Abstract 917: AKT-Runx2 Signaling Regulates Oxidative Stress-induced Vascular Smooth Muscle Cell Calcification
Oxidative stress plays a critical role in the pathogenesis of atherosclerosis, including the development of atherovascular calcification, a prominent feature of atherosclerosis. Enhanced osteogenic differentiation of vascular smooth muscle cells (VSMC) is associated with oxidative stress in vitro. In the present studies, we determine the effect of oxidative stress on the osteogenic differentiation of VSMC and the underlying molecular mechanisms. VSMC were explanted from the aorta of C57BL6 mice and sorted by flow cytometry with smooth muscle specific α-actin antibody. Hydrogen peroxide(H2O2), a model oxidant, induced oxidative stress in VSMC as determined by dichlorofluorescein fluorescence, an intracellular probe of oxidative stress. We found that non-toxic amount of H2O2 induced VSMC calcification in a dose-dependent manner. The expression of bone-associated markers including alkaline phosphatase (ALP), type I collagen (Col I) and osteocalcin (OC) were induced under oxidative stress. H2O2 induced Runx2 expression and transactivity. By contrast, the expression of smooth muscle specific markers such as α-actin and SM22α was decreased under oxidative stress, suggesting that oxidative stress induces a phenotypic change of VSMC into osteogenic cells. The essential role of Runx2 in oxidative stress-induced VSMC calcification was further determined with VSMC from Runx2 heterozygous knockout mice (Runx2+/−). Oxidative stress-induced VSMC calcification was dramatically inhibited in the Runx2+/− VSMC. Furthermore, we demonstrated that H2O2 induced activation of phosphatidylinositide 3-kinase (PI3K), AKT, ERK and PLCr signaling. The specific inhibitors of PI3K and AKT(LY274002, AKTIV), but not ERK or PLCr (PD98059, U73122, U73343) blocked VSMC calcification in dose-dependent manners and decreased the expression of bone-associated markers. Inhibition of AKT also decreased H2O2-induced Runx2 expression and transactivity, but did not induce apoptosis. These data demonstrate an important role of AKT-Runx2 signaling in mediating oxidative stress-induced VSMC calcification. Our studies provide important insights into understanding the molecular mechanism of oxidative stress-induced VSMC calcification.