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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.

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

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.

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.

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.

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.