Abstract 465: Heparanase Alters Endothelial Phenotype, Leukocyte Attachment and Atherosclerotic Plaque Formation

Abstract

Little is known about the role of enzymatic regulation of glycosaminoglycans in the formation of atherosclerotic plaques. We tested the hypothesis that heparanase (HPA) could regulate endothelial cell inflammatory responses and immune cell mobilization, promoting atherogenesis. We transduced human endothelial cells with lentiviral vectors for one of four constructs expressing shRNA targeted to HPA or a scrambled control sequence. These shRNA knocked down HPA expression 30-50% of wild type levels. We also overexpressed HPA in these cells using lentiviral constructs. Finally, we transduced cells to express miRNA 1258 known to reduce levels of HPA in endothelial cells. These cell lines were confirmed for gene modulation using PCR and western blotting for HPA, then assayed for the expression of inflammatory markers such as ICAM, VCAM, and IL-8 under static and physiologic levels of shear stress (12 dynes/cm 2 ). Increasing HPA levels led to a corresponding increase in inflammatory cytokines in endothelial cells, while reduction of HPA levels by targeted shRNA or miRNA caused reduced expression. Using fluorescently labeled monocytes we exposed the endothelial cell layer to circulating leukocytes in an in-vitro flow system. Adhesion of monocytes increased in cell lines with enhanced HPA expression and decreased in those with HPA knockdown. We also evaluated the effects of low endothelial sheer stress in transgenic mice overexpressing the human HPA gene crossed with the ApoE knockout mouse line. We surgically implanted an extravascular cuff on the left carotid artery of mice from this HPA-Tg/ApoE line and the ApoE line to create a region of disturbed flow and low shear stress. We used 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. Doppler imaging revealed overexpression of HPA increases the formation and severity of atherosclerosis. The oscillatory flow in the carotid artery due to the cuff placement lead to increased plaque accumulation and increased inflammation in response to the induced shear stress in the HPA-Tg/ApoE mice compared to the ApoE control mice. Our studies support HPA as a potent regulator of inflammation and plaque formation.