Background-Administration of cardiac progenitor cells (CPCs) 4 hours after reperfusion ameliorates left ventricular function in rats with acute myocardial infarction (MI). Clinically, however, this approach is not feasible, because expansion of autologous CPCs after acute MI requires several weeks. Therefore, we sought to determine whether CPCs are beneficial in the more clinically relevant setting of an old MI (scar). Methods and Results-One month after coronary occlusion/reperfusion, rats received an intracoronary infusion of vehicle or enhanced green fluorescent protein-labeled CPCs. Thirty-five days later, CPC-treated rats exhibited more viable myocardium in the risk region, less fibrosis in the noninfarcted region, and improved left ventricular function. Cells that stained positive for enhanced green fluorescent protein that expressed cardiomyocyte, endothelial, and vascular smooth muscle cell markers were observed only in 7 of 17 treated rats and occupied only 2.6% and 1.1% of the risk and noninfarcted regions, respectively. Transplantation of CPCs was associated with increased proliferation and expression of cardiac proteins by endogenous CPCs. Conclusions-Intracoronary administration of CPCs in the setting of an old MI produces beneficial structural and functional effects. Although exogenous CPCs can differentiate into new cardiac cells, this mechanism is not sufficient to explain the benefits, which suggests paracrine effects; among these, the present data identify activation of endogenous CPCs. This is the first report that CPCs are beneficial in the setting of an old MI when given by intracoronary infusion, the most widely applicable therapeutic approach in patients. Furthermore, this is the first evidence that exogenous CPC administration activates endogenous CPCs. These results open the door to new therapeutic applications for the use of autologous CPCs in patients with old MI and chronic ischemic cardiomyopathy. (Circulation. 2010;121:293-305.)Key Words: regeneration Ⅲ progenitor cells Ⅲ myocardial infarction Ⅲ reperfusion Ⅲ stem cells C ell-based therapies have the potential to alleviate left ventricular (LV) dysfunction and remodeling after acute myocardial infarction (MI) in experimental animal models 1 and in humans. 2,3 Among the various cells used, c-kitpositive (c-kit pos ) cardiac progenitor cells (CPCs) are attractive because they normally reside in the heart and presumably are responsible for replenishing the pool of cardiac myocytes and coronary vessels under normal conditions. 4 -6 The practical utility of CPCs is further supported by the fact that these cells can be isolated from small fragments of cardiac tissue and expanded for subsequent autologous administration. 5,6 Clinical Perspective on p 305Transplantation of autologous or syngeneic CPCs has been found to be effective in limiting LV dysfunction and remodeling in rodent models of acute MI, both in the setting of a permanent coronary occlusion 4 -6 and in that of a transient occlusion followed by reperfusion. 7 Clinically, how...

Background-Stromal cell-derived factor-1␣ (SDF-1␣) binding to its cognate receptor, CXCR4, regulates a variety of cellular functions such as stem cell homing, trafficking, and differentiation. However, the role of the SDF-1␣-CXCR4 axis in modulating myocardial ischemia/reperfusion injury is unknown. Methods and Results-In mice subjected to ischemic preconditioning, myocardial SDF-1␣ mRNA was found to be increased 3 hours later (PϽ0.05). Myocardial SDF-1␣ and CXCR4 mRNA and protein were found to be expressed in both cardiac myocytes and fibroblasts. SDF-1␣ production increased significantly after 1 or 4 hours of hypoxia and 18 hours of reoxygenation in cultured myocytes (PϽ0.05) but did not change in fibroblast cultures. In isolated myocytes, CXCR4 activation by SDF-1␣ resulted in increased phosphorylation of both ERK 1/2 and AKT and decreased phosphorylation of JNK and p38. Cultured myocytes pretreated with SDF-1␣ were resistant to hypoxia/reoxygenation damage, exhibiting less lactate dehydrogenase release, trypan blue uptake, and apoptotic cell death (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay) (PϽ0.05). This protective effect was blocked by the CXCR4 selective antagonist AMD3100. In vivo, administration of SDF-1␣ before 30 minutes of coronary occlusion followed by 4 hours of reperfusion decreased infarct size (PϽ0.05). The decrease in infarct size with SDF-1␣ administration also was blocked by AMD3100. Conclusions-We conclude that SDF-1␣ and its receptor, CXCR4, constitute a paracrine or autocrine axis in cardiac myocytes that is activated in response to preconditioning and hypoxic stimuli, recruiting the antiapoptotic kinases ERK and AKT and promoting an antiapoptotic program that confers protection against ischemia/reperfusion damage.

The role of the SDF-1α-CXCR4 axis in response to myocardial infarction is unknown. We addressed it using the CXCR4 antagonist, AMD3100, to block SDF-1α interaction with CXCR4 after chronic coronary artery ligation. Chronic AMD3100 treatment decreased ejection fraction and fractional shortening in mice 20 days after myocardial infarction compared with vehicletreated mice (echocardiography). Morphometric analysis showed hearts of AMD3100-treated infarcted mice to have expanded scar, to be hypertrophic (confirmed by myocyte cross-section area) and dilated, with increased LV end systolic and end diastolic dimensions, and to have decreased scar collagen content; p-AKT levels were attenuated and this was accompanied by increased apoptosis. Despite increased injury, c-kit pos cardiac progenitor cells (CPCs) were increased in the risk region of AMD3100-treated infarcted mice; CPCs were CD34 neg /CD45 neg with the majority undergoing symmetric cell division. c-kit pos /MHC pos CPCs also increased in the risk region of the AMD3100-treated infarcted group. In this group, GSK-3β signaling was attenuated compared to vehicle-treated, possibly accounting for increased proliferation and increased cardiac committed MHC pos CPCs. Increased proliferation following AMD3100 treatment was supported by increased levels of cyclin D1, a consequence of increased prolyl isomerase, Pin1, and decreased cyclin D1 phosphorylation. In summary, pharmacologic antagonism of CXCR4 demonstrates that SDF-1α-CXCR4 signaling plays an important role during and after myocardial infarction and that it exerts pleiotropic salubrious effects, protecting the myocardium from apoptotic cell death, facilitating scar formation, restricting CPC proliferation, and directing CPCs toward a cardiac fate.

Increased levels of extracellular superoxide dismutase (ecSOD) induced by preconditioning or gene therapy protect the heart from ischemia/reperfusion injury. To elucidate the mechanism responsible for this action, we studied the effects of increased superoxide scavenging on nitric oxide (NO) bioavailability in a cardiac myocyte-specific ecSOD transgenic (Tg) mouse. Results indicated that ecSOD overexpression increased cardiac myocyte-specific ecSOD activity 27.5-fold. Transgenic ecSOD was localized to the sarcolemma and, notably, the cytoplasm of cardiac myocytes. Ischemia/reperfusion injury was attenuated in ecSOD Tg hearts, in which infarct size was decreased and LV functional recovery was improved. Using the ROS spin trap, DMPO, electron paramagnetic resonance (EPR) spectroscopy demonstrated a significant decrease in ROS in Tg hearts during the first 20 min of reperfusion. This decrease in ROS was accompanied by an increase in NO production determined by EPR using the NO spin trap, Fe-MGD. Attenuated ROS in ecSOD Tg myocytes was also supported by decreased production of peroxynitrite (ONOO−). Increased NO bioavailability was confirmed by attenuated guanylate cyclase-dependent (p-VASP) signaling. In conclusion, attenuation of ROS levels by cardiac-specific ecSOD overexpression increases NO bioavailability in response to ischemia/reperfusion and protects against reperfusion injury. These findings are the first to demonstrate increased NO bioavailability with attenuation of ROS by direct measurement of these reactive species (EPR, reactive fluorescent dyes) with cardiac-specific ecSOD expression. This is also the first indication that the predominantly extracellular SOD isoform is capable of cytosolic localization that affects myocardial intracellular signal transduction and function.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-012-0305-1) contains supplementary material, which is available to authorized users.