Abstract 462: Ascl3 Protects Vascular Smooth Muscle Cells Against Oxidative Stress

Abstract

Atherosclerosis initiation and progression depends on multiple risk factors and is sustained by self-perpetuating inflammatory cellular and molecular events that ultimately translate into increased oxidative stress inducing endothelial dysfunction, macrophage activation and altering vascular smooth muscle cell (VSMC) homeostasis. Our initial work identified the under-characterized bHLH achaete-scute like 3, ASCL3, as a pro-proliferative anti-apoptotic factor in VSMC and prompted the hypothesis that ASCL3 plays a protective role against oxidative stress in VSMC. We determined that ASCL3 expression is up-regulated in the media and lesions of the atherosclerotic aorta in ApoE-mice fed a high fat diet. In vitro, ASCL3 expression is consistent with an adaptive role in oxidative stress. In primary RASMC, time- and dose-response to H₂O₂ (0.5-2mM, 4-24h) result in marked ASCL3 mRNA increase (~10-fold) at low concentrations or shorter times and is reduced as oxidative stress increases. siRNA knockdown of ASCL3 increases susceptibility of RASMC to 0.25mM H₂O₂ (low dose) as determined by caspase-3 activation. Stable over-expression of ASCL3 in A7r5 cells results in enhanced protection at higher doses of H₂O₂, and is associated with higher AKT and ERK1/2 phosporylation. This protective effect involves the mitochondrial pathway since ASCL3 up-regulates Bcl-2/Bax ratio in basal and induced conditions (0.4mM, 4h), inhibits mitochondrial depolarization, prevents mitochondrial fragmentation and preserves mitochondrial DNA. ROS levels are significantly reduced in ASCL3 over-expressing cells (0.2mM, 2h). ASCL3 over-expression appears to increase mitochondrial density and results in altered mitochondrial and cristae morphology as determined by TEM. Surprisingly, subcellular localization studies indicate that ASCL3 localizes primarily to the mitochondria. In summary, low oxidative stress in VSMC promotes survival, with enhanced proliferation, and as it increases, induces adaptation associated with an antioxidant gene expression profile. Eventually high oxidative stress leads to cell death. Altogether, our data indicate that ASCL3 plays a protective role in the survival and adaptation of vascular smooth muscle cells to oxidative stress.