Abstract 513: Role of Heparanase in Atherosclerotic Plaque Formation

Abstract

Little is know about the role of enzymatic regulation of glycosaminoglycans in the formation of atherosclerotic plaques. Heparanase is the only known mammalian enzyme that degrades the glycosaminoglycan heparan sulfate. We tested the hypothesis that heparanase could regulate atherogenesis and atherosclerotic plaque severity. We transduced human endothelial cells with lentiviruses expressing one of four different constructs expressing shRNA targeted to heparanase or a scrambled control sequence. These shRNA knocked down the expression of heparanase to 30-50% of wild type expression levels. These lines were selected using puromycin and confirmed for gene modulation using immunostaining and western blotting for heparanase. The ability to uptake lipids was tested using fluorescently labeled, oxidized low-density lipoprotein (OxLDL). We found that cells expressing 30% of the heparanase protein in wild type cells had increased uptake of OxLDL in comparison to wild type cells (1.8+0.1 versus 1.00+0.07 of normalized OxLDL uptake; p <0.05) . We also evaluated the formation of atherosclerosis in transgenic mice overexpressing the human heparanase gene (HPA-Tg). This mouse line was crossed with the ApoE knockout mouse line. We exposed mice from this HPA-Tg/ApoE KO line and from the ApoE KO line of 20 weeks of age to 6 weeks of a high fat diet. At week 2 we surgically implanted an extravascular cuff on the left carotid artery to create a region of disturbed flow and low shear stress. Following the surgery, we used a high resolution combined ultrasound and photoacoustic imaging system to image the flow and plaque morphology after 1 day, 2 weeks and 4 weeks post-implantation of the cuff. Overexpression of heparanase increases the formation and severity of atherosclerosis in this model. Color doppler imaging revealed the existence of oscillatory flow in the left common carotid artery due to the formation of atherosclerotic plaque. This analysis confirmed an increase in the inflammatory response and neointimal formation in response to the low endothelial shear stress induced by the cuff in the HPA models compared to the ApoE. Our studies support that heparanase is a powerful regulator of lipid uptake and atherosclerotic plaque formation in an animal model of hyperlipidemia.