence of the variant allele was not associated with alterations in NOm and ET-1 levels in patients with SCD. The presence of the variant allele in the polymorphisms investigated did not reveal any influence on the occurrence priapism. Future studies involving larger samples, as well as investigations including patients in priapism crisis, could contribute to an enhanced understanding of the development of priapism in SCD.

Topics: Adolescent; Anemia, Sickle Cell; Case-Control Studies; Child; Endothelin-1; Fetal Hemoglobin; Genetic Association Studies; Humans; Male; Multivariate Analysis; Nitric Oxide; Nitric Oxide Synthase Type III; Polymorphism, Single Nucleotide; Priapism

Sickle cell disease (SCD) is a monogenic disease associated with multisystem morbidity. Vasculopathy caused by delicate imbalance between coagulation and endothelial systems plays a pivotal role in disease course. As Protein Z and Endothelin-1 genetic polymorphisms may increase the thrombotic risk, the aim of the current work was to verify the possible impact of Protein Z (PROZ G79A) and Endothelin-1 (EDN1 G5665T) polymorphisms on the clinic-laboratory features of the SCD in a cohort of Egyptian pediatric patients.. Genotyping of Protein Z G79A and Endothelin-1 G5665T was carried out by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) assay for 100 SCD patients and 100 controls.. Protein -Z G79A polymorphism was not associated with vascular complications in the studied SCD patients. Endothelin-1 G5665T polymorphism was associated with pulmonary dysfunction (pulmonary artery hypertension and acute chest syndrome) and severe vaso-occlusive crises (VOC).. Endothelin-1 G5665T polymorphism could be considered as a molecular predictor for pulmonary dysfunction and severe VOC in SCD. Further researches with larger cohorts are recommended to understand the pathophysiology of SCD and to explain the inter-patients' variability of disease severity.

Topics: Adolescent; Anemia, Sickle Cell; Blood Proteins; Child; Child, Preschool; Egypt; Endothelin-1; Female; Genetic Predisposition to Disease; Genotype; Humans; Male; Polymorphism, Single Nucleotide

Sickle cell disease is associated with acute painful episodes and chronic intractable pain. Endothelin-1, a known pain inducer, is elevated in the blood plasma of both sickle cell patients and mouse models of sickle cell disease. We show here that the levels of endothelin-1 and its endothelin type A receptor are increased in the dorsal root ganglia of a mouse model of sickle cell disease. Pharmacologic inhibition or neuron-specific knockdown of endothelin type A receptors in primary sensory neurons of dorsal root ganglia alleviated basal and post-hypoxia evoked pain hypersensitivities in sickle cell mice. Mechanistically, endothelin type A receptors contribute to sickle cell disease-associated pain likely through the activation of NF-κB-induced Nav1.8 channel upregulation in primary sensory neurons of sickle cell mice. Our findings suggest that endothelin type A receptor is a potential target for the management of sickle cell disease-associated pain, although this expectation needs to be further verified in clinical settings.

Topics: Anemia, Sickle Cell; Animals; Biomarkers; Disease Models, Animal; Disease Susceptibility; Endothelin-1; Ganglia, Spinal; Hyperalgesia; Male; Mice; Mice, Knockout; Mice, Transgenic; Pain; Posterior Horn Cells; Receptor, Endothelin A

Although the primary origin of sickle cell disease is a hemoglobin disorder, many types of cells contribute considerably to the pathophysiology of the disease. The adhesion of neutrophils to activated endothelium is critical in the pathophysiology of sickle cell disease and targeting neutrophils and their interactions with endothelium represents an important opportunity for the development of new therapeutics. We focused on endothelin-1, a mediator involved in neutrophil activation and recruitment in tissues, and investigated the involvement of the endothelin receptors in the interaction of neutrophils with endothelial cells. We used fluorescence intravital microscopy analyses of the microcirculation in sickle mice and quantitative microfluidic fluorescence microscopy of human blood. Both experiments on the mouse model and patients indicate that blocking endothelin receptors, particularly ET

Topics: Adolescent; Anemia, Sickle Cell; Animals; Calcium; Case-Control Studies; CD11b Antigen; Cell Adhesion; Cell Survival; Child; Child, Preschool; Disease Models, Animal; Endothelial Cells; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Hemodynamics; Humans; Leukocyte Count; Leukocyte Rolling; Macrophage-1 Antigen; Mice; Neutrophil Activation; Neutrophils; Receptor, Endothelin A; Receptor, Endothelin B; Transendothelial and Transepithelial Migration; Tumor Necrosis Factor-alpha

Oxidative stress and haemolysis-associated nitric oxide (NO) depletion plays a crucial role in the development of vasculopathy in sickle cell anaemia (SS). However it remains unknown whether oxidative stress and haemolysis levels influence vascular function in patients with sickle haemoglobin C disease (SC). Microvascular response to heat (using Laser Doppler flowmetry on finger), oxidative stress biomarkers, NO metabolites, endothelin-1 and haematological parameters were compared between patients with SS and SC. Vascular function, oxidative and nitrosative markers were also measured in healthy (AA) children. SS and SC had increased plasma advanced oxidation protein products (AOPP), malondialdehyde, plasma antioxidant activities and NO end products, compared to AA. SC had lower catalase activity compared to AA and SS. Haemolytic rate, glutathione peroxidase and nitrotyrosine concentrations were significantly increased in children with SS compared to SC and AA. SS and SC had impaired microvascular reactivity compared to AA. In SS, the plateau phase of the response to local thermal heating was negatively associated with nitrotyrosine and AOPP. No association between vascular function parameters and oxidative stress markers was observed in SC. Mild haemolysis in SC, compared to SS, may limit oxidative and nitrosative stress and could explain the better preserved microvascular function in this group.

Topics: Adolescent; Advanced Oxidation Protein Products; Anemia, Sickle Cell; Antioxidants; Blood Viscosity; Case-Control Studies; Child; Endothelin-1; Female; Fingers; Hemoglobin SC Disease; Hemolysis; Humans; Laser-Doppler Flowmetry; Male; Malondialdehyde; Microcirculation; Nitric Oxide; Oxidative Stress

Pulmonary hypertension (PH), a serious complication of sickle cell disease (SCD), causes significant morbidity and mortality. Although a recent study determined that hemin release during hemolysis triggers endothelial dysfunction in SCD, the pathogenesis of SCD-PH remains incompletely defined. This study examines peroxisome proliferator-activated receptor γ (PPARγ) regulation in SCD-PH and endothelial dysfunction. PH and right ventricular hypertrophy were studied in Townes humanized sickle cell (SS) and littermate control (AA) mice. In parallel studies, SS or AA mice were gavaged with the PPARγ agonist, rosiglitazone (RSG), 10 mg/kg/day, or vehicle for 10 days. In vitro, human pulmonary artery endothelial cells (HPAECs) were treated with vehicle or hemin for 72 hours, and selected HPAECs were treated with RSG. SS mice developed PH and right ventricular hypertrophy associated with reduced lung levels of PPARγ and increased levels of microRNA-27a (miR-27a), v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1), endothelin-1 (ET-1), and markers of endothelial dysfunction (platelet/endothelial cell adhesion molecule 1 and E selectin). HPAECs treated with hemin had increased ETS1, miR-27a, ET-1, and endothelial dysfunction and decreased PPARγ levels. These derangements were attenuated by ETS1 knockdown, inhibition of miR-27a, or PPARγ overexpression. In SS mouse lung or in hemin-treated HPAECs, activation of PPARγ with RSG attenuated reductions in PPARγ and increases in miR-27a, ET-1, and markers of endothelial dysfunction. In SCD-PH pathogenesis, ETS1 stimulates increases in miR-27a levels that reduce PPARγ and increase ET-1 and endothelial dysfunction. PPARγ activation attenuated SCD-associated signaling derangements, suggesting a novel therapeutic approach to attenuate SCD-PH pathogenesis.

Topics: Anemia, Sickle Cell; Animals; Blood Pressure; Endothelial Cells; Endothelin-1; Gene Knockdown Techniques; Hemin; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Ligands; Lung; Mice; MicroRNAs; Models, Biological; PPAR gamma; Proto-Oncogene Protein c-ets-1; Pulmonary Artery; Rosiglitazone; Systole; Thiazolidinediones; Up-Regulation

Endothelin-1 (ET-1) is increased in patients with sickle cell disease and may contribute to the development of sickle cell nephropathy. The current study was designed to determine whether ET-1 acting via the ETA receptor contributes to renal injury in a mouse model of sickle cell disease.. Adult, humanized HbSS (homozygous for sickle Hb) mice had increased ET-1 mRNA expression in both the cortex and the glomeruli compared with mice heterozygous for sickle and Hb A (HbAS controls). In the renal cortex, ETA receptor mRNA expression was also elevated in HbSS (sickle) mice although ETB receptor mRNA expression was unchanged. Ligand binding assays confirmed that sickle mice had increased ETA receptors in the renal vascular tissue when compared with control mice.. In response to PKC stimulation, reactive oxygen species production by isolated glomeruli from HbSS sickle mice was increased compared with that from HbSA controls, an effect that was prevented by 1 week in vivo treatment with the selective ETA antagonist, ABT-627. Protein and nephrin excretion were both elevated in sickle mice, effects that were also significantly attenuated by ABT-627. Finally, ETA receptor antagonism caused a significant reduction in mRNA expression of NADPH oxidase subunits, which may contribute to nephropathy in sickle cell disease.. These data support a novel role for ET-1 in the progression of sickle nephropathy, specifically via the ETA receptor, and suggest a potential role for ETA receptor antagonism in a treatment strategy.

Topics: Acute Kidney Injury; Anemia, Sickle Cell; Animals; Disease Progression; Dose-Response Relationship, Drug; Endothelin-1; Female; Gene Knock-In Techniques; Humans; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Transgenic; Reactive Oxygen Species; Receptor, Endothelin A

Previous studies showed that high levels of placenta growth factor (PlGF) correlated with increased plasma levels of endothelin-1 (ET-1), a potent vasoconstrictor, in sickle cell disease (SCD). PlGF-mediated transcription of the ET-1 gene occurs by activation of hypoxia inducible factor 1α (HIF-1α) and posttranscriptionally by microRNA 199a2 (miR-199a2), which targets the 3' untranslated region (UTR) of HIF-1α mRNA. However, relatively less is known about how PlGF represses the expression of miR-199a2 located in the DNM3 opposite strand (DNM3os) transcription unit. Here, we show that PlGF induces the expression of activated transcription factor 3 (ATF3), which, in association with accessory proteins (c-Jun dimerization protein 2 [JDP2], ATF2, and histone deacetylase 6 [HDAC6]), as determined by proteomic analysis, binds to the DNM3os promoter. Furthermore, we show that association of HDAC6 with ATF3 at its binding site in this promoter was correlated with repression of miR-199a2 transcription, as shown by DNM3os transcription reporter and chromatin immunoprecipitation (ChIP) assays. Tubacin, an inhibitor of HDAC6, antagonized PlGF-mediated repression of DNM3os/pre-miR-199a2 transcription with a concomitant reduction in ET-1 levels in cultured endothelial cells. Analysis of lung tissues from Berkeley sickle (BK-SS) mice showed increased levels of ATF3 and increased expression of ET-1. Delivery of tubacin to BK-SS mice significantly attenuated plasma ET-1 and PlGF levels. Our studies demonstrated that ATF3 in conjunction with HDAC6 acts as a transcriptional repressor of the DNM3os/miR-199a2 locus.

Topics: Activating Transcription Factor 3; Anemia, Sickle Cell; Animals; Cell Line; Chromatin Assembly and Disassembly; Disease Models, Animal; Down-Regulation; Endothelin-1; Histone Deacetylase 6; Histone Deacetylases; Humans; Lung; Membrane Proteins; Mice; MicroRNAs; Promoter Regions, Genetic; Proteomics; Transcription, Genetic

Pulmonary hypertension (PHT) is associated with high mortality in sickle cell anemia (SCA). Previously, we showed that elevated levels of placenta growth factor (PlGF) in SCA patients correlate with increased levels of the potent vasoconstrictor endothelin-1 (ET-1) and PHT. Moreover, PlGF induced the expression of ET-1 via hypoxia-inducible factor 1α. Here, we show a novel example of ET-1 posttranscriptional regulation by PlGF via action of microRNA 648 (miR-648), which is subject to transcriptional coregulation with its host gene, MICAL3 (microtubule-associated monooxygenase, calponin, and LIM domain containing 3gene). PlGF repressed expression of miR-648 in endothelial cells. Luciferase reporter assays using wild-type and mutant ET-1 3' untranslated region (UTR) constructs, and transfection of miR-648 mimics showed that miR-648 targets the 3' UTR of ET-1 mRNA. Since miR-648 is located in a 5'-proximal intron of MICAL3, we examined which of three potential promoters was responsible for its expression. The MICAL3 distal promoter (P1) was the predominant promoter used for transcription of pre-miR-648, and it was under positive control by PAX5 (paired box protein 5) transcription factor, as demonstrated by the loss and gain of function of PAX5 activity, and chromatin immunoprecipitation analysis. These studies provide a novel link wherein PlGF-mediated downregulation of PAX5 attenuates miR-648 expression leading to increased ET-1 levels that are known to induce PHT in SCA.

Topics: Anemia, Sickle Cell; Cells, Cultured; Endothelial Cells; Endothelin-1; Gene Expression Regulation; Humans; MicroRNAs; Mixed Function Oxygenases; PAX5 Transcription Factor; RNA, Messenger; Transcription Factors

Children with sickle cell disease (SCD) have painful vaso-occlusive episodes (VOEs), which often reoccur across the individual's lifespan. Vaso-constrictive and vaso-dilatory molecules have been hypothesized to play a role in VOEs. Endothelin-1 (ET-1) is a potent vasoconstrictor that is released during VOEs and is correlated with pain history. Apelin is a vaso-dilatory peptide that also has a modulatory role in pain processing. We hypothesize that the ratio between vaso-dilatory and vaso-constrictive tone in children with SCD may be a marker of pain sensitization and vaso-occlusion. Plasma endothelin and apelin levels were measured in 47 children with SCD. Procedural pain and baseline pain were assessed via child- and caregiver-reports and observational distress. Pain history was assessed using retrospective chart review. Plasma apelin was related to age, with decreased levels in older children. The ratio between apelin and ET-1 was negatively correlated to observational baseline pain. The ratio between apelin and Big ET was negatively correlated to caregiver ratings of baseline pain and positively correlated to history of VOEs, which is possibly due to hydroxyurea treatment. These results suggest that an imbalance in the apelin and endothelin systems may contribute to an increasing number of VOEs and baseline pain in children with SCD.

Topics: Adolescent; Age Factors; Anemia, Sickle Cell; Antisickling Agents; Apelin; Biomarkers; Child; Child, Preschool; Endothelin-1; Female; Gene Expression Regulation; Humans; Hydroxyurea; Intercellular Signaling Peptides and Proteins; Male; Pain; Pain Measurement; Signal Transduction; Vasoconstriction; Vasodilation

The anemia of sickle cell disease is associated with a severe inflammatory vasculopathy and endothelial dysfunction, which leads to painful and life-threatening clinical complications. Growing evidence supports the anti-inflammatory properties of ω-3 fatty acids in clinical models of endothelial dysfunction. Promising but limited studies show potential therapeutic effects of ω-3 fatty acid supplementation in sickle cell disease. Here, we treated humanized healthy and sickle cell mice for 6 weeks with ω-3 fatty acid diet (fish-oil diet). We found that a ω-3 fatty acid diet: (i) normalizes red cell membrane ω-6/ω-3 ratio; (ii) reduces neutrophil count; (iii) decreases endothelial activation by targeting endothelin-1 and (iv) improves left ventricular outflow tract dimensions. In a hypoxia-reoxygenation model of acute vaso-occlusive crisis, a ω-3 fatty acid diet reduced systemic and local inflammation and protected against sickle cell-related end-organ injury. Using isolated aortas from sickle cell mice exposed to hypoxia-reoxygenation, we demonstrated a direct impact of a ω-3 fatty acid diet on vascular activation, inflammation, and anti-oxidant systems. Our data provide the rationale for ω-3 dietary supplementation as a therapeutic intervention to reduce vascular dysfunction in sickle cell disease.

Topics: Anemia, Sickle Cell; Animals; Anti-Inflammatory Agents; Blood Vessels; Dietary Supplements; Disease Models, Animal; Endothelin-1; Endothelium, Vascular; Erythrocyte Membrane; Fatty Acids, Omega-3; Humans; Hypoxia; Mice; Mice, Transgenic; Neutrophils; Oxygen

Endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1) play important roles in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show higher levels of placenta growth factor (PlGF) in SCD correlate with increased plasma levels of ET-1, PAI-1, and other physiological markers of PH. PlGF-mediated ET-1 and PAI-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively little is understood regarding post-transcriptional regulation of PlGF-mediated expression of ET-1 and PAI-1. Herein, we show PlGF treatment of endothelial cells reduced levels of miR-301a and miR-454 from basal levels. In addition, both miRNAs targeted the 3'-UTRs of ET-1 and PAI-1 mRNAs. These results were corroborated in the mouse model of SCD [Berkeley sickle mice (BK-SS)] and in SCD subjects. Plasma levels of miR-454 in SCD subjects were significantly lower compared with unaffected controls, which correlated with higher plasma levels of both ET-1 and PAI-1. Moreover, lung tissues from BK-SS mice showed significantly reduced levels of pre-miR-301a and concomitantly higher levels of ET-1 and PAI-1. Furthermore, we show that miR-301a/miR-454 located in the spindle and kinetochore-associated protein-2 (SKA2) transcription unit was co-transcriptionally regulated by both HIF-1α and peroxisome proliferator-activated receptor-α (PPAR-α) as demonstrated by SKA2 promoter mutational analysis and ChIP. Finally we show that fenofibrate, a PPAR-α agonist, increased the expression of miR-301a/miR-454 and SKA2 in human microvascular endothelial cell line (HMEC) cells; the former were responsible for reduced expression of ET-1 and PAI-1. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-301a/miR-454 expression can ameliorate PH and lung fibrosis by reduction in ET-1 and PAI-1 levels in SCD.

Topics: Anemia, Sickle Cell; Animals; Cell Line; Chromosomal Proteins, Non-Histone; Endothelin-1; Fenofibrate; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; MicroRNAs; Placenta Growth Factor; Plasminogen Activator Inhibitor 1; PPAR alpha; Pregnancy Proteins; Promoter Regions, Genetic

Sickle cell disease (SCD) is associated with acute vaso-occlusive crises that trigger painful episodes and frequently involves ongoing, chronic pain. In addition, both humans and mice with SCD experience heightened cold sensitivity. However, studies have not addressed the mechanism(s) underlying the cold sensitization or its progression with age. Here we measured thermotaxis behavior in young and aged mice with severe SCD. Sickle mice had a marked increase in cold sensitivity measured by a cold preference test. Furthermore, cold hypersensitivity worsened with advanced age. We assessed whether enhanced peripheral input contributes to the chronic cold pain behavior by recording from C fibers, many of which are cold sensitive, in skin-nerve preparations. We observed that C fibers from sickle mice displayed a shift to warmer (more sensitive) cold detection thresholds. To address mechanisms underlying the cold sensitization in primary afferent neurons, we quantified mRNA expression levels for ion channels thought to be involved in cold detection. These included the transient receptor potential melastatin 8 (Trpm8) and transient receptor potential ankyrin 1 (Trpa1) channels, as well as the 2-pore domain potassium channels, TREK-1 (Kcnk2), TREK-2 (Kcnk10), and TRAAK (Kcnk4). Surprisingly, transcript expression levels of all of these channels were comparable between sickle and control mice. We further examined transcript expression of 83 additional pain-related genes, and found increased mRNA levels for endothelin 1 and tachykinin receptor 1. These factors may contribute to hypersensitivity in sickle mice at both the afferent and behavioral levels.

Topics: Age Factors; Aging; Anemia, Sickle Cell; Animals; Cold Temperature; Cryopyrin-Associated Periodic Syndromes; Endothelin-1; Ganglia, Spinal; Gene Expression Regulation; Hemoglobin A; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Fibers, Unmyelinated; Pain Threshold; Potassium Channels, Tandem Pore Domain; Sensory Receptor Cells; Skin; TRPC Cation Channels; TRPM Cation Channels

Chronic inflammation is a salient feature of sickle cell disease (SCD) and transgenic-knockout sickle (BERK) mice. Inflammation is implicated in the activation of hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions. We hypothesize that, in SCD, inflammation coupled with nitric oxide (NO) depletion will induce expression of HIF-1α, a transcription factor with wide-ranging effects including activation of genes for vasoactive molecules. To this end, we have examined the expression of HIF-1α in normoxic BERK mice expressing exclusively human α- and β(S)- globins, and evaluated the effect of fetal hemoglobin (HbF) in BERK mice (i.e., <1.0%, 20%, and 40% HbF). HbF exerts antisickling and anti-inflammatory effects. Here, we show that HIF-1α is expressed in BERK mice under normoxic conditions, accompanied by increased expression of its vasoactive biomarkers such as VEGF, heme oxygenase-1 (HO-1), and serum ET-1 levels. In BERK mice expressing HbF, HIF-1α expression decreases concomitantly with increasing HbF, commensurately with increased NO bioavailability, and shows a strong inverse correlation with plasma NO metabolites (NOx) levels. Reduced HIF-1α expression is associated with decreased HO-1, VEGF, and ET-1. Notably, arteriolar dilation, enhanced volumetric blood flow, and low blood pressure in normoxic BERK mice all show a trend toward normalization with the introduction of HbF. Also, arginine treatment reduced HIF-1α, as well as VEGF expression in normoxic BERK mice, supporting a role of NO bioavailability in HIF-1α activation. Thus HIF-1α expression in normoxic sickle mice is likely a consequence of chronic inflammation, and HbF exerts an ameliorating effect by decreasing sickling, increasing NO bioavailability, and reducing inflammation.

Topics: alpha-Globins; Anemia, Sickle Cell; Animals; Arginine; Arterioles; beta-Globins; Blood Pressure; Disease Models, Animal; Down-Regulation; Endothelin-1; Fetal Hemoglobin; Heme Oxygenase-1; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microcirculation; Nitric Oxide; Regional Blood Flow; Vascular Endothelial Growth Factor A; Vasodilation

The impact of pain early in life is a salient issue for sickle cell disease (SCD), a genetic condition characterized by painful vaso-occlusive episodes (VOEs) that can begin in the first year of life and persist into adulthood. This study examined the effects of age and pain history (age of onset and frequency of recent VOEs) on acute procedural pain in children with SCD. Endothelin-1, a vaso-active peptide released during VOEs and acute tissue injury, and its precursor, Big Endothelin, were explored as markers of pain sensitization and vaso-occlusion. Sixty-one children with SCD (ages 2 to 18) underwent venipuncture at routine health visits. Procedural pain was assessed via child and caregiver reports and observational distress. Pain history was assessed using retrospective chart review. Three primary results were found: 1) younger age was associated with greater procedural pain across pain outcomes; 2) higher frequency of VOEs was associated with greater procedural pain based on observational distress (regardless of age); and 3) age was found to moderate the relationship between VOEs and procedural pain for child-reported pain and observational distress for children 5 years of age and older. Associations between the endothelin variables and pain prior to venipuncture were also observed.. For children with SCD, the child's age and recent pain history should be considered in procedural pain management. The endothelin system may be involved in preprocedure pain, but additional research is needed to understand the role of endothelins in pain sensitization.

Topics: Adolescent; Age Factors; Anemia, Sickle Cell; Child; Child, Preschool; Endothelin-1; Female; Humans; Infant; Male; Pain; Pain Management; Pain Measurement; Pain Perception; Phlebotomy; Surveys and Questionnaires

Placenta growth factor (PlGF) is released by immature erythrocytes and is elevated in sickle cell disease (SCD). Previous data generated in vitro suggest that PlGF may play a role in the pathophysiology of SCD-associated pulmonary hypertension (PHT) by inducing the release of the vasoconstrictor, endothelin-1. In this cross-sectional study of 74 patients with SCD, we confirm that PlGF is significantly elevated in SCD compared with healthy control subjects. We found significantly higher levels of PlGF in SCD patients with PHT but observed no association of PlGF with the frequency of acute pain episodes or history of acute chest syndrome. The observed correlation between PlGF and various measures of red cell destruction suggests that hemolysis, and the resultant erythropoietic response, results in the up-regulation of PlGF. Although relatively specific, PlGF, as well as N-terminal pro-brain natriuretic peptide and soluble vascular cell adhesion molecule, has low predictive accuracy for the presence of PHT. Prospective studies are required to conclusively define the contribution of PlGF to the pathogenesis of PHT and other hemolytic complications in SCD.

Topics: Adult; Anemia, Sickle Cell; Case-Control Studies; Cross-Sectional Studies; Endothelin-1; Female; Hemolysis; Humans; Hypertension, Pulmonary; Inflammation; Male; Middle Aged; Placenta Growth Factor; Predictive Value of Tests; Pregnancy Proteins; Prognosis; Young Adult

Pulmonary hypertension is associated with reduced nitric oxide bioavailability and early mortality in sickle cell disease (SCD). We previously demonstrated that placenta growth factor (PlGF), an angiogenic factor produced by erythroid cells, induces hypoxia-independent expression of the pulmonary vasoconstrictor endothelin-1 in pulmonary endothelial cells. Using a lentivirus vector, we simulated erythroid expression of PlGF in normal mice up to the levels seen in sickle mice. Consequently, endothelin-1 production increased, right ventricle pressures increased, and right ventricle hypertrophy and pulmonary changes occurred in the mice within 8 weeks. These findings were corroborated in 123 patients with SCD, in whom plasma PlGF levels were significantly associated with anemia, endothelin-1, and tricuspid regurgitant velocity; the latter is reflective of peak pulmonary artery pressure. These results illuminate a novel mechanistic pathway linking hemolysis and erythroid hyperplasia to increased PlGF, endothelin-1, and pulmonary hypertension in SCD, and suggest that strategies that block PlGF signaling may be therapeutically beneficial.

Topics: Adult; Anemia, Sickle Cell; Animals; Cell Line; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Humans; Hypertension, Pulmonary; Hypertrophy, Left Ventricular; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Myocardium; Placenta Growth Factor; Pregnancy Proteins

Patients with sickle cell disease (SCD) exhibit a chronic inflammatory state manifested by leukocytosis and increased circulating levels of proinflammatory cytochemokines. Our studies show that placenta growth factor levels are high in SCD, and placental growth factor induces the release of the vasoconstrictor endothelin-1 (ET-1) from pulmonary microvascular endothelial cells. In this study, we observed that ET-1 increased the expression of the chemokines MIP-1β or CCL4. ET-1-induced MIP-1β mRNA expression in THP-1 cells and human peripheral blood monocytes occurred via the activation of PI3K, NADPH oxidase, p38 MAPK, and JNK-1 but not JNK-2. ET-1-induced MIP-1β expression involved hypoxia-inducible factor-1α (HIF-1α), independent of hypoxia, as demonstrated by silencing with HIF-1α small interfering RNA, EMSA, and chromatin immunoprecipitation analysis. ET-1-induced MIP-1β promoter luciferase activity was attenuated when any of the five hypoxia-response elements, AP-1, or NF-κB binding motifs in the proximal MIP-1β promoter (-1053/+43 bp) were mutated. Furthermore, ET-1 significantly downregulated the expression of a key microRNA, microRNA-195a, which showed a complementary binding site in the 3' untranslated region of MIP-1β mRNA. Moreover, ET-1-induced MIP-1β mRNA expression in either THP-1 cells or peripheral blood monocytes was reduced upon expression of microRNA-195a. Conversely, transfection of monocytes with anti-microRNA-195a oligonucleotide augmented several-fold ET-1-induced MIP-1β expression. Taken together, these studies showed that ET-1-mediated MIP-1β gene expression is regulated via hypoxia-response elements, AP-1, and NF-κB cis-binding elements in its promoter and negatively regulated by microRNA-195, which targets the 3' untranslated region of MIP-1β RNA. These studies provide what we believe are new avenues, based on targets of HIF-1α and microRNAs, for ameliorating inflammation in SCD.

Topics: 3' Untranslated Regions; Anemia, Sickle Cell; Blotting, Western; Cell Line; Chemokine CCL4; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Endothelin-1; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; MicroRNAs; Monocytes; Mutagenesis, Site-Directed; NF-kappa B; Promoter Regions, Genetic; RNA, Small Interfering; Transcription Factor AP-1; Transfection

Patients with sickle-cell disease (SCD) suffer from tissue damage and life-threatening complications caused by vasoocclusive crisis (VOC). Endothelin receptors (ETRs) are mediators of one of the most potent vasoconstrictor pathways in mammals, but the relationship between vasoconstriction and VOC is not well understood. We report here that pharmacological inhibition of ETRs prevented hypoxia-induced acute VOC and organ damage in a mouse model of SCD. An in vivo ultrasonographic study of renal hemodynamics showed a substantial increase in endothelin-mediated vascular resistance during hypoxia/reoxygenation-induced VOC. This increase was reversed by administration of the dual ETR antagonist (ETRA) bosentan, which had pleiotropic beneficial effects in vivo. It prevented renal and pulmonary microvascular congestion, systemic inflammation, dense rbc formation, and infiltration of activated neutrophils into tissues with subsequent nitrative stress. Bosentan also prevented death of sickle-cell mice exposed to a severe hypoxic challenge. These findings in mice suggest that ETRA could be a potential new therapy for SCD, as it may prevent acute VOC and limit organ damage in sickle-cell patients.

Topics: Anemia, Sickle Cell; Animals; Antihypertensive Agents; Bosentan; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Hemodynamics; Humans; Hypoxia; Kidney; Lung; Mice; Mice, Inbred C57BL; Neutrophils; Receptors, Endothelin; Regional Blood Flow; Renal Circulation; Sulfonamides; Vasoconstriction

As hydroxyurea (HU), sodium phenyl butyrate (SPB) is known to induce fetal hemoglobin (HbF) expression and thus shows potentials for sickle-cell disease (SCD) treatment. More recently, few studies suggested that endothelial cells (ECs), a major pathophysiological actor of SCD, are also a target of SPB. Here, we show that SPB, as HU, reduces endothelin-1 mRNA expression and peptide release by human ECs in culture. SPB increases VCAM-1 and ICAM-1 mRNAs and soluble ICAM-1 release. Both drugs have a cumulative effect on ICAM-1 expression. We conclude that SPB, as HU, also affects the expression of molecules important to the pathophysiology of SCD, in addition to its effect on HbF. Its potential as an alternative or adjuvant drug in SCD treatment warrants further investigations.

Topics: Anemia, Sickle Cell; Cell Line, Transformed; Drug Evaluation, Preclinical; Drug Synergism; Endothelial Cells; Endothelin-1; Gene Expression Regulation; Humans; Hydroxyurea; Intercellular Adhesion Molecule-1; Interferon-gamma; Phenylbutyrates; RNA, Messenger; Solubility; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Elevated plasma levels of cytokines such as endothelin-1 (ET-1) have been shown to be associated with sickle cell disease (SCD). However, the role of ET-1 in the pathophysiology of SCD is not entirely clear. I now show that treatment of SAD mice, a transgenic mouse model of SCD, with BQ-788 (0.33 mg.kg(-1).day(-1) intraperitoneally for 14 days), an ET-1 receptor B (ET(B)) antagonist, induced a significant decrease in Gardos channel activity (1.7 +/- 0.1 to 1.0 +/- 0.4 mmol.10(13) cell(-1).h(-1), n = 3, P = 0.019) and reduced the erythrocyte density profile by decreasing the mean density (D(50); n = 4, P = 0.012). These effects were not observed in mice treated with BQ-123, an ET-1 receptor A (ET(A)) antagonist. A mixture of both antagonists induced a similar change in density profile as with BQ-788 alone that was associated with an increase in mean cellular volume and a decrease in corpuscular hemoglobin concentration mean. I also observed in vitro effects of ET-1 on human sickle erythrocyte dehydration that was blocked by BQ-788 and a mixture of ET(B)/ET(A) antagonists but not by ET(A) antagonist alone. These results show that erythrocyte hydration status in vivo is mediated via activation of the ET(B) receptor, leading to Gardos channel modulation in SCD.

Topics: Anemia, Sickle Cell; Animals; Antihypertensive Agents; Disease Models, Animal; Endothelin A Receptor Antagonists; Endothelin-1; Erythrocytes, Abnormal; Mice; Mice, Transgenic; Oligopeptides; Piperidines; Potassium Channels, Calcium-Activated; Receptor, Endothelin A; Water

The association of endothelin 1 (ET-1) and endothelial constitutive nitric oxide synthase (ecNOS) gene polymorphisms (G5665T and T8002C, VNTR and T-786C respectively) with the occurrence of acute chest syndrome and painful vaso-occlusive crises was evaluated in homozygous SS children. This retrospective study reveals that ET-1 T8002 and ecNOS C-786 alleles are associated with, respectively, an increased and a decreased risk of acute chest syndrome.

Topics: Adolescent; Anemia, Sickle Cell; Chest Pain; Child; Endothelin-1; Female; Genetic Predisposition to Disease; Humans; Male; Nitric Oxide Synthase Type III; Pain; Polymorphism, Genetic; Vascular Diseases

Plasma endothelin-1 (ET-1) is elevated in patients with sickle cell disease (SCD). Hydroxyurea (HU) is the only drug with demonstrated clinical efficacy in SCD. Here we show that treatment with HU results in a decreased concentration of circulating ET-1 which is not correlated with the HU-induced increase in HbF level. Blunting of the ET-1 vasoconstrictive stimulus could contribute to the beneficial effects of HU.

Topics: Anemia, Sickle Cell; Case-Control Studies; Child; Endothelin-1; Fetal Hemoglobin; Humans; Hydroxyurea

To compare the accuracy of biomarkers for identifying acute chest syndrome (ACS) in patients with sickle cell disease presenting to a pediatric emergency department (ED).. We conducted a 13-month-long (2002-2003) cohort study with nested case-control in patients with sickle cell disease presenting to the pediatric ED with vaso-occlusive crises or fever in which we compared levels of secretory phospholipase A2 (sPLA2), endothelin-1, interleukin-6 (IL-6), and peripheral white blood cell count (WBC) in cases that were complicated by ACS and in control subjects with uncomplicated illnesses. For diagnosis, a test was considered to be accurate when the area under its receiver operator characteristic curve (AUC) was >0.70. Laboratory tests with AUC values > or =0.70 were entered into a binary recursive partitioning model for diagnosis.. For the period of study, samples from 72 visits were obtained from 51 patients who presented with vaso-occlusive crises (range: 1-4 visits per patient; 15 were enrolled more than once). ACS complicated 19 of 72 visits (26%, 95% confidence interval: 17%-38%). At an AUC value of 0.79, only the sPLA2 test was accurate for diagnosing ACS. AUC values for peripheral WBC, endothelin-1, and IL-6 were 0.68, 0.51, and 0.52, respectively. Binary recursive partitioning retained only sPLA2 at a cutoff of 13.7 ng/mL to be accurate for diagnosis. This cutoff had a sensitivity of 74% (14 of 19), a specificity of 87% (46 of 53), a positive likelihood ratio of 5.6, and a negative likelihood ratio of 0.18.. Secretory phospholipase A2 but not endothelin-1, IL-6, or WBC is an accurate test for identifying present or incipient ACS in young patients who present to the ED with sickle cell pain crises.

Topics: Acute Disease; Adolescent; Anemia, Sickle Cell; Area Under Curve; Biomarkers; Chest Pain; Child; Endothelin-1; Female; Humans; Interleukin-6; Leukocyte Count; Lung Diseases; Male; Phospholipases A; Phospholipases A2; Sensitivity and Specificity; Syndrome

Sickle cell disease (SCD) affects the kidney by acute mechanisms as well as by insidious renal medullary/papillary necrosis, resulting in tubular defects, which increase the risk of dehydration and subsequent sickle crisis. Hypoxia has been reported to stimulate endothelin-1 (ET-1) synthesis by endothelial cells and also in the renal tubule.. This case-control study measured ET-1 in urine as a marker of its renal synthesis in asymptomatic SCD patients. Baseline plasma and urinary ET-1 levels were measured and followed during a water deprivation study and a subsequent administration of desmopressin.. Urine and plasma levels of ET-1 were elevated in patients with SCD, compared with carefully matched African-French and African controls, and urine ET-1 excretion was associated with a marked urine-concentrating defect. Moreover, urinary ET-1 output was correlated with microalbuminuria in SCD patients.. ET-1 is known to antagonize the tubular effects of vasopressin and to promote renal scarring; increased renal production of ET-1 could produce nephrogenic diabetes insipidus and dehydration in SCD patients through a combination of fibrosis and functional resistance to vasopressin. This study provides a rationale for trials with endothelin receptor antagonists in sickle cell disease nephropathy.

Topics: Adult; Albuminuria; Anemia, Sickle Cell; Biomarkers; Case-Control Studies; Diabetes Insipidus, Nephrogenic; Disease Progression; Endothelin-1; Female; Humans; Kidney Tubules; Male; Radioimmunoassay; Severity of Illness Index

While systemic plasma endothelin-1 (ET-1) levels are increased during acute crisis in sickle cell disease, the relative levels of potent vasoactive factors that contribute to the regulation of vascular function, such as ET-1, NO, and cell-free hemoglobin, during the course of periodic vaso-occlusive episodes remain unclear. Moreover, whether and to what extent sickling-induced release of ET-1 alters vascular tone is not completely understood. To investigate the sequential changes in circulating vasoactive factors, we measured plasma ET-1, NO metabolites (NOx), and cell-free hemoglobin (Hb) before (steady-state), during (crisis), and after a vaso-occlusive (post-crisis) episode. Steady-state ET-1 levels (fmol/mL) increased from 2.3 +/- 0.4 to 11.0 +/- 1.4 and 4.2 +/- 1.0 during crisis and post-crisis periods, respectively. There was no significant difference in plasma NOx levels. Cell-free Hb levels were significantly higher in sickle cell patients in all phases as compared to the control group, and especially during crisis cell-free Hb levels were elevated by 4-fold (209,000 +/- 31,000 vs. 46,000 +/- 5,300 ng/mL in steady-state). Conditioned medium from human pulmonary artery endothelial cells exposed to sickled erythrocytes prepared by deoxygenation induced contraction of aortic rings, and this effect was blocked by an ET(A) receptor antagonist. These findings indicate that ET-1 is the predominant contractile factor released by cultured endothelial cells upon exposure to deoxygenated sickled SS erythrocytes and ET-1-NO-NO scavenger balance is altered in favor of vasoconstriction during an acute episode in SCD.

Topics: Anemia, Sickle Cell; Aorta; Endothelial Cells; Endothelin A Receptor Antagonists; Endothelin-1; Erythrocytes, Abnormal; Hemoglobins; Humans; Nitric Oxide; Oxygen; Pulmonary Artery; Vasoconstriction

The clinical efficacy of oral hydroxyurea (HU) in adults and children with sickle cell anemia (SCA) cannot solely be explained by its ability to enhance fetal hemoglobin (HbF) expression. Since increased adherence of sickle red blood cells to vascular endothelium is a possible contributing factor to vaso-occlusive crisis (VOC), we explored the effect of HU on human endothelial cell (EC) lines (TrHBMEC and EA-hy 926). We demonstrated that HU, in a dose-dependent and reversible manner, significantly decreased (up to three-fold) the release of endothelin-1 (ET-1), a vasoconstrictor peptide through downregulation (up to three-fold) of ET-1 gene expression. This finding is of therapeutic relevance as SCA patients exhibit elevated serum levels of ET-1 during episodes of VOC and levels correlate with disease severity. Unexpectedly, HU upregulated (up to three-fold) the expression of membrane-bound intercellular cell adhesion molecule 1 (mbICAM-1) and its soluble form (sICAM-1) with a parallel increase in ICAM-1 mRNA expression. Although ICAM-1 does not appear to be involved in the sickle cell adhesion to vascular endothelium, it may exacerbate vaso-occlusion by promoting leukocyte adhesion. The HU-induced increase in mbICAM-1 may appear inconsistent with the clinical benefits confered by HU. However, both the increase in sICAM-1- and HU-induced leukocyte reduction in patients, may counteract the potentially detrimental effect of elevated mbICAM-1 expression. Also HU reduces the expression of vascular cell adhesion molecule (VCAM-1) on EC. Since HU reduces the very late antigen 4-positive reticulocytes in SCA patients, a ligand for VCAM-1, HU-induced downregulation of VCAM-1 on EC will very likely decrease the reticulocyte-endothelium adhesion. Thus, HU, apart from inducing HbF expression in the red cell, also affects the expression profile of EC compartment.

Topics: Anemia, Sickle Cell; Antisickling Agents; Cell Line; Dose-Response Relationship, Drug; Down-Regulation; Endothelial Cells; Endothelin-1; Flow Cytometry; Gene Expression; Humans; Hydroxyurea; Intercellular Adhesion Molecule-1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

We studied levels of plasma endothelin-1 (ET-1) in 25 beta-thalassemia/Hb E patients before and after blood transfusion. Baseline ET-1 levels in these patients were significantly higher than in normal controls (10.17 +/- 2.1 pg/mL vs. 8.9 +/- 2.0 pg/mL, P < 0.05). After blood transfusion, levels of plasma ET-1 tend to slightly increase during the first 24 hr but significantly decline at the 7th day to levels (8.01 +/- 1.7 pg/mL) which do not differ from those of controls. This study highlights a different alteration of plasma ET-1 in patients with beta-thalassemia compared to that of patients with sickle-cell anemia.

Topics: Adolescent; Anemia, Sickle Cell; beta-Thalassemia; Blood Transfusion; Child; Child, Preschool; Endothelin-1; Female; Hematocrit; Hemoglobin E; Hemoglobins; Humans; Male

It has recently been shown that the Gardos channel activity of mouse erythrocytes can be modified by endothelins, suggesting a functional linkage between endothelin receptors and the Gardos channel. Using (86)Rubidium ((86)Rb) influx, effects were estimated of proinflammatory molecules such as platelet activator factor (PAF), endothelin-1 (ET-1), interleukin-10 (IL-10), and regulated on activation normal T cells expressed and secreted (RANTES) on the Gardos channel activity in human normal and sickle red cells. It was found that PAF (EC(50): 15 +/- 7 nM), RANTES (EC(50), 9 +/- 6 ng/mL [1.2 +/- 0.8 nM]), IL-10 (EC(50), 11 +/- 8 ng/mL [204 +/- 148 nM]), and ET-1 (EC(50), 123 +/- 34 nM) induce a significant increase in Gardos channel activity-between 28% and 84%-over the control. In addition, these agents modify the Gardos channel affinity for internal Ca(++) (K(0.5)) by 2- to 6-fold. Biochemical evidence is provided for the presence of ET receptor subtype B in sickle and normal red cells. Furthermore, it was found that ET-1, PAF, RANTES, and IL-10 induce a significant increase in red cell density (P <.05). These data suggest that activation of the Gardos channel is functionally coupled to receptor motifs such as C-X-C (PAF), C-C (RANTES), and ET receptor subtype B. Thus, cell volume regulation or erythrocyte hydration states might be altered by activation of the Gardos channel by cytokines in vivo. The role of these mediators in promoting sickle cell dehydration in vivo is under investigation.

Topics: Anemia, Sickle Cell; Calcium; Cell Size; Chemokine CCL5; Cytokines; Endothelin Receptor Antagonists; Endothelin-1; Erythrocyte Count; Erythrocytes, Abnormal; Humans; Interleukin-10; Kinetics; Oligopeptides; Piperidines; Platelet Activating Factor; Platelet Membrane Glycoproteins; Potassium Channels, Calcium-Activated; Protein Kinase C; Receptor, Endothelin B; Receptors, CCR5; Receptors, Cell Surface; Receptors, Chemokine; Receptors, Endothelin; Receptors, G-Protein-Coupled; Rubidium Radioisotopes

We investigated the effects of sickle erythrocytes on the production of vasotone mediators in endothelial cells (ECs) using an in vitro recirculating flow system. Sickle erythrocytes increased the EC production of two important vasoactivators, prostacyclin and endothelin-1, under venous wall shear stress conditions of 1dyncm2. The presence of interleukin-1 in the perfusion system, as a model for inflammatory cytokine effects, enhanced the overall amounts of released prostacyclin but did not affect the production of endothelin-1. This study demonstrates the effects of sickle erythrocytes on the function and metabolism of ECs under vascular flow environments. The altered production of vasoactivators may contribute to the vasotone instability and vasoocclusive crises in sickle cell anemia.

Topics: 6-Ketoprostaglandin F1 alpha; Adult; Anemia, Sickle Cell; Cells, Cultured; Endothelin-1; Endothelium, Vascular; Epoprostenol; Erythrocytes, Abnormal; Hemolysis; Hemorheology; Humans; Interleukin-1; Kinetics

The aim of this study was to determine the response of inflammatory and vasoactive mediators to 3 consecutive days of exercise in African-American women with and without sickle cell anemia (SCA). Circulating inflammatory mediators [C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha)] were measured before, and vasoactive mediators [endothelin-1 (ET-1), nitric oxide metabolites (NOx)] before and after each exercise bout in ten subjects with SCA and ten controls. Exercise did not affect ET-1, IL-6 or CRP concentrations (p >.05). TNFalpha was higher in SCA than controls (p < or = .0005) at all times; however, the response pattern was similar for the groups: no change from day 1 to day 2, but a decrease from day 2 to day 3 (p < or = .05). NOx increased significantly after exercise (p < or = .0001) but returned to baseline by 24 h afterward. On the 3rd day, NOx increased after exercise in SCA but not in the controls (p < or = .05). In conclusion, exercise did not cause a harmful inflammatory response in these individuals with SCA. However, NOx increased after exercise on all 3 days in SCA but appeared attenuated after 2 days in controls.

Topics: Adult; Anemia, Sickle Cell; Biomarkers; C-Reactive Protein; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Exercise; Female; Fluorescence Polarization; Heart Rate; Hematocrit; Hemoglobins; Humans; Immunoassay; Interleukin-6; Nitric Oxide; Pain; Time Factors; Tumor Necrosis Factor-alpha

Endothelin-1 (Et-1) is a vasoconstrictor produced by endothelial and vascular smooth muscle cells in response to hypoxia, which induces hypertrophy in cultured cardiac myocytes. We measured plasma Et-1 levels and left ventricular dimensions in 13 patients with sickle cell anemia (SCD) and in 12 African-American controls ages 16-29 years. Endothelin-1 concentrations are significantly higher in SCD subjects than controls (10.6 +/- 1.9 vs. 3.0 +/- 1.3 pmol/L). There was a negative correlation between oxygen saturation and Et-1 levels in SCD patients (r = -0.71, P = 0.01). SCD subjects have more dilated and hypertrophied hearts corrected for body surface area than controls as evidenced by significant increases in left ventricular end diastolic dimension (31 +/- 0.8 vs. 24 +/- 0.9 mm/m2, P < 0.001), left ventricular end systolic dimension (20 +/- 0.9 vs. 16 +/- 0.8 mm/m2, P = 0.002), left ventricular posterior wall thickness (5.0 +/- 0.1 vs. 4.0 +/- 0.1 mm/m2, P < 0.001), and left ventricular mass (125 +/- 7.2 vs. 69 +/- 5.1 g/m2, P < 0.001). The index of left ventricular function, the shortening fraction, was not different between groups (34 +/- 1.2% in SCD vs. 35 +/- 1.5% in controls). The correlation between left ventricular mass and levels of Et-1 in SCD subjects was not significant (r = 0.47, P = 0.121).

Topics: Adolescent; Adult; Anemia, Sickle Cell; Body Surface Area; Echocardiography; Endothelin-1; Female; Humans; Hypertrophy, Left Ventricular; Male; Organ Size; Oxygen

The relative contributions of microvascular inflammation and vasomotor dysregulation to the development of acute vaso-occlusive crisis in sickle cell disease have been intensely studied. The present observational study was designed to examine the levels of circulating proinflammatory cytokines, anti-inflammatory cytokines, and vasoactive mediators during and after acute painful crisis. In symptomatic sickle cell patients, plasma levels of endothelin-1 and prostaglandin E2 were elevated during crises compared with healthy African-American controls. These levels had decreased, but not normalized, when patients were seen 1 to 3 weeks after discharge from hospital. Other mediators (tumor necrosis factor alpha [TNFalpha], interleukin-1beta [IL-1beta], IL-6, IL-8, and IL-10) were neither elevated in asymptomatic sickle cell disease nor in acute vaso-occlusive crisis. As a potent long-acting mediator of vasoconstriction and inflammation, endothelin-1 may play a key role in the cycle of ischemia and inflammation that initiates and sustains pain of crisis. The downregulatory effects of prostaglandin E2 on immune cell function may contribute to the increased susceptibility to infection observed in patients with sickle cell disease.

Topics: Acute Disease; Adult; Anemia, Sickle Cell; Cell Adhesion Molecules; Cytokines; Dinoprostone; Endothelin-1; Humans; Ischemia; Microcirculation; Middle Aged; Monocytes; Pain; Vascular Diseases; Vasculitis; Vasoconstriction

Topics: Acute Disease; Anemia, Sickle Cell; Endothelin-1; Endothelium, Vascular; Humans

To investigate the role of the endothelial-derived vasoactive mediator endothelin (ET-1) in the acute chest syndrome (ACS), we incubated bovine pulmonary artery endothelial cells (BPAEC) with red blood cells (equivalent to a hematocrit of 20%) and/or autologous plasma (1:10 dilution) from two patients during ACS and during routine clinic visits. Cellular RNA was analyzed for ET-1 transcripts by Northern analysis and ET-1 protein levels in BPAEC supernatants and in plasma measured by radioimmunoassay. ET-1 mRNA expression and protein levels increased in BPAEC exposed to plasma obtained during ACS; in contrast, exposure to plasma obtained during routine clinic visits did not alter BPAEC ET-1 mRNA expression or protein levels. Plasma ET-1 level was elevated during ACS, decreased during resolution, and remained slightly elevated during routine clinic visits. Plasma obtained from one patient 4 d prior to hospitalization for vasoocclusive crisis contained the highest ET-1 level and markedly increased BPAEC ET-1 mRNA expression and protein levels. In both patients, BPAEC ET-1 mRNA and protein expression in vitro and plasma ET-1 levels in vivo correlated with stage of disease and occurred in the absence of direct erythrocyte contact in vitro. These observations suggest that ET-1 production contributes to development of ACS.

Topics: Acute Disease; Adult; Anemia, Sickle Cell; Animals; Cattle; Endothelin-1; Female; Humans; Lung Diseases; Pulmonary Artery; RNA; RNA, Messenger

The molecular defect in sickle cell disease resides in the beta globin gene, with consequent defects in erythrocytes only, suggesting that the vascular occlusion and vasomotor instability which characterize this disease are the result of interactions between abnormal sickle erythrocytes and cells of the blood vessel wall. We explored whether sickle erythrocytes may have effects on vascular tone, exclusive of adhesion events. Exposure of human endothelial cells in culture to previously sickled sickle erythrocytes resulted in a four to eight-fold transcriptional induction of the gene encoding the potent vasoconstrictor endothelin-1 (ET-1). Unsickled sickle erythrocytes or normal erythrocytes exposed to "sickling" conditions had no effect on ET-1 gene induction. Contact of the sickled erythrocytes with the endothelium was not required. Elevations in the ET-1 transcript peaked at 3 h after exposure and persisted for up to 24 h. Four to sixfold increases in the amount of ET-1 peptide was released into the medium surrounding the endothelial cells after exposure to sickled sickle erythrocytes. This is the first demonstration of the regulation of gene expression in endothelial cells as a result of interaction with sickle cells, with induction of genes encoding vasoconstrictors. Furthermore, these findings suggest that sickle erythrocytes may have the capacity to affect local vasomotor tone directly.

Topics: Anemia, Sickle Cell; Cell Hypoxia; Cells, Cultured; Endothelin-1; Endothelins; Endothelium, Vascular; Erythrocytes, Abnormal; Gene Expression Regulation; Humans; Protein Precursors; RNA, Messenger; Transcriptional Activation; Umbilical Veins; Vasoconstriction