fibrin and Hypoxia

What is the topic of this review? Overview of the coagulation abnormalities, including elevated D-dimers widely reported with COVID-19, often labelled as COVID coagulopathy. What advances does it highlight? The review highlights the changes in bronchoalveolar haemostasis due to apoptosis of alveolar cells, which contributes to acute lung injury and acute respiratory distress syndrome; the pathophysiological mechanisms, including endothelial dysfunction and damage responsible for thrombosis of pulmonary microcirculation and potential contribution to the hypoxaemia of COVID-19 acute lung injury; and changes in coagulation proteins responsible for the hypercoagulability and increased risk of thrombosis in other venous and arterial beds. The rationale for anticoagulation and fibrinolytic therapies is detailed, and potential confounders that might have led to less than expected improvement in the various randomised controlled trials are considered.. Coronavirus disease 19 (COVID-19) causes acute lung injury with diffuse alveolar damage, alveolar-capillary barrier disruption, thrombin generation and alveolar fibrin deposition. Clinically, hypoxaemia is associated with preserved lung compliance early in the disease, suggesting the lack of excessive fluid accumulation typical of other lung injuries. Notably, autopsy studies demonstrate infection of the endothelium with extensive capillary thrombosis distinct from the embolic thrombi in pulmonary arteries. The inflammatory thrombosis in pulmonary vasculature secondary to endothelial infection and dysfunction appears to contribute to hypoxaemia. This is associated with elevated D-dimers and acquired hypercoagulability with an increased risk of deep vein thrombosis. Hypercoagulability is secondary to elevated plasma tissue factor levels, von Willebrand factor, fibrinogen, reduced ADAMTS-13 with platelet activation and inhibition of fibrinolysis. Multi-platform randomised controlled studies of systemic therapeutic anticoagulation with unfractionated and low molecular mass heparins demonstrated a survival benefit over standard care with full-dose anticoagulation in patients with non-severe disease who require supplemental oxygen, but not in severe disease requiring ventilatory support. Late intervention and the heterogeneous nature of enrolled patients can potentially explain the apparent lack of benefit in severe disease. Improvement in oxygenation has been demonstrated with intravenous fibrinolytics in small studies. Inhaled anticoagulants, thrombolytic agents and non-specific proteolytic drugs in clinical trials for decreasing alveolar fibrin deposition might benefit early disease. Essentially, COVID-19 is a multi-system disorder with pulmonary vascular inflammatory thrombosis that requires an interdisciplinary approach to combination therapies addressing both inflammation and intravascular thrombosis or alveolar fibrin deposits to improve outcomes.

Topics: Acute Lung Injury; Anticoagulants; COVID-19; Fibrin; Humans; Hypoxia; SARS-CoV-2; Thrombophilia; Thrombosis

The pathomechanisms of hypoxemia and treatment strategies for type H and type L acute respiratory distress syndrome (ARDS) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced coronavirus disease 2019 (COVID-19) have not been elucidated.. SARS-CoV-2 mainly targets the lungs and blood, leading to ARDS, and systemic thrombosis or bleeding. Angiotensin II-induced coagulopathy, SARS-CoV-2-induced hyperfibrin(ogen)olysis, and pulmonary and/or disseminated intravascular coagulation due to immunothrombosis contribute to COVID-19-associated coagulopathy. Type H ARDS is associated with hypoxemia due to diffuse alveolar damage-induced high right-to-left shunts. Immunothrombosis occurs at the site of infection due to innate immune inflammatory and coagulofibrinolytic responses to SARS-CoV-2, resulting in microvascular occlusion with hypoperfusion of the lungs. Lung immunothrombosis in type L ARDS results from neutrophil extracellular traps containing platelets and fibrin in the lung microvasculature, leading to hypoxemia due to impaired blood flow and a high ventilation/perfusion (VA/Q) ratio. COVID-19-associated ARDS is more vascular centric than the other types of ARDS. D-dimer levels have been monitored for the progression of microvascular thrombosis in COVID-19 patients. Early anticoagulation therapy in critical patients with high D-dimer levels may improve prognosis, including the prevention and/or alleviation of ARDS.. Right-to-left shunts and high VA/Q ratios caused by lung microvascular thrombosis contribute to hypoxemia in type H and L ARDS, respectively. D-dimer monitoring-based anticoagulation therapy may prevent the progression to and/or worsening of ARDS in COVID-19 patients.

Topics: Anticoagulants; Biomarkers; Blood Platelets; COVID-19; COVID-19 Drug Treatment; Extracellular Traps; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinolysis; Hemostasis; Humans; Hypoxia; Lung; Microvessels; Phenotype; Respiratory Distress Syndrome; SARS-CoV-2; Thromboinflammation; Thrombosis

Hypoxemia has long been associated with vascular fibrin formation leading to thrombosis. This review describes a pathway through which mononuclear phagocytes and vascular smooth muscle cells upregulate tissue factor under hypoxic conditions. Increased expression of tissue factor triggers events leading to vascular fibrin deposition, providing insight into a novel mechanism potentially underlying thrombosis in ischemic vasculature.

Topics: Animals; Blood Vessels; Fibrin; Humans; Hypoxia; Thromboplastin; Thrombosis

Topics: Adenosine Diphosphate; Blood Platelets; Coronary Vessels; Dyspnea; Fibrin; Histamine; Humans; Hypertension; Hypoxia; Platelet Factor 4; Prostaglandins; Pulmonary Embolism; Serotonin

During the last 25 years, cutaneous biologists have been particularly interested in abnormal cutaneous vascular patterns, the profusion of capillary anastomoses, the leakiness of venules, clotting, fibrinolysis, and blood viscosity. As a result, the effects of hypoxia and the factors that encourage new vessel proliferation are better understood than before. Only when the biologic behavior of the two extremes of growth from hypoplasia to hyperplasia is studied and compared can the blood supply of a tissue be understood. Hyperplastic tissues are seen in wounds, psoriasis, cancer, and in selected sites of chronic stasis and hypoxia where the vessels are extremely permeable, where blood cells easily escape, and where lymphatics dilate and proliferate. The proliferation of other tissues, such as endothelium, epithelium, mast cells, and probably of locally infective organisms, is also encouraged in hyperplasia. Moreover, fibrinolysis does not occur and fibrin is deposited, the electrostatic charge on the internal vascular surface becomes more positive, and the organ is more vulnerable to subsequent injury. Atrophic or hypoplastic tissues have a reduced cellular turnover and are less hypoxic. The vessels are less permeable, blood cells do not escape, there is only a slight tendency to clot, and fibrinolysis is often increased. Lymphatics are sparse and infection is not a feature. The electrostatic charge on the internal surface of the vessel is negative.

Topics: Animals; Blood Viscosity; Capillaries; Cell Membrane; Disseminated Intravascular Coagulation; Fibrin; Fibrinolysis; Humans; Hypoxia; Ischemia; Skin; Skin Neoplasms; Telangiectasis

Topics: Animals; Antifibrinolytic Agents; Autopsy; Blood Coagulation Factors; Dogs; Fibrin; Fibrinolysis; Fibrinolytic Agents; Heart Failure; Humans; Hypoxia; Kidney; Lung; Lymph; Plasminogen Activators; Plasminogen Inactivators; Prostaglandins; Pulmonary Embolism; Pulmonary Veins; Respiration; Respiratory Insufficiency; Syndrome; Thrombin; Wounds and Injuries

Recent evidence supports the - rare - occurrence of vertical transplacental SARS-CoV-2 transmission. We previously determined that placental expression of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor, and associated viral cell entry regulators is upregulated by hypoxia. In the present study, we utilized a clinically relevant model of SARS-CoV-2-associated chronic histiocytic intervillositis/massive perivillous fibrin deposition (CHIV/MPFVD) to test the hypothesis that placental hypoxia may facilitate placental SARS-CoV-2 infection.. We performed a comparative immunohistochemical and/or RNAscope in-situ hybridization analysis of carbonic anhydrase IX (CAIX, hypoxia marker), ACE2 and SARS-CoV-2 expression in free-floating versus fibrin-encased chorionic villi in a 20-weeks' gestation placenta with SARS-CoV-2-associated CHIV/MPVFD.. The levels of CAIX and ACE2 immunoreactivity were significantly higher in trophoblastic cells of fibrin-encased villi than in those of free-floating villi, consistent with hypoxia-induced ACE2 upregulation. SARS-CoV-2 showed a similar preferential localization to trophoblastic cells of fibrin-encased villi.. The localization of SARS-CoV-2 to hypoxic, fibrin-encased villi in this placenta with CHIV/MPVFD suggests placental infection and, therefore, transplacental SARS-CoV-2 transmission may be promoted by hypoxic conditions, mediated by ACE2 and similar hypoxia-sensitive viral cell entry mechanisms. Understanding of a causative link between placental hypoxia and SARS-CoV-2 transmittability may potentially lead to the development of alternative strategies for prevention of intrauterine COVID-19 transmission.

Topics: Adult; Angiotensin-Converting Enzyme 2; Carbonic Anhydrase IX; Chorionic Villi; COVID-19; Female; Fibrin; Gestational Age; Histiocytes; Humans; Hypoxia; Infectious Disease Transmission, Vertical; Necrosis; Placenta; Pregnancy; Pregnancy Complications, Infectious; SARS-CoV-2; Stillbirth; Trophoblasts

Perinatal death is an increasingly important problem as the coronavirus disease 2019 (COVID-19) pandemic continues, but the mechanism of death has been unclear.. To evaluate the role of the placenta in causing stillbirth and neonatal death following maternal infection with COVID-19 and confirmed placental positivity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).. Case-based retrospective clinicopathologic analysis by a multinational group of 44 perinatal specialists from 12 countries of placental and autopsy pathology findings from 64 stillborns and 4 neonatal deaths having placentas testing positive for SARS-CoV-2 following delivery to mothers with COVID-19.. Of the 3 findings constituting SARS-CoV-2 placentitis, all 68 placentas had increased fibrin deposition and villous trophoblast necrosis and 66 had chronic histiocytic intervillositis. Sixty-three placentas had massive perivillous fibrin deposition. Severe destructive placental disease from SARS-CoV-2 placentitis averaged 77.7% tissue involvement. Other findings included multiple intervillous thrombi (37%; 25 of 68) and chronic villitis (32%; 22 of 68). The majority (19; 63%) of the 30 autopsies revealed no significant fetal abnormalities except for intrauterine hypoxia and asphyxia. Among all 68 cases, SARS-CoV-2 was detected from a body specimen in 16 of 28 cases tested, most frequently from nasopharyngeal swabs. Four autopsied stillborns had SARS-CoV-2 identified in internal organs.. The pathology abnormalities composing SARS-CoV-2 placentitis cause widespread and severe placental destruction resulting in placental malperfusion and insufficiency. In these cases, intrauterine and perinatal death likely results directly from placental insufficiency and fetal hypoxic-ischemic injury. There was no evidence that SARS-CoV-2 involvement of the fetus had a role in causing these deaths.

Topics: COVID-19; Female; Fibrin; Humans; Hypoxia; Infant, Newborn; Infectious Disease Transmission, Vertical; Perinatal Death; Placenta; Pregnancy; Pregnancy Complications, Infectious; Retrospective Studies; SARS-CoV-2; Stillbirth

The Coronavirus disease 2019 (COVID)-19 pandemic has already affected millions worldwide, with a current mortality rate of 2.2%. While it is well-established that severe acute respiratory syndrome-coronavirus-2 causes upper and lower respiratory tract infections, a number of neurological sequelae have now been reported in a large proportion of cases. Additionally, the disease causes arterial and venous thromboses including pulmonary embolism, myocardial infarction, and a significant number of cerebrovascular complications. The increasing incidence of large vessel ischemic strokes as well as intracranial hemorrhages, frequently in younger individuals, and associated with increased morbidity and mortality, has raised questions as to why the brain is a major target of the disease. COVID-19 is characterized by hypercoagulability with alterations in hemostatic markers including high D-dimer levels, which are a prognosticator of poor outcome. Together with findings of fibrin-rich microthrombi, widespread extracellular fibrin deposition in affected various organs and hypercytokinemia, this suggests that COVID-19 is more than a pulmonary viral infection. Evidently, COVID-19 is a thrombo-inflammatory disease. Endothelial cells that constitute the lining of blood vessels are the primary targets of a thrombo-inflammatory response, and severe acute respiratory syndrome coronavirus 2 also directly infects endothelial cells through the ACE2 (angiotensin-converting enzyme 2) receptor. Being highly heterogeneous in their structure and function, differences in the endothelial cells may govern the susceptibility of organs to COVID-19. Here, we have explored how the unique characteristics of the cerebral endothelium may be the underlying reason for the increased rates of cerebrovascular pathology associated with COVID-19.

Topics: Angiotensin-Converting Enzyme 2; Blood Coagulation; Brain; Brain Ischemia; COVID-19; Cytokines; Endothelial Cells; Fibrin; Fibrin Fibrinogen Degradation Products; Hemostasis; Humans; Hypoxia; Incidence; Inflammation; Ischemic Stroke; Myocardial Infarction; Pandemics; Prognosis

The aim of this study was to analyze the three-dimensional distribution of hypoxia in the arteriovenous (AV) loop model in rats, by examining the distribution of hypoxia-inducible factor-1 alpha (HIF-1α).. AV loops were created from the femoral artery and vein of male Lewis rats and an interpositional graft from the contralateral femoral vein. This AV fistula was embedded in a fibrin-filled isolation chamber and subcutaneously implanted into the thigh. The specimens were harvested after 7 days (n = 4), 10 days (n = 5), and 14 days (n = 4). The fibrin clots were stained for lectin, HIF-1α, and ectodysplasin 1 (ED1). The distribution of positive and negative cells was analyzed in three dimensions and at different points in time.. The HIF-1α-positive rate increased from the proximity of the central vessel to the distant regions. From day 7 to 10, we noted a decrease in the HIF-1α-positive rate in the proximity of the vessels and an increase in the periphery. A global decrease in positive cells was seen at day 14. HIF-1α and macrophage (ED1) double staining indicated that macrophages accounted for a significant fraction of the cells. Double staining for endothelium (with lectin) demonstrated that no HIF-1α was detectable in well-vascularized areas.. In the AV loop model, the HIF-1α-positive cell distribution is highly related to the vascularization process. The onset of rapid vessel outgrowth follows the increase of the HIF-1α rate closely, indicating that HIF-1α may be a driving force for vascularization.

Topics: Animals; Fibrin; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Neovascularization, Pathologic; Rats; Rats, Inbred Lew; Tissue Engineering

Conceptually, premature initiation of post-wound angiogenesis could interfere with hemostasis, as it relies on fibrinolysis. The mechanisms facilitating orchestration of these events remain poorly understood, however, likely due to limitations in discerning the individual contribution of cells and extracellular matrix. Here, we designed an in vitro Hemostatic-Components-Model (HCM) to investigate the role of the fibrin matrix as protein factor-carrier, independent of its cell-scaffold function. After characterizing the proteomic profile of HCM-harvested matrix releasates, we demonstrate that the key pro-/anti-angiogenic factors, VEGF and PF4, are differentially bound by the matrix. Changing matrix fibrin mass consequently alters the balance of releasate factor concentrations, with differential effects on basic endothelial cell (EC) behaviors. While increasing mass, and releasate VEGF levels, promoted EC chemotactic migration, it progressively inhibited tube formation, a response that was dependent on PF4. These results indicate that the clot's matrix component initially serves as biochemical anti-angiogenic barrier, suggesting that post-hemostatic angiogenesis follows fibrinolysis-mediated angiogenic disinhibition. Beyond their significance towards understanding the spatiotemporal regulation of wound healing, our findings could inform the study of other pathophysiological processes in which coagulation and angiogenesis are prominent features, such as cardiovascular and malignant disease.

Topics: Angiogenesis Inducing Agents; Blood Coagulation; Cell Movement; Endothelial Cells; Extracellular Matrix; Fibrin; Hemostasis; Humans; Hypoxia; Neovascularization, Physiologic; Oxygen; Protein Binding; Signal Transduction; Wound Healing

Metastasis is the cause of over 90% of all human cancer deaths. Early steps in the metastatic process include: the formation of new blood vessels, the initiation of epithelial-mesenchymal transition (EMT), and the mobilization of tumor cells into the circulation. There are ongoing efforts to replicate the physiological landscape of human tumor tissue using three-dimensional in vitro culture models; however, few systems are able to capture the full range of authentic, complex in vivo events such as neovascularization and intravasation. Here we introduce the Prevascularized Tumor (PVT) model to investigate early events of solid tumor progression. PVT spheroids are composed of endothelial and tumor cells, and are embedded in a fibrin matrix containing fibroblasts. The PVT model facilitates two mechanisms of vessel formation: robust sprouting angiogenesis into the matrix, and contiguous vascularization within the spheroid. Furthermore, the PVT model enables the intravasation of tumor cells that is enhanced under low oxygen conditions and is also dependent on the key EMT transcription factor Slug. The PVT model provides a significant advance in the mimicry of human tumors in vitro, and may improve investigation and targeting of events in the metastatic process.

Topics: Blotting, Western; Cell Line, Tumor; Endothelial Cells; Epithelial-Mesenchymal Transition; Fibrin; Humans; Hypoxia; In Vitro Techniques; Microscopy, Fluorescence; Neoplasms; Neovascularization, Pathologic; RNA, Small Interfering; Snail Family Transcription Factors; Transcription Factors

It has been demonstrated that co-treatment of rats with amiodarone (AMD) and bacterial lipopolysaccharide (LPS) produces idiosyncrasy-like liver injury. In this study, the hypothesis that the hemostatic system and neutrophils contribute to AMD/LPS-induced liver injury was explored. Rats were treated with AMD (400 mg/kg, ip) or vehicle and 16 h later with LPS (1.6×10⁶ endotoxin units/kg, iv) or saline (Sal). AMD did not affect the hemostatic system by itself but significantly potentiated LPS-induced coagulation activation and fibrinolysis impairment. Increased hepatic fibrin deposition and subsequent hypoxia were observed only in AMD/LPS-treated animals, starting before the onset of liver injury. Administration of anticoagulant heparin abolished AMD/LPS-induced hepatic fibrin deposition and reduced AMD/LPS-induced liver damage. Polymorphonuclear neutrophils (PMNs) accumulated in liver after treatment with LPS or AMD/LPS, but PMN activation was only observed in AMD/LPS-treated rats. Rabbit anti-rat PMN serum, which reduced accumulation of PMNs in liver, prevented PMN activation and attenuated AMD/LPS-induced liver injury in rats. PMN depletion did not affect hepatic fibrin deposition. Anticoagulation prevented PMN activation without affecting PMN accumulation. In summary, both the hemostatic system alteration and PMN activation contributed to AMD/LPS-induced liver injury in rats, in which fibrin deposition was critical for the activation of PMNs.

Topics: Amiodarone; Animals; Anticoagulants; Blood Coagulation; Chemical and Drug Induced Liver Injury; Fibrin; Fibrinolysis; Hemostasis; Heparin; Hypoxia; Lipopolysaccharides; Liver; Male; Neutrophil Activation; Neutrophils; Rats; Rats, Sprague-Dawley; Time Factors

Hypoxemia in the circulation can lead to venous thrombosis (VT) through tissue factor (TF) activation, but the mechanism of TF activation in hypoxia remains obscure. Ligands released from damaged tissues or cells due to hypoxia are identified by various pattern-recognition receptors (PRR), including Toll-like receptor3 (TLR3). In the present study, we investigated the mechanism of TF activation during acute hypoxia in a rat model. The expression of TLR3 and TF was analyzed by immunoblotting and RT-PCR. The TF activity was evaluated by two-stage chromogenic assay and fibrin deposition was detected by immunohistochemistry. The expression of TLR3, TF, and TF activity was increased significantly 6 h post acute hypoxia and then decreased gradually. The contribution of TLR3 in TF activation was investigated by poly I:C and TLR3 neutralizing antibody. We also found increased ERK phosphorylation both in acute hypoxia and poly I:C treatment. We further showed that the pre-treatment of TLR3 neutralizing antibody or ERK inhibitor (PD98059) 2 h prior to acute hypoxia or poly I:C treatment completely abrogated ERK phosphorylation and TF activation. The pre-treatment of TLR3 neutralizing antibody also inhibited fibrin deposition in lung vasculature. These data indicate that acute hypoxia induced TF activation is mediated through TLR3-ERK1/2 pathway.

Topics: Animals; Antibodies, Neutralizing; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Fibrin; Flavonoids; Gene Expression Regulation; Hypoxia; Male; MAP Kinase Signaling System; Phosphorylation; Poly I-C; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Thromboplastin; Toll-Like Receptor 3

To report on the histopathologic findings of affected skin in consecutively collected biopsy specimens from 49 patients with erythromelalgia (EM).. Skin biopsy specimens were obtained from the foot arch and analyzed by light microscopy, immunofluorescence microscopy, and electron microscopy.. Oslo University Hospital-Gaustad, University of Oslo, Oslo, Norway.. Thirty-one patients had primary EM, 17 patients had secondary EM, and 1 patient had erythromelalgic syndrome.. Evidence of microvascular abnormalities in skin biopsy specimens.. Light microscopy showed evidence of capillary proliferation in 10 of 31 patients with primary EM and in 1 of 17 patients with secondary EM. The biopsy specimen from the patient with erythromelalgic syndrome showed numerous capillary nests with endothelial cell defects and a slight perivascular inflammatory reaction. Among the 17 secondary EM cases, sparse perivascular lymphocyte infiltrations were observed in the biopsy specimens from 2 patients with chronic myelogenous leukemia and 1 patient with diabetes mellitus. Eleven patients also had signs of vasculopathy based on findings of immunodeposits of C3 and fibrin. Six of 30 patients with primary EM showed endothelial abnormalities on electron microscopy. All 3 investigations showed unremarkable biopsy results in 16 cases.. Histopathologic analysis is not useful as a routine diagnostic tool in EM because no morphological changes are specific to EM. The capillary proliferation and vasculopathy are assumed to be a consequence of intermittent skin hypoxia (vascular hypothesis of pathogenesis). Whether the proliferation is a consequence of EM or a pathogenic factor in the development of the disease is uncertain.

Topics: Adult; Aged; Biopsy; Capillaries; Complement C3; Erythromelalgia; Female; Fibrin; Humans; Hypoxia; Male; Microscopy; Microscopy, Electron; Microscopy, Fluorescence; Middle Aged; Norway; Prospective Studies; Skin; Syndrome

Oxygen-induced retinopathy (OIR) is a well-characterized model for retinopathy of prematurity, a disorder that results from rapid microvascular proliferation after exposure of the retina to high oxygen levels. Here, we report that the proliferative phase of OIR requires transcriptional induction of the annexin A2 (A2) gene through the direct action of the hypoxia-inducible factor-1 complex. We show, in addition, that A2 stabilizes its binding partner, p11, and promotes OIR-related angiogenesis by enabling clearance of perivascular fibrin. Adenoviral-mediated restoration of A2 expression restores neovascularization in the oxygen-primed Anxa2(-/-) retina and reinstates plasmin generation and directed migration in cultured Anxa2(-/-) endothelial cells. Systemic depletion of fibrin repairs the neovascular response to high oxygen treatment in the Anxa2(-/-) retina, whereas inhibition of plasminogen activation dampens angiogenesis under the same conditions. These findings show that the A2 system enables retinal neoangiogenesis in OIR by enhancing perivascular activation of plasmin and remodeling of fibrin. These data suggest new potential approaches to retinal angiogenic disorders on the basis of modulation of perivascular fibrinolysis.

Topics: Animals; Annexin A2; Aorta; Blotting, Western; Cell Movement; Cells, Cultured; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Fibrin; Fibrinolysin; Fibrinolysis; Flow Cytometry; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoenzyme Techniques; Immunoprecipitation; Luciferases; Mice; Mice, Inbred C57BL; Mice, Knockout; Neovascularization, Pathologic; Oxygen; Plasminogen Activators; Promoter Regions, Genetic; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction; RNA Stability; RNA, Messenger; Transfection; Umbilical Veins

Disruption of microenvironment and decrease in oxygen supply during isolation and culture lead to pancreatic islet injury and their poor survival after transplantation. This study aimed to create a matrix for culturing islets, using fibrin as scaffold and perfluorodecalin as oxygen diffusion enhancing medium. Human pancreatic islets were divided in four groups: control, islets cultured in fibrin, islets in fibrin containing non-emulsified perfluorodecalin, and finally islets in fibrin supplemented with emulsified perfluorodecalin. After an overnight culture, cell damage (viability, proinsulin and insulin unregulated release, apoptosis (caspase-3 activation), secretory function, and presence of hypoxia markers (HIF-1a and VEGF expression) were assessed. Islets cultured in a matrix, had similar islet viability to controls (no matrix) but decreased levels of active caspase-3 and unregulated hormone release, but high level of hypoxia markers expression. Although the supplementation of fibrin with non-emulsified perfluorodecalin improves secretory response, there was no decrease in hypoxia markers expression. In contrast, emulsified perfluorodecalin added to the matrix improved islet function, islet viability and maintained level of hypoxia markers similar to control. Fibrin matrix supplemented with emulsified perfluorodecalin can provide a beneficial physical and chemical environment for improved pancreatic human islet function and viability in vitro.

Topics: Cell Nucleus; Extracellular Matrix; Fibrin; Fluorocarbons; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin; Islets of Langerhans; Organ Culture Techniques; Protein Transport; Tissue Survival; Vascular Endothelial Growth Factor A

Evidence suggests a role of both innate and adaptive immunity in the development of pulmonary arterial hypertension. The complement system is a key sentry of the innate immune system and bridges innate and adaptive immunity. To date there are no studies addressing a role for the complement system in pulmonary arterial hypertension.. Immunofluorescent staining revealed significant C3d deposition in lung sections from IPAH patients and C57Bl6/J wild-type mice exposed to three weeks of chronic hypoxia to induce pulmonary hypertension. Right ventricular systolic pressure and right ventricular hypertrophy were increased in hypoxic vs. normoxic wild-type mice, which were attenuated in C3-/- hypoxic mice. Likewise, pulmonary vascular remodeling was attenuated in the C3-/- mice compared to wild-type mice as determined by the number of muscularized peripheral arterioles and morphometric analysis of vessel wall thickness. The loss of C3 attenuated the increase in interleukin-6 and intracellular adhesion molecule-1 expression in response to chronic hypoxia, but not endothelin-1 levels. In wild-type mice, but not C3-/- mice, chronic hypoxia led to platelet activation as assessed by bleeding time, and flow cytometry of platelets to determine cell surface P-selectin expression. In addition, tissue factor expression and fibrin deposition were increased in the lungs of WT mice in response to chronic hypoxia. These pro-thrombotic effects of hypoxia were abrogated in C3-/- mice.. Herein, we provide compelling genetic evidence that the complement system plays a pathophysiologic role in the development of PAH in mice, promoting pulmonary vascular remodeling and a pro-thrombotic phenotype. In addition we demonstrate C3d deposition in IPAH patients suggesting that complement activation plays a role in the development of PAH in humans.

Topics: Animals; Arterioles; Biomarkers; Cell Proliferation; Chronic Disease; Complement C3; Complement C3a; Complement C5a; Endothelium, Vascular; Fibrin; Gene Deletion; Humans; Hypertension, Pulmonary; Hypoxia; Mice; Mice, Inbred C57BL; Myocytes, Smooth Muscle; Platelet Activation; Pulmonary Artery; Thromboplastin; Up-Regulation

Assessing state of various hemostasis levels in patients having pulmonary diseases caused by dust, in accordance with respiratory failure severity, enables to diagnose early changes confirming hypoxia. The most reliable parameters are changes in vascular and platelet hemostasis and in fibrinolysis system.

Topics: Biomarkers; Bronchitis; Disease Progression; Early Diagnosis; Fibrin; Hemostasis; Humans; Hypoxia; Lung Diseases; Middle Aged; Occupational Exposure; Particulate Matter; Platelet Function Tests; Respiratory Insufficiency; Thrombin

As one of the most important endogenous chemotactic factors for neutrophils, the chemokine CXCL8 (IL-8) is involved in the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), characterized by massive neutrophil infiltration in the lung. Since neutralization of CXCL8 with polyclonal antibody has been shown to reduce the severity of ALI/ARDS in animal models, we explored the potential of humanized anti-CXCL8 antibody as a preventive or therapeutic agent for ALI. We used a 'two-hit' protocol to induce ALI in rabbits that showed extensive edema in the alveolar lumina, marked infiltration of neutrophils in the lung tissue, fibrin deposition in alveolar space, and destruction of pulmonary architecture, culminating in severe hypoxemia. Concomitant challenge with endotoxin after priming with oleic acid (OA) induced a marked elevation of CXCL8 level in bronchoalveolar lavage fluid. Treatment of the rabbits with a humanized anti-CXCL8 antibody prevented neutrophil infiltration in the lung in association with alleviated ALI syndrome. Our results indicate a promising future for utilization of humanized anti-CXCL8 antibody in the prevention and treatment of ALI and ARDS in human.

Topics: Acute Lung Injury; Animals; Antibodies, Monoclonal; Bronchoalveolar Lavage; Capillary Permeability; Edema; Endotoxins; Female; Fibrin; Hypoxia; Interleukin-1; Interleukin-8; Lung; Male; Mice; Neutrophils; Oleic Acid; Rabbits; Respiratory Distress Syndrome; Tumor Necrosis Factor-alpha

Sulindac (SLD) is a nonsteroidal anti-inflammatory drug (NSAID) that has been associated with a greater incidence of idiosyncratic hepatotoxicity in human patients than other NSAIDs. One hypothesis regarding idiosyncratic adverse drug reactions is that interaction of a drug with a modest inflammatory episode precipitates liver injury. In this study, we tested the hypothesis that lipopolysaccharide (LPS) interacts with SLD to cause liver injury in rats. SLD (50 mg/kg) or its vehicle was administered to rats by gavage 15.5 h before LPS (8.3 x 10(5) endotoxin unit/kg) or its saline vehicle (i.v.). Thirty minutes after LPS treatment, SLD or vehicle administration was repeated. Rats were killed at various times after treatment, and serum, plasma, and liver samples were taken. Neither SLD nor LPS alone caused liver injury. Cotreatment with SLD/LPS led to increases in serum biomarkers of both hepatocellular injury and cholestasis. Histological evidence of liver damage was found only after SLD/LPS cotreatment. As a result of activation of hemostasis induced by SLD/LPS cotreatment, fibrin and hypoxia were present in liver tissue before the onset of hepatotoxicity. Heparin treatment reduced hepatic fibrin deposition and hypoxia and protected against liver injury induced by SLD/LPS cotreatment. These results indicate that cotreatment with nontoxic doses of LPS and SLD causes liver injury in rats, and this could serve as a model of human idiosyncratic liver injury. The hemostatic system is activated by SLD/LPS cotreatment and plays an important role in the development of SLD/LPS-induced liver injury.

Topics: Analysis of Variance; Animals; Chemical and Drug Induced Liver Injury; Cholestasis; Dose-Response Relationship, Drug; Fibrin; Hemostasis; Heparin; Hypoxia; Lipopolysaccharides; Liver; Male; Rats; Sulindac; Tumor Necrosis Factor-alpha

This study investigated the connection among HER-2 gene amplification, HER-2 protein expression, and markers of tumor angiogenesis and oxygenation in patients with operable, invasive breast tumors.. From 1988 to 1995, 425 patients with metastatic breast cancer were enrolled in a study of high-dose chemotherapy with autologous transplant. Primary tumor blocks were obtained and evaluated using immunohistochemistry (IHC) staining of vessels with von Willebrand factor antibody. Mean microvessel densities (MVD) were determined by counting von Willebrand factor stained cells in three separate "vascular hot spots" using image analysis. Tumor samples were also stained for HER-2 by IHC, HER-2 gene amplification by fluorescence in situ hybridization, carbonic anhydrase 9 by IHC, and vascular endothelial growth factor (VEGF) by IHC. Plasma from 36 patients with primary tumor samples had VEGF (R&D Systems, MN) and d-dimer (American Diagnostica, Greenwich, CT) levels determined.. There was a significant positive correlation between HER-2 gene amplification and both maximum and average MVD (Spearman coefficient = 0.51 and 0.50; P = 0.03 and 0.05, respectively). There was an inverse correlation with HER-2 gene amplification and expression of the tumor hypoxia marker CA-9 (chi(2) P = 0.02). The level of HER-2 gene amplification correlated with plasma d-dimer levels (Spearman coefficient = 0.43; P = 0.021). Interestingly, tumors with HER-2 by IHC had decreased amounts of VEGF staining (chi(2) = 5.81; P = 0.01). There was no correlation between HER-2 by IHC and MVD or d-dimer. Of all of the variables examined, only average (P = 0.0016) and maximum MVD (P = 0.0128) predicted disease-free survival (Cox univariate model).. HER-2-amplified breast cancers have increased amounts of angiogenesis, decreased amounts of hypoxia, and increased markers of fibrin degradation. These findings have prognostic, predictive, and therapeutic implications in breast cancer treatment.

Topics: Breast Neoplasms; Carbonic Anhydrases; Disease-Free Survival; Enzyme-Linked Immunosorbent Assay; Female; Fibrin; Fibrin Fibrinogen Degradation Products; Humans; Hypoxia; Immunohistochemistry; In Situ Hybridization, Fluorescence; Microcirculation; Neoplasms; Neovascularization, Pathologic; Oxygen; Prognosis; Receptor, ErbB-2; Time Factors; Vascular Endothelial Growth Factor A; von Willebrand Factor

Coadministration of nonhepatotoxic doses of the histamine 2-receptor antagonist ranitidine (RAN) and bacterial lipopolysaccharide (LPS) results in hepatocellular injury in rats, the onset of which occurs in 3 to 6 hours. This reaction resembles RAN idiosyncratic hepatotoxicity in humans. Early fibrin deposition occurs in livers of rats cotreated with LPS/RAN. Accordingly, we tested the hypothesis that the hemostatic system contributes to liver injury in LPS/RAN-treated rats. Rats were given either LPS (44.4 x 10(6) EU/kg) or its vehicle, then RAN (30 mg/kg) or its vehicle 2 hours later. They were killed 2, 3, 6, 12, or 24 hours after RAN treatment, and liver injury was estimated from serum alanine aminotransferase activity. A modest elevation in serum hyaluronic acid, which was most pronounced in LPS/RAN-cotreated rats, suggested altered sinusoidal endothelial cell function. A decrease in plasma fibrinogen and increases in thrombin-antithrombin dimers and in serum concentration of plasminogen activator inhibitor-1 occurred before the onset of liver injury. Hepatic fibrin deposition was observed in livers from LPS/RAN-cotreated rats 3 and 6 hours after RAN. Liver injury was abolished by the anticoagulant heparin and was significantly attenuated by the fibrinolytic agent streptokinase. Hypoxia, one potential consequence of sinusoidal fibrin deposition, was observed in livers of LPS/RAN-treated rats. In conclusion, the results suggest that the hemostatic system is activated after LPS/RAN cotreatment and that fibrin deposition in liver is important for the genesis of hepatic parenchymal cell injury in this model.

Topics: Animals; Anti-Ulcer Agents; Blood Coagulation; Chemical and Drug Induced Liver Injury; Endothelial Cells; Fibrin; Fibrinolytic Agents; Heparin; Hypoxia; Lipopolysaccharides; Liver; Male; Plasminogen Activator Inhibitor 1; Ranitidine; Rats; Rats, Sprague-Dawley; Streptokinase

Angiogenesis, or the formation of new microvessels, is often encountered in pathological situations. A fibrinous exudate can often act as a temporary matrix for the ingrowth of these new microvessels. This matrix consists mainly of fibrin, but is mingled with other plasma components and interstitial collagen fibres. In vitro, capillary-like tube formation can be mimicked by exposing human microvascular endothelial cells (hMVECs), seeded on top of a three-dimensional fibrin matrix, to an angiogenic growth factor (e.g. fibroblast growth factor (FGF)-2) and the cytokine tumour necrosis factor (TNF)-alpha. Plasmin activity is required in this process. We investigated whether the angiogenic potential of hMVECs was altered by the presence of collagen. The addition of type I collagen to fibrin matrices dose-dependently inhibited tube-formation. Tube-formation in these fibrin/collagen matrices by hMVECs required matrix metalloprotease (MMP) activity, as well as plasmin activity. On a pure collagen type I matrix, hMVECs were not able to form tube-like structures in the matrix but formed sprouts. This sprouting required MMP activity and was, in contrast to the tube-like structures in a fibrin matrix, not influenced by hypoxia. These data indicate that the interaction between endothelial cells and different matrix components is of importance for the angiogenic potential of these cells.

Topics: Cell Culture Techniques; Collagen Type I; Dose-Response Relationship, Drug; Endothelium, Vascular; Fibrin; Humans; Hypoxia; Matrix Metalloproteinases; Microcirculation; Neovascularization, Physiologic; Urokinase-Type Plasminogen Activator

Fibrin deposition is a salient feature of hypoxemic vasculature and results from induction of tissue factor. Such tissue factor expression in an oxygen deficient environment is driven by the transcription factor Early Growth Response (Egr)-1. Using homozygous null mice for the protein kinase C beta-isoform gene (PKCbeta null), PKCbeta is shown to be upstream of Egr-1 in this oxygen deprivation-mediated pathway for triggering procoagulant events. Whereas wild-type mice exposed to hypoxia (6%) displayed a robust increase in tissue factor transcripts and antigen, and vascular fibrin deposition, PKCbeta null animals showed a markedly blunted response. Consistent with a central role for Egr-1 in hypoxia-induced expression of tissue factor, PKCbeta null mice subjected to oxygen deprivation displayed at most a minor elevation in Egr-1 transcripts, antigen, and intensity of the gel shift band by electrophoretic mobility shift assay, compared with normoxic animals. These data firmly establish PKCbeta as a trigger for events leading to induction of Egr-1 and tissue factor under hypoxic conditions, and provide insight into a biologic cascade whereby oxygen deprivation recruits targets of PKCbeta and Egr-1, thereby amplifying the cellular response.

Topics: Animals; DNA-Binding Proteins; Early Growth Response Protein 1; Endothelium, Vascular; Enzyme Activation; Fibrin; Gene Expression Regulation, Enzymologic; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Immediate-Early Proteins; Isoenzymes; Lung; Macrophages, Alveolar; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nuclear Proteins; Oxygen Consumption; Protein Kinase C; Protein Kinase C beta; Transcription Factors

Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix. hMVECs stimulated with fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor (VEGF) together with tumor necrosis factor-alpha (TNF-alpha) formed 2- to 3-fold more tubular structures under hypoxic conditions than in normoxic (20% oxygen) conditions. In both conditions the in-growth of capillary-like tubular structures into fibrin required cell-bound urokinase-type plasminogen activator (uPA) and plasmin activities. The hypoxia-induced increase in tube formation was accompanied by a decrease in uPA accumulation in the conditioned medium. This decrease in uPA level was completely abolished by uPA receptor-blocking antibodies. During hypoxic culturing uPA receptor activity and messenger RNA (mRNA) were indeed increased. This increase and, as a consequence, an increase in plasmin formation contribute to the hypoxia-induced stimulation of tube formation. A possible contribution of VEGF-A to the increased formation under hypoxic conditions is unlikely because there was no increased VEGF-A expression detected under hypoxic conditions, and the hypoxia-induced tube formation by FGF-2 and TNF-alpha was not inhibited by soluble VEGFR-1 (sVEGFR-1), or by antibodies blocking VEGFR-2. Furthermore, although the alpha(v)-integrin subunit was enhanced by hypoxia, blocking antibodies against alpha(v)beta(3)- and alpha(v)beta(5)-integrins had no effect on hypoxia-induced tube formation. Hypoxia increases uPA association and the angiogenic response of human endothelial cells in a fibrin matrix; the increase in the uPA receptor is an important determinant in this process. (Blood. 2000;96:2775-2783)

Topics: Antigens, CD; Cell Hypoxia; Cell Survival; Cells, Cultured; Culture Media; Culture Media, Conditioned; DNA, Complementary; Endothelial Growth Factors; Endothelium, Vascular; Extracellular Matrix; Fibrin; Fibrinolysin; Fibroblast Growth Factor 2; Gene Expression Regulation; Humans; Hypoxia; Integrin alphaV; Integrins; Lymphokines; Morphogenesis; Neovascularization, Pathologic; Oxygen; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptors, Cell Surface; Receptors, Growth Factor; Receptors, Urokinase Plasminogen Activator; Receptors, Vascular Endothelial Growth Factor; Receptors, Vitronectin; RNA, Messenger; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors

To investigate changes of vWF in vascular endothelial cells with acute hypoxia and assess its significance in the study of vascular injury and hypercoagulability.. vWF in vascular endothelial cells and in blood were measured by immunohistochemical staining image analysis and ELISA technique. Fibrin was displayed by phosphotungstic hematoxylin staining.. The amount of vWF in vascular endothelial cells decreased progressively, while in blood increased progressively with hypoxia. Fibrin prominently increased with hypoxia.. Acute hypoxia caused severe injury of vascular endothelial cells which resulted in release of vWF into blood from endothelial cells. The vWF amounts in blood may reflect the hypercoagulability, and may serve as a useful indicator of vascular injury and hypercoagulability.

Topics: Animals; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Fibrin; Hypoxia; Immunohistochemistry; Male; Rats; Rats, Wistar; von Willebrand Factor

Local hypoxemia and stasis trigger thrombosis. We have demonstrated previously that in a murine model of normobaric hypoxia pulmonary fibrin deposition is a result of expression of tissue factor, especially in oxygen-deprived mononuclear phagocytes (MPs). We now show that transcription factor early-growth-response gene product (Egr-1) is rapidly activated in hypoxia, both in vitro and in vivo, and is responsible for transcription and expression of tissue factor in hypoxic lung. MPs and HeLa cells subjected to hypoxia (pO2 approximately 13 torr) had increased levels of tissue factor transcripts (approximately 18-fold) and an increased rate of transcription (approximately 15-fold), based on nuclear run-on analysis. Gel-shift analysis of nuclear extracts from hypoxic MPs and HeLa cells demonstrated increased DNA-binding activity at the serum response region (SRR; -111/+14 bp) of the tissue factor promoter at Egr-1 motifs. Using 32P-labeled Egr consensus oligonucleotide, we observed induction of DNA-binding activity in nuclear extracts from hypoxic lung and HeLa cells because of activation of Egr-1, by means of supershift analysis. Transient transfection of HeLa cells with chimeric plasmids containing wild-type or mutant SRR from the tissue factor promoter showed that intact Sp1 sites are necessary for basal promoter activity, whereas the integrity of Egr-1 sites was required for hypoxia-enhanced expression. A central role for Egr-1 in hypoxia-mediated tissue factor expression was confirmed by experiments with homozygous Egr-1 null mice; wild-type mice subjected to oxygen deprivation expressed tissue factor and showed fibrin deposition, but hypoxic homozygous Egr-1 null mice displayed neither tissue factor nor fibrin. These data delineate a novel biology for hypoxia-induced fibrin deposition, in which oxygen deprivation-induced activation of Egr-1, resulting in expression of tissue factor, has an unexpected and central role.

Topics: Animals; DNA-Binding Proteins; Early Growth Response Protein 1; Fibrin; HeLa Cells; Humans; Hypoxia; Immediate-Early Proteins; Leukocytes, Mononuclear; Lung; Mice; Mice, Knockout; Oxygen; Thromboplastin; Transcription Factors; Transcription, Genetic

Topics: Animals; Cyclic AMP; Fibrin; Heart; Heart Transplantation; Hypoxia; Interleukin-6; Leukostasis; Mice; Myocardial Reperfusion Injury; Myocardium; Organ Preservation; P-Selectin; Rats; Second Messenger Systems; Translocation, Genetic

Angiogenesis and coronary artery collateral formation can improve blood flow and thereby prevent myocardial ischemia. The role of perivascular fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibroblasts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between different cell types, we either stained endothelial cells or fibroblasts in the living with 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine-perchlorate (DiI). In cocultures, low numbers of heart fibroblasts stimulated endothelial sprouting, and capillary growth was also induced by fibroblast-conditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultures, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, in cell death. Depending on the local ratio of fibroblasts to endothelial cell numbers, fibroblasts determined the location of capillary growth and the size of developing capillaries and thereby contributed to capillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of endothelial sprouts. NIH fibroblasts were frequently seen in close association with endothelial capillaries, resembling pericytes. Contact-dependent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-beta1 antibodies, by addition of serum, of medium conditioned by hypoxic tumor cells or myocardium, by various cytokines or by growing cocultures under hypoxic conditions. Our results implicate a pivotal role of periendothelial mesenchymal cells for the regulation of microvascular network remodeling and collateral formation.

Topics: Animals; Carbocyanines; Cell Communication; Coculture Techniques; Collateral Circulation; Coronary Circulation; Coronary Vessels; Culture Media, Conditioned; Culture Media, Serum-Free; Endothelium, Vascular; Fibrin; Fibroblasts; Gels; Humans; Hypoxia; Myocardium; Neovascularization, Physiologic; Swine

Oxygen deprivation, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. To investigate the effects of low ambient oxygen tension on the fibrinolytic system, mice were placed in a hypoxic environment with pO2 < 40 Torr. Plasma levels of plasminogen activator inhibitor-1 (PAI-1) antigen, detected by ELISA, increased in a time-dependent fashion after hypoxic exposure (increased as early as 4 h, P < 0.05 vs. normoxic controls), and were accompanied by an increase in plasma PAI-1 activity by 4 h (P < 0.05 vs. normoxic controls). Northern analysis of hypoxic murine lung demonstrated an increase in PAI-1 mRNA compared with normoxic controls; in contrast, transcripts for both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) decreased under hypoxic conditions. Immunocolocalization studies identified macrophages as the predominant source of increased PAI-1 within hypoxic lung. Using a transformed murine macrophage line, striking induction of PAI-1 transcripts occurred under hypoxic conditions, due to both increased de novo transcription as well as increased mRNA stability. Consistent with an important role of the fibrinolytic system in hypoxia-induced fibrin accumulation, PAI-1 +/+ mice exposed to hypoxia exhibited increased pulmonary fibrin deposition based upon a fibrin immunoblot, intravascular fibrin identified by immunostaining, and increased accumulation of 125I-fibrinogen/fibrin in hypoxic tissue. In contrast, mice deficient for the PAI-1 gene (PAI-1 -/-) similarly exposed to hypoxic conditions did not display increased fibrin accumulation compared with normoxic PAI-1 +/+ controls. Furthermore, homozygous null uPA (uPA -/-) and tPA (tPA -/-) mice subjected to oxygen deprivation showed increased fibrin deposition compared with wild-type controls. These studies identify enhanced expression of PAI-1 as an important mechanism suppressing fibrinolysis under conditions of low oxygen tension, a response which may be further amplified by decreased expression of plasminogen activators. Taken together, these data provide insight into an important potential role of macrophages and the fibrinolytic system in ischemia-induced thrombosis.

Topics: Animals; Cell Line; Fibrin; Fibrinolysis; Gene Expression Regulation; Hypoxia; Immunohistochemistry; Lung; Macrophages; Male; Mice; Mice, Inbred Strains; Mice, Transgenic; Oxygen; Plasminogen Activator Inhibitor 1; Plasminogen Activators; RNA, Messenger; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator

We consecutively inactivated both alleles of the thrombomodulin (TM) gene in murine embryonic stem (ES) cells and generated TM-deficient (TM-/-) chimeric mice. Quantitation of an ES-cell marker and protein C cofactor activity indicates that up to 50% of pulmonary endothelial cells are ES-cell derived and therefore TM deficient. Infusions of 125I-fibrinogen into mice show a significant increase (fourfold, P <.005) in radiolabeled cross-linked fibrin in TM-/- chimeric mouse lung as compared with wild-type mice. However, only chimeric mice that exhibit at least a 30% reduction in protein C cofactor activity and are at least 15 months old display this phenotype. Immunocytochemical localization of TM in chimeras shows a mosaic pattern of expression in both large and small blood vessels. Colocalization of cross-linked fibrin and neo (used to replace TM) reveals that fibrin is deposited in TM-/- regions. However, the fibrin deposits were largely restricted to pulmonary vessels with a lumenal area greater than 100 micrometer2. The hypercoagulable phenotype can be induced in younger chimeric mice by exposure to hypoxia, which causes a fivefold increase in beta-fibrin levels in lung. Our findings show that TM chimerism results in spontaneous, intravascular fibrin deposition that is dependent on age and the magnitude of the TM deficiency.

Topics: Age Factors; Animals; Disease Models, Animal; Erythrocyte Aggregation; Fibrin; Hypoxia; Mice; Mice, Mutant Strains; Pulmonary Artery; Thrombomodulin

Clinical conditions associated with local or systemic hypoxemia can lead to prothrombotic diatheses. This study was undertaken to establish a model of whole-animal hypoxia wherein oxygen deprivation by itself would be sufficient to trigger tissue thrombosis. Furthermore, this model was used to test the hypothesis that hypoxia-induced mononuclear phagocyte (MP) recruitment and tissue factor (TF) expression may trigger the local deposition of fibrin which occurs in response to oxygen deprivation. Using an environmental chamber in which inhaled oxygen tension was lowered to 6%, hypoxic induction of thrombosis was demonstrated in murine pulmonary vasculature by 8 h based upon: (a) immunohistologic evidence of fibrin formation in hypoxic lung tissue using an antifibrin antibody, confirmed by 22.5-nm strand periodicity by electron microscopy; (b) immunoblots revealing fibrin gamma-gamma chain dimers in lungs from hypoxic but not normoxic mice or hypoxic mice treated with hirudin; (c) accelerated deposition of 125I-fibrin/fibrinogen and 111In-labeled platelets in the lung tissue of hypoxic compared with normoxic animals; (d) reduction of tissue 125I-fibrin/fibrinogen accumulation in animals which had either been treated with hirudin or depleted of platelets before hypoxic exposure. Because immunohistochemical analysis of hypoxic pulmonary tissue revealed strong MP staining for TF, confirmed by increased TF RNA in hypoxic lungs, and because 111In-labeled murine MPs accumulated in hypoxic pulmonary tissue, we evaluated whether recruited MPs might be responsible for initiation of hypoxia-induced thrombosis. This hypothesis was supported by several lines of evidence: (a) MP depletion before hypoxia reduced thrombosis, as measured by reduced 125I-fibrin/fibrinogen deposition and reduced accumulation of cross-linked fibrin by immunoblot; (b) isolated murine MPs demonstrated increased TF immunostaining when exposed to hypoxia; and (c) administration of an anti-rabbit TF antibody that cross-reacts with murine TF decreased 125I-fibrin/fibrinogen accumulation and cross-linked fibrin accumulation in response to hypoxia in vivo. In summary, these studies using a novel in vivo model suggest that MP accumulation and TF expression may promote hypoxia-induced thrombosis.

Topics: Animals; Fibrin; Fibrinogen; Hypoxia; Lung; Mice; Monocytes; Neutrophils; Thromboplastin; Thrombosis

Alveolar and interstitial fibrin deposition is a prominent pathologic feature in many acute lung injury syndromes. Previous studies have suggested that ischemic lung preservation has a stimulatory effect on donor alveolar macrophages (Mphis) during transplantation. An animal model of lung preservation was developed to examine the hypothesis that ischemia enhances Mphi procoagulant activity (PCA) as a potential mechanism contributing to lung reperfusion injury. Histologic examination of ischemic lungs reperfused ex vivo revealed evidence of alveolar fibrin deposition. Mphis lavaged from lungs stored for at least 8 h at 21 degrees C exhibited increased PCA. The use of factor-deficient human plasma characterized this Mphi procoagulant as tissue factor (TF). Since increased PCA correlated with decreased airspace pO2 at the end of preservation, the effect of various O2 concentrations on PCA induction in vivo and in vitro was examined. Lung inflation during ischemia with decreasing O2 concentrations confirmed that hypoxia was associated with a rise in Mphi PCA in situ. However, in vitro exposure of Mphis to hypoxia did not increase Mphi PCA, suggesting that hypoxia alone was not responsible for induction of this procoagulant effect. Northern blot analysis demonstrated an increase in TF mRNA levels from in situ but not in vitro Mphis, thereby confirming transcriptional TF induction in this group. In addition, enhanced PCA was observed when Mphis were suspended in the bronchoalveolar lavage supernatant from the ischemic lungs stored at 21 degrees C. This suggests that in situ lung ischemia and hypoxia may produce soluble factors that either directly or indirectly stimulate Mphi TF expression. These factors may contribute to Mphi-mediated ischemic lung injury.

Topics: Animals; Blotting, Northern; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Fibrin; Hypoxia; Ischemia; Macrophages, Alveolar; Male; Microscopy, Electron, Scanning; Pulmonary Alveoli; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Thromboplastin; Tubulin

Endothelial damage caused by hypoxia and the local administration of Varicocid, or by using vascular clamps on the jugular vein of rabbits, was studied under a scanning electron microscope. Adhering platelets and fibrin were found only in the acutely damaged veins with endothelial defects. Swelling and vacuolization of endothelial cells did not induce intravascular coagulation. Adhering thrombocyte aggregates and fibrin strands can be found on the venous wall after acute changes under a scanning microscope only if part or all of the endothelial lining has been lost. Intravascular coagulation is not induced by swelling or vacuolization. The jugular vein of rabbits was studied after hypoxia, the administration of Varicocid and the use of vascular clamps.

Topics: Animals; Blood Platelets; Constriction; Endothelium, Vascular; Fatty Acids; Fibrin; Hypoxia; Jugular Veins; Macrophages; Microscopy, Electron, Scanning; Platelet Adhesiveness; Rabbits; Sclerosing Solutions; Thrombosis

Blood coagulation, fibrinolysis, and arterial blood gases were examined in 66 nonacclimatized mountaineers at 4,557 m. Subjects were classified according to a clinical score as healthy (n = 25), having mild acute mountain sickness (AMS) (n = 24), showing severe AMS (n = 13), and suffering from high-altitude pulmonary edema (HAPE) (n = 4). Coagulation times, euglobulin lysis time, and fibrin(ogen) fragment E were normal in all groups without significant changes. Fibrinopeptide A (FPA), a molecular marker of in vivo fibrin formation, was elevated in HAPE to 4.2 +/- 2.7 ng/ml (P less than 0.0001) compared with the other groups showing mean values between 1.6 +/- 0.4 and 1.8 +/- 0.7 ng/ml. FPA was normal in one patient with HAPE, however. Severe AMS was accompanied by a significant decrease in arterial PO2 due to an increase in alveolar-arterial O2 difference, whereas arterial PCO2 did not change significantly. We conclude that activation of blood coagulation is not involved in the pathogenesis of AMS and the impairment of gas exchange in this disease. Fibrin generation occurring in HAPE is probably an epiphenomenon of edema formation.

Topics: Adult; Altitude Sickness; Arteries; Blood Coagulation; Blood Gas Analysis; Female; Fibrin; Humans; Hypoxia; Male; Pulmonary Edema

Topics: Blood Proteins; Capillaries; Capillary Permeability; Fibrin; Fibrinolysis; Humans; Hyperemia; Hypoxia; Leg; Rheology; Varicose Ulcer; Veins; Venous Insufficiency; Venous Pressure

The role of fibrinolysis in the pathogenesis of hyaline membrane disease was studied in newborn rabbits at 28- or 29-day gestation. Hyaline membrane was recognized in the lungs of rabbits that were exposed to intrauterine hypoxia and received trans-4-aminomethylcyclohexane carboxylic acid (t-AMCHA), a plasmin inhibitor found to cause a marked decrease in the fibrinolytic activity of lung extracts. In the newborn rabbits subjected to intrauterine hypoxia alone, surface activity of lung extracts was reduced, but hyaline membrane was not seen. By immunofluorescent examination the hyaline membrane was found to be rich in fibrinogen and its derivatives. Electron microscopic examination revealed that they contained various amounts of cellular debris, bundles of fibrils and electron-dense, finely granular deposits and polymerized fibrin with 230 A periodicity. Disintegration of pulmonary epithelial cells was also seen. These findings indicate that diminished fibrinolytic activity of lung tissue contributes to intra-alveolar accumulation of fibrinogen, fibrin and their degradation products, thereby bringing about morphological and physiological disintegration of the terminal airways.

Topics: Animals; Fibrin; Fibrinogen; Fibrinolysis; Humans; Hyaline Membrane Disease; Hypoxia; Infant, Newborn; Lung; Rabbits; Tranexamic Acid

Topics: Animals; Capillary Permeability; Clone Cells; Education, Medical, Graduate; Epithelial Cells; Epithelium; Fibrin; Humans; Hypoxia; Intestinal Mucosa; Mice; Necrosis; Neoplasms; Radiation Dosage; Radiation Effects; Radiation Injuries, Experimental; Radiobiology; Radiology; Radiotherapy; Rats; Research; Serum Albumin, Radio-Iodinated; United Kingdom

Topics: Animals; Burns; Fibrin; Fractures, Bone; Humans; Hypoxia; Lung; Microcirculation; Pulmonary Edema; Pulmonary Embolism; Wounds and Injuries

Topics: Animals; Blood Coagulation; Blood Platelets; Bone Marrow; Bone Marrow Cells; Cell Movement; Fibrin; Hindlimb; Hypoxia; Male; Mice; Microscopy, Electron; Reticulocytes

Topics: Animals; Biopsy; Blood Coagulation Tests; Bone and Bones; Collagen; Disease Models, Animal; Disseminated Intravascular Coagulation; Dogs; Embolism, Fat; Exudates and Transudates; Fibrin; Fractures, Bone; Hindlimb; Hypoxia; Leukocytes; Lung; Microscopy; Microscopy, Electron; Muscles; Musculoskeletal System; Pulmonary Edema; Pulmonary Embolism; Triolein

Topics: Animals; Arteries; Benzopyrans; Fibrin; Hypoxia; Mesenteric Arteries; Microscopy, Electron; Muscle, Smooth; Necrosis; Phosphotungstic Acid; Rats; Staining and Labeling

Topics: Apgar Score; Fibrin; Humans; Hypoxia; Infant, Newborn; Infant, Newborn, Diseases; Time Factors

Topics: Animals; Capillaries; Cell Membrane Permeability; Cytoplasm; Fibrin; Hypoxia; Mesenteric Arteries; Microscopy, Electron; Muscle, Smooth; Necrosis; Oxygen Consumption; Rats

Topics: Adult; Anti-Bacterial Agents; Autoimmune Diseases; Blood Viscosity; Capillaries; Cell Membrane; Fibrin; Gas Poisoning; Glucocorticoids; Humans; Hypoxia; Male; Nitrogen Dioxide; Pulmonary Edema; Shock

Arterial hypoxia has been accepted as a potent stimulus to increased fibrinolytic activity in vivo. This study has shown that significant levels of arterial hypoxaemia induced in healthy volunteers by breathing a hypoxic gas mixture have failed to induce changes in the fibrinolytic enzyme system.

Topics: Adult; Arteries; Blood Coagulation Tests; Fibrin; Fibrinogen; Fibrinolysis; Fibrinolytic Agents; Humans; Hypoxia; Oxygen; Plasminogen; Serum Globulins

Topics: Basement Membrane; Female; Fetal Death; Fibrin; Humans; Hypertension; Hypoxia; Photomicrography; Placenta; Pre-Eclampsia; Pregnancy

Topics: Aminocaproates; Aminocaproic Acid; Animals; Autoimmune Diseases; Cartilage; Caseins; Cathepsins; Connective Tissue; Fibrin; Fibrinolysis; Glycosaminoglycans; Goats; Humans; Hypoxia; Kidney; Lysosomes; Rabbits; Research; Sheep; Surface-Active Agents; Synovial Fluid; Synovial Membrane

Topics: Blood Coagulation; Cardiovascular Abnormalities; Fibrin; Heart Diseases; Heparin; Hypoxia