Effects And Underlying Mechanisms Of GATA-4Transduction Promotes Myocardial Transdifferentiation And Angiogenesis Of Mesenchymal Stem Cells

Part ⅠIsolation and Culture of Mesenchymal Stem Cells from Rat BoneMarrow and Transduction of GATA-4GeneObjective: Gene modified mesenchymal stem cells (MSCs) is a new direction toincrease cell resistance to hypoxia and increase myocardial transdifferentiation. Isolation,culture and purification of mesenchymal stem cells from rat bone marrow. Establishmentof stable cell model of GATA-4gene modified MSCs through retroviral transduction ofGATA-4gene into MSC.Methods: MSCs were isolated and cultured from male rat bone marrow. GATA-4(MSCGATA-4) was overexpressed in MSC by using a murine stem cell virus (pMSCV)retroviral expression system at the third passage. Control cells were infected with emptyvector (MSCNull). Gene and protein expression of GATA-4in MSCGATA-4were analyzedusing real-time quantitative polymerase chain reaction (real-time PCR), Western blottingand immunofluorescence.Results: Compared with control cells (MSCNull), mRNA and protein level ofGATA-4were enhanced in MSCGATA-4, as shown by real-time PCR and Western blot,P<0.05. Real-time PCR data indicated that the mRNA expression level of GATA-4was258-fold higher in MSCGATA-4vs. MSCNull. Whereas both MSCGATA-4and MSCNullwereGFP immunopositive, only MSCGATA-4stained intensely for GATA-4immunofluorescence. Conclusions: Retroviral-mediated transduction and expression of bicistronicGATA-4/GFP construct were evaluated by the methods of immunostaining, real-time PCR,and Western blotting, as a basis for further exploration of GATA-4in regulating MSCsmyocardial transdifferentiation and angiogenesis.Part IITransduction of GATA-4Promotes Myocardial Transdifferentiationof Mesenchymal Stem Cells and Its Related MechanismObjective: GATA-4is a cardiac transcription factor and plays an important role incell lineage differentiation during development. We investigated whether retroviraltransduction of GATA-4increased adult mesenchymal stromal cell (MSC)transdifferentiation into a cardiac phenotype in vitro.Methods：（1）Native cardiomyocytes (CM) were isolated from ventricles of neonatal rats.Cardiomyocytes were indentified through the methods of light-electricity microscopy,transmission electron microscopy and immunocytochemistry.（2）The expression of cardiac genes, including brain natriuretic peptide (BNP),Islet-1and α-sarcomeric actinin (α-SA), was detected by real-time PCR and Western blotafter MSC were co-cultured with native CM for7days.（3）Myocardial transdifferentiation of MSCGATA-4and MSCNullwere determined byα-sarcomeric actinin immunostaining.（4）Electrophysiological properties of ion channels were assessed in MSC usingpatch-clamp technology. TTX-sensitive Na+current (INa.TTX), L-type calcium current (ICa.L),transient outward K+current (Ito), delayed rectifier K+current (IKDR), the inwardlyrectifying K+current (IK1) and action potential (AP) were recorded in MSCGATA-4andMSCNullafter MSC were co-cultured with native CM.（5）The transdifferentiation rate was calculated directly from flow cytometry in MSCGATA-4and MSCNull.（6）The gene expression level of insulin-like growth factor binding protein-4(IGFBP-4）in MSCGATA-4and MSCNullwere analyzed using real-time PCR and Westernblotting and Microarray.（7）To study the role of IGFBP-4in GATA-4-mediated myocardialtransdifferentiation, IGFBP-4activity was knocked down in MSCGATA-4by siRNA genesilencing. Non-silencing, scrambled siRNA was used as a negative control. IGFBP-4geneexpression was analyzed using real-time PCR and Western blotting.（8）Myocardial transdifferentiation rate of MSCGATA-4after knockdown of IGFBP-4was observed.Results:(1) The expression of cardiac genes, including brain natriuretic peptide (BNP),Islet-1and α-sarcomeric actinin (α-SA), was up-regulated in MSCGATA-4compared withcontrol cells that were transfected with Green Fluorescent Protein (GFP) only (MSCNull).(2) MSCGATA-4exhibited higher levels of INa.TTX, ICa.L, Ito, IKDRand IK1channelactivities reflective of electrophysiological characteristics of CM.(3) Low amplitude of action potential was recorded in MSCGATA-4after MSC wereco-cultured. No action potential was recorded in MSCNull.(4) Some GFP+cells were positive for α-actinin staining after MSC were co-culturedwith native CM for7days. The transdifferentiation rate was significantly higher inMSCGATA-4(34%±4%), compared with MSCNull(15%±2%), P<0.05.(5) At the same time, IGFBP-4was significantly up-regulated in MSCGATA-4, asshown by real-time PCR and Western blot and microarray, compared with MSCNull,P<0.05.（6）After knocking down IGFBP-4in MSCGATA-4, real-time PCR showed that theexpression level of IGFBP-4in IGFBP-4-siRNA-MSCGATA-4(IG4-siRNA) was only38%of that in MSCGATA-4(P<0.05). IGFBP-4protein was reduced in IG4-siRNA cells by28%compared with MSCGATA-4(P<0.05). (7) The effect of GATA-4on expressing cardiac markers (BNP、α-actinin和Islet-1) inMSC was suppressed when IGFBP-4-siRNA was transfected in MSCGATA-4, compared withMSCGATA-4, P<0.05.(8) The transdifferentiation of various MSC was quantified using fluorescencea-activated cell sorting (FACS). The transdifferentiation rate was higher in MSCGATA-4(34.09±4.65%) than in MSCNull(14.54±1.62%; P<0.05). However, after IGFBP-4was knockeddown in the MSCGATA-4, the transdifferentiation rate was lower in IGFBP-4-siRNA (16.87±1.98%) than in MSCGATA-4(P<0.05).（9）Functional studies indicated that the differentiation potential of MSCGATA-4wasdecreased by knockdown of IGFBP-4.Conclusions: Retroviral transduction of GATA-4significantly increases MSCdifferentiation into a myocardial phenotype, which might be associated with theup-regulation of IGFBP-4.Part ⅢTransduction of GATA-4Improved in vivo and in vitroAngiogenesis of Mesenchymal Stem Cells and Its RelatedMechanismObjective: Transplanted mesenchymal stem cells (MSC) release soluble factors thatcontribute to cardiac repair and vascular regeneration. We hypothesized that retroviraltransduction of GATA-4enhances the MSC secretome, thereby increasing angiogenesisand cell survival and promoting postinfarction cardiac angiogenesis.Methods：(1) The concentration of VEGF, IGF-1, and basic FGF in conditioned medium (CdM)of MSC were measured using ELISA.(2) The gene expression levels of VEGF, IGF-1, and basic FGF in transduced MSC were detected using quantitative real-time PCR.(3) To study the resistance of MSC to oxidative stress, MSCs were exposed tohypoxia for24h,48h,72h and (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) intake was assessed.(4) Human umbilical vein endothelial cells (HUVECs）were labeled with PKH67celltracker dye using PKH67Green Fluorescent Cell Linker kit. Conditioned medium wereadded into HUVECs and capillary-like structure formation was assessed. IGF-1-and/orVEGF neutralizing antibodies were added to CdMGATA-4. After12h of incubation,capillary-like structure formation was assessed.(5) Spheroids of HUVEC were then embedded in3D Collagen Cell Culture Systemfor24h in the presence or absence of CdM. IGF-1-and/or VEGF neutralizing antibodieswere added to CdMGATA-4. The number of sprouts and cumulative sprout length in eachspheroid were detected and calculated.(6) MSCs were injected into the peri-infarct region in an acute myocardial infarctionmodel in Sprague-Dawley rats developed by ligation of the left anterior descendingcoronary artery.(7) Survival of male donor MSCs that expressed Sry gene in the female recipienthearts was assessed using real-time PCR. MSC survival in ischemic myocardium wasassayed.(8) Cardiac function was assessed by echocardiography. Infarction size andangiogenesis were detected.Results:(1) Expression of IGF-1and VEGF-A in MSC was significantly upregulated inMSCGATA-4compared with MSCNull, P<0.05.(2) levels of IGF-1and VEGF-A in CdM were assessed by ELISA and shown to besignificantly increased in CdMGATA-4, P<0.05, but b-FGF was unchanged, P＞0.05.(3) MTT intake was significantly reduced when MSCs were exposed to hypoxia for48or72h. Overexpression of GATA-4partially prevented this reduction. (4) HUVEC treated with MSCGATA-4conditioned medium (CdMGATA-4) exhibitedincreased formation of capillary-like structures and promoted migration (HUVECmigration was investigated by measuring both the number of sprouts per spheroid andcumulative sprout length), compared with HUVECs treated with MSCNullconditionedmedium, P<0.05. This CdMGATA-4-stimulated increase in formation of capillary-likestructures and migration were diminished by treatment with neutralizing antibodies againstIGF-1, VEGF, or both, compared with HUVECs treated with only MSCGATA-4conditionedmedium, P<0.05.(5) Sry gene in peri-infarcted and infarcted myocardium was significantly higher inMSCGATA-4-transplanted hearts, compared with MSCNullor MSCbas, P<0.05.Overexpression of GATA-4significantly increased MSC survival in ischemic myocardium.(6) MSCGATA-4-treated animals showed significantly improved cardiac function asassessed by echocardiography, compared with MSCNullor MSCbas-treated animals, P<0.05.(7) Histological studies revealed increased blood vessel density and reducedinfarction size in MSCGATA-4-treated animals, compared with MSCNullor MSCbas-treatedanimals, P<0.05.Conclusions: Transduction of GATA-4in MSCs increased both MSC survival andangiogenic potential in ischemic myocardium and may therefore represent a novel andefficient therapeutic approach for postinfarct remodeling.