Role of Argininosuccinate Synthase 1 ‐Dependent L‐Arginine Biosynthesis in the Protective Effect of Endothelial Sirtuin 3 Against Atherosclerosis

Abstract Atherosclerosis is initiated with endothelial cell (EC) dysfunction and vascular inflammation under hyperlipidemia. Sirtuin 3 (SIRT3) is a mitochondrial deacetylase. However, the specific role of endothelial SIRT3 during atherosclerosis remains poorly understood. The present study aims to study the role and mechanism of SIRT3 in EC function during atherosclerosis. Wild‐type Sirt3f/f mice and endothelium‐selective SIRT3 knockout Sirt3f/f; Cdh5Cre/+ (Sirt3 EC‐KO ) mice are injected with adeno‐associated virus (AAV) to overexpress PCSK9 and fed with high‐cholesterol diet (HCD) for 12 weeks to induce atherosclerosis. Sirt3 EC‐KO mice exhibit increased atherosclerotic plaque formation, along with elevated macrophage infiltration, vascular inflammation, and reduced circulating L‐arginine levels. In human ECs, SIRT3 inhibition resulted in heightened vascular inflammation, reduced nitric oxide (NO) production, increased reactive oxygen species (ROS), and diminished L‐arginine levels. Silencing of SIRT3 results in hyperacetylation and deactivation of Argininosuccinate Synthase 1 (ASS1), a rate‐limiting enzyme involved in L‐arginine biosynthesis, and this effect is abolished in mutant ASS1. Furthermore, L‐arginine supplementation attenuates enhanced plaque formation and vascular inflammation in Sirt3 EC‐KO mice. This study provides compelling evidence supporting the protective role of endothelial SIRT3 in atherosclerosis and also suggests a critical role of SIRT3‐induced deacetylation of ASS1 by ECs for arginine synthesis.