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Elderly patients are more susceptible to ischemic injury. N6-methyladenosine (m6A) modification is the most abundant reversible epitranscriptomic modification in mammalian RNA and plays a vital role in many biological processes. However, it is unclear whether age difference impacts m6A RNA methylation in hearts and their response to acute myocardial ischemia/reperfusion (I/R) injury. In this study, we measured the global level of m6A RNA methylation as well as the expression of m6A RNA “writers” (methylation enzymes) and “erasers” (demethylation enzymes) in the hearts of young and elderly female mice undergone sham surgery or acute MI/R injury. We found that m6A RNA level and associate modifier gene expression was similar in intact young and old female hearts. However, young hearts show a significant reduction in m6A RNA while elderly hearts showed only a slight reduction in m6A RNA in response to acute I/R injury. To explore the mechanism of differential level of m6A RNA modification, we use qRT-PCR and Western blotting to compare the mRNA and protein expression of major m6A-related “writers” (Mettl3, Mettl14, and WTAP) and ‘erasers” (ALKBH5 and FTO). Mettl3 mRNA and protein expression were significantly reduced in both young and elderly hearts. However, the levels of FTO’s mRNA and protein were only significantly reduced in ischemic elderly hearts, and age-related downregulation of FTO may offset the effect of reduced Mettl3 on reduced m6A RNA level in the hearts of aging mice hearts with acute I/R injury, indicating aging-related differences in epitranscriptomic m6A regulation in hearts in response to acute I/R injury. To further investigate specific I/R related targets of Mettl3, we overexpressed Mettl3 in cardiomyocyte line (HL1) using lentiviral vector, and the m6A enrichment of Bcl2, Bax and PTEN were quantified with m6A RIP-qPCR, we found that m6A modification of PTEN mRNA decreased after in vitro hypoxia/reperfusion injury (iH/R) while Mettl3 augments m6A levels of both Bax and PTEN after iH/R, indicating that Bax and PTEN are target genes of Mettl3 under iH/R stress.

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.

On March 1 and 2, 2018, the National Institutes of Health 2018 Progenitor Cell Translational Consortium (PCTC) and Cardiovascular Bioengineering Symposium (CVBE) was held at the University of Alabama at Birmingham. Convergence of life sciences and engineering to advance the understanding and treatment of heart failure was the theme of the meeting. Over 150 attendees were present for more than 40 scientists presenting their latest works on engineering human functional myocardium for disease modeling, drug development, and heart failure research. The scientists, engineers and physicians in the field of cardiovascular sciences, met and discussed on the most recent advances in their works and propose future strategies in overcoming the major roadblocks of cardiovascular bioengineering and therapy. Particular emphasis was given for manipulation and using of stem/progenitor cells, biomaterials, and methods to provide molecular, chemical and mechanical cues to cells in order to influence their identity and fate in vitro and in vivo. Collectively, these works are profoundly impacting and progressing toward deciphering the mechanisms and developing novel treatments for left ventricular dysfunction of failing hearts. Here we present some important perspectives that emerged from this meeting.

MicroRNA-150 (miR-150) is downregulated in patients with multiple cardiovascular diseases and in diverse mouse models of heart failure (HF). miR-150 is significantly associated with HF severity and outcome in humans. We previously reported that miR-150 is activated by β-blocker carvedilol (Carv) and plays a protective role in the heart using a systemic miR-150 KO mouse model. However, mechanisms that regulate cell-specific miR-150 expression and function in HF are unknown. Here, we demonstrate that potentially novel conditional cardiomyocyte-specific (CM-specific) miR-150 KO (miR-150 cKO) in mice worsens maladaptive cardiac remodeling after myocardial infarction (MI). Genome-wide transcriptomic analysis in miR-150 cKO mouse hearts identifies small proline-rich protein 1a (Sprr1a) as a potentially novel target of miR-150. Our studies further reveal that Sprr1a expression is upregulated in CMs isolated from ischemic myocardium and subjected to simulated ischemia/reperfusion, while its expression is downregulated in hearts and CMs by Carv. We also show that left ventricular SPRR1A is upregulated in patients with HF and that Sprr1a knockdown in mice prevents maladaptive post-MI remodeling. Lastly, protective roles of CM miR-150 are, in part, attributed to the direct and functional repression of proapoptotic Sprr1a. Our findings suggest a crucial role for the miR-150/SPRR1A axis in regulating CM function post-MI.

Background: Exosomes, key mediators of cell-cell communication, derived from type 2 diabetes mellitus (T2DM) have detrimental effects. Exercise not only improves endothelial dysfunction and angiogenesis in T2DM but also induces secretion of exosomes into circulation. Extracellular superoxide dismutase (ecSOD) is a major secretory Cu containing antioxidant enzyme that catalyzes dismutation of O 2 •- to H 2 O 2 and its full activity requires Cu transporter ATP7A. We reported that ecSOD-derived H 2 O 2 in endothelial cells (ECs) enhances angiogenesis while impaired ATP7A-ecSOD axis in diabetes induces endothelial dysfunction. Here we examined whether exercise-derived exosomes (Exe-Exo) may have pro-angiogenic effects via regulating ATP7A-ecSOD axis in T2DM. Results: Two weeks of voluntary wheel exercise of control C57Bl6 mice increased plasma exosome levels (6.2-fold) characterized by Nanosight, TEM and exosome markers (CD63, CD81, Tsg101). Treatment of HUVECs with equal number of exosomes revealed that angiogenic responses such as EC migration (1.8-fold) and tube formation (1.7-fold) were significantly enhanced by Exe-Exo compared to sedentary-derived exosomes (Sed-Exo). This was associated with increased ATP7A (2.9-fold) and ecSOD (1.4-fold) expression in Exe-Exo. Sed-Exo from high fat-induced T2DM mice significantly decreased EC migration (40%) and tube formation (10%) as well as ATP7A expression (28%) compared to Sed-Exo from control mice, which were restored by T2DM Exe-Exo, but not by T2DM/ecSOD KO Exe-Exo. Furthermore, exosomes overexpressing ecSOD (ecSOD-Exo) which mimic exercise increased angiogenesis and H2O2 levels in ECs, which were inhibited by overexpression of catalase. In vivo, skin wound healing model showed that direct application of T2DM Sed-Exo delayed while T2DM Exe-Exo enhanced wound healing of control mice. Furthermore, defective wound healing in T2DM mice or ecSOD KO mice were rescued by ecSOD-Exo application. Conclusion: Exercise training improves pro-angiogenic function of circulating exosomes in T2DM via increasing ATP7A-ecSOD axis, which may provide an effective therapy for promoting angiogenesis and wound repair in metabolic and cardiovascular diseases.

Background. Ischemic heart disease (IHD) is the major cause of death in patients with cardiovascular disease. Cardiac remodeling is a common pathological change following myocardial infarction (MI), and cardiomyocyte apoptosis plays a key role in this change. Transcription factor recombination signal-binding protein-J (RBP-J)-mediated Notch signaling pathway has been implicated in several inherited cardiovascular diseases, including aortic valve diseases, but whether the RBP-J-mediated Notch signaling pathway plays a role in cardiomyocyte apoptosis after MI is unclear. Method. We crossed RBP-Jfl/fl mice and Myh6-Cre/Esr1 transgenic mice to delete RBP-J in vivo and to partly inhibit the canonical Notch signaling pathway. MI was induced in mice by permanent ligation of the left anterior descending coronary artery followed by the knockout of RBP-J. Cardiac function and morphology were assessed by echocardiography and histological analysis 4 weeks after infarction. In addition, the expression and regulation of apoptosis-related molecules were examined by real time PCR and western blot. Results. RBP-J knockout decreased the survival rate and deteriorated post-MI remodeling and function in mice, and this effect was associated with increased cardiomyocyte apoptosis. The potential mechanisms might be related to the downregulated expression of bcl-2, upregulated expression of bax, and cleaved-caspase 3 to exacerbate cardiomyocyte apoptosis. Conclusion. These findings show that the RBP-J-mediated Notch signaling pathway in cardiomyocytes limits ventricular remodeling and improves cardiac function after MI. The RBP-J-mediated Notch signaling pathway has a protective role in cardiomyocyte apoptosis following cardiac injury.

Ischemic cardiac preconditioning protects the heart during myocardial infarction by activating critical cardioprotective genes such as eNOS, SOD, and HO-1. Clinical trials only show marginal effects of conventional preconditioning strategies, however, in part due to transient activation of cardioprotective genes. Recent studies have shown that N6-methyladenosine (m6A) mRNA methylation is the most abundant RNA modification in eukaryotes, and governs mRNA stability and, in turn, the level of protein expression. We hypothesize that regulation of m6A mRNA methylation levels of cardioprotective mRNAs will result in stable expression of the cardioprotective proteins, rendering ischemic cardiac preconditioning more robust and reducing infarct size. To test this hypothesis, we will test the effects of introducing m6A methylases/demethylases into ischemic preconditioned/post conditioned hearts and subjecting them to myocardial infarction. We will assess the half-life of key cardioprotective mRNAs (e.g., eNOS, SOD, and HO-1) and cardiac apoptosis to determine which m6A methylases/demethylases have a synergistic effect on cardiac preconditioning.

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, and as the disease progresses, the patient develops symptoms of dilated cardiomyopathy, arrhythmia, and congestive heart failure. The DMD(MDX) mutant mice do not express dystrophin, and are commonly used as a mouse model of DMD. In our recent study, we observed that intramyocardial injection of wide type (WT)-myogenic progenitor cells-derived exosomes (MPC-Exo) transiently restored the expression of dystrophin in the myocardium of DMD(MDX) mutant mice, which was associated with a transient improvement in cardiac function suggesting that WT-MPC-Exo may provide an option to relieve the cardiac symptoms of DMD. This article describes the technique of MPC-Exo purification and transplantation into hearts of DMD(MDX) mutant mice.

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 death rate from cancer in the USA has declined steadily over the past 2 decades (1). Improved cancer survival associated with anticancer agents has led to a greater recognition of the late effects of cardiotoxicity including left ventricular dysfunction, heart failure and coronary heart disease.

Our previous study demonstrated the beneficial effects of exosomes secreted by cardiac mesenchymal stem cells (C-MSC-Exo) in protecting acute ischemic myocardium from reperfusion injury. Here, we investigated the effect of exosomes from C-MSC on angiogenesis in ischemic myocardium. We intramyocardially injected C-MSC-Exo or PBS into the infarct border zone after induction of acute mouse myocardial infarction (MI). We observed that hearts treated with C-MSC-Exo exhibit improved cardiac function compared to control hearts treated with PBS at one month after MI. Capillary density and Ki67-postive cells were significantly higher following treatment with C-MSC-Exo as compared with PBS. Moreover, C-MSC-Exo treatment increased cardiomyocyte proliferation in infarcted hearts. In conclusion, intramyocardial delivery of C-MSC-Exo after myocardial infarction enhances cardiac angiogenesis, promotes cardiomyocyte proliferation, and preserves heart function. C-MSC-Exo constitute a novel form of cell-free therapy for cardiac repair.

We demonstrated the effects of exosomes secreted by cardiac mesenchymal stem cells (C-MSC-Exo) in protecting acute ischemic myocardium from reperfusion injury. To investigate the effect of exosomes from C-MSC on angiogenesis, we injected C-MSC-Exo or PBS intramuscularly into ischemic hind limb. Blood perfusion of limb was evaluated by laser Doppler Imaging. We observed that ischemic limb treated with C-MSC-Exo exhibits improved blood perfusion compared to ischemic limb treated with PBS at 2 weeks and 1 month after induction of limb ischemia. To explore the potential mechanisms underlying C-MSC-Exo’s angiogenetic effect, we performed microRNA array analysis and identify mmu-miR-7116-5p as the most abundant enriched miRNA detected in C-MSC-Exo. Bioinformatics’ analysis shows that miR-7116-5p negatively regulates protein polyubiquitination. In conclusion, our study demonstrated that intramuscular delivery of C-MSC-Exo after limb ischemia improves blood perfusion, and we identified the most abundant miRNAs that are preferentially enriched in C-MSC-Exo.

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.

Objective:To verify the influence of 3 potential factors on the accuracy of non-invasive central BP measurement, namely the calibration peripheral BP (brachial or radial), calibration methods (systolic/diastolic BP or mean/diastolic BP), and devices.Design and method:50 participants (aged 62.4 ± 8.9

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.

Objective— Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results— We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet–fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed α-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions— These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1–dependent mechanisms.

Aims Acute myocardial infarction (MI) leads to cardiac remodelling and development of heart failure. Insufficient myocardial capillary density after MI is considered a critical determinant of this process. MicroRNAs (miRs), negative regulators of gene expression, have emerged as important players in MI. We previously showed that miR-532-5p (miR-532) is up-regulated by the β-arrestin-biased β-adrenergic receptor antagonist (β-blocker) carvedilol, which activates protective pathways in the heart independent of G protein-mediated second messenger signalling. Here, we hypothesize that β2-adrenergic receptor/β-arrestin-responsive miR-532 confers cardioprotection against MI. Methods and results Using cultured cardiac endothelial cell (CEC) and in vivo approaches, we show that CECs lacking miR-532 exhibit increased transition to a fibroblast-like phenotype via endothelial-to-mesenchymal transition (EndMT), while CECs over-expressing miR-532 display decreased EndMT. We also demonstrate that knockdown of miR-532 in mice causes abnormalities in cardiac structure and function as well as reduces CEC proliferation and cardiac vascularization after MI. Mechanistically, cardioprotection elicited by miR-532 is in part attributed to direct repression of a positive regulator of maladaptive EndMT, prss23 (a protease serine 23) in CECs. Conclusions In conclusion, these findings reveal a pivotal role for miR-532-prss23 axis in regulating CEC function after MI, and this novel axis could be suitable for therapeutic intervention in ischemic heart disease.

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

Background Recent evidence has demonstrated that cardiac progenitor cells play an essential role in the induction of angiomyogenesis in infarcted myocardium. We and others have shown that engraftment of c-kit+ cardiac stem cells (CSCs) into infarcted hearts led to myocardium regeneration and neovascularization, which was associated with an improvement of ventricular function. The purpose of this study is aimed at investigating the functional role of transcription factor (TF) Oct3/4 in facilitating CSCs to promote myocardium regeneration and preserve cardiac performance in the post-MI heart. Methods c-kit+ CSCs were isolated from adult hearts and re-introduced into the infarcted myocardium in which the mouse MI model was created by permanent ligation of the left anterior descending artery (LAD). The Oct3/4 of CSCs was inhibited by transfection of Oct3/4 siRNA, and transfection of CSCs with control siRNA serves as control groups. Myocardial functions were evaluated by echocardiographic measurement. Histological analysis was employed to assess newly formed cardiogenesis, neovascularization, and cell proliferations. Terminal deoxynucleotidyltransferase (TdT) nick-end labeling (TUNEL) was carried out to assess apoptotic cardiomyocytes. Real time polymerase chain reaction and Western blot were carried out to evaluate the level of Oct 3/4 in CSCs. Results Two weeks after engraftment, CSCs increased ventricular functional recovery as shown by a serial echocardiographic measurement, which is concomitant with the suppression of cardiac hypertrophy and attenuation of myocardial interstitial fibrosis. Suppression of Oct 3/4 of CSCs abrogated functional improvements and mitigated the hypertrophic response and cardiac remodeling. Transplantation of c-kit+ CSCs into MI hearts promoted cardiac regeneration and neovascularization, which were abolished with the knockdown of Oct3/4. Additionally, suppression of Oct3/4 abrogated myocyte proliferation in the CSC-engrafted myocardium. Conclusion Our results indicate that CSCs-derived cardiac regeneration improves the restoration of cardiac function and is mediated through Oct 3/4.

To determine the effect of intracoronary transfer of superparamagnetic iron oxide (SPIO) labeled heme oxygenase-1 (HO-1) overexpressed bone marrow stromal cells (BMSCs) in a porcine myocardial ischemia/reperfusion model. Cell apoptosis was assayed and supernatant cytokine concentrations were measured in BMSCs that underwent hypoxia/reoxygen in vitro. Female mini-swines that underwent 1 h LAD occlusion followed by 1 h reperfusion were randomly allocated to receive intracoronary saline (control), 1 x 10(7) SPIO-labeled BMSCs transfected with pcDNA3.1-Lacz plasmid (Lacz-BMSCs), pcDNA3.1-human HO-1 (HO-1-BMSCs), pcDNA3.1-hHO-1 pretreated with a HO inhibitor, tin protoporphyrin (SnPP, n = 10 each). MRI and postmortem histological analysis were made at 1 week or 3 months thereafter. Post hypoxia/reoxygen in vitro, apoptosis was significantly reduced, supernatant VEGF significantly increased while TNF-alpha and IL-6 significantly reduced in HO-1-BMSCs group compared with Lacz-BMSCs group (all p0.05). Myocardial expression of VEGF was significantly higher in HO-1-BMSCs than in Lacz-BMSCs group at 1 week post transplantation (all p0.05). Signal voids induced by the SPIO were detected in the peri-infarction region in all BMSC groups at 1 week but not at 3 months post transplantation and the extent of the hypointense signal was the highest in HO-1-BMSCs group, and histological analysis showed that signal voids represented cardiac macrophages that engulfed the SPIO-labeled BMSCs. Pretreatment with SnPP significantly attenuated the beneficial effects of HO-1-BMSCs. Transplantation of HO-1-overexpressed BMSCs significantly enhanced the beneficial effects of BMSCs on improving cardiac function in this model.

Myocardial infarction rapidly depletes the endogenous cardiac progenitor cell pool, and the inefficient recruitment of exogenously administered progenitor cells limits the effectiveness of cardiac cell therapy. Recent reports indicate that interactions between the CXC chemokine stromal cell–derived factor 1 and its receptor CXC chemokine receptor 4 (CXCR4) critically mediate the ischemia-induced recruitment of bone marrow–derived circulating stem/progenitor cells, but the expression of CXCR4 in cardiac progenitor cells is very low. Here, we studied the influence of hypoxia on CXCR4 expression in cardiac progenitor cells, on the recruitment of intravenously administered cells to ischemic heart tissue, and on the preservation of heart function in a murine myocardial infarction model. We found that hypoxic preconditioning increased CXCR4 expression in CLK (cardiosphere-derived, Lin − c-kit + progenitor) cells and markedly augmented CLK cell migration (in vitro) and recruitment (in vivo) to the ischemic myocardium. Four weeks after surgically induced myocardial infarction, infarct size and heart function were significantly better in mice administered hypoxia-preconditioned CLK cells than in mice treated with cells cultured under normoxic conditions. Furthermore, these effects were largely abolished by the addition of a CXCR4 inhibitor, indicating that the benefits of hypoxic preconditioning are mediated by the stromal cell–derived factor 1/CXCR4 axis, and that therapies targeting this axis may enhance cardiac-progenitor cell–based regenerative therapy.

Cell therapy, particularly with stem cells, has created great interest as a solution to the fact that there are limited treatments for postischemic heart disease and none that can regenerate damaged heart cells to strengthen cardiac performance. From the first efforts with myoblasts to recent clinical trials with bone marrow-derived stem cells, early reports of cell therapy suggest improvement in cardiac performance as well as other clinical end points. Based on these exciting but tentative results, other stem cell types are being explored for their particular advantages as a source of adult stem cells. Autologous adipose-derived stem cells are multilinear and can be obtained relatively easily in large quantities from patients; cardiac-derived stem cells are highly appropriate for engraftment in their natural niche, the heart. Human umbilical cord blood cells are potentially forever young and allogenic adult mesenchymal stem cells appear not to evoke the graft versus host reaction. Human embryonic stem cells are effective and can be scaled up for supply purposes. The recent discovery of induced pluripotentcy in human adult stem cells, with only three transcription factor genes, opens a whole new approach to making autologous human pluripotent stem cells from skin or other available tissues. Despite the excitement, stem cells may have to be genetically modified with heme oxygenase, Akt or other genes to survive transplantation in a hypoxic environment. Homing factors and hormones secreted from transplanted stem cells may be more important than cells if they provide the necessary stimulus to trigger cardiac regrowth to replace scar tissue. As we await results from larger and more prolonged clinical trials, the science of stem cell therapy in cardiac disease keeps progressing.

OBJECTIVES Beta-blockers are widely used and effective for treating hypertension, acute myocardial infarction (MI) and heart failure, but they present side-effects mainly due to antagonism of beta2-adrenergic receptor (AR). Currently available beta-blockers are at best selective but not specific for beta1 or beta2-AR. METHODS To specifically inhibit the expression of the beta1-AR, we developed a small interfering RNA (siRNA) targeted to beta1-AR. Three different sequences of beta1 siRNA were delivered into C6-2B cells with 90% efficiency. RESULTS One of the three sequences reduced the level of beta1-AR mRNA by 70%. The siRNA was highly specific for beta1-AR inhibition with no overlap with beta2-AR. To test this in vivo, systemic injection of beta1 siRNA complexed with liposomes resulted in efficient delivery into the heart, lung, kidney and liver, and effectively reduced beta1-AR expression in the heart without altering beta2-AR. beta1 siRNA significantly lowered blood pressure of spontaneously hypertensive rats (SHR) for at least 12 days and reduced cardiac hypertrophy following a single injection. Pretreatment with beta1 siRNA 3 days before induction of MI in Wistar rats significantly improved cardiac function, as demonstrated by dP/dt and electrocardiogram following the MI. The protective mechanism involved reduction of cardiomyocyte apoptosis in the beta1 siRNA-treated hearts. CONCLUSIONS The present study demonstrates the possibility of using siRNA for treating cardiovascular diseases and may represent a novel beta-blocker specific for beta1-AR.

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.

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.

Objectives: The effect of a cardiac specific, hypoxia-regulated, human heme oxygenase-1 (hHO-1) vector to provide cardioprotection from ischemia-reperfusion injury was assessed. Background: When myocardial ischemia and reperfusion is asymptomatic, the damaging effects are cumulative and patients miss timely treatment. A gene therapy approach that expresses therapeutic genes only when ischemia is experienced is a desirable strategy. We have developed a cardiac-specific, hypoxia-regulated gene therapy “vigilant vector” system that amplifies cardioprotective gene expression. Methods: Vigilant hHO-1 plasmids, LacZ plasmids, or saline (n = 40 per group) were injected into mouse heart 2 days in advance of ischemia-reperfusion injury. Animals were exposed to 60 minutes of ischemia followed by 24 hours of reperfusion. For that term (24 hours) effects, the protein levels of HO-1, inflammatory responses, apoptosis, and infarct size were determined. For long-term (3 week) effects, the left ventricular remodeling and recovery of cardiac function were assessed. Results: Ischemia-reperfusion resulted in a timely overexpression of HO-1 protein. Infarct size at 24 hours after ischemia-reperfusion was significantly reduced in the HO-1-treated animals compared with the LacZ-treated group or saline-treated group ( P < .001). The reduction of infarct size was accompanied by a decrease in lipid peroxidant activity, inflammatory cell infiltration, and proapoptotic protein level in ischemia-reperfusion-injured myocardium. The long-term study demonstrated that timely, hypoxia-induced HO-1 overexpression is beneficial in conserving cardiac function and attenuating left ventricle remodelling. Conclusions: The vigilant HO-1 vector provides a protective therapy in the heart for reducing cellular damage during ischemia-reperfusion injury and preserving heart function.

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.

Hypoxia represents an endogenous pathophysiological signal underlying cell growth, adaptation and death in a variety of diseases, including ischemic heart diseases, stroke and solid tumors. A vigilant vector system depends on a gene switch which can sense the hypoxia signal occurring in ischemic events and turn on/off protective gene expressions when necessary. This system uses the oxygen-dependent degradation domain derived from hypoxia-inducible factor 1alpha as the hypoxia sensor and a double-vector system as signal amplifier. For treating ischemic heart diseases, a cardiac-specific MLC-2v promoter is used to deliver transgenes specifically to the heart. When tested in cardiomyocyte cultures, it produced a rapid and robust gene induction upon exposure to low oxygen. In a mouse model for myocardial infarction, the vigilant vectors turned on therapeutic genes such as heme oxygenase-1 in response to ischemia, significantly reduced apoptosis in the infarct area and improved cardiac functions. The hypoxia-regulated gene transfer afforded by the vigilant vectors may provide a powerful tool for delivering therapeutic proteins specifically to ischemic tissues with optimal physiological control.

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.

The miR-17-92 cluster is an important microRNA cluster in the animals. It was mainly investigated as an oncogene in tumors but never studied in cardiovascular disease. On one aspect, miR-17-92 cluster is documented to play anti-apoptotic roles in tumor cells, including hypoxia-induced apoptosis. The families of miR-17, miR-19, and miR-92 promote resistance to apoptosis by directly inhibiting pro-apoptotic protein, and by the two major cell survival signaling pathways—PI3K/AKT, and MAPK/ERK. On the other aspect, the component members of miR-17-92 cluster are high expressed in the hearts of canine and mice, suggesting that there are effect targets of the cluster in the myocardium. So that, we hypothesized that the miR-17-92 cluster may protect the heart by diminishing the apoptosis and alleviating ischemia/reperfusion injury. This may be a new regulating target for patients with myocardial infarction and undergoing cardiac surgery.

Objective The objective was to analyze completed trials assessing the effect of oral L-arginine supplementation on clinical outcomes of patients with acute myocardial infarction (AMI). Background Prior trials suggest that oral L-arginine administration improves endothelial function in patients with stable coronary artery disease (CAD). However, it is still unclear whether oral supplementation of L-arginine has any effect on clinical outcomes in patients with unstable CAD, such as AMI. Methods We systematically searched PubMed, Cochrane Library, Embase, reviews, and reference lists of relevant articles. The search strategy paired the term “arginine” with the following: “coronary heart disease,” “myocardial infarction,” “cardiovascular disease,” “ischemia,” and “trial.” We conducted a meta-analysis of randomized, placebo-controlled L-arginine supplementation trials that evaluated clinical outcomes in AMI patients. Two reviewers independently assessed the trials. Differences were resolved by consensus. Results Only 2 trials (927 participants) were included. None of the 2 studies showed a significant difference in event rate between the L-arginine and placebo groups. In an overall pooled estimate, there was a 7% reduction in mortality in the L-arginine treatment group (105/459, 22.9%) compared with the control group (111/455, 24.4%), which did not reach statistical significance (risk ratio [RR]: 0.93, 95% confidence interval [CI]: 0.74–1.17; P = 0.54). Conclusion Oral L-arginine supplementation has no effect on the clinical outcomes of patients with AMI. Copyright © 2009 Wiley Periodicals, Inc.

Cardiomyocytes are final differentiated cells that lose the ability to regenerate. Autologous cellular transplantation for cardiac repair has recently emerged as a promising new approach for end-stage heart failure that avoids the risk of immune rejection and ethical problems. Skeletal myoblasts (or satellite cells) are committed progenitor cells located under the basal lamina of adult skeletal muscle; they are committed to multiply after injury. They are highly resistant to ischemia and possess a considerable potential for division in culture. The cardiac milieu might alter the developmental program of grafted myoblast and facilitate their conversion to the slow-twitch phenotype, which is capable of performing cardiac work. We have used autologous cultured myoblast grafts for mouse and rat myocardial infarction in more than 200 cases. From these grafting experiments, it has been shown that cultured autologous myoblast grafting is a useful technique in the treatment of ischemic heart failure. This chapter offers a step-by-step guide to a successful research project on cultured myoblasts for the treatment of myocardial infarction, as well as a set of special techniques for yielding large numbers of skeletal myoblasts and studying postimplantation myocardial repair.

Cell transplantation is a novel therapy for patients with postinfarction ventricular dysfunction and congestive heart failure. The potential of mesenchymal stem cells (MSCs) to be used in the injured myocardium is unlimited because of their ability to self-renew and differentiate into cardiomyocytes in vitro and in vivo. In addition, MSCs are easy to harvest and have a high proliferation capability. Finally, autologous MSCs can survive permanently and integrate with healthy host tissue to form new myocardium after implantation in vivo. We have used autologous cultured MSCs grafts for mouse and rat myocardial infarction in more than 500 cases. From these grafting experiments, it has been shown that cultured autologous MSCs grafting is a useful approach in the treatment of ischemic heart failure. This chapter offers a step-by-step guide for successful research on cultured MSCs for the treatment of myocardial infarction, as well as a set of techniques for evaluating postimplantation myocardial repair.

Neovascularization induced by vascular endothelial growth factor (VEGF) represents an appealing approach for treating ischemic heart disease. However, VEGF therapy has been associated with transient therapeutic effects and potential risk for hemangioma growth. Adult mesenchymal stem cells (MSCs) derived from bone marrow are a promising source for tissue regeneration and repair. In order to achieve a safe and persistent angiogenic effect, we have explored the potential of autologous MSCs transplantation to enhance angiogenesis and cardiac function of ischemic hearts. One week after myocardial infarction induced by occlusion of left anterior descending artery, autologous MSCs expanded in vitro was administrated intramyocardially into the infarct area of the same donor rats. By 2 months, MSCs implantation significantly elevated VEGF expression levels, accompanied by increased vascular density and regional blood flow in the infarct zone. The neovascularization resulted in a decreased apoptosis of hypertrophied myocytes and markedly improved the left ventricular contractility (ejection fraction: 79.9+/-7.6% vs. 37.2+/-6.9% in control animals). Therefore, mechanisms underlying MSCs improvement of cardiac functions may involve neovascularization induced by differentiation of MSCs to endothelial cells and para-secretion of growth factors, in addition to the apoptosis reduction and previously reported cardiomyocytes regeneration. Two months after cell transplantation, there are significant improvement of left ventricular function. Hence, autologous MSCs transplantation may represent a promising therapeutic strategy free of ethical concerns and immune rejection, for neovascularization in ischemic heart diseases.

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.

Introduction: Autologous mesenchymal stem cells (MSCs) can improve postinfarcted cardiac function. However, progress in cell therapy is hampered by poor viability of implanted cells and the vulnerability of regenerated tissue to repeated bouts of ischemia. We developed a novel vigilant vector system, which can sense the hypoxia and regulate human heme-oxygenase(hHO-1) expression. We hypothesize that engineering MSCs with a vigilant system could increase the survival of engrafted cells and protect regenerated myocardium from further injury. Methods: Mouse MSCs were transfected with the vigilant vector. We assessed the effect of in-vitro hypoxia and in-vivo ischemia on cell apoptosis and cardiac function. In the study, 1x106 of vigilant hHO-1 transfected-MSCs (MSC-VHO-1), vigilant lacZ transfected MSCs (MSC-VlacZ), or MSCs with 50μl control medium were injected into syngeneic adult BALB/c mouse hearts 1 hour after induction of myocardial infarction. Results: The implanted MSC-VHO-1 expressed hHO-1 in the ischemic region. MSC-VHO-1 hearts had a 2-fold reduction in the number of apoptotic MSCs compared to the MSC-VlacZ group (p = 0.001) and control MSCs (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.