Abstract 242: MMP-3 Promotes SMC Transformation During Medial Artery Calcification

Objective: Medial artery calcification is associated with increased cardiovascular morbidity and mortality. It occurs with diabetes and chronic kidney disease. We have previously demonstrated that the matrix metallopeptidase 3 (MMP-3) is strongly induced in arterial calcification. We also showed that MMP-3 inhibition decreases calcium accumulation in vascular SMCs and that MMP-3 deficient mice develop less medial calcification than wildtype controls. In this series of continuing experiments, we evaluate the effects of MMP-3 on SMCs phenotypic transformation in vitro and in vivo. Methods and Results: Confluent rat aortic smooth muscle cells (RASMCs) cultured in calcification medium containing elevated calcium and phosphate levels for 7 days showed increased MMP-3 activity, decreased expression of the SMC markers SM-actin and SM-MHC, and increased expression of the bone markers alkaline phosphatase (ALP) and osterix (Osx). Cells were next exposed a selective MMP-3 inhibitor 2(EMD Millipore). In the presence of inhibitor, MMP-3 activity (MMP-3 activity assay kit, Abcam) was significantly decreased. Additionally, SMC osteogenic transformation was prevented as demonstrated by maintenance of SM22α and SM-MHC expression with reduction of ALP and Osx expression. In confirmatory experiments, MMP-3 reduction with siRNA inhibited the calcification of SMC that were exposed to calcification medium. We next evaluated phenotypic marker expression in MMP-3 knockout and wild-type mice injected with vitamin D 3 , a model of medial artery calcification. At 7 days after injection, expression of the SMC marker gene SM-MHC was significantly greater in MMP-3 KO mice than controls, while bone cell marker genes (Runx2, ALP, Osx) were decreased as measured by qPCR. Deletion of MMP-3 thus inhibited the osteogenic transformation of medial SMCs in vitamin D 3 -treated mice suggesting that it may control calcification via local effects within the arterial wall. Conclusion: Together these findings suggest that MMP-3 promotes medial artery calcification through local effects on the phenotypic state of vascular SMCs, and further, that it may serve as a therapeutic target to reduce calcification and improve outcomes in our PAD patient population.

Abstract 370: Matrix Metalloproteinase-3 Regulates Arterial Calcification

Objective: Medial artery calcification is associated with increased cardiovascular morbidity and mortality in patients with peripheral artery disease (PAD). It is highly prevalent in diabetes and chronic kidney disease, and it is an important risk factor for cardiovascular events. Accumulating evidences suggest that matrix degrading enzymes are prominently involved in this process. Clinical studies have shown that the matrix metallopeptidase3 (MMP-3, stromelysin-1) is correlated with prevalent arterial calcification in high risk human populations. In this study, we sought to determine whether reducing MMP-3 activity could decrease arterial calcification in smooth muscle cells, organ culture, and in vivo models. Methods and Results: Confluent human aortic smooth muscle cells (HASMCs) were cultured in calcification medium containing high phosphate (Pi) for 7 days in the absence or presence of an MMP-3 specific inhibitor. We found that adding MMP-3 inhibitor dose-dependently reduced Pi-induced calcium deposition as demonstrated by the O-cresolphthalein complexone assay. To further determine the importance of MMP-3 in arterial calcification, we utilized ex vivo aortic rings. In aortic rings from MMP-3 knockout mice, Pi-induced medial calcification was significantly reduced compared with wild-type mice. We next evaluated the effects of MMP-3 deletion in vivo . 12-week-old MMP-3 knockout and wild type mice were injected with 8mg/kg cholecalciferol and arteries were collected after 8 days, Calcium levels were significantly lower in MMP-3 knockout mice. Deletion of MMP-3 also reduced serum calcium levels, but not changed serum phosphate level. In concurrent studies, we showed by immunohistochemical staining that MMP-3 was highly expressed in calcified lesion from human tibial arteries. Conclusion: Together these findings suggest that MMP-3 promotes medial artery calcification, and that it may serve as a potential therapeutic target aimed at improving the poor outcomes of our patients with PAD.