Abstract 19106: Diabetes Induced Actin S-glutathiolation Increases Vein Graft Accelerated Atherosclerosis Through Delayed Endothelial Regeneration

Abstract

Endothelial cell (EC) loss and regeneration are important aspects of the response to vascular injury leading to atherosclerosis in vein grafts (VG). EC regeneration is known to be impaired in diabetes mellitus (DM) leading to an accelerated atherosclerosis, however the mechanism remains unclear. Given the increased oxidative stress in DM, we hypothesized that protein s-glutathiolation, the reversible covalent addition of glutathione to cysteine on target proteins was a candidate mechanism. Overall s-glutathiolation of proteins measured by immunoblot was significantly increased in human DM atherosclerotic plaque compared to non-DM (4-fold, P<0.01; n =10) with the increase in protein intensity at ≈ 40kDa. Mass spectrometry peptide fingerprint screening identified actin (42Kda) and the actin regulatory protein annexin A2 (39Kda) as the s-glutathiolated proteins. In cultured ECs in which s-glutathiolation was induced, proliferation (22%, P<0.05) and motility (37%, P<0.05) were significantly decreased compared to control. Assessment of actin conformational state identified a significant decrease in the ratio of filamentous to globular actin (P<0.05) supporting our findings of decreased motility. As an in vivo model of acute vascular injury we constructed VGs in apolipoprotein-E knockout (ApoE-KO) mice with expression of an EC reporter gene (LacZ) in which DM was induced by streptoztocin (n=6-10/group). In VGs harvested 28 days post-operatively, DM increased VG total vessel wall area by 51% (P=0.001) and reduced endothelialization by 44% (P=0.001). S-glutathiolation levels were increased 10-fold in primary cultured ECs from these mice. Although there was no differences in baseline EC content or number of isolated cells, following 3 days of culture, total EC number, colony size and colony number from ApoE-KO/LacZ DM mice were decreased (4-fold P=0.05; 3-fold P=0.02; 3-fold P=0.04 respectively, n=6) as were putative EC progenitor levels from bone-marrow derived mononuclear cells by culture (20%,P=0.03) and FACS (22%, P=0.02). These observations indicate an important role for actin s-glutathiolation in EC regeneration following vascular injury and identify a mechanism by which accelerated atherosclerosis develops in patients with DM.