Endothelial, cultured

Increases in plasma S-AA levels have previously been reported in patients with coronary disease (57). S-AA and plasma intracellular adhesion molecule-1 were elevated in patients with CAD and hyperhomocysteinemia, but only S-AA decreased after vitamin supplementation (35). Homocysteine activates nuclear factor- in endothelial cells, possibly via oxidative stress (58), and increases monocyte chemoattractant protein-1 expression in vascular smooth muscle cells (59). Additionally, it stimulates interleukin-8 expression in human endothelial cultures (60). These inflammatory factors are known to participate in the development of atherosclerosis. Taken together, these reports suggest an association of elevated tHcy and low-grade inflammation in CAD. 

Cells can be obtained from biopsy samples after extensive washing in PBS containing antibiotics and antimycotics. 1 cm squares of tissue can be incubated with dispase as described for endothelial cultures. ( 6.9) or with PBS-A supplemented with 1 mM EDTA. Cells are plated out onto feeder layers ( 8.1.4). 

We further tested this effect by inducing " " chemical hypoxia (20, 21). Potassium cyanide (KCN, 2.5 mM) was used in endothelial culture for 2 h. In the study group (JST=6), ATP-vesicles were added while no ATP-vesicles were added into the control group (N=6). After 2 h, the cells in the study group maintained more than 90% viability as determined by trypan blue exclusion. In the control, no more than 10% of the cells were still alive (Fig. 3). 

Van Iwaarden F, de Groot PG, Bouma BN The binding of high molecular weight kininogen to cultured human endothelial cells. J Biol Chem 1988 263 4698-4703. 

Schaefer U, Schneider A, Rixen D, Neugebauer E (1998) Neutrophil adhesion to histamine stimulated cultured endothelial cells is primarily mediated via activation of phospholipase C and nitric oxide synthase isozymes. Inflamm Res 47(6) 256-264 Schaefer U, Schmitz V, Schneider A, Neugebauer E (1999) Histamine induced homologous and heterologous regulation of histamine receptor subtype mRNA expression in cultured endothelial ceUs. Shock 12(4) 309-315... 

Evidence from cellular studies in vitro initially showed how oxidative processes could play a central role in the pathological changes involved in the genesis of atherosclerosis. LDL can be oxidatively modified in culture by a range of cell types including endothelial cells (Henriksen et a.1., 1981), arterial smooth muscle cells... 

Henriksen, T., Mahoney, E.M. and Steinberg, D. (1981). Enhanced macrophage degradation of low density lipoproteins previously incubated with cultured endothelial... 

Oxidatively modified LDL up-regulates the surfece expression of VCAM-1 and intracellular adhesion molecule-1 (ICAM-1) in cultured endothelial cells, promoting the interactions between both cell types (Kume et al., 1992). This may play a pivotal role in the development of atherosclerosis by promoting the penetration of circulating monocytes into the suben-dothelial space whilst inhibiting the mobility of resident macrophages. It has been previously demonstrated that ICAM-1, E-selectin, and VCAM-1 are up-regulated in the microvasculature of rheumatoid but not control synovium (Corkill et al., 1991 Koch et al., 1991). The association between ox-LDL and increased expression of adhesion molecules in the inflamed synovium has yet to be studied. 

Kume, N., Cybulsky, M.I. and Gimbrone, M.A. (1992). Lysophosphatidylcholine, a component of atherogenic lipoproteins, mediates mononuclear leucocyte adhesion in cultured human and rabbit endothelial cells. J. Clin. Invest. 90, 1138-1144. 

The cytotoxicity of LDL can also be inferred from the study by Blake et al. (1985). In this study of human cultured endothelial cells, stored sera from patients with necrotizing arteritis demonstrated an enhanced tendency to develop oxidized LDL, which correlated closely with endothelial cell cytotoxicity. This process appears to require the presence of both oxygen and transition metal ions such as iron in the presence of a reducing agent (Gebicki ef /., 1991). There is considerable evidence that transition metals are involved in cell-induced modifications of LDL including the inhibitory effects of EDTA and desfer-rioxamine (Hiramatsu et 1987). A role for Of in LDL modification by endothelial cells and fibroblasts comes from studies showing inhibition of LDL oxidation by SOD (Steinbrecher, 1988). 

Kaneko et al. (1993) have described a group of lipophilic ascorbic-acid analogues that have been studied in cultured human umbilical vein endothelial cells that were first incubated with test drug and then exposed to lipid hydroperoxides. Although ascorbate itself did not protect the endothelial cells, derivatives like CV3611 protected. Pretreatment was necessary. CV3611 was synergistic with vitamin E. The authors concluded that these lipophilic antioxidants incorporate into endothelial cell membranes where they are effective inhibitors of lipid peroxidation. In contrast, lipophobic antioxidants were not effective in their hands (Kaneko et al., 1993). 

Rosolowsky, M. and Campbell, W. B., Synthesis of hydroxyeicosatetraenoic (HETES) and epoxyeicosatrienoic acids (EETS) by cultured bovine coronary artery endothelial cells, Biochim. Biophys. Acta, 1299, 267, 1996. 

Growing clinical data also points to the importance of IL-8 in atherogenesis. IL-8 has been found in atheromatous lesions from patients with atherosclerotic disease including carotid artery stenosis (103), CAD (118), abdominal aortic aneurysms (AAA) (103,104,114), and peripheral vascular disease (PVD) (104). Furthermore, studies using plaque explant samples have yielded more direct evidence for IL-8 involvement. Media from cultured AAA tissue induced IL-8-dependent human aortic endothelial cell (HAEC) chemotaxis (122). Homocysteine, implicated as a possible biomarker for CAD, is also capable of inducing IL-8 (123-125) by direct stimulation of endothelial cells (123,124) and monocytes (125). When patients with hyperhomocysteinemia were treated with low-dose folic acid, decreases in homocysteine levels correlated with decreases in IL-8 levels (126). Statins significantly decrease serum levels of IL-6, IL-8, and MCP-1, as well as expression of IL-6, IL-8, and MCP-1 mRNA by peripheral blood monocytes and HUVECs (127). Thus, IL-8 may be an underappreciated factor in the pathogenesis of atherosclerosis. 

B47. Brigham, K. L Meyrick, B., Berry, L. C Jr., and Repine, J. E., Antioxidants protect cultured bovine lung endothelial cells from injury by endotoxin. J. Appl. Physiol. 63, 840-850 (1987). 

C8. Carlos, T Swartz, R Kovach, N. L., Yee, E., Rosso, M., Osbom, L. C Rosso, G., Newman, B Lobb, R., and Harlan, J., Vascular cell adhesion molecule-1 (VCAM-1) mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells. Blood 76, 965-970 (1990). 

Cultured human endothelial cells generate tissue factor in response to endotoxin. J. Clin. Invest. 71,1893-1896 (1983). 

P13. Pober, J. S., Bevilacqua, M. P., Mendrick, D. L., Lapierre, L. A., Fiers, W., and Gimbrone, M., Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells. J. Immunol. 137,1893-1896 (1986). 

R2. Rainger, E. S., Wautier, M. P., Nash, G. B., and Wautier, J. L., Prolonged E-selectin induction by monocytes potentiates the adhesion of flowing neutrophil to cultured endothelial cells. Br. J. Haematol. 92,192-199 (1996). 

K Audus, R Bartel, I Hidalgo, R Borchardt. The use of cultured epithelial and endothelial cells for drug transport and metabolism studies. Pharm Res 7 435, 1990. 

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