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Recent studies have presented evidence that the processes of hypoxaemia and reperfusion are involved in several pathogenetic mechanisms of atherosclerotic lesions. The ability of hypoxaemia to activate circulating white blood cells (WBCs) and enhance WBC-endothelial cell (EC) interactions is suspected to be a major factor in deleterious processes in the blood vessel wall. Various groups have suggested that cell adhesion molecules (CAMs), such as ICAM-1, VCAM-1 and E-selectin and their leukocyte ligands are involved in intercellular activities of the relevant cell types. We studied the effects of different oxygen tensions, simulating normoxic conditions, hypoxia and hyperoxia in vitro with the help of an umbilical vein EC model in order to determine the effects of oxygenation on CAM presentation on vascular ECs with and without further cytokine and endotoxin (lipopolysaccharides; LPS) stimulation. Semiquantitative analysis of ICAM-1, E-selectin and VCAM-1 was performed using cell enzyme immunoassay techniques. The presentation of ICAM-1, E-selectin and VCAM-1 remained on the whole unaffected by both hypoxia and hyperoxic conditioning after both 7 and 24 h. Stimulation of ICAM-1 by cytokines and LPS was only marginally influenced by the oxygen tension. Cytokine induction of E-selectin was not affected after 7 h and was even reduced under hypoxia, compared to the control culture after 24 h, while stimulation was increased by hyperoxia. VCAM-1 was reduced in both the hypoxic and hyperoxic culture, while being maximally stimulated by cytokines and LPS after 7 h. In general, an effect of hypoxia was not found without any further stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial cells (EC) are very responsive to proinflammatory cytokines, e.g. interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha), as well as to bacterial lipopolysaccharide. EC are stimulated by these substances to secrete chemotactic factors and to increase expression of cell adhesion molecules (CAM), leading to dramatically altered interactions with leukocytes, e.g. granulocytes and monocytes. In these interactions E-selectin, ICAM-1 and VCAM-1 are known to play an important role, as they are presented by the EC and interact with corresponding ligands on the white blood cell membranes. These adhesion molecules have been studied worldwide in a variety of in vitro experiments using cultured EC. Different passages and mixtures of passages have been used in these experiments, often without any regard to the comparability of the results. In this study the expression of E-selectin, ICAM-1 and VCAM-1 on cultured human umbilical vein EC (HUVEC) obtained from different passages (passages 1-6) was studied after 4, 8 and 24 h of exposure to IL-1 beta and TNF alpha. In previous studies, we have shown that IL-1 beta and TNF alpha increase the expression of E-selectin and ICAM-1 on the cytoplasmatic membranes of HUVEC and human adult EC from the saphenous vein and femoral artery in a similar fashion. Using a comparative quantitative cell enzyme immunoassay, we found that the expression of the adhesion molecules was significantly reduced with increasing passages. There was also a decreased persistence of CAM comparing different periods of stimulation between 6 and 24 h in the different passages. These data indicate that the number of passages plays an important role in the expression of adhesion molecules on EC. The results are relevant for the meaningful planning of comparative in vitro studies on EC presentation of CAM.

Cultured mesothelial cells (HOMES) are very responsive to the proinflammatory cytokines, interleukin (IL)-1 and tumor necrosis factor-alpha (TNF-alpha). E-selectin, ICAM-1 and VCAM-1 are known to play an important role, because they are presented by diverse cell types, for example endothelial cells (ECs), and interact with co-responding ligands on white blood cell membranes. In this study, the expression of ICAM-1, VCAM-1, E-selectin as well as PECAM-1 on cultured HOMES was studied over 5, 24, 48 and 72 h exposure to IL-1 beta, interferon-gamma and TNF-alpha. In previous studies we have shown that IL-1 beta and TNF-alpha increase the expression of ICAM-1, E-selectin and VCAM-1 on the cytoplasmatic membranes of HUVECs, HSVECs and HAFECs (ECs from human umbilical vein, saphenous vein and femoral artery, respectively). Using a comparative quantitative cell enzyme immunoassay, we found that expression of the adhesion molecules ICAM-1 and VCAM-1 was significantly increased on HOMES in a dose- and time-dependent manner, compared to nonstimulated cells. Thus, ICAM-1 increased dramatically after 5 h incubation with TNF-alpha. Values of about 450% of the control level were measured. VCAM-1 was similarly stimulated after 24 h incubation with the same cytokine, although its level of expression was significantly lower than that of ICAM-1. In contrast to findings in the literature, VCAM-1 was not found to be expressed constitutively. E-selectin was neither constitutively expressed nor markedly inducible on HOMES. Only weak expression was found after 24 h incubation with high-dose IL-1 beta. PECAM-1 was expressed constitutively, as became evident in antibody dilution studies. These data indicate that HOMES respond to inflammatory stimuli, in some ways in a similar fashion to vascular endothelial cells, but also show a specific pattern of antigen presentation. The results are important for a better understanding of inflammatory processes in serous cavities. The data are also relevant for the improvement of antithrombogenous surfaces of the lumina of vascular prostheses by cell seeding.

Monocyte-endothelial interactions are of particular importance in the regulation of inflammation. The aim of the present study was to investigate the adhesion of functional different monocyte subsets to human umbilical vein endothelial cells treated with various cytokines or a glucocorticoid. The adherence of monocyte subset 27E10, which is associated with inflammatory processes, increased after endothelial activation with interleukin-1 beta (IL-1 beta), interferon-gamma (IFN gamma), tumor necrosis factor-alpha (TNF alpha), and the glucocorticoid prednylidene (Pred). The adherence at IFN gamma-treated endothelial cells was strong after a coculture duration of 10 min with a slight increase up to 60 min. The peak value after TNF alpha stimulation was reached after 15 min, thereafter quickly decreasing. IL-1 and Pred treatment caused a maximal adherence between 15 and 30 min followed by a slow decrease. TNF alpha and particularly interleukin-6 (IL-6) enhanced the endothelial adhesion of the monocyte subtype RM3/1, which is associated with the downregulation of inflammation. The maximal adherence was found after 15 and 30 min of coculture, respectively. The results show that, through modulation of the adhesive properties of endothelial cells, cytokines and glucocorticoids affect the adherence of monocyte subsets differently. They also suggest that IL-6 plays a role in the downregulation of acute inflammation.

Recent studies have presented evidence that the processes of hypoxaemia and reperfusion are involved in several pathogenetic mechanisms of atherosclerotic lesions. The ability of hypoxaemia to activate circulating white blood cells (WBCs) and enhance WBC-endothelial cell (EC) interactions is suspected to be a major factor in deleterious processes in the blood vessel wall. Various groups have suggested that cell adhesion molecules (CAMs), such as ICAM-1, VCAM-1 and E-selectin and their leukocyte ligands are involved in intercellular activities of the relevant cell types. We studied the effects of different oxygen tensions, simulating normoxic conditions, hypoxia and hyperoxia in vitro with the help of an umbilical vein EC model in order to determine the effects of oxygenation on CAM presentation on vascular ECs with and without further cytokine and endotoxin (lipopolysaccharides; LPS) stimulation. Semi-quantitative analysis of ICAM-1, E-selectin and VCAM-1 was performed using cell enzyme immunoassay techniques. The presentation of ICAM-1, E-selectin and VCAM-1 remained on the whole unaffected by both hypoxia and hyperoxic conditioning after both 7 and 24 h. Stimulation of ICAM-1 by cytokines and LPS was only marginally influenced by the oxygen tension. Cytokine induction of E-selectin was not affected after 7 h and was even reduced under hypoxia, compared to the control culture after 24 h, while stimulation was increased by hyperoxia. VCAM-1 was reduced in both the hypoxic and hyperoxic culture, while being maximally stimulated by cytokines and LPS after 7 h. In general, an effect of hypoxia was not found without any further stimulation. Moreover, evidence is presented that reoxygenation might be the more important aspect in the mechanisms of ischaemia/reperfusion.

Understanding the mechanisms involved in maintaining the integrity of the vascular endothelium is fundamental to studies on atherosclerosis, thrombosis, inflammation and tumor invasion. One of the essential aspects is the relationship between the endothelial cell (EC) layer and the underlying components of the basement membrane (BM). The importance of the biological role of the individual components of the BM in the promotion of EC adhesion is investigated. In this study suspensions of bovine corneal ECs (BCECs; 5 × 104/ml) were used to investigate the adhesion of EC to collagen type IV and a mixture of fragments of the tetrameric molecule (IV-F, consisting of 75, 120 and 140 kD fragments), as well as collagen types I and III, coated at a 10-µg/ml concentration onto glass coverslips in vitro. Adhesion was quantified after 2 h of interaction by direct counting in the light microscope following fixation of the adherent cells. Collagens type IV and IV-F markedly promoted BCEC adhesion both in the presence or absence of 10 or 50% fetal calf serum, indicating that the integrity of the tetrameric molecule is not required for EC adhesion to collagen type IV, but can be replaced by high molecular weight fragments. Collagens type I and HI increased EC adhesion in the absence of serum, although not in the presence of serum. Indirect evidence for a possible role of fibronectin in EC adhesion to type-IV collagen is given by the ability of the tetrapeptide (Arg-Gly-Asp-Ser (10 µg/ml) to temporarily block (15–30 min) the adhesion-promoting effect of type-IV collagen. The nature of the adhesion sequences on the fragments of type-IV collagen remains to be elucidated.