Effect Of Rat Bone Marrow Mesenchymal Stem Cell On Matrix Metalloproteinase Synthesis Of Cardiac Fibroblasts And The Underlying Mechanisms

Background:Ventricular remodeling secondary to myocardial infarction (MI) is characterized by compensatory hypertrophy of myocardial cells, cell necrosis or apoptosis, and component changes of extracellular matrix; changes in ventricular size, shape, wall thickness and a series of changes in organizational structure. Ventricular remodeling after MI often leads to cardiac contractile dysfunction, arrhythmias and causes heart failure. Congestive heart failure is one of the major diseases with significant morbidity and mortality on a global scale, since the incidence increases annually.Despite great strides in medical strategies aimed at post-infarct remodeling such as interventional diagnosis and therapy, as well as common drug therapy, but still they can not repair the necrotic myocardium. The identification of stem cells capable of contributing to tissue regeneration has raised the possibility that cell therapy could be employed for repair of damaged myocardium. Mesenchymal stem cells (MSCs) are pluripotent and have the self-renewal capacity. They can differentiate into endothelial cells, vascular smooth muscle cells and cardiac cell-like cells, and promote angiogenesis. Previous basic research and preclinical studies demonstrated that MSCs are capable of ameliorating the cardiac function after MI by modulating ventricular. However, the underlying molecular mechanism of MSCs modulating ventricular remodeling is still unclear.Previous studies have shown that MSCs transplantation in the post-MI myocardium reduce the synthesis and activity of matrix metalloproteinases (MMPs) to improve ventricular remodeling. MMPs is an enzyme family degrading extracellular matrix (ECM) and increased MMPs can damage collagen structure which lead to size and shape changes of left ventricule, ultimately reducing the myocardial contractility. Tissue inhibitor of metalloproteinases (TIMPs)is a specific endogenous inhibitor of MMPs. Cardiac fibroblasts (CFs) is the dominant cell type secreting MMPs in the heart. Therefore, we speculate, MSCs may affect MMPs activity and expression of CFs through paracrine effects, which play a role in improvement of ventricular remodeling.Objectives:To evaluate MSCs paracrine effect on MMPs activity and expression of CFs in vitro, and to explore the possible mechanisms.Methods:In vitro hypoxia model was used to simulated the environment during myocardial infarction. CFs were divided into four groups:control group (CFs), hypoxia group (H-CFs), MSCs co-culture group (H-CFs-MSCs) and hypoxia preconditioned MSCs co-cultured group (H-CFs-HMSCs). Zymography was used to detect MMP-2 and MMP-9 activity. Western-blot detected MMP-2, MMP-9, MT1-MPMP and TIMP-1 protein expression. Next, to inhibit erythropoietin (EPO) secreted from MSCs, erythropoietin soluble receptor (EPOsR) or erythropoietin neutralizing antibodies (EPOAb) was added in the co-culture medium of H-CFs-MSCs group and H-CFs-HMSCs group. Zymography and western-blot detected changes of MMPs level. We further detected ERK1/2 and AKT signaling pathway which is closely related to MMPs synthesis, hypoxia and EPO related signaling pathway by Western-blot.Results:Zymography analysis showed that, after 24 hours of hypoxia, MMP-2 activity of H-CFs was significantly increased compared with CFs group; MMP-2 activity of H-CFs was reduced after co-cultured with MSCs or HMSCs. Western-blot results demonstrated that the expression of MMP-2 and MT1-MMP in H-CFs was significantly upregulated compared with CFs group and downregulated after co-culture with MSCs or HMSCs. TIMP-1 protein level in H-CFs was lower than that in CFs and was upregulated by co-culture with MSCs or HMSCs. MMP-9 activity and protein expression in the CFs, H-CFs, H-CFs-MSCs and H-CFs-HMSCs groups were not significantly changed. Both EPOsR and EPOAb reversed MSCs and HMSCs effect on MMPs activity and expression in H-CFs. The phosphorylation ERK1/2 level of CFs was significantly increseased by hypoxia and significantly decreased by co-culture with MSCs or HMSCs; EPO inhibitors EPOsR and EPOAb reversed the change of phosphorylation ERK1/2 in H-CFs induced by co-culture with MSCs or HMSCs. There was no change in AKT signaling pathway in above groups. In our study, there was no significant differences between MSCs and HMSCs effect on s MMPs activity and experssion in CFs.Conclusions:MSCs modulate MMP/TIMP expression in CFs induced by hypoxia through paracrine function and EPO act as an important cytokine secreted by MSCs through ERK 1/2 signaling pathway during this process.