Shi Zuo

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.

Mobilized bone marrow progenitor cells serve as donors of cytoprotective genes for cardiac repair

We proposed here that mobilized progenitor cells (MPCs) from the bone marrow are special cell types which carry cytoprotective proteins for cardiac repair following ischemia. Myocardial ischemia was induced by ligation of the left anterior descending coronary artery (LAD) in mice. Progenitor cells in peripheral blood were analyzed by fluorescence-activated cell sorting (FACS). The expression of cytoprotective genes was assayed by ELISA, RT-PCR, and/or real-time PCR. G-CSF was markedly up-regulated in the ischemic myocardium. A good correlation was observed between serum G-CSF and progenitor cells in circulation following LAD ligation. MPCs overexpressed cardiac transcription factor, GATA-4, and anti-apoptotic factor, Bcl-2, besides expression of the surface markers of bone marrow stem cells (BMSCs). Transplantation of cultured MPCs into the ischemic border area significantly improved cardiac function by reducing infarction size. More importantly, MPCs significantly protected cardiomyocytes against apoptosis when co-cultured with cardiomyocytes. The cardiac protection by MPCs was blocked by Bcl-2 neutralizing antibody and GATA-4 siRNA. In contrast, transfection of BMSCs with GATA-4 provided increased protection of myocytes against apoptosis. It is concluded that MPCs are highly cytoprotective and carry protective genes responsible for cardiac repair.

Paracrine Effect of Wnt11-Overexpressing Mesenchymal Stem Cells on Ischemic Injury

Our previous studies have suggested that transduction of Wnt11 directly increases bone marrow-derived mesenchymal stem cells (MSCs) differentiation into cardiac phenotypes. In this study, we investigated whether Wnt11 enhances MSC-mediated cardioprotection via paracrine fashion after acute ischemia. MSCs were harvested from male rat bone marrow and transduced with Wnt11 (MSC(Wnt11)). An acute myocardial infarction model in rats was developed by ligation of the left anterior descending coronary artery. MSC(Wnt11) were transplanted into the peri-infarct region after acute myocardial infarction. To mimic ischemic injury, cultured cardiomyocytes (CMs) isolated from neonatal ventricles were exposed to hypoxia. ELISA studies indicated that the release of Wnt11 (3.45-fold) as well as transforming growth factor-β2 (TGFβ2) (1.5-fold) was significantly increased from MSC(Wnt11) compared with transduced control MSC (MSC(Null)). Hypoxia-induced apoptosis and cell death was significantly reduced when CM were co-cultured with MSC(Wnt11) in a dual chamber system. The cell protection mediated by MSC(Wnt11) was mimicked by treating CM with conditioned medium obtained from MSC(Wnt11) and abrogated by Wnt11- and TGFβ2 neutralizing antibodies. Further, animals receiving MSC(Wnt11) showed a significant improvement in cardiac contractile function as assessed by echocardiography. Masson trichrome and TUNEL staining showed a significant reduction in infarct size and apoptosis of CM in MSC(Wnt11)-treated animals. Transplantation of MSC(Wnt11) improved cardiac function. The release of Wnt11 and other factors from transplanted MSC(Wnt11) is more likely responsible for protection of native CM at risk.

Transduction of Wnt11 promotes mesenchymal stem cell transdifferentiation into cardiac phenotypes

Transplantation of mesenchymal stem cells (MSCs) has emerged as a potential treatment for ischemic heart repair. Previous studies have suggested that Wnt11 plays a critical role in cardiac specification and morphogenesis. In this study, we examined whether transduction of Wnt11 directly increases MSC differentiation into cardiac phenotypes. MSCs harvested from rat bone marrow were transduced with both Wnt11 and green fluorescent protein (GFP) (MSC(Wnt11)) using the murine stem cell virus (pMSCV) retroviral expression system; control cells were only GFP-transfected (MSC(Null)). Compared with control cells, MSC(Wnt11) was shown to have higher expression of Wnt11 by immunofluorescence, real-time polymerase chain reaction, and western blotting. MSC(Wnt11) shows a higher expression of cardiac-specific genes, including GATA-4, brain natriuretic peptide (BNP), islet-1, and α-actinin, after being cultured with cardiomyocytes (CMs) isolated from ventricles of neonatal (1-3 day) SD rats. Some MSC(Wnt11) were positive for α-actinin when MSCs were cocultured with native CMs for 7 days. Electron microscopy further confirmed the appearance of sarcomeres in MSC(Wnt11). Connexin 43 was found between GFP-positive MSCs and neonatal rat CMs labeled with red fluorescent probe PKH26. The transdifferentiation rate was significantly higher in MSC(Wnt11) than in MSC(Null), as assessed by flow cytometry. Functional studies indicated that the differentiation of MSC(Wnt11) was diminished by knockdown of GATA-4 with GATA-4-siRNA. Transduction of Wnt11 into MSCs increases their differentiation into CMs by upregulating GATA-4.

GATA-4 promotes myocardial transdifferentiation of mesenchymal stromal cells via up-regulating IGFBP-4

BACKGROUND AIMS GATA-4 is a cardiac transcription factor and plays an important role in cell lineage differentiation during development. We investigated whether overexpression of GATA-4 increases adult mesenchymal stromal cell (MSC) transdifferentiation into a cardiac phenotype in vitro. METHODS MSC were harvested from rat bone marrow (BM) and transduced with GATA-4 (MSC(GATA-4)) using a murine stem cell virus (pMSCV) retroviral expression system. Gene expression in MSC(GATA-4) was analyzed using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Native cardiomyocytes (CM) were isolated from ventricles of neonatal rats. Myocardial transdifferentiation of MSC was determined by immunostaining and electrophysiologic recording. The transdifferentiation rate was calculated directly from flow cytometery. RESULTS The expression of cardiac genes, including brain natriuretic peptide (BNP), Islet-1 and α-sarcomeric actinin (α-SA), was up-regulated in MSC(GATA-4) compared with control cells that were transfected with Green Fluorescent Protein (GFP) only (MSC(Null)). At the same time, insulin-like growth factor-binding protein (IGFBP)-4 was significantly up-regulated in MSC(GATA-4). A synchronous beating of MSC with native CM was detected and an action potential was recorded. Some GFP (+) cells were positive for α-SA staining after MSC were co-cultured with native CM for 7 days. The transdifferentiation rate was significantly higher in MSC(GATA-4). Functional studies indicated that the differentiation potential of MSC(GATA-4) was decreased by knockdown of IGFBP-4. CONCLUSIONS Overexpression of GATA-4 significantly increases MSC differentiation into a myocardial phenotype, which might be associated with the up-regulation of IGFBP-4.