Abstract 707: L-arginine Metabolism Regulated By Sirt3 During Endothelial Cell Dysfunction And Atherosclerosis

Abstract

Background: Atherosclerosis is the chronic inflammation of the vascular wall initiated by endothelial cell dysfunction. SIRT3 is a mitochondrial protein deacetylase with a better defined role in regulating mitochondrial metabolism. There are also some indirect evidence suggesting its function in protecting endothelial cell function. However, there lacks information on the effect of SIRT3 in the endothelium in vivo during atherogenesis and also the underlying mechanisms. Methods: Endothelial cell (EC) selective knockout of Sirt3 was achieved by crossing the Sirt3 floxP/floxP mice (kindly provided by Prof Johan Auwerx) with Cdh5 Cre/+ mice (from Jackson Laboratory) to generate Sirt3f/f (or Sirt3 EC-WT ) as wild type, and Sirt3f/f; Cdh5Cre/+ (or Sirt3 EC-KO ) as endothelium-selective SIRT3 knockout mice. Mice were injected with adeno-associated virus to overexpress PCSK9 for decreasing hepatic LDLR and fed with high cholesterol diet to induce hypercholesterolemic atherosclerosis mouse model. Vascular reactivity was measured in aorta by wire myograph (DMT). Human umbilical vein endothelial cells (HUVECs) were used for in vitro experiments. Results: Endothelium-selective knockout of Sirt3 enhanced plaque formation, with increased levels of infiltrating macrophages, endothelial adhesion molecules in the plaque area, and expression of inflammatory cytokines. Sirt3 EC-KO mice also had reduced plasma level of L-arginine. Further analysis in HUVECs showed that silencing of SIRT3 caused hyperacetylation of mitochondrial proteins, including ASS1 (Argininosuccinate Synthase 1), responsible for recycling L-arginine. L-arginine supplementation reduced plaque size, vascular inflammation in Sirt3 EC-KO mice. L-arginine also attenuated endothelial activation in HUVECs, and improved endothelium-dependent vasodilation in the aorta from Sirt3 EC-KO mice. Conclusion: Our data demonstrated the important role of SIRT3 in protecting endothelial function and identified ASS1-dependent metabolism of arginine regulated by SIRT3 as an underlying mechanism (supported by GRF14109519).