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15-Lipoxygenase (15-LO) expression in artery wall cells has been demonstrated during the development of atherosclerosis in various animal models. We examined whether the expression of 15-LO in aortic endothelial cells affects the gene expression of the adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1). Transient transfection of human 15-LO cDNA into bovine aortic endothelial cells led to the expression of 15-LO protein and enzymatic activity. We studied the induction of VCAM-1 mRNA in these cells. 15-LO expressing cells showed no detectable levels of VCAM-1 message. However, when TNF was added to these cells there was a synergistic increase in VCAM-1 expression relative to cells that were transfected with control plasmid pcDNA I. Our data suggest that 15-LO expression in aortic endothelium may amplify the expression of VCAM-1 induced by inflammatory stimulus during atherogenesis.

The effect of bovine lipoprotein lipase (LPL) on very low density lipoprotein (VLDL) binding to subendothelial matrix was studied. Without LPL, VLDL bound poorly to the matrix. However, decreasing NaCl or elevating Ca++ concentration increased matrix VLDL binding. With LPL, VLDL binding was markedly increased. Since LPL is a normal constituent of the artery wall and is elevated in atherosclerotic lesions, we postulate two potential mechanisms for the involvement of VLDL and LPL in atherogenesis. First, VLDL acquisition is attenuated by the increased matrix LPL content in the developing atheroma. Secondly, elevated plasma levels of VLDL (and VLDL remnants) such as in Type II or III dyslipidemia could enhance such interactions. These events likely accelerate the rate of atherosclerosis lesion development.

Lipoprotein lipase (LPL), bound to the luminal surface of vascular endothelium catalyzes lipoprotein triglyceride hydrolysis. Studies were performed to identify human aortic endothelial (HAEC) cell-surface proteins having high affinity for LPL. LPL-sepharose affinity chromatography of [35S]O4 labeled HAEC proteins identified a 220-kDa proteoglycan. Ligand blotting of HAEC plasma membrane proteins with LPL revealed two specific binding proteins of MW 116 kDa and 85 kDa, respectively, which were not released from the cell-surface by heparin treatment. Since the 220-kDa and 116-kDa proteins have been reported previously in bovine endothelial cells, we focused on the 85-kDa protein. The 85-kDa protein was not labelled by incubation of the cells with [35S]O4, suggesting that it is not a sulfated proteoglycan. Treatment of HAEC with tunicamycin markedly decreased detection of the 85-kDa protein, suggesting that it is likely a glycoprotein synthesized by HAEC. We conclude that HAEC cell surface has three specific LPL binding proteins, a 220-kDa proteoglycan, a 116-kDa protein and a novel 85-kDa protein.

The effects of a novel anti-inflammatory agent, 5-methoxy-3-(1-methyl-ethoxy)benzo[b]thiophene-2-carboxamide-1-oxide (PD 144795) on adhesion molecule expression in tumor necrosis factor (TNF) stimulated human aortic endothelial cells (HAEC) were examined. PD 144795 treatment markedly inhibited the TNF-induced cell expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein and mRNA. Gel shift assays using nuclear extracts from HAEC treated with PD 144795 failed to show a decrease in the activation of NFkB by this compound, whereas pyrrolidine dithiocarbamate (PDTC), an antioxidant, markedly inhibited the activation of this transcription factor. Thus, PD 144795 inhibits agonist-stimulated VCAM-1 and ICAM-1 expression likely via an NFkB independent mechanism, distinct from that of PDTC. Such agents may provide a novel approach for control of adhesion molecule gene expression in inflammation.

Lipoprotein lipase (LPL) bound to vascular endothelial cells hydrolyses triglycerides in plasma lipoproteins. To explore the role of LPL in atherogenesis, the effect of LPL-mediated lipolysis of very low density lipoproteins (VLDL) on monocyte adhesion to endothelial cells was examined. Adhesion of U937 monocytes to porcine aortic endothelial cells that were incubated with VLDL and purified bovine milk LPL was markedly higher than endothelial cells that were incubated with VLDL alone. The increase in monocyte adhesion obtained with VLDL was dependent on the concentration of the lipoprotein, monocyte dose and time of incubation. The increase in adhesion correlated with generation of free fatty acids from the hydrolysis of triglycerides in VLDL by LPL. Furthermore, direct addition of oleic acid to endothelial cells also increased adhesion of monocytes. We postulate that LPL-derived lipolytic products increase monocyte adhesion to vascular endothelium and thereby promote atherogenesis.

Rat serum phosphorylcholine binding protein (PCBP) is characterized by its Ca2+ dependent property to bind phosphorylcholine ligand. PCBP immobilized on sepharose has been shown to selectively bind human plasma apo B and E containing lipoproteins. The present report describes an inhibitory effect of PCBP on the binding of human 125I-LDL to LDL receptors on estradiol treated rat liver membranes. Pre-incubation of liver membranes with PCBP did not affect the binding of 125I-LDL to the membranes. Gel filtration analysis of the incubation products from the LDL-receptor assay showed a concentration dependent binding of 125I-PCBP to LDL. The inhibitory effect of PCBP is likely due to the formation of LDL-PCBP complex and not due to the binding of PCBP to the LDL receptor site.