Genshan Ma

Cardiac progenitor-derived exosomes protect ischemic myocardium from acute ischemia/reperfusion injury

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

Hypoxic Preconditioning Improves Survival of Cardiac Progenitor Cells: Role of Stromal Cell Derived Factor-1α–CXCR4 Axis

Background Cardiac progenitor cells (CPCs) have been shown to be suitable in stem cell therapy for resurrecting damaged myocardium, but poor retention of transplanted cells in the ischemic myocardium causes ineffective cell therapy. Hypoxic preconditioning of cells can increase the expression of CXCR4 and pro-survival genes to promote better cell survival; however, it is unknown whether hypoxia preconditioning will influence the survival and retention of CPCs via the SDF-1α/CXCR4 axis. Methods and Results CPCs were isolated from adult mouse hearts and purified by magnetic activated cell sorting using c-kit magnetic beads. These cells were cultured at various times in either normoxic or hypoxic conditions, and cell survival was analyzed using flow cytometry and the expression of hypoxia-inducible factor-1α (HIF-1α), CXCR4, phosphorylated Akt and Bcl-2 were measured by Western blot. Results showed that the expression of pro-survival genes significantly increased after hypoxia treatment, especially in cells cultured in hypoxic conditions for six hours. Upon completion of hypoxia preconditioning from c-kit+ CPCs for six hours, the anti-apoptosis, migration and cardiac repair potential were evaluated. Results showed a significant enhancement in anti-apoptosis and migration in vitro, and better survival and cardiac function after being transplanted into acute myocardial infarction (MI) mice in vivo. The beneficial effects induced by hypoxia preconditioning of c-kit+ CPCs could largely be blocked by the addition of CXCR4 selective antagonist AMD3100. Conclusions Hypoxic preconditioning may improve the survival and retention of c-kit+ CPCs in the ischemic heart tissue through activating the SDF-1α/CXCR4 axis and the downstream anti-apoptosis pathway. Strategies targeting this aspect may enhance the effectiveness of cell-based cardiac regenerative therapy.

Transforming Growth Factor β1 Induces the Expression of Collagen Type I by DNA Methylation in Cardiac Fibroblasts

Transforming growth factor-beta (TGF-β), a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1) expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs) were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression.

Clinical Implication of Coronary Tortuosity in Patients with Coronary Artery Disease

Background Coronary tortuosity (CT) is a common coronary angiography finding. The exact pathogenesis, clinical implication and long-term prognosis of CT are not fully understood. The purpose of this study is to investigate the clinical characteristics of CT in patients with suspected coronary artery disease(CAD) in a Chinese population. Methods A total of 1010 consecutive patients underwent coronary angiography with complaints of chest pain or related symptoms were included in the present study (544 male, mean age: 64±11 years). CT was defined by the finding of ≥3 bends (defined as ≥45° change in vessel direction) along main trunk of at least one artery in systole and in diastole. Patients with or without CAD were further divided into CT-positive and CT-negative groups, all patients were followed up for the incidence of major adverse cardiovascular events (MACE) for 2 to 4 years. Results The prevalence of CT was 39.1% in this patient cohort and incidence of CT was significantly higher in female patients than that in male patients (OR = 2.603, 95%CI 1.897, 3.607, P<0.001). CT was positively correlated with essential hypertension (OR = 1.533, 95%CI 1.131, 2.076, P = 0.006) and negatively correlated with CAD (OR = 0.755, 95%CI 0.574, 0.994, P = 0.045). MACE during follow up was similar between CAD patients with or without CT. Conclusions CT is more often seen in females and positively correlated with hypertension and negatively correlated with coronary atherosclerosis.

Erythropoietin attenuates cardiac dysfunction by increasing myocardial angiogenesis and inhibiting interstitial fibrosis in diabetic rats

BackgroundRecent studies revealed that erythropoietin (EPO) has tissue-protective effects in the heart by increasing vascular endothelial growth factor (VEGF) expression and attenuating myocardial fibrosis in ischemia models. In this study, we investigated the effect of EPO on ventricular remodeling and blood vessel growth in diabetic rats.MethodsMale SD rats were randomly divided into 3 groups: control rats, streptozotocin (STZ)-induced diabetic rats, and diabetic rats treated with 1000 U/kg EPO by subcutaneous injection once per week. Twelve weeks later, echocardiography was conducted, and blood samples were collected for counting of peripheral blood endothelial progenitor cells (EPCs). Myocardial tissues were collected, quantitative real-time PCR (RT-PCR) was used to detect the mRNA expression of VEGF and EPO-receptor (EPOR), and Western blotting was used to detect the protein expression of VEGF and EPOR. VEGF, EPOR, transforming growth factor beta (TGF-β), and CD31 levels in the myocardium were determined by immunohistochemistry. To detect cardiac hypertrophy, immunohistochemistry of collagen type I, collagen type III, and Picrosirius Red staining were performed, and cardiomyocyte cross-sectional area was measured.ResultsAfter 12 weeks STZ injection, blood glucose increased significantly and remained consistently elevated. EPO treatment significantly improved cardiac contractility and reduced diastolic dysfunction. Rats receiving the EPO injection showed a significant increase in circulating EPCs (27.85 ± 3.43%, P < 0.01) compared with diabetic untreated animals. EPO injection significantly increased capillary density as well as EPOR and VEGF expression in left ventricular myocardial tissue from diabetic rats. Moreover, EPO inhibited interstitial collagen deposition and reduced TGF-β expression.ConclusionsTreatment with EPO protects cardiac tissue in diabetic animals by increasing VEGF and EPOR expression levels, leading to improved revascularization and the inhibition of cardiac fibrosis.

Mesenchymal stem cell transplantation enhancement in myocardial infarction rat model under ultrasound combined with nitric oxide microbubbles.

Objective This study evaluated the effects of ultrasound combined with the homemade nitric oxide (NO) micro-bubble destruction on the in vitro proliferation, apoptosis, and migration of mesenchymal stem cells (MSCs). Furthermore, we studied whether or not irradiation of the NO micro-bubble combined with bone-marrow derived MSC infusion had a better effect on treating myocardial infarction. The possible mechanism of MSC delivery into the infarcted myocardium was also investigated. Methods The murine bone marrow-derived MSCs were isolated, cultured, irradiated, and combined with different concentrations of NO microbubbles. MTT proliferation assay, annexin V-FITC apoptosis detection, migration assay, and RT-PCR were performed 24 h after the irradiation. The NO micro-bubbles was a intravenously injected, followed by the infusion of MSCs, which were labeled by CM-Dil. Myocardium was harvested 48 h later and the distribution of MSCs was observed by laser scanning confocal microscope after frozen sectioning. Echocardiography, histological examination, RT-PCR, and western blotting were performed four weeks after the cell transplantation. Results Ultrasound combined with 1:70 NO micro-bubbles had no significant impact on the proliferation or apoptosis of MSCs. Transwell chamber findings demonstrated that MSCs migrated more efficiently in group that underwent ultrasound combined with 1:70 NO micro-bubbles. The Real-time PCR results indicated that the expression of CXCR4 was much higher in the group undergoing ultrasound combined with 1:70 NO micro-bubbles. The normalized fluorescence intensity greatly increased in the group of US+NO micro-bubbles and the cardiac function was also markedly improved. Immunohistochemical staining showed that the capillary density was much greater in the group of US+NO micro-bubbles as compared to that of the other groups. RT-PCR and western blotting also revealed a higher SDF-1 and VEGF expression in the group of US+NO micro-bubbles. Conclusions NO micro-bubbles could be used in the cell transplantation, which efficiently promoted the MSC homing into the infarcted myocardium.

Dual-modal tracking of transplanted mesenchymal stem cells after myocardial infarction.

Purpose: Results for implantation efficiency and effective improvement of cardiac function in the field of mesenchymal stem cells (MSCs) are controversial. To attempt to clarify this debate, we utilized magnetic resonance imaging (MRI) and near-infrared optical imaging (OI) to explore the effects of different delivery modes of mesenchymal stem cells on cell retention time and cardiac function after myocardial infarction (MI). Methods: Rat MSCs were labeled with superparamagnetic iron oxide nanoparticles and 1, 1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) for noninvasive cell tracking in a rat MI model. Rats underwent coronary artery ligation and were randomized into three experimental groups: intravenous (IV), intramyocardial (IM), and a control group. The first two groups referred to the route of delivery of the transplanted dual-labeled MSCs; whereas the control group was given an IV injection of serum-free medium one day post-MI. Cellular engraftment was determined 1 day and 7 days post cell delivery by measuring the iron and optical signals in explanted organs. Prussian blue staining and fluorescent microscopy were performed on histological sections for iron and DiD, respectively. Cardiac function was measured by echocardiography on day 7. Results: The cardiac function of the IM group increased significantly compared to the IV and control groups at day 7. In the IM group, labeled cells were visualized in the infracted heart by serial MRI, and the intensity by OI was significantly higher on day 1. In the IV group, the heart signals were significantly attenuated by dual-modal tracking at two time points, but the lung signals in OI were significantly stronger than the IM group at both time points. Conclusion: IM injection of MSCs increased cell engraftment within infarcted hearts and improved cardiac function after MI. However, IV infusion has a low efficacy due to the cell trapping in the lung. Therefore, direct injection may provide an advantage over IV, with regard to retention of stem cells and protection of cardiac function.

Advanced glycation endproducts increase EPC apoptosis and decrease nitric oxide release via MAPK pathways.

OBJECTIVE Previous studies have shown that advanced glycation endproducts (AGE) can induce endothelial progenitor cells (EPC) apoptosis, which contributes to the pathogenesis of diabetes mellitus. Nitric oxide (NO) signaling is closely associated with apoptosis. We therefore investigated the effects of AGE on human EPC apoptosis, NO release and related signal transduction pathways. METHODS EPC isolated from healthy human subjects were cultured with various concentrations of AGE (0, 2, 20 and 200mg/L) for 0, 24, 48 and 72 h in the presence or absence of various MAPK (ERK/P38/JNK) inhibitors, respectively. EPC apoptosis (detected by flow cytometric analyses) and NO concentration in culture supernatant were determined. The mRNA levels of eNOS, COX-2, Bcl-2 and Bax were assessed by RT-PCR and the protein expressions of NF-kappaB and Caspase-3 assessed by Western blot. RESULTS Increased EPC apoptosis and reduced NO release were induced by 200mg/L AGE, accompanied by a downregulation of eNOS and Bcl-2 expressions as well as an elevation in COX-2, Bax, NF-kappaB and Caspase-3 expressions in a time-dependent manner (all P<0.05). These changes were significantly attenuated by pretreatment with various MAPK (ERK/P38/JNK) inhibitors (P<0.05). CONCLUSIONS AGE can promote EPC apoptosis and decrease NO release via MAPK pathways.

Exposure to supernatants of macrophages that phagocytized dead mesenchymal stem cells improves hypoxic cardiomyocytes survival.

OBJECTIVE To observe the impact of supernatants from macrophages that phagocytized dead MSCs (pMΦ) on the survival of hypoxic cardiomyocytes. METHODS MSCs were isolated from bone marrow of mice and dead MSCs were harvested after 6h hypoxia. Macrophages were obtained from thioglycolate-elicited peritoneal cavity. Macrophages and dead MSCs were co-cultured for 2 days in the presence or absence of LPS (1 μg/ml). Cardiomyocytes obtained from neonatal mice were exposed to various medium including supernatants from pMΦ. MTT cell proliferation assay and mitochondria membrane potential were used to evaluate the viability of cardiomyocytes. Cytokines and chemokines (TNF-α, IFN-γ, IL-6, IL-12, PGE2, VEGF-α, Ang-1, KGF, IGF-1, PDGF-BB, and EPO) in culture medium of macrophages, MSCs and pMΦ were detected by ELISA and Real-Time-PCR. RESULTS phagocytic activity of macrophages to dMSC was significantly enhanced by LPS. PGE2, VEGF-α, Ang-1, KGF, IGF-1, PDGF-BB, and EPO levels were significantly increased in supernatants of pMΦ. Exposure to supernatants of pMΦ significantly improved viability and survival time of hypoxic cardiomyocytes. CONCLUSION Exposure to supernatants of pMΦ significantly improved viability and survival time of hypoxic cardiomyocytes, which might be linked to increased cytokines and chemokines secretion by pMΦ.

Effects of aerobic cycling training on cardiovascular fitness and heart rate recovery in patients with chronic stroke

OBJECTIVE Using heart rate recovery (HRR) after exercise as an index of autonomic function, we evaluate the effects of aerobic cycling training on HRR and cardiovascular fitness (peak VO2) in chronic stroke patients and investigate the relationship between changes in HRR and those in peak VO2. METHODS 128 participants with chronic stroke were randomized to a 12-week (5×/week) progressive aerobic cycling training group (n = 65) or a control group (n = 63). Peak VO2, muscle strength, 6-minute walking distance (6MWD) and HRR were measured before and after the intervention. RESULTS Cycling training leads to significant increase in peak VO2, HRR, muscle strength and 6MWD. In the cycling group, percent changes in peak VO2 were positively associated with those in paretic and nonparetic muscle strength and HRR. Linear regression revealed that percent increases in peak VO2 were significantly correlated with percent changes in HRR when controlling for pre-peak VO2, age, gender, duration since stroke and improved muscle strength. CONCLUSION Aerobic cycling training can favorably modify HRR in stroke survivors. Rapid HRR, as an indicator of enhanced autonomic function, is useful for predicting gains in cardiovascular fitness. These findings indicate the underlying importance of autonomic modulation on cardiovascular adaptations to stroke exercise rehabilitation.