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Duchenne muscular dystrophy (DMD) is a progressive myopathic disease caused by mutations in the gene encoding dystrophin protein that eventually leads to the exhaustion of myogenic progenitor cells (MPC). Autologous induced pluripotent stem cells (iPSCs) provide an endless source of MPC, which can potentially replenish the progenitor cell pool, repair muscle damage, and prevent DMD progression. Deletion of mutant exon 23 (ΔEx23) with clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology can correct dystrophin gene expression in iPSCs. However, successful exon23 deletion and clonal isolation are very inefficient (~3%), and manual selection of each iPSC clone and genotyping to identify ΔEx23 is labor-intensive. To overcome these obstacles, we added a homology-directed repair (HDR) donor vector, which carries floxed fluorescent protein and antibiotic selection genes, thus allowing us to identify ΔEx23 iPSC with donor selective gene integration. Our results indicate that the HDR-mediated targeted integration enables ΔEx23 iPSC identification; the HDR donor vector increased the recognition efficiency of clonal isolation (>90% as confirmed by Sanger sequencing). After removal of the inserted genes by Cre-mediated recombination followed by doxycycline (Dox)-induced MyoD induction, ΔEx23 iPSC differentiated into MPC with restored dystrophin expression in vitro. Importantly, transplanted ΔEx23 iPSC-MPC express dystrophin in the muscles of a mouse model of DMD (Mdx mice). In conclusion, the use of HDR donor vector increased the efficiency of ΔEx23 gene correction by CRISPR/Cas9, and facilitate the identification of successfully edited iPSC clones for cell therapy of DMD.

Cardiac mesenchymal stem cells (C-MSCs) are endogenous cardiac stromal cells that play a crucial role in maintaining normal cardiac function. Rab27b is a member of the small GTPase Rab family that controls membrane trafficking and the secretion of exosomes. However, its role in regulating energy metabolism in C-MSC is unclear. In this study, we analyzed mitochondrial oxidative phosphorylation by quantifying cellular oxygen consumption rate (OCR) and quantified the extracellular acidification rate (ECAR) in C-MSC with/without Rab27b knockdown. Knockdown of Rab27b increased glycolysis, but significantly reduced mitochondrial oxidative phosphorylation (OXPHOS) with loss of mitochondrial membrane potential in C-MSC. Furthermore, knockdown of Rab27b reduced H3k4me3 expression in C-MSC and selectively decreased the expression of the essential genes involved in β-oxidation, tricarboxylic acid cycle (TCA), and electron transport chain (ETC). Taken together, our findings highlight a novel role for Rab27b in maintaining fatty acid oxidation in C-MSCs.

Myocardial infarction (MI) is the primary cause of cardiovascular mortality, and therapeutic strategies to prevent or mitigate the consequences of MI are a high priority. Cardiac progenitor cells (CPCs) have been used to treat cardiac injury post-MI, and despite poor engraftment, they have been shown to inhibit apoptosis and to promote angiogenesis through poorly understood paracrine effects. We previously reported that the direct injection of exosomes derived from CPCs (CPCexo) into mouse hearts provides protection against apoptosis in a model of acute ischemia/reperfusion injury. Moreover, we and others have reported that reactive oxygen species (ROS) derived from NADPH oxidase (NOX) can enhance angiogenesis in endothelial cells (ECs). Here we examined whether bioengineered CPCexo transfected with a pro-angiogenic miR-322 (CPCexo-322) can improve therapeutic efficacy in a mouse model of MI as compared to CPCexo. Systemic administration of CPCexo-322 in mice after ischemic injury provided greater protection post-MI than control CPCexo, in part, through enhanced angiogenesis in the border zones of infarcted hearts. Mechanistically, the treatment of cultured human ECs with CPCexo-322 resulted in a greater angiogenic response, as determined by increased EC migration and capillary tube formation via increased Nox2-derived ROS. Our study reveals that the engineering of CPCexo via microRNA (miR) programing can enhance angiogenesis, and this may be an effective therapeutic strategy for the treatment of ischemic cardiovascular diseases.

Progressive cardiomyocyte loss in Duchenne muscular dystrophy (DMD) leads to cardiac fibrosis, cardiomyopathy, and eventually heart failure. In the present study, we observed that myogenic progenitor cells (MPC) carry mRNA for the dystrophin gene. We tested whether cardiac function can be improved in DMD by allograft transplantation of MPC-derived exosomes (MPC-Exo) into the heart to restore dystrophin protein expression. Exo from C2C12 cells (an MPC cell line) or vehicle were delivered locally into the hearts of MDX mice. After 2 days of treatment, we observed that MPC-Exo restored dystrophin expression in the hearts of MDX mice, which correlated with improved myocardial function in dystrophin-deficient MDX mouse hearts. In conclusion, this study demonstrated that allogeneic WT-MPC-Exo transplantation transiently restored dystrophin gene expression and improved cardiac function in MDX mice, suggesting that allogenic exosomal delivery may serve as an alternative treatment for cardiomyopathy of DMD.

The aryl hydrocarbon receptor (AhR) is a DNA binding protein that acts as a nuclear receptor mediating xenobiotic metabolism and environmental responses. Owing to the evolutionary conservation of this gene and its widespread expression in the immune and circulatory systems, AhR has for many years been almost exclusively studied by the pharmacological/toxicological field for its role in contaminant toxicity. More recently, the functions of AhR in environmental adaption have been examined in the context of the occurrence, development, and therapy of cardiovascular diseases. Increasing evidence suggests that AhR is involved in maintaining homeostasis or in triggering pathogenesis by modulating the biological responses of critical cell types in the cardiovascular system. Here, we describe the structure, distribution, and ligands of AhR and the AhR signaling pathway and review the impact of AhR on cardiovascular physiology. We also discuss the potential contribution of AhR as a new potential factor in the targeted treatment of cardiovascular diseases.

Despite substantial advances in the development of medical and interventional strategies in ischemic and non-ischemic heart diseases, cardiovascular diseases (CVDs) remain the leading cause of mortality and morbidity worldwide. Stem cell therapy for heart disease has gained traction over the past two decades and is an emerging option for the treatment of myocardial dysfunction. In this review, we summarize the current literature on different types of stem cells and their potential usage in ischemic and non-ischemic heart diseases. We emphasize the clinical utility of stem cells to improve myocardial structural and function, promote microvascular angiogenesis, and diminish scar size and major adverse cardiovascular events. We also discuss the therapeutic potential of microvesicles, such as exosomes, in the treatment of CVDs, which may open novel avenues for further clinical studies.

Rationale MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate target genes. First transcribed as primary miR transcripts (pri-miRs), they are enzymatically processed by Drosha into premature miRs (pre-miRs) and further cleaved by Dicer into mature miRs. Initially discovered to desensitize β-adrenergic receptor (βAR) signaling, β-arrestins are now well-appreciated to modulate multiple pathways independent of G protein signaling, a concept known as biased signaling. Using the β-arrestin-biased βAR ligand carvedilol, we previously showed that β-arrestin1 (not β-arrestin2)-biased β1AR (not β2AR) cardioprotective signaling stimulates Drosha-mediated processing of six miRs by forming a multi-protein nuclear complex, which includes β-arrestin1, the Drosha microprocessor complex and a single-stranded RNA binding protein hnRNPA1. Objective Here, we investigate whether β-arrestin-mediated βAR signaling induced by carvedilol could regulate Dicer-mediated miR maturation in the cytoplasm and whether this novel mechanism promotes cardioprotective signaling. Methods and results In mouse hearts, carvedilol indeed upregulates three mature miRs, but not their pre-miRs and pri-miRs, in a β-arrestin 1- or 2-dependent manner. Interestingly, carvedilol-mediated activation of miR-466g or miR-532-5p, and miR-674 is dependent on β2ARs and β1ARs, respectively. Mechanistically, β-arrestin 1 or 2 regulates maturation of three newly identified βAR/β-arrestin-responsive miRs (β-miRs) by associating with the Dicer maturation RNase III enzyme on three pre-miRs of β-miRs. Myocardial cell approaches uncover that despite their distinct roles in different cell types, β-miRs act as gatekeepers of cardiac cell functions by repressing deleterious targets. Conclusions Our findings indicate a novel role for βAR-mediated β-arrestin signaling activated by carvedilol in Dicer-mediated miR maturation, which may be linked to its protective mechanisms.

Rationale: Vascular calcification (VC) is a marker of the severity of atherosclerotic disease. Hormones play important roles in regulating calcification; estrogen and parathyroid hormones exert opposing effects, the former alleviating VC and the latter exacerbating it. To date no treatment strategies have been developed to regulate clinical VC. Objective: The objective of this study was to investigate the effect of growth hormone–releasing hormone (GHRH) and its agonist (GHRH-A) on the blocking of VC in a mouse model. Methods and Results: Young adult osteoprotegerin-deficient mice were given daily subcutaneous injections of GHRH-A (MR409) for 4 weeks. Significant reductions in calcification of the aortas of MR409-treated mice were paralleled by markedly lower alkaline phosphatase activity and a dramatic reduction in the expression of transcription factors, including the osteogenic marker gene Runx2 and its downstream factors, osteonectin and osteocalcin. The mechanism of action of GHRH-A was dissected in smooth muscle cells isolated from human and mouse aortas. Calcification of smooth muscle cells induced by osteogenic medium was inhibited in the presence of GHRH or MR409, as evidenced by reduced alkaline phosphatase activity and Runx2 expression. Inhibition of calcification by MR409 was partially reversed by MIA602, a GHRH antagonist, or a GHRH receptor–selective small interfering RNA. Treatment with MR409 induced elevated cytosolic cAMP and its target, protein kinase A which in turn blocked nicotinamide adenine dinucleotide phosphate oxidase activity and reduced production of reactive oxygen species, thus blocking the phosphorylation of nuclear factor κB (p65), a key intermediate in the ligand of receptor activator for nuclear factor-κ B-Runx2/alkaline phosphatase osteogenesis program. A protein kinase A–selective small interfering RNA or the chemical inhibitor H89 abolished these beneficial effects of MR409. Conclusions: GHRH-A controls osteogenesis in smooth muscle cells by targeting cross talk between protein kinase A and nuclear factor κB (p65) and through the suppression of reactive oxygen species production that induces the Runx2 gene and alkaline phosphatase. Inflammation-mediated osteogenesis is thereby blocked. GHRH-A may represent a new pharmacological strategy to regulate VC.

Cardiovascular disease is the leading cause of death in the United States, accounting for nearly one in every seven deaths. Over the last decade, various targeted therapeutics have been introduced, but there has been no corresponding improvement in patient survival. Since the mortality rate of cardiovascular disease has not been significantly decreased, efforts have been made to understand the link between heart disease and novel therapeutic targets such as non-coding RNAs. Among multiple non-coding RNAs, long non-coding RNA (lncRNA) has emerged as a novel therapeutic in cardiovascular medicine. LncRNAs are endogenous RNAs that contain over 200 nucleotides and regulate gene expression. Recent studies suggest critical roles of lncRNAs in modulating the initiation and progression of cardiovascular diseases. For example, aberrant lncRNA expression has been associated with the pathogenesis of ischemic heart failure. In this article, we present a synopsis of recent discoveries that link the roles and molecular interactions of lncRNAs to cardiovascular diseases. Moreover, we describe the prevalence of circulating lncRNAs and assess their potential utilities as biomarkers for diagnosis and prognosis of heart disease.

Myogenic fate determination is important in skeletal muscle development, growth and repair. A variety of factors regulate myogenic cell determination via transcriptional and non-transcriptional mechanisms. Amongst these factors, long noncoding RNAs (lncRNAs) have gained considerable attention for their important roles in regulating myogenic differentiation and function. Many classes of lncRNAs have been discovered; various lncRNAs have been implicated in the regulation of myogenic cell fate determination and are the subject of this brief review.

Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disease caused by a lack of dystrophin, which eventually leads to apoptosis of muscle cells and impaired muscle contractility. Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR associated protein 9 (CRISPR/Cas9) gene editing of induced pluripotent stem cells (IPSC) offers the potential to correct the DMD gene defect and create healthy IPSC for autologous cell transplantation without causing immune activation. However, IPSC carry a risk of tumor formation, which can potentially be mitigated by differentiation of IPSC into myogenic progenitor cells (MPC). We hypothesize that precise genetic editing in IPSC using CRISPR-Cas9 technology, coupled with MPC differentiation and autologous transplantation, can lead to safe and effective muscle repair. With future research, our hypothesis may provide an optimal autologous stem cell-based approach to treat the dystrophic pathology and improve the quality of life for patients with DMD.

Background Diabetic cardiomyopathy is a major risk factor in diabetic patients but its pathogenesis remains poorly understood. The ubiquitin-proteasome system (UPS) facilitates protein quality control by degrading unnecessary and damaged proteins in eukaryotic cells, and dysfunction of UPS is implicated in various cardiac diseases. However, the overall functional status of the UPS and its pathophysiological role in diabetic cardiomyopathy have not been determined. Methods and results Type I diabetes was induced in wild-type and transgenic mice expressing a UPS functional reporter (GFPdgn) by injections of streptozotocin (STZ). STZ-induced diabetes progressively impaired cardiac UPS function as evidenced by the accumulation of GFPdgn proteins beginning two weeks after diabetes induction, and by a buildup of total and lysine (K) 48-linked polyubiquitinated proteins in the heart. To examine the functional role of the UPS in diabetic cardiomyopathy, cardiac overexpression of PA28α (PA28αOE) was used to enhance proteasome function in diabetic mouse hearts. PA28αOE diabetic mice displayed exhibited restoration of cardiac UPS function, as demonstrated by the diminished accumulation of GFPdgn and polyubiquitinated proteins. Moreover, PA28αOE diabetic mice exhibited reduced myocardial collagen deposition, decreased cardiomyocyte apoptosis, and improved cardiac systolic and diastolic function. Conclusion Impairment of cardiac UPS function is an early event in STZ-induced diabetes. Overexpression of PA28α attenuates diabetes-induced proteotoxic stress and cardiomyopathy, suggesting a potential therapeutic role for enhancement of cardiac proteasome function in this disorder.

Objective— The efficiency of cell therapy is limited by poor cell survival and engraftment. Here, we studied the effect of the growth hormone–releasing hormone agonist, JI-34, on mesenchymal stem cell (MSC) survival and angiogenic therapy in a mouse model of critical limb ischemia. Approach and Results— Mouse bone marrow–derived MSCs were incubated with or without 10 –8 mol/L JI-34 for 24 hours. MSCs were then exposed to hypoxia and serum deprivation to detect the effect of preconditioning on cell apoptosis, migration, and tube formation. For in vivo tests, critical limb ischemia was induced by femoral artery ligation. After surgery, mice received 50 μL phosphate-buffered saline or with 1×10 6 MSCs or with 1×10 6 JI-34–reconditioned MSCs. Treatment of MSCs with JI-34 improved MSC viability and mobility and markedly enhanced their capability to promote endothelial tube formation in vitro. These effects were paralleled by an increased phosphorylation and nuclear translocation of signal transducer and activator of transcription 3. In vivo, JI-34 pretreatment enhanced the engraftment of MSCs into ischemic hindlimb muscles and augmented reperfusion and limb salvage compared with untreated MSCs. Significantly more vasculature and proliferating CD31 + and CD34 + cells were detected in ischemic muscles that received MSCs treated with JI-34. Conclusions— Our studies demonstrate a novel role for JI-34 to markedly improve therapeutic angiogenesis in hindlimb ischemia by increasing the viability and mobility of MSCs. These findings support additional studies to explore the full potential of growth hormone–releasing hormone agonists to augment cell therapy in the management of ischemia.

Injured or ischemic cardiac tissue has limited intrinsic capacity for regeneration. While stem cell transplantation is a promising approach to stimulating cardiac repair, its success in humans has thus far been limited. Harnessing the therapeutic benefits of stem cells requires a better understanding of their mechanisms of action and methods to optimize their function. Cardiac stem cells (CSC) represent a particularly effective cellular source for cardiac repair, and pre-conditioning CSC with electrical stimulation (EleS) was demonstrated to further enhance their function, although the mechanisms are unknown. Recent studies suggest that transplanted stem cells primarily exert their effects through communicating with endogenous tissues via the release of exosomes containing cardioprotective molecules such as miRNAs, which upon uptake by recipient cells may stimulate survival, proliferation, and angiogenesis. Exosomes are also effective therapeutic agents in isolation and may provide a feasible alternative to stem cell transplantation. We hypothesize that EleS enhances CSC-mediated cardiac repair through its beneficial effects on production of cardioprotective exosomes. Moreover, we hypothesize that the beneficial effects of biventricular pacing in patients with heart failure may in part result from EleS-induced preconditioning of endogenous CSC to promote cardiac repair. With future research, our hypothesis may provide applications to optimize stem cell therapy and augment current pacing protocols, which may significantly advance the treatment of patients with heart disease.

Although thousands of potentially disease-causing mutations have been identified in a handful of genes, the genetic heterogeneity has led to diagnostic confusions, stemming directly from the limitations in our arsenal of genetic tools.We discuss the genetic basis of cardiac ion channelopathies, the gaps in our knowledge and how Next-generation sequencing technology (NGS) and can be used to bridge them, and how induced pluripotent stem cell (iPSC) derived-cardiomyocytes can be used for drug discovery.Univariate, arrhythmogenic arrhythmias can explain some congenital arrhythmias, however, it is far from a comprehensive understanding of the complexity of many arrhythmias. Mutational screening is a critical step in personalized medicine and is critical to the management of patients with arrhythmias. The success of personalized medicine requires a more efficient way to identify a high number of genetic variants potentially implicated in cardiac arrhythmogenic diseases than traditional sequencing methods (eg, Sanger sequencing). Next-generation sequencing technology provides us with unprecedented opportunities to achieve high-throughput, rapid, and cost-effective detection of congenital arrhythmias in patients. Moreover, in personalized medicine era, IPSC derived-cardiomyocytes can be used as 'cardiac arrhythmia in a dish' model for drug discovery, and help us improve management of arrhythmias in patients by developing patient-specific drug therapies with target specificity.

Extracellular microvesicles (MVs) and exosomes (EXs) can mark the changes of their origin cells. They can transfer their carried messages (proteins, mRNAs, and microRNAs) to target cells to modulate the functions of the recipient cells. Therefore, MVs and EXs are recognized as potential biomarkers and important therapeutic targets or tools for a wide spectrum of diseases including vascular and neurological diseases and cancers [1–4]. While research in stem cells has achieved significant advances during the past decades, there is less information regarding the roles of MVs and EXs released from stem cells. The current special issue includes the selected reviews and original articles focusing on novel studies of stem cell-released MVs and EXs as biomarkers and treatments of cardiovascular and neurological diseases and cancers. There is an urgent need to identify sensitive and specific methods for the isolation and identification of MVs and EXs, which is very important and challenging. J. Wang et al. [5] have developed novel methods for analysis of exosomes released from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads and fluorescence quantum dots (Q-dots®) techniques. The authors showed that anti-CD105/anti-CD144 and anti-CD34/anti-KDR had the highest sensitivity and specificity for isolating and detecting EC-EXs and EPC-EXs, respectively. A. Fricke et al. [6] evaluated whether MVs shed by synovial sarcoma cells carry the tumor-specific fusion gene SYT-SSX transcripts and revealed that these MVs might serve as a diagnostic biomarker for synovial sarcoma. They also noted that more sensitive assays are needed to detect cancer specific microvesicles in the peripheral blood of cancer patients. Leukemia stem cells (LSCs) are able to self-renew, proliferate, and differentiate extensively, which are crucial for the initiation and maintenance of acute myeloid leukemia (AML). AML is an aggressive disease characterized by rapid proliferation of immature myeloid cells. Y. Wang et al. [7] reported that LSCs-released MVs support AML cell malignance and targeting of miR34a could offer a new approach for the management of AML. Exosome-based therapy could overcome many limitations of cell-based strategy in safety, potency, efficacy, and scalability of stem cell products. The article by J. Burke et al. [8] reviewed the existing literature to highlight the current advances of stem cell-derived exosomes with particular attention to regenerative medicine in orthopaedics. A. Luarte et al. [9] provided a review on the potential therapies of stem cell-derived exosomes in CNS diseases by focusing on the neurogenic niche. This article discusses how the modulation of the adult neurogenic niches may be a therapeutic target. F. Alcayaga-Miranda et al. [10] provided a review article which discusses the current knowledge related to the angiogenic potential of exosomes of mesenchymal stem cells and methods to enhance their biological activities. H. Zhang et al. [11] performed meta-analysis based on the reports published between 2000 and 2015 to assess the clinical effectiveness of using exosomes in ischemia/reperfusion injury. They revealed that exosomes secreted from mesenchymal stem cells inhibit myocardial ischemia/reperfusion injury. Hepatic stellate cells (HSCs), also known as liver-specific mesenchymal stem cells (MSCs), contribute to liver regeneration. R. Huang et al. [12] showed that HSC-derived MVs dose-dependently protect hepatocytes from toxicant-induced injury. Although the molecular mechanisms have not been elucidated, the beneficial effects of MVs are likely through maintaining the proliferative activity and the antiapoptotic and antiautophagic abilities of hepatocytes. B. Dai et al. [13] investigated the effects of biomaterial multilayer membranes of hyaluronic acid (HA) or chondroitin sulfate (CS) and Collagen I (Col I) on the functions and MVs release of EPCs. They found that multilayer composite membranes composed of HA/CS and Collagen I composed multilayer composite membranes can promote EPCs functions and release of miR-126 rich EPCs-MVs, which provides a novel strategy for tissue repair treatment. Therefore, combined use of biomaterials and stem cells may present a novel approach for promoting wound healing and tissue regeneration. However, further investigations in animal models and patients are required. Stem cell-derived exosomes and MVs could be used as biomarkers of various diseases, as well as novel approaches to combat diseases. However, further experimentation must be done and more discoveries must be made in order for them to become available for clinical use. We hope that the original research articles and review articles presented in this special issue represent the current advances in this field, which will stimulate further in-depth exploration. Yanfang Chen Yaoliang Tang Weiwen Long Chunxiang Zhang

Induced pluripotent stem cells (iPS) exhibit enhanced survival and proliferation in ischemic tissues. However, the therapeutic application of iPS cells is limited by their tumorigenic potential. We hypothesized that iPS cells can transmit cytoprotective signals to cardiomyocytes via exosomes/microvesicles.Exosomes/microvesicles secreted from mouse cardiac fibroblast (CF)-derived iPS cells (iPS-exo) were purified from conditioned medium and confirmed by electron micrograph, size distribution and zeta potential by particle tracking analyzer and protein expression of the exosome markers CD63 and Tsg101.We observed that exosomes are at low zeta potential, and easily aggregate. Temperature affects zeta potential (-14 to -15 mV at 23 °C vs -24 mV at 37 °C). The uptake of iPS-exo protects H9C2 cells against H2O2-induced oxidative stress by inhibiting caspase 3/7 activation (P0.05, n = 6). Importantly, iPS-exo treatment can protect against myocardial ischemia/reperfusion (MIR) injury via intramyocardial injection into mouse ischemic myocardium before reperfusion. Furthermore, iPS-exo deliver cardioprotective miRNAs, including nanog-regulated miR-21 and HIF-1α-regulated miR-210, to H9C2 cardiomyocytes in vitro.Exosomes/microvesicles secreted by iPS cells are very effective at transmitting cytoprotective signals to cardiomyocytes in the setting of MIR. iPS-exo thus represents novel biological nanoparticles that offer the benefits of iPS cell therapy without the risk of tumorigenicity and can potentially serve as an "off-the-shelf" therapy to rescue ischemic cardiomyocytes in conditions such as MIR.

Aims Cardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs). For example, miR-150 is down-regulated in patients with acute myocardial infarction, atrial fibrillation, dilated and ischaemic cardiomyopathy as well as in various mouse heart failure (HF) models. Circulating miR-150 has been recently proposed as a better biomarker of HF than traditional clinical markers such as brain natriuretic peptide. We recently showed using the β-arrestin-biased β-blocker, carvedilol that β-arrestin1-biased β1-adrenergic receptor cardioprotective signalling stimulates the processing of miR-150 in the heart. However, the potential role of miR-150 in ischaemic injury and HF is unknown. Methods and results Here, we show that genetic deletion of miR-150 in mice causes abnormalities in cardiac structural and functional remodelling after MI. The cardioprotective roles of miR-150 during ischaemic injury were in part attributed to direct repression of the pro-apoptotic genes egr2 (zinc-binding transcription factor induced by ischaemia) and p2x7r (pro-inflammatory ATP receptor) in cardiomyocytes. Conclusion These findings reveal a pivotal role for miR-150 as a regulator of cardiomyocyte survival during cardiac injury.

Aims The E2F transcription factors are best characterized for their roles in cell-cycle regulation, cell growth, and cell death. Here we investigated the potential role of E2F1 in cardiac neovascularization. Methods and results We induced myocardial infarction (MI) by ligating the left anterior descending artery in wild-type (WT) and E2F1−/− mice. E2F1−/− mice demonstrated a significantly better cardiac function and smaller infarct sizes than WT mice. At infarct border zone, capillary density and endothelial cell (EC) proliferation were greater, apoptotic ECs were fewer, levels of VEGF and placental growth factor (PlGF) were higher, and p53 level was lower in E2F1−/− than in WT mice. Blockade of VEGF receptor 2 (VEGFR2) signalling with the selective inhibitor SU5416 or with the VEGFR2-blocking antibody DC101 abolished the differences between E2F1−/− mice and WT mice in cardiac function, infarct size, capillary density, EC proliferation, and EC apoptosis. In vitro , hypoxia-induced VEGF and PlGF up-regulation was significantly greater in E2F1−/− than in WT cardiac fibroblasts, and E2F1 overexpression suppressed PlGF up-regulation in both WT and p53−/− cells; however, VEGF up-regulation was suppressed only in WT cells. E2F1 interacted with and stabilized p53 under hypoxic conditions, and both E2F1 : p53 binding and the E2F1-induced suppression of VEGF promoter activity were absent in cells that expressed an N-terminally truncated E2F1 mutant. Conclusion E2F1 limits cardiac neovascularization and functional recovery after MI by suppressing VEGF and PlGF up-regulation through p53-dependent and -independent mechanisms, respectively.

Stem cell therapy has a potential for regenerating damaged myocardium. However, a key obstacle to cell therapy's success is the loss of engrafted cells due to apoptosis or necrosis in the ischemic myocardium. While many strategies have been developed to improve engrafted cell survival, tools to evaluate cell efficacy within the body are limited. Traditional genetic labeling tools, such as GFP-like fluorescent proteins (eGFP, DsRed, mCherry), have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent these limitations, a near-infrared fluorescent mutant of the DrBphP bacteriophytochrome from Deinococcus radiodurans, IFP1.4, was developed for in vivo imaging, but it has yet to be used for in vivo stem/progenitor cell tracking. In this study, we incorporated IFP1.4 into mouse cardiac progenitor cells (CPCs) by a lentiviral vector. Live IFP1.4-labeled CPCs were imaged by their near-infrared fluorescence (NIRF) using an Odyssey scanner following overnight incubation with biliverdin. A significant linear correlation was observed between the amount of cells and NIRF signal intensity in in vitro studies. Lentiviral mediated IFP1.4 gene labeling is stable, and does not impact the apoptosis and cardiac differentiation of CPC. To assess efficacy of our model for engrafted cells in vivo, IFP1.4-labeled CPCs were intramyocardially injected into infarcted hearts. NIRF signals were collected at 1-day, 7-days, and 14-days post-injection using the Kodak in vivo multispectral imaging system. Strong NIRF signals from engrafted cells were imaged 1 day after injection. At 1 week after injection, 70% of the NIRF signal was lost when compared to the intensity of the day 1 signal. The data collected 2 weeks following transplantation showed an 88% decrease when compared to day 1. Our studies have shown that IFP1.4 gene labeling can be used to track the viability of transplanted cells in vivo.

Inflammatory cross talk between perivascular adipose tissue and the blood vessel wall has been proposed to contribute to the pathogenesis of atherosclerosis. We previously reported that human perivascular (PV) adipocytes exhibit a proinflammatory phenotype and less adipogenic differentiation than do subcutaneous (SQ) adipocytes. To gain a global view of the genomic basis of biologic differences between PV and SQ adipocytes, we performed genome-wide expression analyses to identify differentially expressed genes between adipocytes derived from human SQ vs. PV adipose tissues. Although >90% of well-expressed genes were similarly regulated, we identified a signature of 307 differentially expressed genes that were highly enriched for functions associated with the regulation of angiogenesis, vascular morphology, inflammation, and blood clotting. Of the 156 PV upregulated genes, 59 associate with angiogenesis, vascular biology, or inflammation, noteworthy of which include TNFRSF11B (osteoprotegerin), PLAT, TGFB1, THBS2, HIF1A, GATA6, and SERPINE1. Of 166 PV downregulated genes, 21 associated with vascular biology and inflammation, including ANGPT1, ANGPTL1, and VEGFC. Consistent with the emergent hypothesis that PV adipocytes differentially regulate angiogenesis and inflammation, cell culture-derived adipocyte-conditioned media from PV adipocytes strongly enhanced endothelial cell tubulogenesis and monocyte migration compared with media from SQ adipocytes. These findings demonstrate that PV adipocytes have the potential to significantly modulate vascular inflammatory crosstalk in the setting of atherosclerosis by their ability to signal to both endothelial and inflammatory cells.

Circulating endothelial progenitor cells (circEPCs) of bone marrow (BM) origin contribute to postnatal neovascularization and represent a potential therapeutic target for ischemic disease. Statins are beneficial for ischemia disease and have been implicated to increase neovascularization via mechanisms independent of lipid lowering. However, the effect of Statins on EPC function is not completely understood. Here we sought to investigate the effects of Rosuvastatin (Ros) on EPC mobilization and EPC-mediated neovascularization during ischemic injury. In a mouse model of surgically-induced hindlimb ischemia (HLI), treatment of mice with low dose (0.1 mg/kg) but not high dose (5 mg/kg) significantly increased capillary density and accelerated blood flow recovery, as compared to saline-treated group. When HLI was induced in mice that had received Tie2/LacZ BM transplantation, Ros treatment led a significantly larger amount of endothelial cells (ECs) of BM origin incorporated at ischemic sites than saline. After treatment of mice with a single low dose of Ros, circEPCs significantly increased from 2 h, peaked at 4 h, declined until 8 h. In a growth-factor reduced Matrigel plug-in assay, Ros treatment for 5 d induced endothelial lineage differentiation in vivo. Interestingly, the enhanced circEPCs and post-HLI neovascularization stimulated by Ros were blunted in mice deficient in endothelial nitric oxide synthase (eNOS), and Ros increased p-Akt/p-eNOS levels in EPCs in vitro, indicating these effects of Ros are dependent on eNOS activity. We conclude that Ros increases circEPCs and promotes their de novo differentiation through eNOS pathway.

Cardiosphere derived cells (CDC) are present in the human heart and include heterogeneous cell populations of cardiac progenitor cells, multipotent progenitors that play critical roles in the physiological and pathological turnover of heart tissue. Little is known about the molecular pathways that control the differentiation of CDC. In this study, we examined the role of Notch 1/J kappa-recombining binding protein (RBPJ) signaling, a critical cell-fate decision pathway, in CDC differentiation. We isolated CDC from mouse cardiospheres and analyzed the differentiation of transduced cells expressing the Notch1 intracellular domain (N1-ICD), the active form of Notch1, using a terminal differentiation marker polymerase chain reaction (PCR) array. We found that Notch1 primarily supported the differentiation of CDC into smooth muscle cells (SMC), as demonstrated by the upreguation of key SMC proteins, including smooth muscle myosin heavy chain (Myh11) and SM22α (Tagln), in N1-ICD expressing CDC. Conversely, genetic ablation of RBPJ in CDC diminished the expression of SMC differentiation markers, confirming that SMC differentiation CDC is dependent on RBPJ. Finally, in vivo experiments demonstrate enhanced numbers of smooth muscle actin-expressing implanted cells after an injection of N1-ICD-expressing CDC into ischemic myocardium (44±8/high power field (hpf) vs. 11±4/high power field (hpf), n=7 sections, P

Background Sorafenib, a multi-kinase inhibitor, is used as a standard therapy for advanced hepatocellular carcinoma (HCC). However, complete remission has not been achieved and the molecular basis of HCC resistance to sorafenib remains largely unknown. Previous studies have shown that fibroblast growth factor 19 (FGF19) expression correlates with tumor progression and poor prognosis of HCC. Here, we demonstrate the novel role of FGF19 in HCC resistance to sorafenib therapy. Methods FGF19 Knockdown cells were achieved by lentiviral-mediated interference, and FGFR4 knockout cells were achieved by CRISPR-Cas9. Protein levels of FGF19, FGFR4 and c-PARP in various HCC cell lines were measured by Western blotting analysis. Cell viability was determined by MTS assay, apoptosis was determined by DAPI nuclear staining and Western blot of c-PRAP, and ROS generation was determined by DCFH-DA staining and electrochemical biosensor. Results We showed that FGF19, when overexpressed, inhibited the effect of sorafenib on ROS generation and apoptosis in HCC. In contrast, loss of FGF19 or its receptor FGFR4 led to a remarkable increase in sorafenib-induced ROS generation and apoptosis. In addition, knockdown of FGF19 in sorafenib-resistant HCC cells significantly enhanced the sensitivity to sorafenib. Importantly, targeting FGF19/FGFR4 axis by ponatinib, a third-generation inhibitor of chronic myeloid leukemia, overcomes HCC resistance of sorafenib by enhancing ROS-associated apoptosis in sorafenib-treated HCC. Conclusion Our results provide the first evidence that inhibition of FGF19/FGFR4 signaling significantly overcomes sorafenib resistance in HCC. Co-treatment of ponatinib and sorafinib may represent an effective therapeutic approach for eradicating HCC. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0478-9) contains supplementary material, which is available to authorized users.