Abstract Body (Do not enter title and authors here): Introduction Hyperlipidemia promotes atherosclerosis, a leading cause of myocardial infarction and stroke. Atherosclerosis is commonly associated with endothelial dysfunction. Elevated levels of p66Shc and sumoylated proteins have been implicated in hyperlipidemia-induced endothelial dysfunction. Recently, we discovered that SUMO2 modifies p66Shc at lysine-81, regulating its function. However, the role of endogenous p66ShcK81 sumoylation remains unclear.
Methods We used LDLr-/- mice and p66ShcK81R knock-in mice on an LDLr-/- background (p66ShcK81R-KI x LDLr-/-). Hyperlipidemia was induced by feeding the mice a high-fat diet for 4 weeks. We monitored body weight gain and measured serum total cholesterol levels. Endothelial function, oxidative stress, and sumoylation were assessed in the aortas. Additionally, we performed global mass spectrometry to identify the molecular signaling pathways regulated by p66ShcK81-SUMO2ylation in endothelial cells.
Results and Discussion: To determine the contribution of SUMO2 to this sumoylated pool of proteins, we treated human umbilical vein endothelial cells with oxidized LDL and knocked down SUMO2 with siRNA. The majority of the sumoylated proteins were found to be SUMO2-conjugated, suggesting that SUMO2 is primarily affected in hyperlipidemia. In LDLr-/- mice on a high-fat diet, we observed increased expression of SUMO2/3-conjugated proteins compared to controls. Feeding a high-fat diet induced a similar increase in serum cholesterol levels as well as weight gain in both LDLr-/- and p66ShcK81R-KI x LDLr-/- mice. Vascular reactivity revealed significant impairment of endothelium-dependent vasorelaxation of aortic rings in LDLr-/- mice. However, aortic rings from p66ShcK81R-KI x LDLr-/- mice displayed vasorelaxation similar to control p66ShcK81R-KI mice. We also noted increased oxidative stress (8-OHdG levels) in LDLr-/- mice, which was significantly reduced in p66ShcK81R-KI x LDLr-/- mice. Lastly, mass spectrometry data indicated that p66ShcK81-SUMO2ylation promotes mitochondrial dysfunction and impairs Rho-GTPase signaling in endothelial cells.
Conclusion Our findings demonstrate that p66ShcK81 mediates hyperlipidemia-induced endothelial dysfunction and oxidative stress. Investigating the role of specific molecules regulated by p66ShcK81-SUMO2ylation may lead to the discovery of novel tools to manage endothelial dysfunction caused by hyperlipidemia.