Maria Mirotsou

MicroRNA-Mediated In Vitro and In Vivo Direct Reprogramming of Cardiac Fibroblasts to Cardiomyocytes

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 micro (mi)RNAs 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 miRNAs 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 miRNAs 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 on JAK inhibitor I treatment. Importantly, administration of miRNAs 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 noncardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin. Conclusions: The findings from this study provide 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.

Secreted frizzled related protein 2 (Sfrp2) is the key Akt-mesenchymal stem cell-released paracrine factor mediating myocardial survival and repair

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 β-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.

Genetic Modification of Mesenchymal Stem Cells Overexpressing CCR1 Increases Cell Viability, Migration, Engraftment, and Capillary Density in the Injured Myocardium

Rationale: Although mesenchymal stem cell (MSC) transplantation has been shown to promote cardiac repair in acute myocardial injury in vivo, its overall restorative capacity appears to be restricted mainly because of poor cell viability and low engraftment in the ischemic myocardium. Specific chemokines are upregulated in the infarcted myocardium. However the expression levels of the corresponding chemokine receptors (eg, CCR1, CXCR2) in MSCs are very low. We hypothesized that this discordance may account for the poor MSC engraftment and survival. Objective: To determine whether overexpression of CCR1 or CXCR2 chemokine receptors in MSCs augments their cell survival, migration and engraftment after injection in the infarcted myocardium. Methods and Results: Overexpression of CCR1, but not CXCR2, dramatically increased chemokine-induced murine MSC migration and protected MSC from apoptosis in vitro. Moreover, when MSCs were injected intramyocardially one hour after coronary artery ligation, CCR1-MSCs accumulated in the infarcted myocardium at significantly higher levels than control-MSCs or CXCR2-MSCs 3 days postmyocardial infarction (MI). CCR1-MSC–injected hearts exhibited a significant reduction in infarct size, reduced cardiomyocytes apoptosis and increased capillary density in injured myocardium 3 days after MI. Furthermore, intramyocardial injection of CCR1-MSCs prevented cardiac remodeling and restored cardiac function 4 weeks after MI. Conclusions: Our results demonstrate the in vitro and in vivo salutary effects of genetic modification of stem cells. Specifically, overexpression of chemokine receptor enhances the migration, survival and engraftment of MSCs, and may provide a new therapeutic strategy for the injured myocardium.

Exogenously administered secreted frizzled related protein 2 (Sfrp2) reduces fibrosis and improves cardiac function in a rat model of myocardial infarction

Secreted frizzled related protein 2 (Sfrp2) is known as an inhibitor for the Wnt signaling. In recent studies, Sfrp2 has been reported to inhibit the activity of Xenopus homolog of mammalian Tolloid-like 1 metalloproteinase. Bone morphogenic protein 1 (Bmp1)/Tolloid-like metalloproteinase plays a key role in the regulation of collagen biosynthesis and maturation after tissue injury. Here, we showed both endogenous Sfrp2 and Bmp1 protein expressions were up-regulated in rat heart after myocardial infarction (MI). We hypothesize that Sfrp2 could inhibit mammalian Bmp1 activity and, hence, the exogenous administration of Sfrp2 after MI would inhibit the deposition of mature collagen and improve heart function. Using recombinant proteins, we demonstrated that Sfrp2, but not Sfrp1 or Sfrp3, inhibited Bmp1 activity in vitro as measured by a fluorogenic peptide based procollagen C-proteinase activity assay. We also demonstrated that Sfrp2 at high concentration inhibited human and rat type I procollagen processing by Bmp1 in vitro. We further showed that exogenously added Sfrp2 inhibited type I procollagen maturation in primary cardiac fibroblasts. Two days after direct injection into the rat infarcted myocardium, Sfrp2 inhibited MI-induced type I collagen deposition. As early as 2 wk after injection, Sfrp2 significantly reduced left ventricular (LV) fibrosis as shown by trichrome staining. Four weeks after injection, Sfrp2 prevented the anterior wall thinning and significantly improved cardiac function as revealed by histological analysis and echocardiographic measurement. Our study demonstrates Sfrp2 at therapeutic doses can inhibit fibrosis and improve LV function at a later stage after MI.

Genetic Engineering of Mesenchymal Stem Cells and Its Application in Human Disease Therapy

The use of stem cells for tissue regeneration and repair is advancing both at the bench and bedside. Stem cells isolated from bone marrow are currently being tested for their therapeutic potential in a variety of clinical conditions including cardiovascular injury, kidney failure, cancer, and neurological and bone disorders. Despite the advantages, stem cell therapy is still limited by low survival, engraftment, and homing to damage area as well as inefficiencies in differentiating into fully functional tissues. Genetic engineering of mesenchymal stem cells is being explored as a means to circumvent some of these problems. This review presents the current understanding of the use of genetically engineered mesenchymal stem cells in human disease therapy with emphasis on genetic modifications aimed to improve survival, homing, angiogenesis, and heart function after myocardial infarction. Advancements in other disease areas are also discussed.

MicroRNA Induced Cardiac Reprogramming In Vivo Evidence for Mature Cardiac Myocytes and Improved Cardiac Function

Rationale: A major goal for the treatment of heart tissue damaged by cardiac injury is to develop strategies for restoring healthy heart muscle through the regeneration and repair of damaged myocardium. We recently demonstrated that administration of a specific combination of microRNAs (miR combo) into the infarcted myocardium leads to direct in vivo reprogramming of noncardiac myocytes to cardiac myocytes. However, the biological and functional consequences of such reprogramming are not yet known. Objective: The aim of this study was to determine whether noncardiac myocytes directly reprogrammed using miRNAs in vivo develop into mature functional cardiac myocytes in situ, and whether reprogramming leads to improvement of cardiac function. Methods and Results: We subjected fibroblast-specific protein 1-Cre mice/tandem dimer Tomato (tdTomato) mice to cardiac injury by permanent ligation of the left anterior descending coronary artery and injected lentiviruses encoding miR combo or a control nontargeting miRNA. miR combo significantly increased the number of reprogramming events in vivo. Five to 6 weeks after injury, morphological and physiological properties of tdTomato− and tdTomato+ cardiac myocyte–like cells were analyzed ex vivo. tdTomato+ cells expressed cardiac myocyte markers, sarcomeric organization, excitation–contraction coupling, and action potentials characteristic of mature ventricular cardiac myocytes (tdTomato− cells). Reprogramming was associated with improvement of cardiac function, as analyzed by serial echocardiography. There was a time delayed and progressive improvement in fractional shortening and other measures of ventricular function, indicating that miR combo promotes functional recovery of damaged myocardium. Conclusions: The findings from this study further validate the potential use of miRNA-mediated reprogramming as a therapeutic approach to promote cardiac regeneration after myocardial injury.Rationale: A major goal for the treatment of heart tissue damaged by cardiac injury is to develop strategies for restoring healthy heart muscle through the regeneration and repair of damaged myocardium. We recently demonstrated that administration of a specific combination of microRNAs (miR combo) into the infarcted myocardium leads to direct in vivo reprogramming of noncardiac myocytes to cardiac myocytes. However, the biological and functional consequences of such reprogramming are not yet known. Objective: The aim of this study was to determine whether noncardiac myocytes directly reprogrammed using miRNAs in vivo develop into mature functional cardiac myocytes in situ, and whether reprogramming leads to improvement of cardiac function. Methods and Results: We subjected fibroblast-specific protein 1-Cre mice/tandem dimer Tomato (tdTomato) mice to cardiac injury by permanent ligation of the left anterior descending coronary artery and injected lentiviruses encoding miR combo or a control nontargeting miRNA. miR combo significantly increased the number of reprogramming events in vivo. Five to 6 weeks after injury, morphological and physiological properties of tdTomato− and tdTomato+ cardiac myocyte–like cells were analyzed ex vivo. tdTomato+ cells expressed cardiac myocyte markers, sarcomeric organization, excitation–contraction coupling, and action potentials characteristic of mature ventricular cardiac myocytes (tdTomato− cells). Reprogramming was associated with improvement of cardiac function, as analyzed by serial echocardiography. There was a time delayed and progressive improvement in fractional shortening and other measures of ventricular function, indicating that miR combo promotes functional recovery of damaged myocardium. Conclusions: The findings from this study further validate the potential use of miRNA-mediated reprogramming as a therapeutic approach to promote cardiac regeneration after myocardial injury. # Novelty and Significance {#article-title-18}

MicroRNA Induced Cardiac Reprogramming In Vivo

Rationale: A major goal for the treatment of heart tissue damaged by cardiac injury is to develop strategies for restoring healthy heart muscle through the regeneration and repair of damaged myocardium. We recently demonstrated that administration of a specific combination of microRNAs (miR combo) into the infarcted myocardium leads to direct in vivo reprogramming of noncardiac myocytes to cardiac myocytes. However, the biological and functional consequences of such reprogramming are not yet known. Objective: The aim of this study was to determine whether noncardiac myocytes directly reprogrammed using miRNAs in vivo develop into mature functional cardiac myocytes in situ, and whether reprogramming leads to improvement of cardiac function. Methods and Results: We subjected fibroblast-specific protein 1-Cre mice/tandem dimer Tomato (tdTomato) mice to cardiac injury by permanent ligation of the left anterior descending coronary artery and injected lentiviruses encoding miR combo or a control nontargeting miRNA. miR combo significantly increased the number of reprogramming events in vivo. Five to 6 weeks after injury, morphological and physiological properties of tdTomato− and tdTomato+ cardiac myocyte–like cells were analyzed ex vivo. tdTomato+ cells expressed cardiac myocyte markers, sarcomeric organization, excitation–contraction coupling, and action potentials characteristic of mature ventricular cardiac myocytes (tdTomato− cells). Reprogramming was associated with improvement of cardiac function, as analyzed by serial echocardiography. There was a time delayed and progressive improvement in fractional shortening and other measures of ventricular function, indicating that miR combo promotes functional recovery of damaged myocardium. Conclusions: The findings from this study further validate the potential use of miRNA-mediated reprogramming as a therapeutic approach to promote cardiac regeneration after myocardial injury.Rationale: A major goal for the treatment of heart tissue damaged by cardiac injury is to develop strategies for restoring healthy heart muscle through the regeneration and repair of damaged myocardium. We recently demonstrated that administration of a specific combination of microRNAs (miR combo) into the infarcted myocardium leads to direct in vivo reprogramming of noncardiac myocytes to cardiac myocytes. However, the biological and functional consequences of such reprogramming are not yet known. Objective: The aim of this study was to determine whether noncardiac myocytes directly reprogrammed using miRNAs in vivo develop into mature functional cardiac myocytes in situ, and whether reprogramming leads to improvement of cardiac function. Methods and Results: We subjected fibroblast-specific protein 1-Cre mice/tandem dimer Tomato (tdTomato) mice to cardiac injury by permanent ligation of the left anterior descending coronary artery and injected lentiviruses encoding miR combo or a control nontargeting miRNA. miR combo significantly increased the number of reprogramming events in vivo. Five to 6 weeks after injury, morphological and physiological properties of tdTomato− and tdTomato+ cardiac myocyte–like cells were analyzed ex vivo. tdTomato+ cells expressed cardiac myocyte markers, sarcomeric organization, excitation–contraction coupling, and action potentials characteristic of mature ventricular cardiac myocytes (tdTomato− cells). Reprogramming was associated with improvement of cardiac function, as analyzed by serial echocardiography. There was a time delayed and progressive improvement in fractional shortening and other measures of ventricular function, indicating that miR combo promotes functional recovery of damaged myocardium. Conclusions: The findings from this study further validate the potential use of miRNA-mediated reprogramming as a therapeutic approach to promote cardiac regeneration after myocardial injury. # Novelty and Significance {#article-title-18}

C3orf58, a Novel Paracrine Protein, Stimulates Cardiomyocyte Cell-Cycle Progression Through the PI3K–AKT–CDK7 Pathway

Rationale: The regenerative capacity of the heart is markedly diminished shortly after birth, coinciding with overall withdrawal of cardiomyocytes from cell cycle. Consequently, the adult mammalian heart has limited capacity to regenerate after injury. The discovery of factors that can induce cardiomyocyte proliferation is, therefore, of high interest and has been the focus of extensive investigation throughout the past years. Objective: We have recently identified C3orf58 as a novel hypoxia and Akt induced stem cell factor (HASF) secreted from mesenchymal stem cells, which can promote cardiac repair through cytoprotective mechanisms. Here, we tested the hypothesis that HASF can also contribute to cardiac regeneration by stimulating cardiomyocyte division and proliferation. Methods and Results: Neonatal ventricular cardiomyocytes were stimulated in culture for 7 days with purified recombinant HASF protein. Compared with control untreated cells, HASF-treated neonatal cardiomyocytes exhibited 60% increase in DNA synthesis as measured by bromodeoxyuridine incorporation. These results were confirmed by immunofluorescence confocal microscopy showing a 50% to 100% increase in the number of cardiomyocytes in the mitotic and cytokinesis phases. Importantly, in vivo cardiac overexpression of HASF in a transgenic mouse model resulted in enhanced level of DNA synthesis and cytokinesis in neonatal and adult cardiomyocytes. These proliferative effects were modulated by a phosphoinositide 3-kinase–protein kinase B–cycle-dependent kinase 7 pathway as revealed by the use of phosphoinositide 3-kinase -pathway–specific inhibitors and silencing of the Cdk7 gene. Conclusions: Our studies support the hypothesis that HASF induces cardiomyocyte proliferation via a phosphoinositide 3-kinase–protein kinase B–cycle-dependent kinase 7 pathway. The implications of this finding may be significant for cardiac regeneration biology and therapeutics.

MicroRNAs and Cardiac Regeneration

The human heart has a limited capacity to regenerate lost or damaged cardiomyocytes after cardiac insult. Instead, myocardial injury is characterized by extensive cardiac remodeling by fibroblasts, resulting in the eventual deterioration of cardiac structure and function. Cardiac function would be improved if these fibroblasts could be converted into cardiomyocytes. MicroRNAs (miRNAs), small noncoding RNAs that promote mRNA degradation and inhibit mRNA translation, have been shown to be important in cardiac development. Using this information, various researchers have used miRNAs to promote the formation of cardiomyocytes through several approaches. Several miRNAs acting in combination promote the direct conversion of cardiac fibroblasts into cardiomyocytes. Moreover, several miRNAs have been identified that aid the formation of inducible pluripotent stem cells and miRNAs also induce these cells to adopt a cardiac fate. MiRNAs have also been implicated in resident cardiac progenitor cell differentiation. In this review, we discuss the current literature as it pertains to these processes, as well as discussing the therapeutic implications of these findings.

Adult Renal Mesenchymal Stem Cell–Like Cells Contribute to Juxtaglomerular Cell Recruitment

The renin-angiotensin-aldosterone system (RAAS) regulates BP and salt-volume homeostasis. Juxtaglomerular (JG) cells synthesize and release renin, which is the first and rate-limiting step in the RAAS. Intense pathologic stresses cause a dramatic increase in the number of renin-producing cells in the kidney, termed JG cell recruitment, but how this occurs is not fully understood. Here, we isolated renal CD44(+) mesenchymal stem cell (MSC)-like cells and found that they differentiated into JG-like renin-expressing cells both in vitro and in vivo. Sodium depletion and captopril led to activation and differentiation of these cells into renin-expressing cells in the adult kidney. In summary, CD44(+) MSC-like cells exist in the adult kidney and can differentiate into JG-like renin-producing cells under conditions that promote JG cell recruitment.