To investigate the effects of treatment with peripherally-derived EPCs on myocardial infarction in vitro, we evaluated whether PB-EPCs treatment might increase myocardial repair after acute coronary occlusion in two rat models. PB-EPCs were shown to reduce infarct size, decrease ischemia/reperfusion injury, attenuate inflammation, and improve post-infarction cardiac function.
PB-EPCs reduced infarct size and decreased ischemia-reperfusion injury
In the present study, Tβ4-transfected PB-EPCs reduced infarct size and increased regional myocardial contraction reserve in an LAD occlusion model. The impact of Tβ4 or EPCs on infarct size was assessed as the ratio of nonviable myocardium to the AAR, revealing that transplantation of PB-EPCs decreased ischemia/reperfusion injury in a rat model. The ability of Tβ4 or EPCs to prevent cell death within 72 hours after coronary ligation likely leads to the decreased infarct size and explains the improved regional myocardial contraction reserve observed in rats in this study. Incubation of PB-EPCs with Tβ4 resulted in an increase in cell viability and proliferation and had an inhibitory effect on EPCs apoptosis. Our findings are consistent with those of Bock-Marquette et al. (2004) in which Tβ4 stimulated migration of cardiomyocytes and endothelial cells, showing enhanced survival of embryonic and postnatal cardiomyocytes cultured with Tβ4 . Investigators explained that the LIM proteins ILK and PINCH, which are both essential in cell migration and survival, form a complex with Tβ4 that results in phosphorylation of Akt; in mice, this activity enhanced early myocyte survival and improved cardiac function, which may point to activation of Akt as the dominant mechanism by which Tβ4 promotes cell survival . Activation of ILK and subsequent stimulation of Akt may, in part, explain the enhanced cardiomyocyte survival induced by Tβ4 in the present study, although further experiments are required to confirm this. Besides the ability of Tβ4 to mediate angiogenesis in a long-term course , Tβ4 is integrally involved in cell motility, migration and survival in the process of cardiac morphogenesis. Results of our experiments and others indicate that this protein can be reintroduced in order to reduce post-infarction loss of cardiomyocytes and may ultimately reduce the impact of prolonged ischemic injury. However, because we did not use Tβ4 and its blocker in chronically infarcted rats, we cannot conclude that Tβ4 is involved in the chronic phase of myocardial infarction.
PB-EPCs promote expression of proteins involved in cardiac repair and regeneration
In this study, we observed that PB-EPCs increased or activated the expression of proteins essential for cardiac repair and regeneration. Considering the multifaceted roles of VEGF, PDGF, β-Catenin, FGFR-2, FGF-17 and Flk-1 in vessel formation [24–27], our findings suggest that EPCs may promote cardiac regeneration after cardiac injury, especially given that EPC transplantation significantly increased the expression level of VEGF in the infarct region. We detected significant changes in PDGF, VEGF, and Flk-1 in the group receiving EPCs alone. Upregulated expression of VEGF has previously been found to be associated with a significant increase in the number of both capillaries and arterioles after cardiac injury [28, 29]. Therefore, our finding is consistent with that previously reported in a swine model, which showed that EPCs increased neovascularization in the infarct region border zone . Those investigators described a novel mechanism of cytoprotective gene expression that can potentially extend cell survival at the genomic level, and further suggested that a repetitive pattern of ischemia-reperfusion may result in progressive cell protection and survival. These results and ours suggest that activation and increased expression of individual genes plays an important role in cardioprotection.
PB-EPCs attenuate inflammation
In this study, we observed a decrease in post-ischemic inflammation and endothelial apoptosis and significantly enhanced cardiomyocyte survival after intramyocardial injection of PB-EPCs transfected with scrambled SC shRNA and of Tβ4 alone. Tβ4 alone significantly reduced MPO activity, and similar inhibition in MPO activity was observed in the EPCs + SC shRNA group, resembling the effect of EPCs alone. In another study, Tβ4-expressing cells found in cardiac valve precursors known as “endocardial cushions” also expressed Akt as a possible interacting protein and part of the dominant mechanism by which Tβ4 increases cell survival . The Tβ4 protein was also found in the ventricular septum, the compact layer of the trabecular region and the outflow tract myocardium, indicating its presence in migrating cells . We believe that the effect of injected Tβ4 to stimulate migration of myocardial cells was also true in the present study.
Inflammation is now recognized as an important contributing factor to atherosclerosis and heart disease . A strong positive correlation also has been observed between the progression of coronary artery disease (CAD) and circulating EPC levels, which are significantly lower in patients with angiographic CAD progression . In fact, reduced levels of circulating EPCs may predict cardiovascular disease progression. Ischemia-reperfusion injury also is noted to have a marked inflammatory response, including the release of cytotoxic substances, oxygen-derived free radicals and proteases . The Tβ4 protein is angiogenic, helping to form new vessels from existing vasculature, and by this mechanism has been shown to accelerate wound healing and reduce inflammation . Does it also have other antiinflammatory effects? In a rat model of ischemia-reperfusion injury, daidzein, an isoflavone found in soybeans, was administered in vivo, attenuating myocardial damage by inhibiting nuclear factor Kappa-b (NF-kB) activation, which in turn suppressed activation of pro-inflammatory cytokines . Infarct size and inflammation were reduced and cardiac function was significantly improved in the daidzen-treated rats; conversely, reperfusion after ischemia activated NF-kB and increased levels of pro-inflammatory cytokines. Another study showed that flavonoids protect against ischemia-reperfusion injury by scavenging reactive oxygen species (ROS), demonstrating protective efficacy in cardiomyocytes . However, it remains to be seen whether antiinflammatory effects alone are sufficient to reduce infarct size and improve cardiac function.
PB-EPCs improve post-infarction cardiac function
Transplantation of different cell populations, including purified cardiomyocytes , human induced pluripotent stem cells (iPS cells) , and EPCs  have all shown improved cardiac contractile functioning. In the present study, which used EPCs isolated from autologous peripheral blood to treat cardiomyocyte infarction, random-blind ultrasonography with multiple measurements of cardiac contraction was performed in all surviving adult rats in the myocardial infarction model. At 4 weeks post-infarction, the LV fractional shortening in the EPCs-treated group was larger than that of rats in the control group. Two-dimensional echocardiographic measurements of ventricular function also revealed that the mean ejection fraction from the left ventricle in the EPCs-treated group rats after coronary ligation was larger than that of rats in the control group. ESDs and EDDs in rats receiving EPC treatment were significantly improved, suggesting that EPCs treatment resulted in decreased cardiac dilation after myocardial infarction—a sign of improved cardiac function. Reduced remodeling of the heart after ischemic injury followed transplantation of undifferentiated human iPS cells into immunosuppressed mice; although it resulted in tumor development, the cells further differentiated into cardiac cells in vivo, with a trend of declining EF compared to control rats with infarction . A study investigating post-infarction contractile function of the heart via 2D echocardiography, significantly impaired LV function was improved by 3% after EPCs infected with stromal-cell derived factor-1α (SDF-1α) were transplanted; SDF-1α promoted the proliferation and migration of the EPCs into injured tissue . EPCs maintained their protection against progressive LV dilatation and dysfunction compared to the MI control group. The major mechanism appears to be paracrine pathways of intramyocardial injection EPCs, resulting in the prevention of apoptosis and enhanced neovascularization of ischemic myocardium . Applying this understanding, we can infer that the ability of PB-EPCs to prevent cell death after coronary ligation probably leads to decreased infarct size and improved ventricular function as observed in rats in the present study.