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We previously reported that intramyocardial injection of bone marrow-derived mesenchymal stem cells overexpressing Akt (Akt-MSCs) inhibits ventricular remodeling and restores cardiac function measured 2 wk after myocardial infarction. Here, we report that the functional improvement occurs in < 72 h. This early remarkable effect cannot be readily attributed to myocardial regeneration from the donor cells. Thus, we hypothesized that paracrine actions exerted by the cells through the release of soluble factors might be important mechanisms of tissue repair and functional improvement after injection of the Akt-MSCs. Indeed, in the current study we demonstrate that conditioned medium from hypoxic Akt-MSCs markedly inhibits hypoxia-induced apoptosis and triggers vigorous spontaneous contraction of adult rat cardiomyocytes in vitro. When injected into infarcted hearts, the Akt-MSC conditioned medium significantly limits infarct size and improves ventricular function relative to controls. Support to the paracrine hypothesis is provided by data showing that several genes, coding for factors (VEGF, FGF-2, HGF, IGF-I, and TB4) that are potential mediators of the effects exerted by the Akt-MSC conditioned medium, are significantly up-regulated in the Akt-MSCs, particularly in response to hypoxia. Taken together, our data support Akt-MSC-mediated paracrine mechanisms of myocardial protection and functional improvement.

Rationale Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ. Objective The aim of this study was to develop a strategy that identified and evaluated the potential of specific miRNAs capable of inducing reprogramming of cardiac fibroblasts directly to cardiomyocytes in vitro and in vivo. Methods and Results Using a combinatorial strategy, we identified a combination of microRNAs (miRNA) 1, 133, 208, and 499 capable of inducing direct cellular reprogramming of fibroblasts to cardiomyocyte-like cells in vitro. Detailed studies of the reprogrammed cells, demonstrated that a single transient transfection of the microRNAs can direct a switch in cell fate as documented by expression of mature cardiomyocyte markers, sarcomeric organization, and exhibition of spontaneous calcium flux characteristic of a cardiomyocyte-like phenotype. Interestingly, we also found that miRNA-mediated reprogramming was enhanced 10 fold upon JAK inhibitor I treatment. Importantly, administration of microRNAs into ischemic mouse myocardium resulted in evidence of direct conversion of cardiac fibroblasts to cardiomyocytes in situ. Genetic tracing analysis using Fsp1Cre-traced fibroblasts from both cardiac and non-cardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin. Conclusion The findings from this study provide the first proof-of-concept that miRNAs have the capability of directly converting fibroblasts to a cardiomyocyte-like phenotype in vitro. Also of significance is that this is the first report of direct cardiac reprogramming in vivo. Our approach may have broad and important implications for therapeutic tissue regeneration in general.

The type 1 angiotensin II (AT1) receptor is well characterized but the type 2 (AT2) receptor remains an enigma. We tested the hypothesis that the AT2 receptor can modulate the growth of vascular smooth muscle cells by transfecting an AT2 receptor expression vector into the balloon-injured rat carotid artery and observed that overexpression of the AT2 receptor attenuated neointimal formation. In cultured smooth muscle cells, AT2 receptor transfection reduced proliferation and inhibited mitogen-activated protein kinase activity. Furthermore, we demonstrated that the AT2 receptor mediated the developmentally regulated decrease in aortic DNA synthesis at the latter stages of gestation. These results suggest that the AT2 receptor exerts an antiproliferative effect, counteracting the growth action of AT1 receptor.

Stem cell therapy has emerged as a promising tool for the treatment of a variety of diseases. Previously, we have shown that Akt-modified mesenchymal stem cells mediate tissue repair through paracrine mechanisms. Using a comprehensive functional genomic strategy, we show that secreted frizzled related protein 2 (Sfrp2) is the key stem cell paracrine factor that mediates myocardial survival and repair after ischemic injury. Sfrp2 is known to modulate Wnt signaling, and we demonstrate that cardiomyocytes treated with secreted frizzled related protein increase cellular ␤-catenin and up-regulate expression of antiapoptotic genes. These findings reveal the key role played by Sfrp2 in mediating the paracrine effects of Akt-mesenchymal stem cells on tissue repair and identify modulation of Wnt signaling as a therapeutic target for heart disease.D espite major advances in our understanding and therapy of coronary artery disease, myocardial infarction remains a major cause of mortality and morbidity in the United States. The limited ability of the damaged heart to regenerate and replace dead myocardium leads to the devastating sequelae of congestive heart failure. The recent interests in the development of cell-based therapeutic strategies are aimed at replenishing the diminished myocyte mass (1). Such cell-based strategies have used a variety of stem and progenitor cells, including skeletal muscle myoblasts, embryonic stem cells, resident cardiac stem cells, mesenchymal stem cells (MSCs), endothelial progenitor cells, and bone marrowderived mononuclear cells (2). Although the majority of animal and preliminary human studies of cell-based therapy shows an overall improvement in cardiac function when administered to hearts after acute infarction, the effects generally are modest, and the mechanisms underlying such an observed improvement are far from clear (3). Postulated mechanisms include differentiation of transplanted cells or resident cardiac stem cells into cardiomyocytes (4), fusion between donor cells and host cardiomyocytes (5), and/or improved tissue perfusion attributable to enhanced donor cell-derived angiogenesis (6).We recently have reported that intracardiac implantation of genetically engineered MSCs overexpressing the Akt gene (AktMSCs) yielded dramatic diminution of infarct size and restoration of cardiac function in rodent hearts after myocardial injury (7). We observed that these salutary effects occurred as early as 72 h after Akt-MSC implantation (8). Moreover, we demonstrated that Akt-MSC-conditioned medium provided survival signal to adult ventricular myocytes against hypoxia-induced apoptosis in vitro and upon injection into infarcted hearts, dramatically limited infarct size, and prevented ventricular dysfunction in vivo (8). Accordingly, we hypothesized that Akt-MSCs achieve their beneficial effects in part through enhancing early survival of the ischemic myocardium. Furthermore, we postulated that the prosurvival effects of AktMSCs on ischemic myocardium are paracrine in nature and mediated by ...

It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as 8-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessel. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor ofvascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.The process of intimal hyperplasia is common to various forms of vascular diseases such as atherosclerosis, transplant vasculopathy, and restenosis following balloon angioplasty. The use of in vivo gene therapy for the treatment of vascular disorders has been speculated for several years. Recently, our laboratory and others have shown that neointima formation after balloon injury can be inhibited by antisense oligonucleotide transfection (1, 2). However, there has been no report of a "therapeutic" transfection of a functional gene whose product inhibits neointima formation. This study demonstrates the successful inhibition of neointimal hyperplasia by in vivo gene transfer by introducing the cDNA encoding the endothelial cell nitric oxide synthase (ec-NOS). Our study has two major implications: (i) that the prevention of neointimal hyperplasia by an in vivo gene transfer approach is feasible, and (ii) that NO is an effective in vivo inhibitor of vascular smooth muscle cell (VSMC) accumulation and that its enhanced expression by in vivo gene transfer may be a promising strategy for the treatment of vascular disease.Injury to the endothelium plays an essential role in the "response to injury" hypothesis (3, 4). Experimental studies have shown that vascular injury induces local expression of mitogens and chemotactic factors mediating neointima formation. The lesion is characterized in part by the abnormal migration and proliferation of VSMCs in the intima. In addition, it is postulated that endothelial denudation may result in the loss of constitutively expressed endotheliumderived...

We previously reported that intramyocardial injection of bone marrow-derived mesenchymal stem cells overexpressing Akt (MSC-Akt) efficiently repaired infarcted rat myocardium and improved cardiac function. Controversy still exists over the mechanisms by which MSC contribute to tissue repair. Herein, we tested if cellular fusion of MSC plays a determinant role in cardiac repair. We injected MSC expressing Cre recombinase, with or without Akt, into Cre reporter mice. In these mice, LacZ is expressed only after Cre-mediated excision of a loxP-flanked stop signal and is indicative of fusion. MSC engraftment within infarcted myocardium was transient but significantly enhanced by Akt. MSC fusion with cardiomyocytes was observed as early as 3 days, but was infrequent, and we found a low rate of differentiation of MSC into cardiomyocytes. MSC-Akt decreased infarct size at 3 days and restored early cardiac function. In conclusion, MSC-Akt improved early repair despite transient engraftment, low levels of cellular fusion, and differentiation. These new observations further confirm our recently reported data that early paracrine mechanisms mediated by MSC are responsible for enhancing the survival of existing myocytes and that Akt could alter the secretion of various cytokines and growth factors.

Abstract-Heme oxygenase (HO)-1 degrades the pro-oxidant heme and generates carbon monoxide and antioxidant bilirubin. We have previously shown that in response to hypoxia, HO-1-null mice develop infarcts in the right ventricle of their hearts and that their cardiomyocytes are damaged by oxidative stress. To test whether HO-1 protects against oxidative injury in the heart, we generated cardiac-specific transgenic mice overexpressing different levels of HO-1. By use of a Langendorff preparation, hearts from transgenic mice showed improved recovery of contractile performance during reperfusion after ischemia in an HO-1 dose-dependent manner. In vivo, myocardial ischemia and reperfusion experiments showed that infarct size was only 14.7% of the area at risk in transgenic mice compared with 56.5% in wild-type mice. Hearts from these transgenic animals had reduced inflammatory cell infiltration and oxidative damage. Our data demonstrate that overexpression of HO-1 in the cardiomyocyte protects against ischemia and reperfusion injury, thus improving the recovery of cardiac function. Key Words: heart Ⅲ infarction Ⅲ Langendorff preparation Ⅲ cytoprotection Ⅲ inflammation O xidative stress in the heart caused by ischemia and reperfusion leads to cardiomyocyte death. 1-3 Several studies have shown that increased expression of myocardial stress proteins and/or antioxidant enzymes protects against postischemic injury. 4 -6 In response to stress, elevated expression of heat shock proteins may protect the myocardium. 7 These heat shock proteins are thought to mediate cardioprotection through their biological functions as molecular chaperones by preventing protein denaturation. 7 Heme oxygenase (HO)-1, a stress response and cytoprotective protein, also known as hsp32, protects cells from death due to pathophysiological stress. 8 -12 By degrading the pro-oxidant heme and generating the antioxidant bilirubin, 13,14 HO-1 may protect cells against oxidative injury. In addition, carbon monoxide (CO), another HO-1 reaction product, contributes to the regulation of vascular tone and is reported to have antiinflammatory properties, which may contribute to the cytoprotective action of HO-1. 15,16 HO-1 is upregulated in the heart and blood vessels in response to hemodynamic stress in rats 17,18 and ischemia/ reperfusion injury in pigs, 19,20 implicating an important role for HO-1 in cardiovascular homeostasis. We have recently shown that in response to hypoxia, HO-1-null mice develop right ventricular infarcts with organized mural thrombi. Furthermore, increased lipid peroxidation and oxidative damage occur in right ventricular cardiomyocytes from HO-1-null but not wild-type mice. 12 Thus, we hypothesized that HO-1 may play a central role in cardiac homeostasis by protecting cardiomyocytes from ischemia/reperfusioninduced injury and secondary oxidative damage. To gain insight into the cardioprotective role of HO-1 in vivo, we generated transgenic mice overexpressing HO-1 specifically in the heart. We measured cardiac performance during ...