Endothelial Thrombomodulin Research Articles

Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease

Endothelial protein C receptor (EPCR) and thrombomodulin (TM) are expressed at high levels in the resting microvasculature and convert protein C (PC) into its activated form, which is a potent anticoagulant and antiinflammatory molecule. Here we provide evidence that in Crohn disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD), there was loss of expression of endothelial EPCR and TM, which in turns caused impairment of PC activation by the inflamed mucosal microvasculature. In isolated human intestinal endothelial cells, administration of recombinant activated PC had a potent antiinflammatory effect, as demonstrated by downregulated cytokine-dependent cell adhesion molecule expression and chemokine production as well as inhibited leukocyte adhesion. In vivo, administration of activated PC was therapeutically effective in ameliorating experimental colitis as evidenced by reduced weight loss, disease activity index, and histological colitis scores as well as inhibited leukocyte adhesion to the inflamed intestinal vessels. The results suggest that the PC pathway represents a new system crucially involved in governing intestinal homeostasis mediated by the mucosal microvasculature. Restoring the PC pathway may represent a new therapeutic approach to suppress intestinal inflammation in IBD.

Pravastatin induces thrombomodulin expression in TNFα‐treated human aortic endothelial cells by inhibiting Rac1 and Cdc42 translocation and activity

Expression of functionally active thrombomodulin (TM) on the luminal surface of endothelial cells is critical for vascular thromboresistance. The 3-hydroxyl-3-methyl coenzyme A reductase inhibitor, pravastatin, can protect the vasculature in a manner that is independent of its lipid-lowering activity. We examined the effect of pravastatin on TM expression by human aortic endothelial cells (HAECs) with subsequent tumor necrosis factor alpha (TNFalpha) stimulation and investigated the signaling pathways involved. TNFalpha treatment attenuated TM expression in HAECs in a time-dependent manner. Pravastatin upregulated TM levels in TNFalpha-treated HAECs. Specific inhibition of geranylgeranyltransferase-I or the Rho family by GGTI-286 or TcdB, respectively, enhanced TM expression in TNFalpha-treated HAECs, whereas MAP kinase inhibitors, inactivation of Rho by Clostridium botulinum C3 exoenzyme, or the Rho kinase inhibitor, Y-27632, had no effect. In TNFalpha-treated HAECs, pravastatin inhibited Rac1 and Cdc42 activation and their translocation to the cell membrane. Blocking the transcriptional activation of NF-kappaB prevented the TNFalpha-induced downregulation of TM. The pravastatin-induced increase in TM expression in TNFalpha-treated HAECs was mediated through inhibition of NF-kappaB activation. Pravastatin regulates TM expression by inhibiting the activation of the Rho family proteins, Rac1 and Cdc42, and the transcription factor, NF-kappaB. The increase in endothelial TM activity in response to pravastatin constitutes a novel pleiotropic (nonlipid-related) effect of this commonly used compound and may be of clinical significance in disorders in which deficient endothelial TM plays a pathophysiological role.

Plasma level of stromal derived factor-1 (SDF-1) is increased in disseminated intravascular coagulation patients who have poor outcomes: In vitro effect of SDF-1 on coagulopathy

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that activates and directs the migration of leukocytes that have CXCR4, which is the unique receptor for SDF-1. Although SDF-1/CXCR4 interaction has been implicated in various inflammatory conditions, its role in modulating coagulation has not been determined. We studied the plasma SDF-1 levels in 90 patients with suspected disseminated intravascular coagulation (DIC) and we found that circulating SDF-1 was significantly increased in the overt DIC patients and was also increased in overt DIC patients who have a poor outcome. We then tested in vitro whether SDF-1 can affect the expression of monocyte tissue factor (TF) and endothelial thrombomodulin (TM), and both of these play important roles in coagulopathy. SDF-1 did not affect the expression of surface TF protein and its function and the TF mRNA level in both monocytes and the monocytic leukemia cell line THP-1. SDF-1 also did not change the surface TM expression of endothelial cells. SDF-1 could enhance low-dose ADP induced platelet aggregation, although it failed by itself to induce aggregation. These findings suggest that plasma SDF-1 might be closely associated with hypercoagulability though its action as a platelet activator.

Significance of endothelial dysfunction in the pathogenesis of early and delayed radiation enteropathy.

This review summarizes the current state of knowledge regarding the role of endothelial dysfunction in the pathogenesis of early and delayed intestinal radiation toxicity and discusses various endothelial-oriented interventions aimed at reducing the risk of radiation enteropathy. Studies published in the biomedical literature during the past four decades and cited in PubMed, as well as clinical and laboratory data from our own research program are reviewed. The risk of injury to normal tissues limits the cancer cure rates that can be achieved with radiation therapy. During treatment of abdominal and pelvic tumors, the intestine is frequently a major dose-limiting factor. Microvascular injury is a prominent feature of both early (inflammatory), as well as delayed (fibroproliferative) radiation injuries in the intestine and in many other normal tissues. Evidence from our and other laboratories suggests that endothelial dysfunction, notably a deficiency of endothelial thrombomodulin, plays a key role in the pathogenesis of these radiation responses. Deficient levels of thrombomodulin cause loss of vascular thromboresistance, excessive activation of cellular thrombin receptors by thrombin, and insufficient activation of protein C, a plasma protein with anticoagulant, anti-inflammatory, and cytoprotective properties. These changes are presumed to be critically involved in many aspects of early intestinal radiation toxicity and may sustain the fibroproliferative processes that lead to delayed intestinal dysfunction, fibrosis, and clinical complications. In conclusion, injury of vascular endothelium is important in the pathogenesis of the intestinal radiation response. Endothelial-oriented interventions are appealing strategies to prevent or treat normal tissue toxicity associated with radiation treatment of cancer.

Safe Interruption of Ongoing Thrombosis by the Protein C Activator Thrombin Analog, W215A/E217A; a Comparison to Enoxaparin in Primates.

Antithrombotic agents are effective but pose significant bleeding risk, thus safer new agents are needed. Since the infused double mutant human thrombin (W215A/E217A; WE) binds endothelial thrombomodulin, activates protein C locally in the intraluminal boundary layer, and prevents thrombus formation without hemostasis impairment, we investigated whether WE could also interrupt the growth of existing thrombi. WE was compared to standard interventional doses of the low molecular weight heparin, enoxaparin (Sanofi) in awake baboons. Thrombosis was initiated by interposing a two part device consisting of a 2 cm, 4 mm ID polyethylene terephthalate vascular graft (DVG) followed by a 2 cm silicone rubber expansion chamber, 9 mmID. Upon initiation of blood flow at 100 mL/min, wall shear rate of 265 sec-1 in the 4 mm ID segment (arterial type flow) and 29 sec-1 in the expansion chamber (venous type flow), platelet thrombus was monitored by gamma camera imaging of autologous 111In-labelled platelets for 60 min or 90 min. Fibrin accumulation was quantified by homologous 125I-labelled fibrinogen. Hemostasis was assessed as template bleeding time prolongation. APTT was also monitored. Treatments started 25 min after the initiation of thrombosis by enoxaparin, 0.5 mg/kg (LD) and 1 mg/kg (HD) IV bolus or WE, 7.5 μg/kg/hr (LD) and 15 μg/kg/hr (HD) infusion (1/3 dose given as a bolus followed by IV infusion over 60min) and compared to saline infused controls. All interventions were given systemically, downstream from the device and 5–6 studies comprised each study group. Treatment with enoxaparin at 25 min after initiation of thrombus growth resulted in a reduction of platelet deposition of 19 % with the LD and 76 % with the HD after 35 min of therapy in the expanded venous type segment. WE reduced venous thrombosis by 45 % and 65 % at the two doses studied and was further reduced to 65 % and 80 % following 65 min of WE infusion as compared to controls. Arterial type thrombosis was reduced by WE by 34 % and 39 % respectively for the two doses studied. HD enoxaparin reduced arterial type thrombosis by 37 %. Fibrin accumulation paralleled the platelet deposition. APTT was prolonged 1.57- and 1.87-fold respectively for the WE doses without any bleeding time prolongation. Enoxaparin prolonged the APTT 1.4- and 1.75-fold, similar to WE but with a 1.13- and 1.75-fold prolongation of the bleeding time. In conclusion, the double mutant human thrombin, WE, effectively interrupted both venous and arterial type growing thrombi at very low doses without detectable compromise to hemostasis. Likewise, enoxaparin at the doses tested had similar efficacy but with a significantly prolonged bleeding time at the 1 mg/kg bolus dose. WE might provide a safer alternative to heparins in the treatment of acute progressive thrombosis.

Biochemical Markers of Endothelial Activation in Primary Hyperparathyroidism

Patients with primary hyperparathyroidism (PHPT) have impaired vasodilation both dependent and independent of endothelium. The aims of our study were to measure three different biochemical markers of endothelial activation, i. e., plasma thrombomodulin, soluble(s) E-selectin, and von Willebrand factor, in PHPT patients before and one year after successful parathyroidectomy, and to distinguish the potential effect of hypercalcemia and/or high parathyroid hormone from that of major cardiovascular risk factors (diabetes mellitus, hyperlipidemia, hypertension, obesity, smoking habit) on endothelial function. Twenty consecutive patients with PHPT subdivided into two groups according to the absence (n = 8) or presence (n = 12) of one or more risk factors, and fifteen healthy normocalcemic subjects were studied. Baseline thrombomodulin levels were similar in the groups with and without risk factors, and in controls. In contrast, sE-selectin and von Willebrand factor were higher in PHPT patients with risk factors than in those without risk factors (p < 0.05 and p < 0.01, respectively) and controls (p < 0.01). Neither thrombomodulin nor sE-selectin changed after parathyroidectomy in either PHPT group. Plasma von Willebrand factor decreased (p < 0.01) in patients without risk factors, while persisting at high levels in patients with risk factors. In conclusion, in spite of a limitation due to the small number of patients, our study suggests that classic cardiovascular risk factors seem to be the main determinants for the high plasma levels of sE-selectin and vWF in PHPT. Together with unaltered thrombomodulin and sE-selectin levels, a plasma vWF decrease after parathyroidectomy might reflect a specific mechanism of its endothelial calcium- and/or PTH-stimulated secretion in some PHPT patients without risk factors. Whether a vWF reduction after parathyroidectomy may be used as a biochemical index for improved endothelial function in PHPT patients without risk factors has yet to be demonstrated in larger studies.

Regulation of endothelial thrombomodulin expression by inflammatory cytokines is mediated by activation of nuclear factor-kappa B

AbstractInflammation and thrombosis are increasingly recognized as interrelated biologic processes. Endothelial cell expression of thrombomodulin (TM), a key component of the anticoagulant protein C pathway, is potently inhibited by inflammatory cytokines. Because the mechanism underlying this effect is largely unknown, we investigated a potential role for the inflammatory transcription factor nuclear factor-kappa B (NF-κB). Blocking NF-κB activation effectively prevented cytokine-induced down-regulation of TM, both in vitro and in a mouse model of tumor necrosis factor-α (TNF-α)–mediated lung injury. Although the TM promoter lacks a classic NF-κB consensus site, it does contain tandem Ets transcription factor binding sites previously shown to be important for both constitutive TM gene expression and cytokine-induced repression. Using electrophoretic mobility shift assay and chromatin immunoprecipitation, we found that multiple Ets species bind to the TNF-α response element within the TM promoter. Although cytokine exposure did not alter Ets factor binding, it did reduce binding of p300, a coactivator required by Ets for full transcriptional activity. Overexpression of p300 also prevented TM repression by cytokines. We conclude that NF-κB is a critical mediator of TM repression by cytokines. Further evidence suggests a mechanism involving competition by NF-κB for limited pools of the transcriptional coactivator p300 necessary for TM gene expression.

Importance of timing of antiaggregant treatment in the prevention of radiation induced enteropathy

Chronic radiation enteropathy (CRE) is an undesirable radiation-induced toxicity and a common health problem in patients with pelvic or abdominal malignancies. Damage to microvascular endothelial cells and connective tissue is blamed to cause this adverse effect. It is shown that platelets are the first cellular elements that initiate the homeostatic and inflammatory responses and release of several proinflammatory and fibrinogenic mediators. Antiplatelet agents such as ticlopidine and clopidogrel were shown to prevent CRE and this effect is believed to be directed by their activities against thrombocytes. However, recent studies have shown that these drugs also induce apoptosis in endothelial cells and may lead to decreased expression of endothelial prostacyclin and thrombomodulin (TM) and increased release of von Willebrand factor which are shown to be major contributors of coagulation process. Assuming that radiation induced apoptosis occur 6-10h after irradiation, we think that timing of these antiaggregant drugs with irradiation is important and a 6-10h interval between these may be beneficial to avoid this adverse interaction.

Dynamic Defense System Against HMGB1, a Proinflammatory and Lethal Mediator in Severe Infections and Malignancies.

High Mobility Group box 1(HMGB1) is an abundant DNA-binding protein that acts as a proinflammatory cytokine when released in the extracellular milieu by necrotic and inflammatory cells. Moreover, an increased HMGB1 in the circulation of septic patients may induce multi-organ failure and lethality. However, very recent observations suggest that the protein also acts as an innate adjuvant, stem cell chemoattractant and growth factor. Thus only systemic and circulatory HMGB1 may induce morbidity and mortality, however, localized HMGB1 may have beneficial effects. Therefore, we serially examined the serum HMGB1 level in patients with various diseases, and also evaluated the significance of the protein.We demonstrate here how HMGB1 is localized and acts as an immune-adjuvant and a repairing factor in damaged tissue. We first established specific ELISA method to measure HMGB1. An increased level of HMGB1 was detected in the serum from patients with sever sepsis, infections, malignancy and so on. However, serum HMGB1 concentrations were fluctuated during the clinical course, and could not be concluded as a lethal mediator as previously reported. Next we investigated the reason of dynamic fluctuations of the protein in the circulation. Based on our findings, we proposed that this fluctuation of HMGB1 concentrations may be mediated by at least following three fashions; 1) proteolytic degradation by plasmin and thrombin, 2) endothelial thrombomodulin(TM) adsorption, and 3) generation of antibody against the protein. We observed that plasmin efficiently degraded HMGB1 into small fragments. However, interestingly the generated fragments of the protein still possess an ability to produce TNFa in macrophages through an undefined pathway. TM binds the protein on its N-terminus lectin-like domain. Binding of HMGB1 to TM resulted in decrement of TM's cofactor activity to activate protein C by thrombin. HMGB1 bound to TM was gradually degraded by thrombin. These may be a system to localize HMGB1 only in injury sites where TM is down-regulated or disappeared through endothelial-loss. This may exert endothelial defense system against extracellular HMGB1 in severe tissue injury. Another possibility is that the generated antibody against HMGB1 may neutralize the proinflammatory action of the protein. In this context, we found that some of the antibodies against HMGB1 have the characteristics of P-ANCA(perinuclear anti-neutrophil cytoplasmic antibody). This may alter the phenotype of the underlining diseases. In conclusion, we suggest that HMGB1 is not merely a lethal mediator, but a kind of “testament” mediator of cell necrosis or invasive attacks to dendritic cells.

Sustained Pharmacological Activation of Protein C (PC) in Baboons.

The introduction of two mutations at the specificity sites S3-S4 of thrombin (W215A and E217A) results in a mutant thrombin (WE) with more than 1000-fold reduced activity towards fibrinogen and PAR1. WE retains its antithrombotic activity via selective activation of the circulating PC pool and elevation of activated PC (APC) levels when administered as a bolus to primates (Gruber et al. Blood 2002). We investigated whether increased APC levels can be sustained and/or the PC pool exhausted by a 4 to 6 hour long infusion of WE in 15 baboons. In order to assess whether intravascular thrombomodulin availability is rate limiting in pharmacological PC activation, the effect of high dose WE administration on circulating APC (enzyme capture assay) and PC activity (Protac-activatable PC) levels were tested with or without co-administration of equimolar doses of recombinant human soluble thrombomodulin (rhsTM). Infusion of a very high dose of WE alone (110 ug/kg bolus followed by 110 ug/kg/h) gave a peak APC concentration of 201 ng/mL by 10 min. The endogenous APC concentration then sharply decreased with an apparent initial half life (T1/2) of 25 min - similar to that of exogenous human APC in baboons and almost returned to baseline (8 ng/mL) by the end of the treatment despite continued infusion of high dose WE. Thrombin-antithrombin (TAT) complex levels gradually increased reaching an apparent equilibrium at 104 ng/mL by the 4th hour. D-Dimer levels increased throughout the treatment and reached a level that was 20-fold of the baseline value by the end of the treatment. Co-administration of rhsTM (180 ug/kg bolus + 180 ug/kg/h infusion) with the high dose WE also resulted in rapid PC activation and an early APC peak (240 ng/ml at 10 min). The enzyme peak was followed by a decline in APC concentration with an apparent initial T1/2 of 51 min to an average equilibrium concentration of 40 ng/mL by the end of the treatment. TAT levels sharply increased from the 3.5 ng/mL baseline level, reaching greater than 240 ng/ml by the 10th min of the treatment, and remained outside the range of the test. There was a nonsignificant increase in D-Dimer levels towards the end of the WE+rhsTM treatment. Approximately 40% and 80% of the Protac-activatable circulating PC pool was consumed by administration of WE and WE+rhsTM, respectively. Based on the findings we propose that: 1) there is sufficient PC substrate for a sustained pharmacological increase in endogenous APC levels; 2) either complex formation of WE with antithrombin is significantly accelerated by rhsTM or clearance of TAT is inhibited by rhsTM; 3) the existing endogenous soluble and/or endothelial thrombomodulin pool appears to be transiently depleted upon WE infusion; and 4) the circulating PC pool might not be homogeneous such that part of the PC pool is not activatable in vivo by infused WE+rhsTM. In summary, significant and sustained levels of endogenous circulating APC can be generated in baboons by infusion of rhsTM with the highly selective WE thrombin mutant.

Radiation injury and the protein C pathway.

To summarize current knowledge regarding the role of the thrombomodulin (TM)-protein C system in acute and chronic radiation responses in normal tissues. Studies published in the biomedical literature during the past three decades and cited in PubMed and unpublished clinical and laboratory data from our own research program. STUDY SUMMARY: The risk of injury to normal tissues limits the cancer cure rates that can be achieved with radiation therapy. Microvascular injury is a prominent feature of normal tissue radiation injury and plays a critical role in both acute (inflammatory) and chronic (fibrotic) radiation responses. Evidence from our own and other laboratories strongly suggests that dysfunction of the TM-protein C system plays a key role in the pathogenesis of radiation-induced injury to normal tissue. Exposure of normal tissues to ionizing radiation causes a pronounced, sustained deficiency of endothelial TM. This is likely to be due to a combination of initial inactivation of TM by reactive oxygen species, reduced transcription of TM, and release of TM into the circulation. Deficient levels of endothelial TM cause loss of local vascular thrombo-resistance, excessive activation of protease-activated receptor-1 by thrombin, and insufficient activation of protein C. These changes are presumed to be critically involved in many aspects of acute radiation toxicity and in sustaining the fibroproliferative processes that lead to chronic radiation-induced organ dysfunction and clinical complications. Injury of vascular endothelium may be key to the acute responses of normal tissues to ionizing radiation and to the progressive nature of chronic radiation fibrosis. Restitution of the TM-protein C pathway is an appealing strategy by which to prevent or treat normal tissue toxicity associated with radiation treatment of cancer.

Genetic variation in the human thrombomodulin promoter locus and prognosis after acute coronary syndrome

Introduction: Endothelial thrombomodulin (TM) plays a critical role in both anticoagulation and antiinflammation. An impaired TM cofactor function or reduced TM gene expression could constitute a prethrombotic abnormality leading to acute coronary events. Mutations in the TM gene occur, but their functional consequences on the expression and activity of the gene are not yet fully understood. Materials and methods: We performed a prospective study investigating the prevalence of TM mutations in the promoter region in 182 patients with acute coronary syndrome as well as in a control group. The patients were followed-up after 30 days and after 2 years for acute myocardial infarction (MI) and mortality. Results and conclusions: We identified 10 point mutations and 2 small deletions: −1861 C/A, −1852 C/G, −1803 G/C, −1752 G/C, −1213/1212 delTT, −1089 C/G, −1088 C/T, −1083/1082 delCC, −1066 A/C, −801 C/G, −651 A/C and −52 G/A. Two of the mutations, −1752 G/C and −1213/1212 delTT, were frequent in the patients as well as in the controls, while all the others were rare. The only significant finding was that both −1752 G/C and −1213/1212 delTT were associated with a lower than normal risk of suffering a clinical event among smokers at 30 days and 2 years. We did not gain any support for the hypothesis that TM mutations confer an increased risk of MI or mortality.

Statins increase thrombomodulin expression and function in human endothelial cells by a nitric oxide-dependent mechanism and counteract tumor necrosis factor alpha-induced thrombomodulin downregulation.

Expression of functionally active thrombomodulin (TM) on the luminal surface of endothelial cells is critical for vascular thromboresistance. TM maintains thrombohemorrhagic homeostasis by forming a complex with thrombin, which subsequently loses its procoagulant properties and instead activates protein C. Acquired deficiency of endothelial TM is of particular pathophysiological significance in sepsis and related disorders. We show here that two different 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), atorvastatin and simvastatin, strongly increase the expression and functional activity of TM in human umbilical vein endothelial cells, human coronary artery endothelial cells, and EA.hy926 endothelial cells. The increase in endothelial TM conferred by statin was prevented by the addition of mevalonic acid, geranylgeranyl-pyrophosphate, and nitric oxide scavenger, and was mimicked by the addition of a specific inhibitor of geranylgeranyl transferase, as well as by nitric oxide donors. Moreover, statin counteracted tumor necrosis factor alpha-induced downregulation of endothelial cell TM. The increase in endothelial cell TM activity in response to statin constitutes a novel pleiotropic (non-lipid-related) effect of these commonly used compounds, and may be of clinical significance in disorders where deficient endothelial TM and protein C activation play a pathophysiological role.

Soluble thrombomodulin and coronary heart disease.

Endothelial thrombomodulin is a major vasoprotective molecule. The membrane thrombomodulin is digested by proteases and the degradation products are detectable in circulating blood. The purpose of this review is to provide recent information regarding the relationship of soluble thrombomodulin with coronary heart disease. Results from a population-based, prospective, coronary heart disease, case-cohort study reveal an inverse relationship between plasma soluble thrombomodulin and the relative risk of coronary heart disease. Participants in this study were healthy subjects without acute thrombotic events. They were followed, and coronary heart disease events were ascertained. Individuals with a high level of soluble thrombomodulin are associated with a significant reduction in the relative risk of coronary heart disease events. There is a significant interaction between soluble thrombomodulin and soluble intercellular adhesion molecule-1 in predicting the risk of coronary heart disease events. Individuals with a high soluble thrombomodulin level do not have an increased risk of coronary heart disease, even when soluble intercellular adhesion molecule-1 is at the highest levels. In contrast, at low soluble thrombomodulin levels, soluble intercellular adhesion molecule-1 has a 'dose-dependent' association with coronary heart disease risk. These results suggest an interplay between vasoprotective and pro-inflammatory endothelial molecules. Soluble thrombomodulin and its parent molecule appear to play a predominant role in determinations of the risk of coronary heart disease events. The soluble thrombomodulin level in plasma is an independent risk factor for coronary heart disease. It is inversely associated with coronary heart disease risk. Combinatorial analysis of soluble thrombomodulin and soluble intercellular adhesion molecule-1 provides a more specific assessment of coronary heart disease risk in middle-aged subjects.

Capillary Endothelial Thrombomodulin Expression and Fibrin Deposition in Rats with Continuous and Bolus Lipopolysaccharide Administration

We studied capillary endothelial injury, as demonstrated by fibrin deposition and changes in thrombomodulin (Tm) expression, in rats receiving continuous or bolus iv lipopolysaccharide (LPS). Rats were continuously infused with iv LPS (0.1, 0.2, 0.5, or 1.0 mg/kg/hr) for up to 6 hours. Others were given a bolus iv dose of LPS (20 mg/kg), and then the same dose of saline as a continuous infusion was administered for up to 3 hours. Harvested lungs, livers, and kidneys were examined immunohistochemically for thrombomodulin expression and fibrin deposition. Tm expression began to diminish dose- and time-dependently in lung, liver, and renal peritubular capillaries within 2 to 4 hours of the start of continuous LPS administration (1.0 mg/kg/hr) and had completely disappeared by 3 hours, although Tm remained in the glomerulus. The amount of fibrin deposition observed varied with the organ, dose, and duration of treatment in rats that received continuous LPS administration, but little was deposited in the lung. After bolus LPS administration, Tm in the endothelia of lung, liver, and peritubular capillaries diminished 20 to 40 minutes after treatment and then recovered 120 to 180 minutes after treatment, but the Tm activity of the glomerulus did not change. Fibrin deposition in the capillaries was observed in the liver, glomerulus, and peritubular capillaries, but not in the lung. Endothelial injury by LPS administration is dependent on the dose of LPS and the duration of treatment. The amount of fibrin deposition differs among organs and with the duration of contact between the endothelium and the endotoxin.

Mikamo Lecture: a radical view on the 'superfamily' of cardiovascular risk factors.

Many risk factors for cardiovascular disease generate superoxide in the blood vessels and thereby impair endothelial function. To emphasize the critical role of oxygen radicals, this is a 'radical view' of those risk factors. It will be useful to organize risk factors into a 'superfamily', with consideration of mediators, mechanisms, and target organs. Studies are summarized which suggest that, in parallel with the impairment of endothelial vasomotor function, the thrombin/thrombomodulin/activated protein C anticoagulant mechanism, which requires endothelial thrombomodulin, is also impaired by atherosclerosis and improves during regression of atherosclerosis. Impairment of the anticoagulant mechanism may contribute to thrombosis in atherosclerotic arteries, and improvement of the anticoagulant mechanism during regression of atherosclerosis may reduce the risk of cardiovascular events.

THROMBOMODULIN DEFICIENCY IN HUMAN DIABETIC NERVE MICROVASCULATURE

Human diabetic neuropathy is multifactorial in etiology, with ischemia as a final common pathology. Although impaired vascular endothelial cell function in diabetic microvascular injury is established, the role of thrombomodulin (TM)‐dependent protein C antithrombotic mechanism in the pathogenesis of neuropathy is unclear. This neuropathologic case‐control study investigated whether vascular endothelial TM expression is deficient in peripheral nerve microvessels in diabetic neuropathy. Sural nerve biopsies from 7 patients with diabetic neuropathy and 10 with axonal neuropathy without vasculopathy were immunostained with anti‐TM and anti‐von Willebrand factor (vWF; an endothelial cell marker) antibodies. The proportion of TM‐positive microvessels was expressed relative to total vWF‐staining vessels, according to vessel caliber and regional distribution within the nerve. In diabetic nerves compared with reference controls, the proportion of TM‐positive endoneurial microvessels was 15‐fold lower (0.02 vs. 0.30 in diabetic nerves vs. controls, P &lt; 0.004), and the proportion of small‐caliber epineurial microvessels was 10‐fold lower (0.04 vs. 0.43, P &lt; 0.001). No TM expression was detected at the perineurium in diabetic or control nerves. We demonstrate a substantial reduction of vascular endothelial TM expression throughout human diabetic neuropathy. These findings suggest that an impaired native TM‐dependent protein C antithrombotic mechanism may contribute to microvascular ischemia in the pathogenesis of diabetic neuropathy.

Reduced thrombomodulin in human peripheral nerve microvasculature.

Thrombomodulin (TM), a vascular endothelial receptor, terminates the actions of thrombin and accelerates activated protein C formation. TM is ubiquitous throughout the systemic microcirculation but is reduced in brain regions predisposed to lacunar infarction. We investigated whether TM is present within human nerve and differentially expressed according to vessel caliber and proximity to the blood-nerve barrier. Vascular endothelial TM was detected on sural nerve biopsies with immunohistochemistry. The proportion of TM-positive microvessels was expressed relative to total von Willebrand factor (vWF)-positive vessels. Although vWF was detectable in all microvessels, TM expression was absent from the perineurial vessels. TM was detected in 47% (15-80%, 95% confidence level) of larger epineurial arterioles, in 43% (30-61%) of smaller epineurial vessels, and in 30% (19-47%) of endoneurial vessels. These findings demonstrate that TM is present in human nerve microvasculature but is regionally deficient in proximity to the blood-nerve barrier, which may predispose nerve to microvascular ischemia in inflammatory/prothrombotic conditions.

Deficiency of Microvascular Thrombomodulin and Up-Regulation of Protease-Activated Receptor-1 in Irradiated Rat Intestine: Possible Link Between Endothelial Dysfunction and Chronic Radiation Fibrosis

Microvascular injury is believed to be mechanistically involved in radiation fibrosis, but direct molecular links between endothelial dysfunction and radiation fibrosis have not been established in vivo. We examined radiation-induced changes in endothelial thrombomodulin (TM) and protease-activated receptor-1 (PAR-1) in irradiated intestine, and their relationship to structural, cellular, and molecular aspects of radiation injury. Rat small intestine was locally exposed to fractionated X-radiation. Structural injury was assessed 24 hours and 2, 6, and 26 weeks after the last radiation fraction using quantitative histology and morphometry. TM, neutrophils, transforming growth factor-beta, and collagens I and III were assessed by quantitative immunohistochemistry. PAR-1 protein was localized immunohistochemically, and cells expressing TM or PAR-1 transcript were identified by in situ hybridization. Steady-state PAR-1 mRNA levels in intestinal smooth muscle were determined using laser capture microdissection and competitive reverse transcriptase-polymerase chain reaction. Radiation caused a sustained, dose-dependent decrease in microvascular TM. The number of TM-positive vessels correlated with all parameters of radiation enteropathy and, after adjusting for radiation dose and observation time in a statistical model, remained independently associated with neutrophil infiltration, intestinal wall thickening, and collagen I accumulation. PAR-1 immunoreactivity and transcript increased in vascular and intestinal smooth muscle cells in irradiated intestine. PAR-1 mRNA increased twofold in irradiated intestinal smooth muscle. Intestinal irradiation up-regulates PAR-1 and causes a dose-dependent, sustained deficiency of microvascular TM that is independently associated with the severity of radiation toxicity. Interventions aimed at preserving or restoring endothelial TM or blocking PAR-1 should be explored as strategies to increase the therapeutic ratio in clinical radiation therapy.

Thrombomodulin deficiency in human diabetic nerve microvasculature.

Human diabetic neuropathy is multifactorial in etiology, with ischemia as a final common pathology. Although impaired vascular endothelial cell function in diabetic microvascular injury is established, the role of thrombomodulin (TM)-dependent protein C antithrombotic mechanism in the pathogenesis of neuropathy is unclear. This neuropathologic case-control study investigated whether vascular endothelial TM expression is deficient in peripheral nerve microvessels in diabetic neuropathy. Sural nerve biopsies from 7 patients with diabetic neuropathy and 10 with axonal neuropathy without vasculopathy were immunostained with anti-TM and anti-von Willebrand factor (vWF; an endothelial cell marker) antibodies. The proportion of TM-positive microvessels was expressed relative to total vWF-staining vessels, according to vessel caliber and regional distribution within the nerve. In diabetic nerves compared with reference controls, the proportion of TM-positive endoneurial microvessels was 15-fold lower (0.02 vs. 0.30 in diabetic nerves vs. controls, P < 0.004), and the proportion of small-caliber epineurial microvessels was 10-fold lower (0.04 vs. 0.43, P < 0.001). No TM expression was detected at the perineurium in diabetic or control nerves. We demonstrate a substantial reduction of vascular endothelial TM expression throughout human diabetic neuropathy. These findings suggest that an impaired native TM-dependent protein C antithrombotic mechanism may contribute to microvascular ischemia in the pathogenesis of diabetic neuropathy.