Abstract 17887: Cystein-rich Angiogenic Protein 61 Promotes Vascular Smooth Muscle Cell Calcification Via Metalloproteinase Activation
Cystein-rich angiogenic protein 61 (CYR61, CCN1) was reported to be regulated by angiotensin II in vascular smooth muscle cells (SMCs) of Cystein-rich angiogenic protein 61 (CYR61, CCN1) was reported to be regulated by angiotensin II in vascular smooth muscle cells (SMCs) of atheromatous plaque. Because CYR61 was reported to induce osteoblastic differentiation of mesenchymal stem cells, we hypothesized that the CYR61 may play a role in vascular calcification. CYR61 expression was induced by 20.3 folds in SMCs harvested from thoracic aortas of male C57BL6 mouse after 16 hours of adenoviral vector (Ad-CYR61, 50 MOI) transfection, which significantly induced SMC calcification by 209.5±54.8% evaluated by Von Kossa staining after 14 days. In order to evaluate the full range of effects of CYR61 on SMC calcification, we performed microarray analysis. Several metalloproteinases such as MMP-13, -3, -10, and -8 were induced by 55.3, 49.9, 6.4 and 5.2 folds, whereas tissue inhibitor of metalloproteinases such as TIMP-3 and -2 were reduced by 52% and 31% after 16 hours of Ad-CYR61 transfection, respectively (all p<0.05). Remarkably, we found overall suppression of procollagen gene expression. Real time PCR confirmed MMP-13 gene induction by 33±13 folds compared with control adenovirus transfected SMCs (p<0.05). Although mRNA or protein expressions of MMP-9 were not found increased, gelatin zymography showed an increased enzymatic activity of MMP-9 (92kDa) by 202±41% by Ad-CYR61, which was completely reversed by MMP-13 siRNA. Inhibition of MMP activity by the global MMP inhibitor, doxycycline completely blocked Ad-CYR61-induced calcification to the 87.0±30.8% of the control adenovirus transfected SMCs. Also, Ad-CYR61 failed to induce calcification in VSMCs harvested from MMP-9 knock out mouse (100.9±7.6% of the control adenovirus transfected SMCs, p=0.90). These findings demonstrate that CYR61 induces SMC calcification through MMP-13 - MMP-9 cascade. Therapies targeting this signaling pathway may regulate vascular calcification.
- © 2012 by American Heart Association, Inc.