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Myocardial ischemia/reperfusion (MI/R) causes loss of cardiomyocytes via oxidative stress-induced cardiomyocyte apoptosis. miR322, orthologous to human miR-424, was identified as an ischemia-induced angiogenic miRNA, but its cellular source and function in the setting of acute MI/R remains largely unknown. Using LacZ-tagged miR322 cluster reporter mice, we observed that vascular endothelial cells are the major cellular source of the miR322 cluster in adult hearts. Moreover, miR322 levels were significantly reduced in the heart at 24 h after MI/R injury. Intramyocardial injection of mimic-miR322 significantly diminished cardiac apoptosis (as determined by expression levels of active caspase 3 by Western blot analysis and immunostaining for TUNEL) and reduced infarct size by about 40%, in association with reduced FBXW7 and increased active Notch 1 levels in the ischemic hearts. FBXW7, which is an ubiquitin ligase that is crucial for activated Notch1 turnover, was identified as a direct target of miR322 via FBXW7 3’UTR reporter assay. Co-injection of FBXW7 plasmid with mimic-miR322 in ischemic hearts abolished the effect of mimic-miR322 to reduce apoptosis and infarct size in MI/R hearts. These data identify FBXW7 as a direct target of miR322 and suggest that miR322 could have potential therapeutic application for cardioprotection against ischemia/reperfusion-induced injury.

Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator that plays a key role in cell differentiation and oncogenesis, was reported to promote adipogenic differentiation in vitro by catalyzing trimethylation of histone 3 lysine 27. However, inhibition of EZH2 induced lipid accumulation in certain cancer and hepatocyte cell lines. To address this discrepancy, we investigated the role of EZH2 in adipogenic differentiation and lipid metabolism using primary human and mouse preadipocytes and adipose-specific EZH2 knockout (KO) mice. We found that the EZH2-selective inhibitor GSK126 induced lipid accumulation in human adipocytes, without altering adipocyte differentiation marker gene expression. Moreover, adipocyte-specific EZH2 KO mice, generated by crossing EZH2 floxed mice with adiponectin-Cre mice, displayed significantly increased body weight, adipose tissue mass, and adipocyte cell size and reduced very low-density lipoprotein (VLDL) levels, as compared with littermate controls. These phenotypic alterations could not be explained by differences in feeding behavior, locomotor activity, metabolic energy expenditure, or adipose lipolysis. In addition, human adipocytes treated with either GSK126 or vehicle exhibited comparable rates of glucose-stimulated triglyceride accumulation and fatty acid uptake. Mechanistically, lipid accumulation induced by GSK126 in adipocytes was lipoprotein-dependent, and EZH2 inhibition or gene deletion promoted lipoprotein-dependent lipid uptake in vitro concomitant with up-regulated apolipoprotein E (ApoE) gene expression. Deletion of ApoE blocked the effects of GSK126 to promote lipoprotein-dependent lipid uptake in murine adipocytes. Collectively, these results indicate that EZH2 inhibition promotes lipoprotein-dependent lipid accumulation via inducing ApoE expression in adipocytes, suggesting a novel mechanism of lipid regulation by EZH2.

Objective Inflammation in adipose tissues in obesity promotes insulin resistance and metabolic disease. The Duffy antigen receptor for chemokines (DARC) is a promiscuous non-signaling receptor expressed on erythrocytes and other cell types that modulates tissue inflammation by binding chemokines such as monocyte chemoattractant protein-1 (MCP-1) and by acting as a chemokine reservoir. DARC allelic variants are common in humans, but the role of DARC in modulating obesity-related metabolic disease is unknown. Methods We examined body weight gain, tissue adiposity, metabolic parameters and inflammatory marker expression in wild-type and DARC knockout mice fed a chow diet (CD) and high fat diet (HFD). Results Compared to wild-type mice, HFD-fed DARC knockout mice developed glucose intolerance and insulin resistance independent of increases in body weight or adiposity. Interestingly, insulin sensitivity was also diminished in lean male DARC knockout mice fed a chow diet. Insulin production was not reduced by DARC gene deletion, and plasma leptin levels were similar in HFD fed wild-type and DARC knockout mice. MCP-1 levels in plasma rose significantly in the HFD fed wild-type mice, but not in the DARC knockout mice. Conversely, adipose tissue MCP-1 levels were higher, and more macrophage crown-like structures were detected, in the HFD fed DARC knockout mice as compared with the wild-type mice, consistent with augmented adipose tissue inflammation that is not accurately reflected by plasma levels of DARC-bound MCP-1 in these mice. Conclusions These findings suggest that DARC regulates metabolic function and adipose tissue inflammation, which may impact obesity-related disease in ethnic populations with high frequencies of DARC allelic variants.

Research into the biology of extracellular vesicles (EVs), including exosomes and microvesicles, has expanded significantly with advances in EV isolation techniques, a better understanding of the surface markers that characterize exosomes and microvesicles, and greater information derived from –omics approaches on the proteins, lipids, mRNAs, and microRNAs (miRNAs) transported by EVs. We have recently discovered a role for exosome-derived miRNAs in age-related bone loss and osteoarthritis, two conditions that impose a significant public health burden on the aging global population. Previous work has also revealed multiple roles for EVs and their miRNAs in muscle regeneration and congenital myopathies. Thus, EVs appear to be involved in a number of degenerative conditions that impact the musculoskeletal system, indicating that the musculoskeletal system is an excellent model for investigating the role of EVs in tissue maintenance and repair. This review highlights the role of EVs in bone, skeletal muscle, and joint health, including both normal tissue metabolism as well as tissue injury repair and regeneration. A consistent theme that emerges from study of musculoskeletal EVs is that various miRNAs appear to mediate a number of key pathological processes. These findings point to a potential therapeutic opportunity to target EV-derived miRNAs as a strategy for improving musculoskeletal function.

Background Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs), small non-coding RNAs that post-transcriptionally regulate target genes. MiR-125b-5p is downregulated in patients with end-stage dilated and ischemic cardiomyopathy, and has been proposed as a biomarker of heart failure. We previously reported that the β-blocker carvedilol promotes cardioprotection via β-arrestin-biased agonism of β1-adrenergic receptor while stimulating miR-125b-5p processing in the mouse heart. We hypothesize that β1-adrenergic receptor/β-arrestin1-responsive miR-125b-5p confers the improvement of cardiac function and structure after acute myocardial infarction. Methods and results Using cultured cardiomyocyte (CM) and in vivo approaches, we show that miR-125b-5p is an ischemic stress-responsive protector against CM apoptosis. CMs lacking miR-125b-5p exhibit increased susceptibility to stress-induced apoptosis, while CMs overexpressing miR-125b-5p have increased phospho-AKT pro-survival signaling. Moreover, we demonstrate that loss-of-function of miR-125b-5p in the mouse heart causes abnormalities in cardiac structure and function after acute myocardial infarction. Mechanistically, the improvement of cardiac function and structure elicited by miR-125b-5p is in part attributed to repression of the pro-apoptotic genes Bak1 and Klf13 in CMs. Conclusions In conclusion, these findings reveal a pivotal role for miR-125b-5p in regulating CM survival during acute myocardial infarction.

Although stem cell therapy is promising for repairing damaged cardiac tissue and improving heart function, there are safety concerns, especially regarding the risk of arrhythmias, which can be life threatening. To address this issue, we propose to develop a novel screening system to evaluate arrhythmic risk associated with stem cell therapy using a high-throughput multielectrode array system that can measure conduction velocity and action potential duration in cardiomyocytes co-cultured with different types of stem cells, such as mesenchymal stem cells, skeletal myoblasts, and resident cardiac stem cells. We will assess the arrhythmic potential of each of these types of stem cells under normoxic and hypoxic conditions, with/without application of oxidative stress or catecholamines. We hypothesize that these methods will prove to be an effective way to screen for arrhythmic risk of cardiac stem cell therapy. Ultimately, our approach can potentially be personalized to develop a robust screening protocol in order to identify which stem cell type carries the least amount of risk for arrhythmia. This system will have great clinical benefit to improve the risk/benefit ratio of human stem cell therapy for heart disease.

Ischemic heart disease has remained the number one killer around the world for over the past twenty years. While stem cell therapy has become a promising new frontier to repair the damaged heart, limited stem cell survivability post-transplantation has precluded widespread use of this therapy. Strategies to genetically modify stem cells to activate pro-survival and anti-apoptotic and anti-inflammatory pathways, such as Akt and heme oxygenase-1, have been shown to improve the lifespan of transplanted stem cells within the ischemic myocardium, but constitutive overexpression of these pathways at high levels has been shown to have side effects. Therefore, more specific and controlled gene activation would be necessary. Current techniques used for gene regulation include zinc finger and TALE proteins, but there are still disadvantages to each of these methods, such as ease and cost of use. Also, those methods use synthesized promoters to express synthesized cDNA, which lack regulatory elements, including introns and 3′ untranslated regions for microRNA mediated post-transcriptional regulation. A new novel technique, the CRISPR/dCas9 system, was recently developed as a simple and efficient method for endogenous gene regulation. With its use of single guide chimeric RNA’s (sgRNA’s), this system has been shown to provide a high level of specificity and efficiency. When targeting different loci, past studies have found that the CRISPR/dCas9 system can activate gene expression at varying levels. In addition, this system makes use of the genome’s endogenous regulatory elements, such as the aforementioned introns and 3′ UTR’s, which can help provide a safer method of gene activation. If targeted to a gene promoting cellular survival or decreasing cell death, it could potentially improve stem cell longevity in a more efficient and controllable manner. As a result, our hypothesis is to use the CRISPR/dCas9 system to activate expression of an anti-inflammatory and anti-apoptotic gene, such as heme oxygenase-1 (HO-1), to an optimal level to increase transplanted stem cell survival while also mitigating its cytotoxic effects due to lack of internal regulation, thus prolonging its effects within the ischemic myocardium leading to greater therapeutic benefit.

Oxidative stress contributes to endothelial cell (EC) dysfunction, which is prevalent in ageing and atherosclerosis. MicroRNAs (miRs) are small, non-coding RNAs that post-transcriptionally regulate gene expression and play a key role in fine-tuning EC functional responses, including apoptosis and angiogenesis. MiR-92a is highly expressed in young endothelial cells in comparison with senescent endothelial cells, which exhibit increased oxidative stress and apoptosis. However, the impact of miR-92a treatment on EC viability and angiogenesis under oxidative stress is unknown. Hydrogen peroxide (H2O2) was used to induce oxidative stress in human umbilical vein endothelial cells (HUVEC). Pre-miR-92a treatment decreased H2O2–induced apoptosis of HUVEC as determined by TUNEL assay. Pre-miR-92a treatment enhanced capillary tube formation by HUVEC under oxidative stress, which was blocked by LY294002, an inhibitor of Akt phosphorylation. Interestingly, we also observed that inhibition of miR-92a by anti-miR-92a antisense can also enhance angiogenesis in HUVEC with and without oxidative stress exposure. Our results show that perturbation of miR-92a levels outside of its narrow “homeostatic” range may trigger endothelial cell angiogenesis, suggesting that the role of miR-92a in regulating angiogenesis is controversial and may vary depending on the experimental model and method of regulating miR-92a.

The growth of new blood vessels after ischemic injury requires endothelial cells (ECs) to divide and proliferate, and the E2F transcription factors are key regulators of the genes responsible for cell-cycle progression; however, the specific roles of individual E2Fs in ECs are largely unknown. To determine the roles of E2F2 and E2F3 in EC proliferation and the angiogenic response to ischemic injury, hind-limb ischemia was surgically induced in E2F2(-/-) mice, endothelial-specific E2F3-knockout (EndoE2F3(∆/∆)) mice, and their littermates with wild-type E2F2 and E2F3 expression. Two weeks later, Laser-Doppler perfusion measurements, capillary density, and endothelial proliferation were significantly greater in E2F2(-/-) mice and significantly lower in EndoE2F3(∆/∆) mice than in their littermates, and EndoE2F3(∆/∆) mice also developed toe and limb necrosis. The loss of E2F2 expression was associated with increases in the proliferation and G1/S-phase gene expression of isolated ECs, while the loss of E2F3 expression led to declines in these parameters. Thus E2F2 impairs, and endothelial E2F3 promotes, the angiogenic response to peripheral ischemic injury through corresponding changes in EC cell-cycle progression.

Highlights: ► Cardiac progenitor-derived (CPC) Exosomes protect H9C2 from apoptosis in vitro. ► CPC-exosomes protect cardiomyoyctes from MI/R induced apoptosis in vivo. ► CPC-exosomes were taken up by H9C2 with high efficiency using PKH26 labeling. ► miR-451, one of GATA4-responsive miRNA cluster, is enriched in CPC-exosomes. -- Abstract: Background: Cardiac progenitors (CPC) mediate cardioprotection via paracrine effects. To date, most of studies focused on secreted paracrine proteins. Here we investigated the CPC-derived-exosomes on protecting myocardium from acute ischemia/reperfusion (MI/R) injury. Methods and results: CPC were isolated from mouse heart using two-step protocol. Exosomes were purified from conditional medium, and confirmed by electron micrograph and Western blot using CD63 as a marker. qRT-PCR shows that CPC-exosomes have high level expression of GATA4-responsive-miR-451. Exosomes were ex vivo labeled with PKH26, We observed exosomes can be uptaken by H9C2 cardiomyoblasts with high efficiency after 12 h incubation. CPC-exosomes protect H9C2 from oxidative stress by inhibiting caspase 3/7 activation invitro. In vivo delivery of CPC-exosomes in an acute mouse myocardial ischemia/reperfusion model inhibited cardiomyocyte apoptosis by about 53% in comparison with PBS control (p < 0.05). Conclusion: Our results suggest, for the first time, the CPC-exosomes can be used as a therapeutic vehicle formore » cardioprotection, and highlights a new perspective for using non-cell exosomes for cardiac disease.« less

Transdifferentiation of stem cells into insulin-producing cells for the treatment of diabetes have shown promising but inconsistent results. We examined the potential for attracting bone marrow stem cells (BMSCs) to the pancreas using a chemokine, stromal cell-derived factor 1 (SDF-1). SDF-1 treatment markedly increased the number of GFP labeled BMSCs in the pancreas, but surprisingly, the majority was observed in liver. The liver cells had typical pancreatic endocrine cell gene expression including insulin I, insulin II, PDX-1, somatostatin, and glucagon. Combined treatment with SDF-1 and BMSC transplant reduced hyperglycemia and prolonged the long-term survival of diabetic mice, and a sub group had complete normoglycemia (

Resident cardiac stem cells (CSCs) are characterized by their capacity to self-renew in culture, and are multipotent for forming normal cell types in hearts. CSCs were originally isolated directly from enzymatically digested hearts using stem cell markers. However, long exposure to enzymatic digestion can affect the integrity of stem cell markers on the cell surface, and also compromise stem cell function. Alternatively resident CSCs can migrate from tissue explant and form cardiospheres in culture. However, fibroblast contamination can easily occur during CSC culture. To avoid these problems, we developed a two-step procedure by growing the cells before selecting the Sca-1+ cells and culturing in cardiac fibroblast conditioned medium, they avoid fibroblast overgrowth.

Objectives The goal of this study was to modify mesenchymal stem cells (MSCs) cells with a hypoxia-regulated heme oxygenase-1 (HO-1) plasmid to enhance the survival of MSCs in acute myocardial infarction (MI) heart. Background Although stem cells are being tested clinically for cardiac repair, graft cells die in the ischemic heart because of the effects of hypoxia/reoxygenation, inflammatory cytokines, and proapoptotic factors. Heme oxygenase-1 is a key component in inhibiting most of these factors. Methods Mesenchymal stem cells from bone marrow were transfected with either HO-1 or LacZ plasmids. Cell apoptosis was assayed in vitro after hypoxia-reoxygen treatment. In vivo, 1 × 10 6 of male MSC HO-1 , MSC LacZ , MSCs, or medium was injected into mouse hearts 1 h after MI (n = 16/group). Cell survival was assessed in a gender-mismatched transplantation model. Apoptosis, left ventricular remodeling, and cardiac function were tested in a gender-matched model. Results In the ischemic myocardium, the MSC HO-1 group had greater expression of HO-1 and a 2-fold reduction in the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling-positive cells compared with the MSC LacZ group. At seven days after implantation, the survival MSC HO-1 was five-fold greater than the MSC LacZ group; MSC HO-1 also attenuated left ventricular remodeling and enhanced the functional recovery of infarcted hearts two weeks after MI. Conclusions A hypoxia-regulated HO-1 vector modification of MSCs enhances the tolerance of engrafted MSCs to hypoxia-reoxygen injury in vitro and improves their viability in ischemic hearts. This demonstration is the first showing that a physiologically inducible vector expressing of HO-1 genes improves the survival of stem cells in myocardial ischemia.

Background There are several reports that engrafted mesenchymal stem cells (MSCs) stimulate angiogenesis in the ischemic heart, but the mechanism remains controversial. We hypothesize that transplantation of MSCs enhances vascular regeneration through a paracrine action. Methods A transmural myocardial infarction was created by ligation of the left anterior descending coronary artery in rats. Those with an ejection fraction less than 0.70 1 week after myocardial infarction were included. Autologous MSCs (1 × 10 7 ; 0.2 mL) or culture medium (0.2 mL) was injected intramyocardially into the periinfarct zone (50 μL/injection at four sites; n=20/group). At 2 weeks after transplantation, Western blot analysis was used to assay the paracrine factors and proapoptotic proteins. Echocardiography to assess heart function was performed on additional groups at 8 weeks after implantation. Results The angiogenic factors basic fibroblast growth factor, vascular endothelial growth factor, and stem cell homing factor (stromal cell-derived factor -1α) increased in the MSC-treated hearts compared with medium-treated hearts. This was accompanied by a downregulation of proapoptotic protein Bax in ischemic myocardium. Similarly, capillary density increased about 40% in MSC-treated hearts compared with medium-treated hearts ( p = 0.001). Left ventricular contractility, indicated by fractional shortening, improved in MSC-treated hearts at 2 months after implantation (MSCs: 48.6% ± 19.9%; medium: 18.7% ± 6.4%; p = 0.004). Conclusions Autologous MSC transplantation attenuates left ventricular remodeling and improves cardiac performance. The major mechanism appears to be paracrine action of the engrafted cells, increasing angiogenesis and cytoprotection.

A concentration gradient of stromal-cell-derived factor-1alpha (SDF-1alpha) is the major mechanism for homing of haematopoietic stem cells (HSCs) in bone marrow. We tested the hypothesis that a gene therapy using SDF-1alpha can enhance HSCs recruiting to the heart upon myocardial infarction (MI). Adult mice with surgically induced myocardial ischemia were injected intramyocardially with either saline (n=12) or SDF-1alpha plasmid (n=12) in 50 microl volume in the ischemic border zone of the infarcted heart 2 weeks after myocardial infarction. Donor Lin-c-kit+ HSCs from isogenic BalB/c mice were harvested, sorted through magnetic cell sorting (MACS) and labeled with PKH26 Red. Three days after plasmid or saline injection, 1x10(5) labeled cells were injected intravenously (i.v.) into saline mice (n=4) and SDF-1alpha plasmid mice (n=4). The hearts and other tissue were removed for Western blot assay 2 weeks after plasmid or saline treatment. The labeled Lin-c-kit+ cells were identified with immunofluoresent staining and endogenous c-kit+ cells were identified by immunohistochemical staining. In mice killed at 1 month postinfarct, Western blot showed higher levels of SDF-1alpha expression in SDF-1alpha-treated mouse ischemic hearts compared to saline-treated hearts and other tissues. In the SDF-1alpha plasmid-treated hearts, SDF-1alpha is overexpressed in the periinfarct zone. The labeled stem cells engrafted to the SDF-1alpha positive site in the myocardium. There was also evidence for endogenous stem cell recruiting. The density of c-kit+ cells in border zone, an index of endogenous stem cell mobilization, was significantly higher in the SDF-1alpha-treated group than in the saline group (14.63+/-1.068 cells/hpf vs. 11.31+/-0.65 cells/hpf, P=0.013) at 2 weeks after SDF-1alpha or saline treatment. Following myocardial infarction, treatment with SDF-1alpha recruits stem cells to damaged heart where they may have a role in repairing and regeneration. The gene therapy with an SDF-1alpha vector offers a promising therapeutic strategy for mobilizing stem cells to the ischemic myocardium.

We aim to track mesenchymal stem cells (MSCs) after magnetically labeling and test the ability of these cells differentiate into cardiomyocytes in vivo. Therefore, 20 swines were divided into four groups, sham-operated group (n=3); acute myocardial infarction (AMI) transplanted with PBS (n=3); labeled MSCs (n=7) and unlabeled MSCs (n=7) group. 10(7) labeled or unlabeled cells were intracoronary delivered after MI (4.8+/-1.3 days), and serial cardiac MR (3.0T) imaging studies were performed at 0, 4 and 8 weeks after transplantation, then the results were confirmed by histological and western blot analysis. We demonstrated that labeled MSCs can be reliably detected and tracked in vivo using MR imaging. In particular, we provided the evidence of regeneration of labeled MSCs in vivo by histological examination and western blot analysis.

Adult bone marrow-derived stem cells (BMSCs) have emerged as a promising source for regeneration of tissues including those traditionally considered terminally differentiated, for instance, heart and brain. Chronic heart failure afflicts 4.8 million Americans, for those at the end stage of this disease heart transplantation remains the only and last resort. Acute myocardial infarction (AMI) is a leading cause of heart failure. Increasing evidence suggests that the body has a natural response for cardiac repair by up-regulating stromal cell-derived factor 1 (SDF-1), a stem cell active chemokine, which leads to recruitment of circulating BMSCs to ischemic hearts. However, up-regulation of endogenous SDF-1 is transient and inadequate to induce functionally meaningful heart regeneration. Therefore, the current study aimed to enhance stem cell-initiated cardiac repair by targeting circulating BMSCs to ischemic myocardium through targeted and regulated SDF-1 gene expression. To accomplish this goal, we have developed a regulatable system that consists of a MLC-2v promoter for cardiac selective expression as well as an oxygen-sensitive gene switch based on the oxygen-dependent degradation domain of hypoxia inducible factor 1α. It delivers genes of interest in a cardiac-selective and hypoxia-regulated manner in vitro and in vivo. AMI was surgically induced in adult BALB/c mice. DNA plasmid encoding hypoxia-regulated human SDF-1 was administered via intra-myocardial injection. PKH26 labeled BMSCs (6 × 106) were systemically injected immediately after induction of AMI. hSDF-1 expression was evaluated by Western blot analysis and the efficiency of hSDF-1 to attract implanted BMSCs was assessed by immunofluorescent staining and confocal microscopy. 1 week post AMI, hSDF-1 expression was markedly increased in the ischemic mouse heart, while less or no expression was detected in spleen, liver, lung, kidney and skeletal muscle nor in the heart of non-MI group. Confocal microscopy showed that hSDF-1 was highly expressed in the peri-infarct zone, with PKH26-labeled BMSCs enriched nearby. Furthermore, infarct size and cardiac fibrosis were significantly reduced in mice treated with hSDF-1 plus BMSCs compared to BMSCs alone. Taken together, our results indicate that cardiac-selective and hypoxia-inducible SDF-1 gene transfer activated mobilization of BMSCs to ischemic hearts and facilitated post-ischemic repair. This combined approach of regulated gene transfer and adult stem cells may provide a novel, safe and effective strategy for regenerative medicine, leading to efficient and timely repair of injured tissues.

Background: Although constitutive expression of human heme oxygenase-1(hHO-1) could provide a useful approach for cellular protection in the ischemic heart. However, uncontrollable over-expression of hHO-1 can lead to unwanted side effects. We developed a novel vigilant vector system, which is composed of myosin light chain-2v promoter (MLC-2v) and a gene switch that is based on an oxygen dependent degradation domain (ODD) from the hypoxia inducible factor-1-alpha(HIF-1alpha). We hypothesis that vigilant system could control hHO-1 expression and protect ischemic myocardium from ischemic damage.