Abstract 15910: Endothelial Lipase is a Critical Determinant of High Density Lipoprotein-Stimulated Sphingosine 1-Phosphate-Dependent Signaling in Vascular Endothelium

Abstract

Objective: The role of high-density lipoprotein (HDL) in reverse cholesterol transport has been extensively characterized, yet other bioactive lipids bound to HDL also elicit responses in vascular tissues. The mechanisms whereby HDL-associated lipids modulate signaling pathways in the vasculature are incompletely understood. The effects of HDL on endothelial cells are mediated in part by HDL-associated sphingosine 1-phosphate (S1P), which binds to S1P1 receptors and promotes activation of eNOS and kinase Akt. Endothelial lipase (EL) has been identified as an enzyme that is critically involved in HDL-modulated lipid metabolism in the vasculature. In these studies, we used a combination of knockout animal models, RNA interference methods, and pharmacological approaches to characterize the role of EL in the control of signaling pathways involving HDL-associated S1P.

Methods and Results: In the endothelial lipase-null (EL-/-) knockout mouse, HDL-induced angiogenesis (assayed using the aortic ring assay) was markedly decreased compared to wild-type mice. We developed and validated a duplex siRNA targeting construct that potently and specifically knocked down EL by >90%. In endothelial cells, siRNA-mediated EL knockdown abrogated HDL-promoted cell migration (using the endothelial scratch assay) and tube formation (analyzed in the Matrigel assay). HDL promoted robust phosphorylation of eNOS1179 and Akt473 (p<0.01 for both responses), but this response to HDL was entirely lost following siRNA-mediated EL knockdown. Importantly, the addition of S1P completely recovered the phenotype of HDL-induced endothelial migration and Akt/eNOS phosphorylation that had been lost following siRNA-mediated EL knockdown. HDL-induced EC migration and Akt/eNOS phosphorylation were completely blocked by the S1P1 antagonist W146 but not by the S1P3 antagonist CAY10444.

Conclusions: Endothelial lipase is a critical determinant of the effects of HDL on S1P-mediated vascular responses and acts on HDL to promote activation of S1P1, leading to Akt/eNOS phosphorylation and to endothelial migration and angiogenesis. Modulation of endothelial lipase activity and of HDL-associated S1P represent potential therapeutic targets for cardiovascular disease states.