Meifeng Xu

Bone Marrow Stem Cells Prevent Left Ventricular Remodeling of Ischemic Heart Through Paracrine Signaling

In this study, we hypothesized that bone marrow stem cells (BMSCs) protect ischemic myocardium through paracrine effects that can be further augmented with preconditioning. In in vitro experiments, cell survival factors such as Akt and eNOS were significantly increased in BMSCs following anoxia. In the second series of experiments following coronary ligation in mice, left ventricles were randomly injected with the following: DMEM (G-1), BMSCs (G-2), and preconditioned BMSCs (G-3). Four days after myocardial infarction, BMSCs were observed within injured myocardium in G-2 and G-3. Apoptotic cardiomyocytes within periinfarct area were significantly reduced in G-3. Four weeks after myocardial infarction, smaller left ventricular (LV) dimension and increased LV ejection fraction were observed in G-3. Infarct area was significantly reduced in G-3. However, GFP+ cardiomyocytes were observed in low numbers within periinfarct area in G-2 and G-3. In conclusion, BMSCs secreted cell survival factors under ischemia, and they prevented apoptosis in cardiomyocytes adjacent to the infarcted area. Preconditioning of BMSCs enhanced their survival and ability to attenuate LV remodeling, which was attributable, in part, to paracrine effects.

Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infarcted myocardium

Bone marrow mesenchymal stem cells (MSCs) participate in myocardial repair following myocardial infarction. However, their in vivo reparative capability is limited due to lack of their survival in the infarcted myocardium. To overcome this limitation, we genetically engineered male rat MSCs overexpressing CXCR4 in order to maximize the effect of stromal cell-derived factor-1alpha (SDF-1alpha) for cell migration and regeneration. MSCs were isolated from adult male rats and cultured. Adenoviral transduction was carried out to over-express either CXCR4/green fluorescent protein (Ad-CXCR4/GFP) or Ad-null/GFP alone (control). Flow cytometry was used to identify and isolate GFP/CXCR4 over-expressing MSCs for transplantation. Female rats were assigned to one of four groups (n=8 each) to receive GFP-transduced male MSCs (2 x 10(6)) via tail vein injection 3 days after ligation of the left anterior descending (LAD) coronary artery: GFP-transduced MSCs (Ad-null/GFP-MSCs, group 1) or MSCs over-expressing CXCR4/GFP (Ad-CXCR4/GFP-MSCs, group 2), or Ad-CXCR4/GFP-MSCs plus SDF-1alpha (50 ng/microl) (Ad-CXCR4/GFP-MSCs/SDF-1alpha, group 3), or Ad-miRNA targeting CXCR4 plus SDF-1alpha (Ad-miRNA/GFP-MSCs+SDF-1alpha treatment, group 4). Cardiodynamic data were obtained 4 weeks after induction of regional myocardial infarction (MI) using echocardiography after which hearts were harvested for immunohistochemical studies. The migration of GFP and Y-chromosome positive cells increased significantly in the peri- and infarct areas of groups 2 and 3 compared to control group (p<0.05), or miRNA-CXCR4 group (p<0.01). The number of CXCR4 positive cells in groups 2, 3 was intimately associated with angiogenesis and myogenesis. MSCs engraftment was blocked by pretreatment with miRNA (group 4). Cardiac function was significantly improved in rats receiving MSCs over-expressing CXCR4 alone or with SDF-1alpha. The up-regulation of matrix metalloproteinases (MMPs) by CXCR4 overexpressing MSCs perhaps facilitated their engraftment in the collagenous tissue of the infarcted area. CXCR4 over-expression led to enhance in vivo mobilization and engraftment of MSCs into ischemic area where these cells promoted neomyoangiogenesis and alleviated early signs of left ventricular remodeling.

Implantation of bone marrow stem cells reduces the infarction and fibrosis in ischemic mouse heart

Myocardial infarction may cause sudden cardiac death and heart failure. Adult cardiac myocytes do not replicate due to lack of a substantive pool of precursor, stem, or reserve cells in an adult heart. Ventricular myocytes following myocardial infarction are replaced by fibrous tissue and this leads to congestive heart failure in severe cases. Anversa et al. described that resident cardiac stem cells are present in the heart, and can repair the damaged mycardium by myocyte regeneration. Recent findings suggest the feasibility of cardiac repair using cell transplantation. However, it remains controversial which cell types are the best for cell transplantation in the ischemic heart. In this study, we demonstrate that cultured bone marrow stromal cells (MSCs) and Lin(-) bone marrow cells upon transplantation differentiate into myocytes and endothelial cells in the ischemic heart, eventually reducing both infarct size and fibrosis.

Hsp20-Engineered Mesenchymal Stem Cells Are Resistant to Oxidative Stress via Enhanced Activation of Akt and Increased Secretion of Growth Factors

Although heat‐shock preconditioning has been shown to promote cell survival under oxidative stress, the nature of heat‐shock response from different cells is variable and complex. Therefore, it remains unclear whether mesenchymal stem cells (MSCs) modified with a single heat‐shock protein (Hsp) gene are effective in the repair of a damaged heart. In this study, we genetically engineered rat MSCs with Hsp20 gene (Hsp20‐MSCs) and examined cell survival, revascularization, and functional improvement in rat left anterior descending ligation (LAD) model via intracardial injection. We observed that overexpression of Hsp20 protected MSCs against cell death triggered by oxidative stress in vitro. The survival of Hsp20‐MSCs was increased by approximately twofold by day 4 after transplantation into the infarcted heart, compared with that of vector‐MSCs. Furthermore, Hsp20‐MSCs improved cardiac function of infarcted myocardium as compared with vector‐MSCs, accompanied by reduction of fibrosis and increase in the vascular density. The mechanisms contributing to the beneficial effects of Hsp20 were associated with enhanced Akt activation and increased secretion of growth factors (VEGF, FGF‐2, and IGF‐1). The paracrine action of Hsp20‐MSCs was further validated in vitro by cocultured adult rat cardiomyocytes with a stress‐conditioned medium from Hsp20‐MSCs. Taken together, these data support the premise that genetic modification of MSCs before transplantation could be salutary for treating myocardial infarction. STEM CELLS 2009;27:3021–3031

Differentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes

Background—Bone marrow stromal cells (BMSCs) have the potential to differentiate into various cells and can transdifferentiate into myocytes if an appropriate cellular environment is provided. However, the molecular signals that underlie this process are not fully understood. In this study, we show that BMSC differentiation is dependent on communication with cells in their microenvironment. Methods and Results—BMSCs were isolated from green fluorescent protein (GFP)–transgenic mice and cocultured with myocytes in a ratio of 1:40. Myocytes were obtained from neonatal rat ventricles. The differentiation of BMSCs in coculture was confirmed by immunohistochemistry, electron microscopy, and reverse transcription–polymerase chain reaction. Before coculturing, the BMSCs were negative for α-actinin and exhibited a nucleus with many nucleoli. After 7-day coculture with myocytes, some BMSCs became α-actinin–positive and formed gap junctions with native myocytes. However, BMSCs separated from myocytes by a semipermeable membrane were still negative for α-actinin. Transdifferentiated myocytes from BMSCs were microdissected from cocultures by laser captured microdissection to determine the changes in gene expression. BMSCs cocultured with myocytes expressed mouse cardiac transcription factor GATA-4. Conclusions—When cocultured with myocytes, BMSCs can transdifferentiate into cells with a cardiac phenotype. Differentiated myocytes express cardiac transcription factors GATA-4 and myocyte enhancer factor-2. The transdifferentiation processes rely on intercellular communication of BMSCs with myocytes.

Exosomes secreted from GATA-4 overexpressing mesenchymal stem cells serve as a reservoir of anti-apoptotic microRNAs for cardioprotection.

BACKGROUND Exosomes play an important role in intercellular signaling and exert regulatory function by carrying bioactive molecules. This study investigated (1) the cardioprotective capabilities of exosomes derived from mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSC(GATA-4)) and (2) its underlying regulatory mechanisms for expression of target proteins in recipient cells. METHODS AND RESULTS Exosomes were isolated and purified from MSC(GATA-4) (Exo(GATA-4)) and control MSCs (Exo(Null)). Cell injury was investigated in primary cultured rat neonatal cardiomyocytes (CM) and in the rat heart. Exosomes contributed to increased CM survival, reduced CM apoptosis, and preserved mitochondrial membrane potential in CM cultured under a hypoxic environment. Direct intramyocardial transplantation of exosomes at the border of an ischemic region following ligation of the left anterior descending coronary artery significantly restored cardiac contractile function and reduced infarct size. Real-time PCR revealed that several anti-apoptotic miRs were highly expressed in Exo(GATA-4). Rapid internalization of Exo(GATA-4) by CM was documented using time-lapse imaging. Subsequent expression of these miRs, particularly miR-19a was higher in CM and in the myocardium treated with Exo(GATA-4) compared to those treated with Exo(Null). The enhanced protective effects observed in CM were diminished by the inhibition of miR-19a. The expression level of PTEN, a predicted target of miR-19a, was reduced in CM treated with Exo(GATA-4), which resulted in the activation of the Akt and ERK signaling pathways. CONCLUSIONS Exo(GATA-4) upon transplantation in the damaged tissue mediate protection by releasing multiple miRs responsible for activation of the cell survival signaling pathway.

Downregulation of Protein Kinase C Inhibits Activation of Mitochondrial KATP Channels by Diazoxide

Background—The mitochondrial KATP (mitoKATP) channel has been shown to confer short- and long-term cardioprotection against prolonged ischemia via protein kinase C (PKC) signaling pathways. However, the exact association between PKC or its isoforms and mitoKATP channels has not yet been clarified. The present study tested the hypothesis that the activity and translocation of PKC to the mitochondria are important for cardiac protection elicited by mitoKATP channels. Methods and Results—PKC was downregulated by prolonged (24-hour) treatment with phorbol 12-myristate 13-acetate (4 &mgr;g/kg body weight) before subsequent experiments in rats. Langendorff-perfused rat hearts were subjected to 40 minutes of ischemia followed by 30 minutes of reperfusion. Effects of PKC downregulation on the activation of mitoKATP channels and other interventions on hemodynamic, biochemical, and pathological changes were assessed. Subcellular localization of PKC isoforms by Western blot analysis and immunocytochemistry demonstrated that PKC-&agr; and PKC-&dgr; were translocated to the sarcolemma and that PKC-&dgr; was translocated to the mitochondria after diazoxide treatment. In hearts treated with diazoxide (80 &mgr;mol/L), a significant improvement in cardiac function and an attenuation of cell injury were observed. In PKC-downregulated hearts, protection was abolished because mitoKATP channels could not be activated by diazoxide. Conclusions—These data suggest that PKC activation is required for the opening of mitoKATP channels during protection against ischemia and that this effect is linked to isoform-specific translocation of PKC-&dgr; to the mitochondria.

Paracrine factors released by GATA-4 overexpressed mesenchymal stem cells increase angiogenesis and cell survival.

Transplanted mesenchymal stem cells (MSC) release soluble factors that contribute to cardiac repair and vascular regeneration. We hypothesized that overexpression of GATA-4 enhances the MSC secretome, thereby increasing cell survival and promoting postinfarction cardiac angiogenesis. MSCs harvested from male rat bone marrow were transduced with GATA-4 (MSC(GATA-4)) using the murine stem cell virus retroviral expression system; control cells were either nontransduced (MSC(bas)) or transduced with empty vector (MSC(Null)). Compared with these control cells, MSC(GATA-4) were shown by immunofluorescence, real-time PCR, and Western blotting to have higher expression of GATA-4. An increased expression of angiogenic factors in MSC(GATA-4) and higher MSC resistance against hypoxia were observed. Human umbilical vein endothelial cells (HUVEC) treated with MSC(GATA-4) conditioned medium exhibited increased formation of capillary-like structures and promoted migration, compared with HUVECs treated with MSC(Null) conditioned medium. MSC(GATA-4) were injected into the peri-infarct region in an acute myocardial infarction model in Sprague-Dawley rats developed by ligation of the left anterior descending coronary artery. Survival of MSC(GATA-4), determined by Sry expression, was increased at 4 days postengraftment. MSC(GATA-4)-treated animals showed significantly improved cardiac function as assessed by echocardiography. Furthermore, fluorescent microsphere and histological studies revealed increased blood flow and blood vessel density and reduced infarction size in MSC(GATA-4)-treated animals. We conclude that GATA-4 overexpression in MSCs increased both MSC survival and angiogenic potential in ischemic myocardium and may therefore represent a novel and efficient therapeutic approach for postinfarct remodeling.

Cardiomyocyte Protection by GATA-4 Gene Engineered Mesenchymal Stem Cells Is Partially Mediated by Translocation of miR-221 in Microvesicles

Introduction microRNAs (miRs), a novel class of small non-coding RNAs, are involved in cell proliferation, differentiation, development, and death. In this study, we found that miR-221 translocation by microvesicles (MVs) plays an important role in cardioprotection mediated by GATA-4 overexpressed mesenchymal stem cells (MSC). Methods and Results Adult rat bone marrow MSC and neonatal rat ventricle cardiomyocytes (CM) were harvested as primary cultures. MSC were transduced with GATA-4 (MSCGATA-4) using the murine stem cell virus (pMSCV) retroviral expression system. Empty vector transfection was used as a control (MSCNull). The expression of miRs was assessed by real-time PCR and localized using in situ hybridization (ISH). MVs collected from MSC cultures were characterized by expression of CD9, CD63, and HSP70, and photographed with electron microscopy. Cardioprotection during hypoxia afforded by conditioned medium (CdM) from MSC cultures was evaluated by lactate dehydrogenase (LDH) release, MTS uptake by CM, and caspase 3/7 activity. Expression of miR-221/222 was significantly higher in MSC than in CM and miR-221 was upregulated in MSCGATA-4. MSC overexpression of miR-221 significantly enhanced cardioprotection by reducing the expression of p53 upregulated modulator of apoptosis (PUMA). Moreover, expression of PUMA was significantly decreased in CM co-cultured with MSC. MVs derived from MSC expressed high levels of miR-221, and were internalized quickly by CM as documented in images obtained from a Time-Lapse Imaging System. Conclusions Our results demonstrate that cardioprotection by MSCGATA-4 may be regulated in part by a transfer of anti-apoptotic miRs contained within MVs.

Exosomes Secreted from CXCR4 Overexpressing Mesenchymal Stem Cells Promote Cardioprotection via Akt Signaling Pathway following Myocardial Infarction

Background and Objective. Exosomes secreted from mesenchymal stem cells (MSC) have demonstrated cardioprotective effects. This study examined the role of exosomes derived from MSC overexpressing CXCR4 for recovery of cardiac functions after myocardial infarction (MI). Methods. In vitro, exosomes from MSC transduced with lentiviral CXCR4 (ExoCR4) encoding a silencing sequence or null vector were isolated and characterized by transmission electron microscopy and dynamic light scattering. Gene expression was then analyzed by qPCR and Western blotting. Cytoprotective effects on cardiomyocytes were evaluated and effects of exosomes on angiogenesis analyzed. In vivo, an exosome-pretreated MSC-sheet was implanted into a region of scarred myocardium in a rat MI model. Angiogenesis, infarct size, and cardiac functions were then analyzed. Results. In vitro, ExoCR4 significantly upregulated IGF-1α and pAkt levels and downregulated active caspase 3 level in cardiomyocytes. ExoCR4 also enhanced VEGF expression and vessel formation. However, effects of ExoCR4 were abolished by an Akt inhibitor or CXCR4 knockdown. In vivo, ExoCR4 treated MSC-sheet implantation promoted cardiac functional restoration by increasing angiogenesis, reducing infarct size, and improving cardiac remodeling. Conclusions. This study reveals a novel role of exosomes derived from MSCCR4 and highlights a new mechanism of intercellular mediation of stem cells for MI treatment.