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Low density lipoprotein (LDL) interactions with the endothelium are thought to play a major role in the development of atherosclerosis. The mechanism(s) involved are not fully understood, although several lines of evidence support the idea that oxidation of LDL increases its atherogenicity. In this study we report for the first time that native LDL (n-LDL) binding to the LDL receptor (100-700 mug/ml) triggers a rise in intracellular calcium which acts as a second messenger to induce vascular cell adhesion molecule-1 (VCAM-1) expression in human coronary artery (HCAEC) and pig aortic endothelial cells (PAEC) and VCAM-1 and E-selectin expression in human aortic (HAEC) endothelial cells. Preincubation of HCAEC with a monoclonal antibody (IgGC7) to the classical LDL receptor or pretreatment with pertussis toxin blocked the n-LDL-induced calcium transients. Preincubation of each of the endothelial cell lines with the calcium chelator 1,-2-bis(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acetomethyl ester (BAPTA/AM) prevented the expression of VCAM-1 and E-selectin. The increase in VCAM-1 by n-LDL results in increased monocyte binding to HCAEC which can be attenuated by inhibiting the intracellular calcium rise or by blocking the VCAM-1 binding sites. These studies in human and pig endothelial cells link calcium signaling conferred by n-LDL to mechanisms controlling the expression of endothelial cell adhesion molecules involved in atherogenesis.

Lp(a) is a major inherited risk factor for premature atherosclerosis. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to interfere with plasminogen activation and its atherogenic potential as a lipoprotein particle after receptor-mediated uptake. We demonstrate that Lp(a) stimulates production of vascular cell adhesion molecule 1 (VCAM-1) and E-selectin in cultured human coronary artery endothelial cells (HCAEC). This effect resulted from a rise in intracellular free calcium induced by Lp(a) and could be inhibited by the intracellular calcium chelator, BAPTA/AM. The involvement of the LDL and VLDL receptors in Lp(a) activation of HCAEC were ruled out since Lp(a) induction of adhesion molecules was not prevented by an antibody (IgGC7) to the LDL receptor or by receptor-activating protein, an antagonist of ligand binding to the VLDL receptor. Addition of alpha2-macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease levels of VCAM-1 and E-selectin stimulated by Lp(a), suggesting that neither the low density lipoprotein receptor-related protein nor cell-surface proteoglycans are involved in Lp(a)-induced adhesion molecule production. Neither does the binding site on HCAEC responsible for adhesion molecule production by Lp(a) appear to involve plasminogen receptors, as levels of VCAM-1 and E-selectin were not significantly decreased by the addition of glu-plasminogen, the lysine analog epsilon-aminocaproic acid, or by trans-4-(aminomethyl)-cyclohexanecarboxymethylic acid (tranexamic acid), which acts by binding to the lysine binding sites carried on the kringle structures in plasminogen. In contrast, recombinant apolipoprotein (a) [r-apo(a)] competed with Lp(a) and attenuated the expression of VCAM-1 and E-selectin. In summary, we have identified a calcium-dependent interaction of Lp(a) with HCAEC capable of inducing potent surface expression of VCAM-1 and E-selectin that does not appear to involve any of the known potential Lp(a) binding sites. Because leukocyte recruitment to the vessel wall appears to represent one of the important early events in atherogenesis, this newly described endothelial cell-activating effect of Lp(a) places it at a crucial juncture in the initiation of atherogenic disease and may lead to a better understanding of the role of Lp(a) in the vascular biology of atherosclerosis.

The expression of adhesion molecules by endothelial cells is crucial in many inflammatory processes and plays an active role in the development of reperfusion injury, acute and chronic rejection. The expression of adhesion molecules in different parts of the coronary tree to cytokine stimulation is not known. We describe here a detailed study of the effects of the inflammatory cytokines TNFalpha and IL-1beta on the expression of adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), E-selectin and intracellular cell adhesion molecule-1 (ICAM-1) on human aortic root (HAEC), coronary artery (HCAEC) and heart microvascular (HHMEC)) endothelial cells in culture, using flow cytometry. We found constitutive levels of both VCAM-1 and E-Selectin on HCAEC and HHMEC (approximately 20%) which were significantly higher compared to HAEC (approximately 3%). There was an extreme sensitivity of HCAEC and HHMEC to 0.002 ng/ml TNFalpha: (VCAM-1 approximately 40%, E-Selectin approximately 25%) respectively, compared to HAEC (VCAM-1 approximately 5%, E-selectin approximately 5%). IL-1beta showed a similar pattern of expression at low doses (5 U/ml), but was less potent. We also observed prolonged expression of these adhesion molecules, especially on the HHMEC (>48 hours) compared to HAEC. There was also increased binding of peripheral blood mononuclear cells (PBMC) to both non-stimulated and TNFalpha stimulated HCAEC and HHMEC compared to HAEC. This data suggest that endothelial cells in different regions of the coronary tree express different patterns of basal and cytokine-stimulated adhesion molecule expression.

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