Effects Of ROCK On The Activity Of Matrix Metalloproteinases 2 (MMP-2) Of Vascular Smooth Muscle Cells Induced By PDGF

Objectives:Migration of VSMCs is believed to play an important role in the pathophysiology of many vascular disorders, such as hypertension, atherosclerosis and restenosis after coronary angioplasty. The role of growth factors such as Platelet-derived growth factor (PDGF) is well documented in neointimal formation. PDGF is a potent growth factor produced by platelets, VSMCs and endothelial cells in the injured vascular wall. It plays an important role in vascular remodeling during cellular and extracellular response to injury. PDGF can induce VSMCs to differentiate, which involves alteration from a contractile to a synthetic phenotype, following which these cells can proliferate and migrate from the medium to the intima layer of the vessel wall.Normal medial VSMCs are surrounded by a dense ECM network of interstitial collagen fibers and laminin, and VSMC migration may require the degradation or remodeling of extracellular matrix (ECM) surrounding the cells. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endoproteinases whose enzymatic activity is directed against components of the ECM. Of the MMPs protein families, MMP-2 and MMP-9 are important for VSMC proliferation and migration into the intima.Rho-kinase, an effector of the small G protein Rho, is activated by several stimuli and mediates various cellular functions including smooth muscle contraction, migration and actin cytoskeleton organization, all of which might be involved in the pathogenesis of vascular remodeling. There are two isoforms of ROCK, ROCKâ… and ROCKâ…¡,that share overall 65% homology at the amino acid level and 92% homology at the kinase domain. Therefore, it is generally believed that they have same functions in the cells. However, recent study found that ROCKâ… and ROCKâ…¡in some cells have different substrates and cell functions. At present, it is not clear that whether they have the same function of two ROCK informs in VSMCs migration. This study is aimed to investigate the effects of ROCKâ… and ROCKâ…¡on the expression and activity of matrix metalloproteinases-2 (MMP-2) of vascular smooth muscle cells (VSMC) induced by PDGF-BB.Methods:(1)The activity of MMP-2 was assessed by Gelatin Zymognaphy. (2)ROCKâ… and ROCKâ…¡genes were down-regulated by siRNA transfection. (3)The expression levels of ROCKâ… /â…¡and MMP-2 were detected by western blot. (4)The effects of down-regulation of ROCKâ… and ROCKâ…¡gene expression and Y-27632 of ROCK inhibitor on PDGF induced A7r5 migration were detected by Boyden chamber method.(5)Remodeing of the actin cytoskeleton detected by immunofluorescence.Results:(1)Gelatinase zymography showed that compared with the non-induced VSMCs, a dose-dependent increase in MMP-2 activity was observed in PDGF-induced VSMCs; (2)By siRNA transfection, the protein expressions of ROCKâ… and ROCKâ…¡were down regulated by 79.8% and 70.1% respectively; (3)The expression and activity of MMP-2 were inhibited by the down-regulated ROCKâ… and ROCKâ…¡, whereas the effect of ROCKâ…¡was more marked. (4)ROCKâ… siRNA and Y-27632 decreased the migration of A7r5 cells, but ROCKâ…¡siRNA had no significant effects at the same condition; (5)The filopodia of PDGF induced cells increased significantly. PDGF induced transfection ROCKâ… siRNA cell filopodia was not obvious, but transfection ROCKâ…¡siRNA cell filopodia increased significantly.Conclusions:(1)ROCKâ… plays a major role in VSMCs migration. (2)Both ROCKâ… and ROCKâ…¡siRNA decreased the expression and activity of MMP-2. In the migration aspects, MMP-2 may be the Rho/ROCK signaling pathway downstream molecule.