Previously we reported that inhibition of glycogen synthase kinase-3␤ (GSK3␤), a key regulator in many intracellular signaling pathways, enhances the survival and migration of vascular endothelial cells. Here we investigated the effect of inhibition of GSK3␤ activity on the angiogenic function of endothelial progenitor cell (EPC) and demonstrated a new therapeutic angiogenesis strategy using genetically modified EPC. As we previously reported, two biologically distinct types of EPC, spindle-shaped "early EPC" and cobblestone-shaped "late EPC" could be cultivated from human peripheral blood. Catalytically inactive GSK3␤ gene was transduced into both EPC. Inhibition of GSK3␤ signaling pathway led to increased nuclear translocation of ␤-catenin and increased secretion of angiogenic cytokines (vascular endothelial growth factor and interleukin-8). It enhanced the survival and proliferation of early EPC, whereas it promoted the survival and differentiation of late EPC. Transplantation of either of these genetically modified EPC into the ischemic hind limb model of athymic nude mouse significantly improved blood flow, limb salvage, and tissue capillary density compared with nontransduced EPC. Inhibition of GSK3␤ signaling of either of these genetically modified EPC augmented the in vitro and in vivo angiogenic potency of these cell populations. These data provide evidence that GSK3␤ has a key role in the angiogenic properties of EPC. Furthermore, the genetic modification of EPC to alter this signaling step can improve the efficacy of cell-based therapeutic vasculogenesis.

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Stem cell transplantation in acute myocardial infarction (AMI) has emerged as a promising therapeutic option. We evaluated the impact of AMI on mesenchymal stem cell (MSC) differentiation into cardiomyocyte lineage. Cord blood-derived human MSCs were exposed to in vitro conditions simulating in vivo environments of the beating heart with acute ischemia, as follows: (a) myocardial proteins or serum obtained from sham-operated rats, and (b) myocardial proteins or serum from AMI rats, with or without application of oscillating pressure. Expression of cardiacspecific markers on MSCs was greatly induced by the infarcted myocardial proteins, compared with the normal proteins. It was also induced by application of oscillating pressure to MSCs. Treatment of MSCs with infarcted myocardial proteins and oscillating pressure greatly augmented expression of cardiac-specific genes. Such expression was blocked by inhibitor of transforming growth factor ␤ 1 (TGF-␤ 1 ) or bone morphogenetic protein-2 (BMP-2). In vitro cellular and electrophysiologic experiments showed that these differentiated MSCs expressing cardiomyocytespecific markers were able to make a coupling with cardiomyocytes but not to selfbeat. The pathophysiologic significance of in vitro results was confirmed using the rat AMI model. The protein amount of TGF-␤ 1 and BMP-2 in myocardium of AMI was significantly higher than that in normal myocardium. When MSCs were transplanted to the heart and analyzed 8 weeks later, they expressed cardiomyocyte-specific markers, leading to improved cardiac function. These in vitro and in vivo results suggest that infarctrelated biological and physical factors in AMI induce commitment of MSCs to cardiomyocyte-like cells through TGF-␤/BMP-2 pathways.

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