New approaches in cardiac regeneration for heart failure treatment

Cardiac failure is the most frequent cause of hospitalization among patients older than 65 years. Current techniques of treatment, percutaneous transluminal coronary angioplasty and coronary artery bypass grafting, are aimed at the revascularization of the remaining viable myocardium and have no effect on contractile mass lost. In the last years efforts have been performed to promote cardiac tissue regeneration. Stem cell transplantation offers a potential therapeutic approach to the repair and regeneration of the damaged heart after acute myocardial infarction (MI). Among different types of stem cells currently being considered for cardiac regeneration, ckit+ cardiac stem cells (ckit+ CSCs) represent a good candidate. These cells are able to stimulate cardiac repair in the infarcted heart by replacing damaged myocardium and/or by the paracrine release of growth factors. The regenerative effects of cardiac ckit+ cells in acute MI have been studied extensively, but little is known about how these cells confer a protective effect to cardiomyocytes. Growing evidences suggest that injury triggers in adult stem cells inflammatory signaling pathways which control tissue repair and regeneration. First, we investigated ¬the mechanisms underlying the cardioprotective effects of ckit+ CSCs following transplantation in a murine model of MI. We used an in vivo murine model of MI and an in vitro colture of murine ckit+ CSCs. Following isolation and in vitro expansion, ckit+ CSCs were subjected to normoxic and hypoxic conditions for different time points. Both q-RT PCR and WB analysis showed that these cells adapted to hypoxia and activated an inflammatory and reparative response (IRR). Specifically, adaptation of these cells to hypoxia occurred through the activation of HIF-1a and the expression of a number of genes, such as VEGF, GLUT1, EPO, HK2, CAR9 and, importantly, alarmin receptors, such as RAGE, P2X7 and Toll-like receptors (TLR2 and TLR4). Activation of these receptors determined a NFkB-dependent inflammatory and reparative gene response. Importantly, following IRR activation, hypoxic ckit+ CSCs increased the secretion of two survival growth factors, i.e. insulin-like growth factor-1 and hepatocyte growth factor, detected by ELISA assay. To verify whether activation of the IRR in a hypoxic microenvironment could exert a beneficial effect in vivo, autologous ckit+ CSCs were transplanted into the damaged heart of an ischemic mouse. Interestingly, transplantation of ckit+ CSCs lowered apoptotic rates and induced autophagy in the peri-infarct area; further, it determined a shift in myosin heavy chain isoform content from beta to alpha and an increase in the expression level of the Muscle Lim Protein (MLP, also known as cysteine rich protein 3-CSRP3). Using a LUMINEX assay, reduced levels of the pro-fibrotic TGF b1 were detected following MI and ckit+ CSC transplantation. Accordingly, a decreased protein expression of collagen I was observed in infarcted transplanted hearts compared to controls. These results were also supported by an increase in MMP9 mRNA and a decrease in TIMP4 mRNA in hypoxic ckit+ CSCs. Second, we investigated whether, in an in vivo murine model of myocardial infarction (MI), allogeneic transplantation of CSCs was able to stimulate endogenous reparative mechanisms without inducing an immune response. Currently, two general approaches are followed to promote cardiac tissue regeneration: transplantation of regeneration-competent stem cells (CSCs) and stimulation of endogenous cardiovascular progenitors (CSCs). The first procedure is affected by several problems related to the time necessary to prepare CSCs once isolated from a patient and their potential to proliferate and differentiate. For this reason, the possibility to use allogeneic CSCs to repair infarcted hearts would be an alternative of paramount importance to autologous procedures. This possibility has been already demonstrated for cardiospheres, another source of CSCs, but not for ckit+ CSCs. For this study we decided to isolate and expand ckit+CSCs from Balb C murine hearts and then to transplant them into the hearts of Balb C mice (to have a syngeneic transplant) or in the hearts of C57 BL6 mice (to have an allogeneic transplant). At three different time points (3,7 and 21 days) hearts were collected from the same mice and used for immunohistochemistry and WB analysis. Specifically, using these techniques, we detected and quantified different markers of primary and secondary inflammation among the different groups of mice to verify the presence of an immune response: CD45RA to identify B monocytes, CD3 for lymphocytes and CD68 for macrophages. Results showed the lack of difference immune response comparing the syngeneic group to the allogeneic group both by WB analysis and by immunofluorescence. Specifically, the presence of monocytes, lymphocytes and macrophages was similar in the two groups at 3, 7 and 21 days following infarction and transplantation. Blood was collected in all animals just before sacrifice to determine the levels of circulating cytokines using the LUMINEX Assay. Plasma quantification of pro- and anti-inflammatory cytokines at 3,7 and 21 days following MI and transplantation, showed that the differences between the syngeneic and allogeneic groups are not statistically significant, thus confirming the data obtained by WB analysis and immunofluorescence. Moreover, in our experimental conditions, we detected lower expression levels of inflammatory cytokines in infarcted hearts transplanted with CSCs compared to infarcted hearts used as controls. In conclusion, we demonstrated the feasibility of an allogeneic transplantation using CSCs as demonstrated by comparable expression levels of inflammatory cytokines between the syngeneic and allogeneic groups both in the cardiac tissue, following MI and transplantation, and in the bloodstream.