Jinghai Chen

Hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells

In recent years, the understanding that regeneration progresses at the level of the myocardium has placed stem cell research at the center stage in cardiology. Despite an increasing interest in cell transplant research, relatively little is known about the biochemical regulation of the stem cell itself after transplantation into an ischemic heart. We demonstrated here, using rat mesenchymal stem cells (MSCs), that cells undergo caspase‐dependent apoptosis in response to hypoxia and serum deprivation (SD), which are both components of ischemia in vivo. In particular, the treated cells exhibited mitochondrial dysfunction, including cytochrome C release, loss in ΔΨm, and Bax accumulation, but in a p53‐independent manner. Although the cells treated by hypoxia/SD possess the activity of caspase‐8, zIEDT‐fmk, a specific caspase‐8 inhibitor, failed to inhibit cell apoptosis induced in our system. Taken together, our findings indicate that MSCs are sensitive to hypoxia/SD stimuli that involve changes in mitochondrial integrity and function but are potentially independent of caspase‐8.

mir-17-92 Cluster is Required for and Sufficient to Induce Cardiomyocyte Proliferation in Postnatal and Adult Hearts

Rationale: Cardiomyocytes in adult mammalian hearts are terminally differentiated cells that have exited from the cell cycle and lost most of their proliferative capacity. Death of mature cardiomyocytes in pathological cardiac conditions and the lack of regeneration capacity of adult hearts are primary causes of heart failure and mortality. However, how cardiomyocyte proliferation in postnatal and adult hearts becomes suppressed remains largely unknown. The miR-17–92 cluster was initially identified as a human oncogene that promotes cell proliferation. However, its role in the heart remains unknown. Objective: To test the hypothesis that miR-17–92 participates in the regulation of cardiomyocyte proliferation in postnatal and adult hearts. Methods and Results: We deleted miR-17–92 cluster from embryonic and postnatal mouse hearts and demonstrated that miR-17–92 is required for cardiomyocyte proliferation in the heart. Transgenic overexpression of miR-17–92 in cardiomyocytes is sufficient to induce cardiomyocyte proliferation in embryonic, postnatal, and adult hearts. Moreover, overexpression of miR-17–92 in adult cardiomyocytes protects the heart from myocardial infarction-induced injury. Similarly, we found that members of miR-17–92 cluster, miR-19 in particular, are required for and sufficient to induce cardiomyocyte proliferation in vitro. We identified phosphatase and tensin homolog, a tumor suppressor, as an miR-17–92 target to mediate the function of miR-17–92 in cardiomyocyte proliferation. Conclusions: Our studies therefore identify miR-17–92 as a critical regulator of cardiomyocyte proliferation, and suggest this cluster of microRNAs could become therapeutic targets for cardiac repair and heart regeneration.

LincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis and Atherosclerosis by Enhancing p53 Activity

Background— Long noncoding RNAs (lncRNAs) have recently been implicated in many biological processes and diseases. Atherosclerosis is a major risk factor for cardiovascular disease. However, the functional role of lncRNAs in atherosclerosis is largely unknown. Methods and Results— We identified lincRNA-p21 as a key regulator of cell proliferation and apoptosis during atherosclerosis. The expression of lincRNA-p21 was dramatically downregulated in atherosclerotic plaques of ApoE−/− mice, an animal model for atherosclerosis. Through loss- and gain-of-function approaches, we showed that lincRNA-p21 represses cell proliferation and induces apoptosis in vascular smooth muscle cells and mouse mononuclear macrophage cells in vitro. Moreover, we found that inhibition of lincRNA-p21 results in neointimal hyperplasia in vivo in a carotid artery injury model. Genome-wide analysis revealed that lincRNA-p21 inhibition dysregulated many p53 targets. Furthermore, lincRNA-p21, a transcriptional target of p53, feeds back to enhance p53 transcriptional activity, at least in part, via binding to mouse double minute 2 (MDM2), an E3 ubiquitin-protein ligase. The association of lincRNA-p21 and MDM2 releases MDM2 repression of p53, enabling p53 to interact with p300 and to bind to the promoters/enhancers of its target genes. Finally, we show that lincRNA-p21 expression is decreased in patients with coronary artery disease. Conclusions— Our studies identify lincRNA-p21 as a novel regulator of cell proliferation and apoptosis and suggest that this lncRNA could serve as a therapeutic target to treat atherosclerosis and related cardiovascular disorders.

MicroRNA-22 Regulates Cardiac Hypertrophy and Remodeling in Response to Stress

Rationale: The adult heart is composed primarily of terminally differentiated, mature cardiomyocytes that express signature genes related to contraction. In response to mechanical or pathological stress, the heart undergoes hypertrophic growth, a process defined as an increase in cardiomyocyte cell size without an increase in cell number. However, the molecular mechanism of cardiac hypertrophy is not fully understood. Objective: To identify and characterize microRNAs that regulate cardiac hypertrophy and remodeling. Methods and Results: Screening for muscle-expressed microRNAs that are dynamically regulated during muscle differentiation and hypertrophy identified microRNA-22 (miR-22) as a cardiac- and skeletal muscle–enriched microRNA that is upregulated during myocyte differentiation and cardiomyocyte hypertrophy. Overexpression of miR-22 was sufficient to induce cardiomyocyte hypertrophy. We generated mouse models with global and cardiac-specific miR-22 deletion, and we found that cardiac miR-22 was essential for hypertrophic cardiac growth in response to stress. miR-22–null hearts blunted cardiac hypertrophy and cardiac remodeling in response to 2 independent stressors: isoproterenol infusion and an activated calcineurin transgene. Loss of miR-22 sensitized mice to the development of dilated cardiomyopathy under stress conditions. We identified Sirt1 and Hdac4 as miR-22 targets in the heart. Conclusions: Our studies uncover miR-22 as a critical regulator of cardiomyocyte hypertrophy and cardiac remodeling.

Cardiac-Specific YAP Activation Improves Cardiac Function and Survival in an Experimental Murine MI Model

Rationale: Yes-associated protein (YAP), the terminal effector of the Hippo signaling pathway, is crucial for regulating embryonic cardiomyocyte proliferation. Objective: We hypothesized that YAP activation after myocardial infarction (MI) would preserve cardiac function and improve survival. Methods and Results: We used a cardiac-specific, inducible expression system to activate YAP in adult mouse heart. Activation of YAP in adult heart promoted cardiomyocyte proliferation and did not deleteriously affect heart function. Furthermore, YAP activation after MI preserved heart function and reduced infarct size. Using adeno-associated virus subtype 9 (AAV9) as a delivery vector, we expressed human YAP (hYAP) in the adult murine myocardium immediately after MI. We found that AAV9:hYAP significantly improved cardiac function and mouse survival. AAV9:hYAP did not exert its salutary effects by reducing cardiomyocyte apoptosis. Rather, AAV9:hYAP stimulated adult cardiomyocyte proliferation. Gene expression profiling indicated that AAV9:hYAP stimulated expression of cell cycle genes and promoted a less mature cardiac gene expression signature. Conclusions: Cardiac-specific YAP activation after MI mitigated myocardial injury, improved cardiac function, and enhanced survival. These findings suggest that therapeutic activation of YAP or its downstream targets, potentially through AAV-mediated gene therapy, may be a strategy to improve outcome after MI.

Omega-3 Fatty Acids Prevent Pressure Overload–Induced Cardiac Fibrosis Through Activation of Cyclic GMP/Protein Kinase G Signaling in Cardiac Fibroblasts

Background— Omega-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) from fish oil ameliorate cardiovascular diseases. However, little is known about the effects of &ohgr;-3 polyunsaturated fatty acids on cardiac fibrosis, a major cause of diastolic dysfunction and heart failure. The present study assessed the effects of &ohgr;-3 polyunsaturated fatty acids on cardiac fibrosis. Methods and Results— We assessed left ventricular fibrosis and pathology in mice subjected to transverse aortic constriction after the consumption of a fish oil or a control diet. In control mice, 4 weeks of transverse aortic constriction induced significant cardiac dysfunction, cardiac fibrosis, and cardiac fibroblast activation (proliferation and transformation into myofibroblasts). Dietary supplementation with fish oil prevented transverse aortic constriction–induced cardiac dysfunction and cardiac fibrosis and blocked cardiac fibroblast activation. In heart tissue, transverse aortic constriction increased active transforming growth factor-&bgr;1 levels and phosphorylation of Smad2. In isolated adult mouse cardiac fibroblasts, transforming growth factor-&bgr;1 induced cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid increased cyclic GMP levels and blocked cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid blocked phospho-Smad2/3 nuclear translocation. DT3, a protein kinase G inhibitor, blocked the antifibrotic effects of eicosapentaenoic acid and docosahexaenoic acid. Eicosapentaenoic acid and docosahexaenoic acid increased phosphorylated endothelial nitric oxide synthase and endothelial nitric oxide synthase protein levels and nitric oxide production. Conclusion— Omega-3 fatty acids prevent cardiac fibrosis and cardiac dysfunction by blocking transforming growth factor-&bgr;1–induced phospho-Smad2/3 nuclear translocation through activation of the cyclic GMP/protein kinase G pathway in cardiac fibroblasts.

Lysophosphatidic acid protects mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis.

Bone marrow‐derived mesenchymal stem cells (MSCs) have shown great promise for cardiac repair. However, poor viability of transplanted MSCs within the ischemic heart has limited their therapeutic potential. Our previous studies have documented that hypoxia and serum deprivation (hypoxia/SD), induced MSCs apoptosis through the mitochondrial apoptotic pathway. Since serum lysophosphatidic acid (LPA) levels are known to be significantly elevated after acute myocardial infarction and that LPA enhanced survival of other cell systems, we embarked on determining whether LPA protects MSCs against hypoxia/SD‐induced apoptosis. We have also investigated the potential mechanism(s) that may mediate such actions of LPA. All experiments were carried out on rat bone marrow MSCs. Apoptosis was induced by exposure of cells to hypoxia/SD in a sealed GENbox hypoxic chamber. Effects of LPA were investigated in the absence and presence of inhibitors that target either Giproteins, the mitogen activated protein kinases ERK1/2, or phosphoinositide 3‐kinase (PI3K). The data obtained showed that hypoxia/SD‐induced apoptosis was significantly attenuated by LPA through Gi‐coupled LPA1 receptors linked to the downstream ERK1/2 and PI3K/Akt signaling pathways that function in parallel. Additional studies have demonstrated that hypoxia/SD‐induced activation of mitochondrial dysfunction was virtually abolished by LPA treatment and that inhibition of the LPA1 receptor, Gi proteins, the PI3K/Akt pathway, or ERKs effectively reversed this protective action of LPA. Taken together, our findings indicate that LPA is a novel, potent survival factor for MSCs and this may prove to be of considerable therapeutic significance in terms of exploiting MSC‐based therapy in the infracted myocardium.

Cardiac-Specific YAP Activation Improves Cardiac Function and Survival in an Experimental Murine Myocardial Infarction Model

Rationale: Yes-associated protein (YAP), the terminal effector of the Hippo signaling pathway, is crucial for regulating embryonic cardiomyocyte proliferation. Objective: We hypothesized that YAP activation after myocardial infarction (MI) would preserve cardiac function and improve survival. Methods and Results: We used a cardiac-specific, inducible expression system to activate YAP in adult mouse heart. Activation of YAP in adult heart promoted cardiomyocyte proliferation and did not deleteriously affect heart function. Furthermore, YAP activation after MI preserved heart function and reduced infarct size. Using adeno-associated virus subtype 9 (AAV9) as a delivery vector, we expressed human YAP (hYAP) in the adult murine myocardium immediately after MI. We found that AAV9:hYAP significantly improved cardiac function and mouse survival. AAV9:hYAP did not exert its salutary effects by reducing cardiomyocyte apoptosis. Rather, AAV9:hYAP stimulated adult cardiomyocyte proliferation. Gene expression profiling indicated that AAV9:hYAP stimulated expression of cell cycle genes and promoted a less mature cardiac gene expression signature. Conclusions: Cardiac-specific YAP activation after MI mitigated myocardial injury, improved cardiac function, and enhanced survival. These findings suggest that therapeutic activation of YAP or its downstream targets, potentially through AAV-mediated gene therapy, may be a strategy to improve outcome after MI.

Pi3kcb Links Hippo-YAP and PI3K-AKT Signaling Pathways to Promote Cardiomyocyte Proliferation and Survival

Rationale: Yes-associated protein (YAP), the nuclear effector of Hippo signaling, regulates cellular growth and survival in multiple organs, including the heart, by interacting with TEA (transcriptional enhancer activator)-domain sequence–specific DNA-binding proteins. Recent studies showed that YAP stimulates cardiomyocyte proliferation and survival. However, the direct transcriptional targets through which YAP exerts its effects are poorly defined. Objective: To identify direct YAP targets that mediate its mitogenic and antiapoptotic effects in the heart. Methods and Results: We identified direct YAP targets by combining differential gene expression analysis in YAP gain- and loss-of-function with genome-wide identification of YAP-bound loci using chromatin immunoprecipitation and high throughput sequencing. This screen identified Pik3cb, encoding p110&bgr;, a catalytic subunit of phosphoinositol-3-kinase, as a candidate YAP effector that promotes cardiomyocyte proliferation and survival. YAP and TEA-domain occupied a conserved enhancer within the first intron of Pik3cb, and this enhancer drove YAP-dependent reporter gene expression. Yap gain- and loss-of-function studies indicated that YAP is necessary and sufficient to activate the phosphoinositol-3-kinase-Akt pathway. Like Yap, Pik3cb gain-of-function stimulated cardiomyocyte proliferation, and Pik3cb knockdown dampened YAP mitogenic activity. Reciprocally, impaired heart function in Yap loss-of-function was significantly rescued by adeno-associated virus–mediated Pik3cb expression. Conclusions: Pik3cb is a crucial direct target of YAP, through which the YAP activates phosphoinositol-3-kinase-AKT pathway and regulates cardiomyocyte proliferation and survival.

Loss of microRNA-155 Protects the Heart from Pathological Cardiac Hypertrophy

Rationale: In response to mechanical and pathological stress, adult mammalian hearts often undergo mal-remodeling, a process commonly characterized as pathological hypertrophy, which is associated with upregulation of fetal genes, increased fibrosis, and reduction of cardiac dysfunction. The molecular pathways that regulate this process are not fully understood. Objective: To explore the function of microRNA-155 (miR-155) in cardiac hypertrophy and remodeling. Methods and Results: Our previous work identified miR-155 as a critical microRNA that repressed the expression and function of the myocyte enhancer factor 2A. In this study, we found that miR-155 is expressed in cardiomyocytes and that its expression is reduced in pressure overload–induced hypertrophic hearts. In mouse models of cardiac hypertrophy, miR-155 null hearts suppressed cardiac hypertrophy and cardiac remodeling in response to 2 independent pathological stressors, transverse aortic restriction and an activated calcineurin transgene. Most importantly, loss of miR-155 prevents the progress of heart failure and substantially extends the survival of calcineurin transgenic mice. The function of miR-155 in hypertrophy is confirmed in isolated cardiomyocytes. We identified jumonji, AT rich interactive domain 2 (Jarid2) as an miR-155 target in the heart. miR-155 directly represses Jarid2, whose expression is increased in miR-155 null hearts. Inhibition of endogenous Jarid2 partially rescues the effect of miR-155 loss in isolated cardiomyocytes. Conclusions: Our studies uncover miR-155 as an inducer of pathological cardiomyocyte hypertrophy and suggest that inhibition of endogenous miR-155 might have clinical potential to suppress cardiac hypertrophy and heart failure.