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We examined the relationship between specific endothelial cell markers soluble E-selectin, von Willebrand factor and soluble thrombomodulin and the location or extent of atherosclerosis by analysing plasma samples from 200 patients with symptomatic peripheral vascular disease and 213 age- and sex-matched asymptomatic control subjects. Using ELISAS, we found increased von Willebrand factor and thrombomodulin (both P < 0.0001) in the patients relative to the control subjects, but no significant change in soluble E-selectin. Soluble thrombomodulin was increased in patients with disease at one locus (i.e. of the carotid or iliac/femoral arteries), with an additional significant increase in patients with disease at multiple loci (i.e. any combination of carotid, coronary or iliac/femoral artery disease). No marker differentiated carotid artery disease from iliac/femoral artery disease. We conclude that von Willebrand factor is a marker of generalized atherosclerosis, but that soluble thrombomodulin is related to the extent of disease. Further research into these endothelial cell products are warranted to explore their diagnostic and/or prognostic potential.

Membrane thrombomodulin (TM) is a very efficient natural anti-thrombin glycoprotein with anticoagulant properties expressed on endothelial cell surface. Circulating plasmatic thrombomodulin (TMp) detected by enzyme immunoassay in plasma is considered as a cell marker of endothelial injury. The TMp levels are increased in many conditions (diabetes mellitus, atheromatous disease...). In cases of collagen vascular diseases, where vascular endothelium damage is suspected, TMp is increased particularly in systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). It is noteworthy that the TMp level is correlated with disease activity. Since TMp is a non specific marker of endothelial damage, it may be of interest as a useful marker for the supervision of these diseases. Further studies are needed on larger series. TMp level change during spontaneous evolution or under treatment will help determine wether TMp is a predictor and prognostic marker of these systemic diseases.

P-selectin is an endothelial adhesion molecule involved in the initial step of the neutrophil recruitment. We investigated the effect of hypoxia (95% N2, 5% CO2) and of hypoxia-reoxygenation (95% air, 5% CO2) on the expression of P-selectin by human umbilical vein endothelial cells (HUVEC). P-selectin expression was detected by immunolabelling and quantified by flow cytometric analysis. Our data indicate that hypoxia induces an increase in P-selectin expression with a maximum reached after 90 minutes. A hypoxic exposure of 90 minutes results in a highly significant increase compared to normoxia (p < 0.001, n=13). Furthermore, when a reoxygenation period follows 90 minutes of hypoxia, the initially elevated levels of P-selectin are dramatically enhanced with a maximum obtained after 60 minutes of reoxygenation.

Circulating levels of the adhesion molecule P-selectin (CD62P) are increased in the plasma of patients with atherosclerosis, but its relationship to the anatomical location of symptomatic disease or extent of symptomatic disease is unknown. The influence of the risk factors for atherosclerosis on soluble P-selectin is also unclear. To clarify these questions we analysed plasma samples from 170 patients with symptomatic peripheral vascular disease and 119 asymptomatic controls who were, as a group, age- and sex-matched. Soluble P-selectin (ELISA) was increased in 83 patients with symptomatic disease of the iliac and/or femoral arteries alone (P < 0.05, ANOVA) but not in 37 patients with symptomatic carotid artery disease alone compared with controls. Soluble P-selectin was equally raised in 120 patients with disease at one arterial site and in 50 patients with disease at two or more arterial sites (both P < 0.05) compared with controls. Smoking and atherosclerosis were both independent predictors of raised soluble P-selectin. We conclude that increased soluble P-selectin may have value as a marker of peripheral vascular disease of the iliac and/or femoral arteries in group comparisons only, as the poor discrimination and wide variation of data make comparisons at the individual level difficult.

Thrombomodulin is a membrane glycoprotein expressed by endothelium and is a receptor for thrombin. The thrombin-thrombomodulin complex inactivates the procoagulant activity of thrombin and catalyzes activation of protein C(1). The decrease in thrombomodulin expression at the surface of the vascular endothelium may contribute to the development of thrombosis. In vitro studies have shown that cytokines (TNF, IL-1) or endotoxins down regulate thrombomodulin expression (2, 3, 4) whereas histamine (5), retinoic acid (6) and cAMP analogs (7, 8) increase its expression. Hypoxia is frequently related to disorders of the vascular system such as atheromatous arterial disease and venous insufficiency (9). Clinically, hypoxia induces an increase in vascular permeability (10) and a prothrombotic tendency (12). In vitro, it has been demonstrated that hypoxia is able to modify endothelial properties. In effect, hypoxia has been shown to down regulate thrombornodulin expression on bovine arterial endothelial cells (BAEC) and microvascular endothelial cells (11, 13, 14). Moreover, it has been demonstrated that hypoxia decreases endothelial cell barrier function by lowering cAMP levels (15). cAMP is the major up regulator of thrombomodulin expression, so we investigated the effect of agents able to increase cAMP levels on membrane thrombomodulin expression in hypoxic conditions on HUVEC

Resume Introduction. — Le facteur de transcription NF-κB est un regulateur essentiel des ganes impliques dans la reponse a l’infection, a l’inflammation et au stress. Il a ete decrit comme une proteine fixee a une region specifique de l’ADN, constituee de dix paires de bases, activatrice du gane de la chaine legare κ des lymphocytes B. Actualites et points forts. — NF-κB est habituellement present dans le cytoplasme de la cellule fixee a une proteine inhibitrice appelee IκB. Le signal de localisation nucleaire (NLS) du NF-κB correspond a une courte sequence d’acides amines impliques dans le transport vers le noyau. Lors de l’activation de NF-κB, la proteine IκB est dissociee et degradee du complexe NF-κB. NF-κB migre alors vers le noyau et se fixe au niveau de son site ADN-specifique. La repression de l’expression du gane dependant de NF-κB serait un des elements majeurs de l’immunodepression induite par les glucocorticoides. Perspectives et projets. — Au niveau d’un site inflammatoire, les cellules endotheliales expriment de nombreux ganes sous le controle du facteur nucleaire NF-κB. Ce facteur de transcription, en presence de ses inhibiteurs, est au centre d’un systame d’autoregulation qui peut etre active par de nombreux stimuli au cours de diverses situations pathologiques vasculaires. NF-κB est ainsi un candidat serieux pour un nouveau type de traitement anti-inflammatoire.

Introduction Some viral infections are associated with deep venous thrombosis. We report a case of deep venous thrombosis in an adult with varicella. He had neither known predisposing factors for thrombosis nor thrombophilia. Exegesis Transient significant level of antiphospholipid antibodies and lupus circulating anticoagulant were observed. There was no evidence of thrombophilia. Deep venous thrombosis has been mostly associated with varicella in children. A transient protein S deficiency was present in almost all cases, though it was sometimes related to an anti-protein S antibody. This association is exceptional in adults. Some viruses such as herpesvirus and HIV are responsible for endothelium dysfunction, but this is still unclear in the case of varicella-zoster virus. Conclusion In our observation, endothelium activation or antiphospholipid antibodies might be responsible for thrombosis.

SummaryPatients infected with HIV are at increased risk of atherosclerosis, and have evidence of endothelium dysfunction. The hypothesis was tested that HIV-related endothelium dysfunction is related to loss of antioxidants. This was done by the supplementation of the antioxidants selenium and beta-carotene. We supplemented the diet of 10 HIV-sero-positive subjects with 100 μg selenium daily, 11 subjects with 30 mg beta-carotene twice daily while 15 subjects were not supplemented. Plasma was obtained at outset and after a year, and tested by ELISA for endothelial cell, platelet and inflammatory markers.The non-supplemented patients experienced increases in von Wille-brand factor and soluble thrombomodulin (both p < 0.01). There were no changes in any of the indices in the patients taking selenium or beta-carotene.Increased von Willebrand factor and soluble thrombomodulin in the non-supplemented patients imply increased damage to the endothelium over the year of the study. Therefore we interpret the lack of increase in the patients taking antioxidants as evidence of the protection of the endothelium by these agents.

To determine if anti-endothelial cell antibodies (AECA) and plasma markers of endothelial cell function are related to disease severity in systemic lupus erythematosus (SLE).We measured AECA by human umbilical vein endothelial cell binding, endothelial markers von Willebrand factor, soluble thrombomodulin, and soluble E-selectin by ELISA, and disease severity by SLEDAI and SLICC/ACR in 35 patients with SLE.Despite high levels of IgG AECA (p = 0.001) and von Willebrand factor (p = 0.0007) compared to 21 healthy controls, we found a positive correlation only between IgG AECA and the SLEDAI index (r = 0.393, p = 0.021).IgG AECA seem to be related to disease activity in SLE, possibly in a pathogenic role. Conversely, plasma markers of endothelial cell damage seem to be an epiphenomenon and may simply be related to excess inflammation.

Background In systemic lupus erythematosus (SLE), disease severity may be related to factors such as endothelial cell damage. Objectives We tested the hypothesis that anti-endothelial cell antibodies (AECA) and plasma markers of endothelial cell dysfunction would be related to disease severity in SLE. Methods Accordingly we measured AECA by human umbilical vein endothelial cell binding, endothelial markers von Willebrand factor, soluble thrombomodulin, soluble E-selectin by ELISA, and disease severity by SLEDAI and SLICC/ACR in 35 patients with SLE. Results Despite high levels of IgG AECA (p Conclusion We conclude that IgG AECA seem to be related to disease activity in SLE, possibly in a pathogenic role. Conversely, plasma markers of endothelial cell damage seem to be an epiphenomenon and may only be related to excess inflammation in these patients.

The transcriptional regulatory protein nuclear factor kappaB (NFkappaB) participates in the control of gene expression of many modulators of the inflammatory and immune responses. Various activators trigger NFkappaB release and nuclear translocation after phosphorylation and proteolytic degradation of IkappaB. This study evaluated the abilities of fluorescence and confocal microscopies, laser scanning cytometry (LSC), electrophoretic mobility-shift assay (EMSA), and Western blotting to detect NFkappaB activation in endothelial cells (ECs) and to investigate the role of homocysteine (Hcy) in NFkappaB activation. ECs were treated with interleukin-1B (10 ng/mL) or Hcy thiolactone (1 and 5 mmol/L) as NFkappaB activators. Hcy, a thiol-containing amino acid, has been shown to directly damage ECs in vitro. Experimental evidence suggests that the atherogenic propensity associated with hyperhomocysteinemia results from EC dysfunction. When ECs were pretreated with an inhibitor (pyrrolidine dithiocarbamate, 100 micromol/L) or with staurosporine (5 microL/mL), no NFkappaB activation was observed. NFkappaB activation in ECs could be detected with all five techniques, clearly showing NFkappaB translocation from the cytoplasm to the nuclei. Confocal microscopy was more sensitive and less subjective than immunofluorescence microscopy. LSC was even more sensitive, specific, and reproducible. EMSA, the reference method, has the disadvantages of being radioactive, expensive, and time consuming. Western blot analysis detected the NFkappaB p50 subunit implicated in NFkappaB activation. The techniques usually used to detect NFkappaB activation in ECs are immunofluorescence microscopy and confocal microscopy, LSC, EMSA, and Western blot analysis, but none of them is ready for routine use.

Hyperhomocysteinaemia is a risk factor for premature atherosclerosis and venous thromboembolic disease. Supplementation with folic acid and vitamin B6 has been shown to decrease plasma homocysteine but data fail to assess an effect on the progression of vascular disease. We measured plasma homocysteine and two markers of endothelial injury (plasma soluble thrombomodulin and von Willebrand factor) at baseline and after 3 months of treatment with folic acid and vitamin B6. After this treatment there was a significant decrease in fasting soluble thrombomodulin (−15 ng/ml, 95%CI 5–22.2). Von Willebrand factor was significantly raised after methionine load at baseline but did not significantly rise after supplementation.

Lejoly-Boisseau, H., Appriou, M., Seigneur, M., Pruvost, A., Tribouley-Duret, J., and Tribouley, J. 1999.Schistosoma mansoni: In vitroadhesion of parasite eggs to the vascular endothelium. Subsequent inhibition by a monoclonal antibody directed to a carbohydrate epitope.Experimental Parasitology91,20–29.Schistosoma mansonieggs come into direct contact with the vascular endothelium, particularly in the postcapillary venules of the mesenteric tract (oviposition site). We investigated the adhesion of eggs to endothelial cells in a staticin vitroassay and in a flow-basedin vitroassay. LiveS. mansonieggs rapidly attached, in a time-dependent manner, to the human endothelial cell line ECV 304, but not KOH-treated eggs. Activation of ECV monolayers with interleukin-1 promoted liveS. mansonieggs adhesion. Anin vitroflow-based assay of human umbilical vein endothelial cells (HUVEC) showed the influence of wall shear stresses on the attachment of eggs to endothelial cells, particularly under postcapillary venule shear stress conditions. Interleukin-1 activation of HUVEC promoted adhesion between live eggs and endothelial cells. Higher wall shear stresses were needed to obtain the detachment of eggs from activated endothelial cells than control cells. Preincubation of interleukin-1-activated HUVEC, in a staticin vitroassay, with monoclonal antibodies specific for intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1 significantly decreased adhesion of live eggs. Previous studies have shown that a monoclonal antibody specific for a schistosome carbohydrate epitope abundant in eggs is related to the Lewis X antigen. In this study, the anti-Lewis X-specific monoclonal antibody was used for adhesion-inhibition assays. Preincubation of eggs with this monoclonal antibody significantly decreased adhesion of live eggs to interleukin-1-activated HUVEC culturedin vitro.These results suggest that surface adhesion molecules, expressed by endothelial cells under conditions of interleukin-1 activation, directly participate in egg adhesion and that egg carbohydrate antigens play an important role in liveS. mansoniegg adhesion to the vascular endothelium.

SummaryEndothelial damage is present in HIV infection but our understanding of markers and mechanisms is incomplete. We found increased levels of markers of endothelial cell damage such as von Willebrand factor (vWf), soluble thrombomodulin (sTM) and adhesion molecule E- selectin in 90 subjects seropositive for HIV relative to healthy controls. sTM was strongly raised in those patients with the lowest CD4+ cell count (p

Atherosclerosis has been reported in some HIV-positive subjects without any known risk factor. The purpose of the present study was to investigate cervical arteries, abdominal aorta and femoral arteries by B-mode ultrasonography and doppler in 30 HIV-positive subjects matched to 18 controls for sex, age, tobacco consumption and arterial hypertension. Although no haemodynamically or clinically relevant lesions were found, plaques occurred more often in patients than in controls (11 patients, 36.7% vs. 2, 11.1%; P = 0.05). Compared to the HIV-positive patients without plaques, those with plaques had a tendency to have decreased lower HDL cholesterol, higher tobacco consumption and lower CD4-cell count (77 +/- 85/mm3 vs. 220 +/- 202/mm3). The patients with plaques (but not those without plaques) had lower HDL cholesterol than controls (P = 0.03). Asymptomatic atherosclerosis seems to be more frequent in HIV-positive patients and is associated to lower HDL cholesterol.

The plasma thrombomodulin (TM) level depends on the integrity of the endothelium and the clearance of the molecule. In several different pathological conditions, plasma TM levels increase with damage to the endothelium. We studied plasma TM levels in patients with various localizations of atheromatous arterial disease who had normal serum creatinine levels. Two groups of patients had a single symptomatic localization, which was either peripheral occlusive arterial disease (POAD) or ischemic heart disease (IHD) and a third group of patients had multiple symptomatic localizations (polyvascular). We compared the plasma TM levels with the plasma levels of other specific markers of endothelial cell activation such as: prostacyclin (PGI2), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1). Plasma TM levels were significantly increased in all three individual groups and when all patients were considered (total patients), as compared with normal controls. When all patients were considered, there was a significant positive correlation between plasma TM levels and t-PA and between plasma TM levels and PGI2. A significant positive correlation was also found between the plasma TM levels and PAI-1 for patients with POAD. Thus, our findings suggest that an increased influx of TM into the plasma may be caused by endothelial cell damage in patients with atheromatous arterial disease. However in our study, the plasma TM levels obtained were similar for all three types of atheromatous arterial disease. Though plasma thrombomodulin is a marker of endothelial cell injury, it cannot be of a clinical interest until its levels are related to the extend of the atheromatous lesions. Moreover, we show that plasma TM is not independent of the usual endothelial cell activation markers.

In vitro studies on the action of buflomedil (BFL) and its derivative CRL 41034 on the polymorphonuclear cells (PMN) has been performed using functional tests and scanning electron microscopy. The two drugs exhibited the same effects. BFL does not change the in vitro chemotaxis of PMN, but exhibits a regulatory effect on ZMS-induced aggregation of these cells. BFL also appeared to decrease superoxide production of PMN, in a dose- and time-dependent way. The cytoskeleton F-actin polymerization, analyzed through the binding of rhodamin-phalloidin, was increased when the total F-actin of the cells was unchanged. When cell extensions were studied morphologically a change in the shape of the pseudopods as well as the general aspect of the PMN (cottonous aspect) was observed as compared to controls. These drug-induced modifications in the shape change may be efficient in adhesion processes. Finally this latter effect and the influence on oxygen metabolite production could be another means of BFL to protect the microvessels during ischemia, in addition to its vasomotion promoting properties.

Soluble intercellular adhesion molecule-1 (sICAM-1) level was measured in sera from 41 patients with Schistosoma mansoni schistosomiasis and compared with the sICAM-1 level in 41 healthy subjects. A significant increase in serum sICAM-1 was observed in patients with schistosomiasis compared with control subjects. As they were inhabitants of the French Antilles, the patients were, however, not settled in a malaria endemic zone, allowing this cause of sICAM-1 enhancement to be eliminated. No correlation was found between the level of sICAM-1 and the schistosomiasis serological titre. Such results favour the hypothesis of an activation of vascular endothelial cells due to egg deposition.

Variations of plasma thrombomodulin levels, which is a putative marker of endothelial damage, were measured in 23 cases of systemic lupus erythematosus (without antiphospholipids and/or lupus anticoagulants) and in 7 dermatopolymyositis. This parameter was significantly increased in dermatopolymyositis suggesting the involvement of ischemia in endothelium injury. No change in plasma thrombomodulin levels was observed in patients with systemic lupus erythematosus.

Plasma thrombomodulin (TMp) is proned as a marker of endothelial cell domage. Significantly elevated levels were found in patients with atherosclerosis. Furthermore a positive correlation was pointed out between TMp and prostacyclin (PG12) and tissue-type plasminogen activator (t-PA) which are markers of endothelial cells activation.