Cellular and molecular mechanisms in myocardial regeneration
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Heart failure is a significant cause of morbidity and mortality worldwide. Currently, the only curative therapy for heart failure is heart transplantation and therefore alternative therapies are needed. Mesenchymal stem cells (MSCs) have been used for heart repair, but clinical trials have thus far demonstrated moderate and inconsistent benefits, indicating an urgent need to improve their therapeutic potency. Clinical trials have largely relied on injections of 1 × 10 6 cells/kg, but according to published preclinical studies it appears that this injection dose is too low to elicit a robust therapeutic response. Repeated cell passaging necessary for large-scale expansion of MSCs causes cellular senescence, which can suppress trophic factor expression by MSCs, resulting in attenuation of their therapeutic potency. Using the RNA mimetic polyinosinic-polycytidylic acid (poly(I:C)) to activate MSC toll-like receptor 3 (TLR3), we found that poly(I:C) induced the expression of cardioprotective trophic factors such us interleukin 6 (IL6)-type cytokines, hepatocyte growth factor (HGF), stromal-derived factor 1 (SDF1), and vascular endothelial growth factor (VEGF). At the clinically suboptimal injection dose of 1 × 10 6 cells/kg, poly(I:C)-conditioned MSCs (MSC-IC), but not unconditioned MSCs injected intramuscularly, increased CD34 + progenitor cells in the heart. These cells were able to proliferate (CD34 + /ki67 + ) and become cardiac cells (CD34 + /GATA4 + ). The upregulation of progenitor cells in the heart was associated with a reduction in fibrosis and apoptosis, and an increase in angiogenesis and cardiomyogenesis, leading to cardiac functional improvement. These functional, histological, and molecular characterizations thus establish the utility of TLR3 engagement for enhancing the low-dose MSC therapy that may be transferred to more efficacious clinical applications. The upregulation of trophic factors by MSCs after poly(I:C) treatment and the subsequent beneficial effects in the failing heart demonstrate that it is not the physical presence of the cells but the trophic factors that the cells release and act in a paracrine fashion that are critical for cardiac repair. Along this line we sought an alternative protein therapy. We have been exploring MSC and VEGF therapies for the failing heart. These studies led to the finding that the failing hamster heart exhibits significantly increased expression of secreted frizzled-related protein 2 (sFRP2) compared to the normal control heart. MSC- and VEGF-mediated cardiac repair each restored function, attenuated myocardial fibrosis and decreased expression of sFRP2, suggesting a potentially harmful effect of dysregulated sFRP2. We used an antibody-based sFRP2 blockade strategy to further decipher the role of sFRP2 in chronic heart failure. After intraperitoneal administration of sFRP2 antibody, fibrosis and apoptosis were decreased, and angiogenesis and cardiomyogenesis were increased, leading to cardiac functional improvement. Notably, VEGF protein levels were significantly increased after sFRP2 blockade. To simulate the effects of sFRP2 in the heart, cardiac fibroblasts were treated with sFRP2 protein in culture. VEGF transcription was not significantly affected by sFRP2, but its protein levels were decreased by more than 50%. This in vitro study suggests that elevated sFRP2 likely downregulates VEGF through a post-translational mechanism. These sFRP2 studies establish that the failing hamster heart can be repaired by antibody-based sFRP2 blockade, and the regenerative mechanism involves stabilization of VEGF protein, a key therapeutic trophic factor. Taken together, our animal and cell culture studies established a poly(I:C) preconditioning strategy for MSCs, which enhances their therapeutic potency and is applicable for clinical trials. Furthermore, we were able to decipher the role of sFRP2 in heart failure, and show the efficiency of antibody-based sFRP2 blockade for heart failure therapy.