Oxidative stress plays a critical role in the pathogenesis of atherosclerosis including the formation of lipid laden macrophages and the development of inflammation. However, oxidative stress-induced molecular signaling that regulates the development of vascular calcification has not been investigated in depth. Osteogenic differentiation of vascular smooth muscle cells (VSMC) is critical in the development of calcification in atherosclerotic lesions. An important contributor to oxidative stress in atherosclerotic lesions is the formation of hydrogen peroxide from diverse sources in vascular cells. In this study we defined molecular signaling that is operative in the H 2 O 2 -induced VSMC calcification. We found that H 2 O 2 promotes a phenotypic switch of VSMC from contractile to osteogenic phenotype. This response was associated with an increased expression and transactivity of Runx2, a key transcription factor for osteogenic differentiation. The essential role of Runx2 in oxidative stress-induced VSMC calcification was further confirmed by Runx2 depletion and overexpression. Inhibition of Runx2 using short hairpin RNA blocked VSMC calcification, and adenovirus-mediated overexpression of Runx2 alone induced VSMC calcification. Inhibition of H 2 O 2 -activated AKT signaling blocked VSMC calcification and Runx2 induction concurrently. This blockage did not cause VSMC apoptosis. Taken together, our data demonstrate a critical role for AKT-mediated induction of Runx2 in oxidative stress-induced VSMC calcification.Atherosclerosis is characterized by the presence of atherosclerotic lesions in the arterial intima that leads to narrowing of the vessel lumen. Vascular calcification, the presence of calcium deposits in the vessel wall, is a feature of advanced atherosclerosis and reduces elasticity and compliance of the vessel wall (1). Hence, the extent of calcification is a key risk factor in the pathogenesis of the disease. Several cell types, such as endothelium, monocytes, and vascular smooth muscle cells (VSMC), 5 are involved in different stages of lesion development. VSMC contribute to the development of atherosclerotic lesions through increased migration, proliferation, secretion of matrix components, osteogenic differentiation, and the associated calcification (1). During this process, the differentiated VSMC undergo de-differentiation, and subsequently osteogenic transition that results in vascular calcification (2).Many factors that have been linked to an increased prevalence of vascular calcification are associated with elevated oxidative stress, including hypercholesterolemia, hypertension, diabetes mellitus, and dialysis-dependent end stage renal disease (3-6). Pro-oxidant events in atherosclerosis include the production of reactive oxygen species (ROS) and nitrogen species by vascular cells (7). Of particular interest is hydrogen peroxide (H 2 O 2 ), which is a cell-permeable ROS that has emerged as a key mediator of intracellular signaling (8 -10). H 2 O 2 is produced in vascular cells by multiple enzyma...
Rationale Vascular calcification is a hallmark of atherosclerosis, a major cause of morbidity and mortality in the United States. We have previously reported that the osteogenic transcription factor Runx2 is an essential and sufficient regulator of calcification of vascular smooth muscle cells (VSMC) in vitro. Objective To determine the contribution of osteogenic differentiation of VSMC to the pathogenesis of vascular calcification and the function of VSMC-derived Runx2 in regulating calcification in vivo. Methods and Results SMC-specific Runx2 deficient mice, generated by breeding SM22α-Cre mice with the Runx2 exon 8 floxed mice, exhibited normal aortic gross anatomy and expression levels of SMC-specific marker genes. Runx2 deficiency did not affect basal SMC markers, but inhibited oxidative stress-reduced expression of SMC markers. High-fat diet-induced vascular calcification in vivo was markedly inhibited in the Runx2-deficient mice compared with their control littermates. Runx2 deficiency inhibited the expression of receptor activator of nuclear factor κB ligand, which was accompanied by decreased macrophage infiltration and formation of osteoclast-like cells in the calcified lesions. Co-culture of VSMC with bone marrow-derived macrophages demonstrated that the Runx2 deficient VSMC failed to promote differentiation of macrophages into osteoclast-like cells. Conclusions These data have determined the importance of osteogenic differentiation of VSMC in the pathogenesis of vascular calcification in mice and defined the functional role of SMC-derived Runx2 in regulating vascular calcification and promoting infiltration of macrophages into the calcified lesion to form osteoclast-like cells. Our studies suggest that the development of vascular calcification is coupled with the formation of osteoclast-like cells, paralleling the bone remodeling process.
Objective Clinical and experimental studies demonstrate the important roles of vascular smooth muscle cells (VSMC) in the pathogenesis of atherosclerosis. We have previously determined that osteogenic transcription factor, Runx2, is essential for VSMC calcification. The present studies characterized Runx2-regulated signals and their potential roles in vascular calcification. Methods and Results In vivo studies with atherogenic ApoE−/− mice demonstrated that increased oxidative stress was associated with upregualtion of Runx2 and receptor activator of nuclear factor κB ligand (RANKL), which colocalized in the calcified atherosclerotic lesions and were juxtaposed to infiltrated macrophages and osteoclast-like cells that are positively stained for an osteoclast marker, tartrate-resistant acid phosphatase (TRAP). Mechanistic studies using RNA interfering, a luciferase reporter system, chromatin immunoprecipitation and electrophoretic mobility shift assays identified that Runx2 regulated the expression of RANKL via a direct binding to the 5'-flanking region of the RANKL. Functional characterization revealed that RANKL did not induce VSMC calcification, nor RANKL was required for oxidative stress-induced VSMC calcification. Using a co-culture system, we demonstrated VSMC-expressed RANKL induced migration as well as differentiation of bone marrow-derived macrophages into multinucleated, TRAP-positive osteoclast-like cells. These effects were inhibited by the RANKL antagonist, osteoprotegerin, and with VSMC deficient in Runx2 or RANKL. Conclusions We demonstrate that Runx2 directly binds to the promoter and controls the expression of RANKL, which mediates the crosstalk between calcifying VSMC and migration and differentiation of macrophages into osteoclast-like cells in the atherosclerotic lesions. Our studies provide novel mechanistic insights into the regulation and function of VSMC-derived RANKL in the pathogenesis of atherosclerosis and vascular calcification.
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