Novel strategies for enhancing myocardial infarction

Abstract

Ischemic heart disease is a leading cause of morbidity and mortality worldwide. Massive cell loss initiated a series of cascade events upon ischemic injury. Enourmous effort has been put to investigate strategies for enhancing myocardial healing. Cell therapy emerges as a promising strategy for regenerating lost cardiomyocytes. Among different sources, cardiac progenitor cells, due to their differentiation capacity toward cardiomyocytes, endothelial cells and smooth muscle cells, is considered as an ideal source for myocardial regeneration. Though promising and effective, the low cell survival and unrequired cell motility away from the injection site restricts the effectiveness of cell therapy. In this thesis, we are investigating the approaches and the underlying mechanism of promoting myocardial healing by using a small RNA molecule, microRNA-155(miR-155), and a pharmacological agent necrostatin-1, a specific inhibitor of necrotic cell death. We demonstrated that miR-155 efficiently prevent oxidative-stress induced necrosis, the dominant type of cell death in CMPCs. In addition, no active caspase activities were observed upon oxidative-stress, suggesting the apoptosis pathway not being activated. RIP1, a target for miR-155 in macrophages, emerged as a potential target candidate for miR-155 regulated inhibition of necrosis. In apoptosis-deficient conditions, RIP1 has been found to be required for the activation of necrosis by death-receptor agonists. We confirmed a direct effect of RIP1 on necrosis by using a specific inhibitor of RIP1, necrostain-1(Nec-1), or via siRNA treatment for RIP1, without affecting apoptosis. We further found that miR-155 attenuated necrosis independent of pro-survival and apoptosis pathways. We also investigated the potential use of increasing miR-155 levels in CMPCs to influence cell migration. Functional assays demonstrated that miR-155 efficiently blocked cell motility mediated by reduced MMP-2 and -9 activities. Furthermore, we found that miR-155 directly down-regulated MMP-16, an activator of MMP-2 and -9, and that inhibition of MMP16 by a blocking antibody or siRNA knockdown reduced CMPC migration to a similar extent as miR-155 over-expression.This confirms the functional relevance of MMP-16 in the down regulation of cell migration. We next intended to test the inhibitive effect of miR-155 on cell migration in vivo. As expected, we were able to visualize SPIO labeled cells two days after injection at longitudinal and axial slices of a mouse heart, suggesting the possibility of utilizing MRI to track CMPCs migration in vivo. We also reported the potential cytoprotective effects of Nec-1 in neonatal mouse cardiomyocytes in vitro and in an ischemia/reperfusion in vivo mouse model. Both in vitro and in vivo, Nec-1 preconditioning led to a significant decrease in PI-positive staining, reflecting its strong effect on inhibiting necrotic cell death. Consequently, Nec-1 administration significantly diminished infarct size by 47% one day after reperfusion. In line with the reduced infarct volume, relative LDH release was much lower in the Nec-1 treated group, confirming reduced ischemic injury upon Nec-1 treatment. Furthermore, Nec-1 infusion efficiently decreased EDV and ESV, accompanied by a 27.1% improvement of EF one month after reperfusion. Consistently, Nec-1 treatment rescued left ventricle wall thinning by 18.1%, suggesting less maladaptive ventricle remodeling with Nec-1 preconditioning