All ETDs from UAB

Advisory Committee Chair

Jay M McDonald

Advisory Committee Members

Yabing Chen

Victor Darley-Usmar

Rosa Serra

Chenbei Chang

Xu Feng

Document Type

Dissertation

Date of Award

2009

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Oxidative stress plays a critical role in pathogenesis of atherosclerosis. However, the effect of oxidative stress-induced molecular signaling in development of vascular calcification, a feature of advanced atherosclerosis, has not been defined. Osteogenic differentiation of vascular smooth muscle cells (VSMC) is critical in development of vascular calcification. We determined hydrogen peroxide (H2O2)-induced oxidative stress promoted a phenotypic switch of mouse primary VSMC from contractile to osteogenic phenotype. This effect was associated with increased expression and transactivity of Runx2, a key transcription factor for osteogenic differentiation. Inhibition of Runx2 using short hairpin RNA blocked oxidative stress-induced VSMC calcification. By contrast, adenovirus-mediated over-expression of Runx2 induced VSMC calcification. Furthermore, inhibition of H2O2-activated AKT signaling blocked VSMC calcification and Runx2 induction concurrently. Runx2 was found to mediate H2O2-induced expression of receptor activator of NF-kappaB ligand (RANKL). Direct binding of Runx2 between -400 and -200 bp of 5'-flanking region of RANKL gene was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift analyses. RANKL is a key regulator for osteoclastogenesis and formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells which have been reported to be present near sites of calcification. Using co-culture system, we demonstrated H2O2-stimulated VSMC induced differentiation of bone marrow-derived macrophages (BMM) into multinucleated TRAP-positive cells and increased migration of BMM in a Runx2/RANKL-dependent manner. Smooth muscle-specific Runx2-deficient mice (deltaRunx2SMC) were generated using Cre-LoxP system by breeding Runx2 floxed mice with SM22alpha-Cre mice, smooth muscle-specific Cre transgenic mice. In vivo characterization of function of Runx2 in vascular calcification was carried out in deltaRunx2SMC mice on atherogenic ApoE-/- background. Histochemical analysis revealed smooth muscle-specific Runx2-deficiency resulted in decreased vascular calcification. Furthermore, RANKL expression and macrophage infiltration were decreased in deltaRunx2SMCApoE-/- mice. Most interestingly, macrophage infiltration was restricted mainly to endothelial layer in deltaRunx2SMCApoE-/- mice, whereas in control mice neointimal/medial layer was main site infiltrated with macrophages. Thus, results from this dissertation support an important role of Runx2 and Runx2-regulated RANKL in modulating vascular calcification as well as macrophage infiltration. Therefore, targeting Runx2 signaling in vessel wall may represent a novel strategy for prevention and therapy of atherosclerosis and vascular calcification.

Share

COinS