Pivotal Role Of Exosomes Secreted From GATA4~+ Cardiac Colony-forming Unit Fibroblasts In The Treatment Of Myocardial Infarction And Its Underlying Mechanisms

BackgroundGATA4 is an early cardiac-specific transcription factor,and endogenous GATA4-positive cells play a critical role in cardioprotection after myocardial injury.As functional paracrine units of therapeutic cells,exosomes can partially reproduce the reparative properties of their parental cells.Here,we investigated the cardioprotective capabilities of exosomes derived from cardiac colony-forming unit fibroblasts(cCFU-Fs)overexpressing GATA4(GATA4⁺cCFU-Fs)and the underlying mechanism through which these exosomes use microRNA(miRNA)delivery to regulate target proteins in myocardial infarction(MI).Part ? Extraction and identification of exosomes derived from GATA4⁺cCFU-Fs Objective:Isolate and purify Sca-1⁺cCFU-Fs,evaluate the effect of hypoxia on the expression of GATA4 in cCFU-Fs.GATA4 was transfected with lentivirus.Extract and identify the exosomes of GATA4⁺cCFU-Fs.Methods:1)cCFU-Fs were isolated from the hearts of 2-month-old male C57BL/6mice according to the method of Lijuan Chen et al.The immunomagnetic bead screening used to remove hematopoietic cells.Secondary sorting using Sca-1 labeled magnetic beads to obtain Sca-1⁺cCFU-Fs,and the expressions of the cell surface markers were analyzed by flow cytometry.RT-q PCR and Western-blot were used to detect the effect of hypoxia on the expression of GATA4 in cCFU-Fs.2)GV358lentivirus carrying GATA4 was used to transduce cCFU-Fs using polybrene as the linker molecule.Cell immunofluorescence and Western blot used to detect transfection efficiency.Transduction with the negative control lentivirus vector using the same protocol was performed as a control.3)Exosomes were obtained from the cCFU-Fs culture supernatants by ultracentrifugation.Immunoelectron microscopy,nanoparticle trace analysis and Western blot were used to detect specific proteins for exosome identification.Immunoelectron microscopy,nanoparticle trace analysis and western blotting were used for the identification of exosomes.Results:1)Using the phase-contrast inverted microscope,we observed that the cells screened by magnetic beads showed clone-like amplification.Flow cytometric analysis indicate that Sca-1 of cCFU-Fs is highly expressed:92.10%,and the CD34 and CD45was 2.44%and 2.36%respective,and Sca-1⁺,CD34^-,CD45^-cCFU-Fs was obtained.RT-q PCR and Western-blot results showed that the expression of GATA4 in cCFU-Fs was significantly reduced after hypoxia for 24h.2)Lentivirus-mediated transduction and expression of the GFP⁺GATA4 bicistronic construct were verified by immunostaining and Western-blot.Both cCFU-Fs^GATA4 and cCFU-Fs transfected with the empty vector control(cCFU-Fs^NC)expressed GFP,and there was no difference in cellular morphology between cCFU-Fs^GATA4 and cCFU-Fs^NC.Western-blot data indicated that the expression of GATA4 was much higher in cCFU-Fs^GATA4.3)The morphology of the cCFU-Fs-derived exosomes was directly acquired under a transmission electron microscope,and the particles were observed to be small,round vesicles with a bilayer membrane structure.Nano Sight analysis demonstrated that the diameters of the particles were within the range of 50-200 nm.We measured the protein levels of the exosome markers TSG101,Alix,and CD9 by Western-blot,and all markers could be detected in the cCFU-Fs-derived exosomes.Conclusion:Hypoxia down-regulates the expression of GATA4 in cCFU-Fs.The cCFU-F-derived particles obtained by ultracentrifugation in our experiments were exosomes.Part ? Anti-apoptotic effects of exosomes derived from cCFU-Fs^GATA4 in vivo and in vitroObjective:Comparing the different effects of exosomes derived from cCFU-Fs ^GATA4and cCFU-Fs^NC on hypoxic H9C2 in vitro and cardiac function after acute myocardial infarction in vivo.Methods:1)We used immunofluorescence to detect the uptake of exosomes after H9C2 incubate with PKH26 labeled exosomes for 12h.To compare the protective effect of the exosomes from cCFU-Fs^GATA4 and cCFU-Fs^NC on hypoxia-induced myocardial cell injury,we measured the cell viability and the apoptotic rate of H9C2cells after coculture with the two kinds of exosomes under hypoxic conditions for 24h using a CCK8 kit and flow cytometry.2)To evaluate the therapeutic effect of GATA4-Exo,we generated a mouse model of acute MI via permanent LAD artery ligation.We intramyocardially injected 100?l of PBS,NC-Exo,or GATA4-Exo into the mice after LAD ligation,and mice in the sham operation groups served as controls.TUNEL staining was used to evaluate the apoptosis.Western-blot was used to detect the expression of apoptosis-related proteins in myocardial tissue.Cardiac function was measured by echocardiography.Mice were sacrificed 4 weeks after exosomes transplantation,heart paraffin sections were prepared,and masson staining was used to observe infarct size.Results:1)After incubating the labeled exosomes with cardiomyocytes for 12 hours,the exosomes pellet shows strong fluorescence in the in the cytoplasm of H9C2.The CCK8 results showed that the cell viability of the H9C2 cells was suppressed after hypoxia treatment.The addition of exosomes(100?g/?l)generated from cultured cCFU-Fs^NC has no pronounced protective effect on cell damage caused by hypoxia,but the GATA4-Exo can significantly increase cell viability.Compared with NC-Exo pretreatment,GATA4-Exo treatment significantly decreased the protein expression of cleaved-caspase-3(c-Cas-3)in hypoxia-stimulated H9C2 cells.The flow cytometry results showed that both NC-Exo and GATA4-Exo pretreatment can reduce the percent of early(Q2)and late(Q3)apoptotic cells caused by hypoxia.Compared with NC-Exo,the reduction caused by GATA4-Exo was more remarkable,especially for the percentage of early apoptotic cells.2)Echocardiography showed that GATA4-Exo treatment preserved LV function,as indicated by the end-systolic chamber volume (ESCV)and end-systolic left ventricular internal diameter(LVID)after myocardial infarction,but NC-Exo treatment did not.Similarly,GATA4-Exo treatment improved systolic function,as indicated by the higher ejection fraction(EF%)and fraction shortening(FS%).Masson trichrome staining was performed 28 days after exosome transplantation.Through quantitative analyses using Image J software,we found that the percentage of the fibrotic area in the entire LV cross-sectional area was distinctly reduced in the GATA4-Exo group compared with the NC-Exo and PBS groups.TUNEL staining results showed that the number of apoptotic cells in the border zone of the ischemic heart tissue was significantly increased in PBS-injected mice compared with mice in the sham operation group.Delivery of GATA4-Exo to the myocardium after LAD artery ligation caused an obviously reduction in TUNEL-positive apoptotic cells compared with those in the hearts of PBS and NC-Exo treated mice.Conclusion:In vitro and in vivo data illustrate that functional improvement in the GATA4Exo group was accompanied by decreased infarct size which was partly due to the inhibition of CM apoptosis.Part ?:The mechanism of GATA4 enhancing the anti-apoptotic effect of cCFU-Fs exosomes in vitroObjective:1)Elucidate the molecular mechanism of GATA4 overexpression to enhance the anti-apoptotic ability of exosomes derived from cCFU-Fs by Affymetrix microRNA chip and luciferase reporter gene.2)Explore the role of PTEN-PI3K/Akt pathway in miR221-mediated GATA4-Exo cardiac protection by miR221 inhibitor and si PTEN.Methods:1)Obtain the totalRNA of NC-Exo and GATA4-Exo.Global miRNA expression patterns were then examined by using Affymetrix Gene Chip miRNA 4.0.RT-q PCR further verified the chip results.2)Target Scan,miRanda,microRNA.ORG were used to predict miRNA target genes.Luciferase activity was measured using the Luciferase Assay System.3)miRNA mimic and inhibitor were constructed to detect the effect of miRNA on target gene expression and on the viability and apoptosis in hypoxic H9C2.4)After H9C2 were treated with GATA4-Exo,miR221 inhibitor and si PTEN after hypoxia stimulation:(1)RT-q PCR and Western-blot were used to detect the expression of PTEN?p-Akt and c-Caspase3;(2)Cell viability and apoptosis were determined by CCK8 and flow cytometry;Results:1)The criteria for significantly differentially expressed miRNAs were a p value<0.05 and a fold change in expression of at least 2.0.The hierarchical clustering of miRNA expression showed that GATA4-Exo had a miRNA expression signature very different from that of NC-Exo.Overall,10 miRNAs,including miR6538,miR2137,miR221,and miR6968-5p et al,showed marked upregulation in GATA4-Exo compared with NC-Exo,whereas 4 miRNAs(miR7651-3p,miR539-3p,miR345-5p,miR7025-5p)showed evident downregulation in GATA4-Exo compared with NC-Exo.RT-q PCR showed all tested miRNAs exhibited an agreement with the array generated expression data.Compared with other miRNAs,miR221 has the most significantly increase in GATA4-Exo.2)Luciferase reporter assays showed that compared with the NC mimics,the relative luciferase activity of the PTEN-3'UTRwt reporter was significantly reduced by the miR221 mimics;however,mutation of the miR221 seed sequence reversed this inhibitory effect.Additionally,the miR221 mimics downregulated the expression of PTEN at both the mRNA and protein levels,and conversely,the inhibitor upregulated the expression of PTEN.3)The results of CCK8 suggested that the miR221 mimics could protect cells against hypoxia-induced H9C2 cell injury,while the miR221 inhibitor aggravated the damage.The treatment of H9C2 cells with the miR221 mimics decreased hypoxia-induced apoptosis.However,the inhibitor increased cell injury,in agreement with the flow cytometry results.Annexin V analysis showed significantly more apoptotic cells among cells transfected with the miR221 inhibitor than among those transfected with the miR221mimics.4)RT-q PCR and Western-blot results showed that GATA4-Exo significantly downregulated the expression of PTEN at both the mRNA and protein levels,which was in contrast with the effect of the miR221 inhibitor.The results of CCK8 assays showed that GATA4-Exo protected H9C2 from apoptosis and injury caused by hypoxia,while the miR221 inhibitor decreased this protective effect.When PTEN was knocked out with siRNA,the H9C2 showed reduced apoptosis and increased cell viability after hypoxia stimulation,which is similar to the effects of GATA4-Exo.Furthermore,hypoxia downregulated the expression of p-Akt but increased c-Caspase3 expression.The expression of p-Akt was increased,while the expression of c-Caspase3 was reduced in cells incubated with GATA4-Exo before hypoxia compared with cells treated with hypoxia alone.The pretreatment of the miRNA221inhibitor abrogated the effect of GATA4-Exo.When cells were pretreated with siPTEN,the changes in p-Akt and c-caspase3 expression were similar to those in the GATA4-Exo-treated group.Conclusion:GATA4 up-regulates the expression of miRNA221 in cCFU-Fs exosomes.The anti-apoptotic effect of miR221 is exerted by down-regulates caspase3 through the PTEN-PI3K/Akt pathway.The cardioprotective effect of GATA4-Exo might be comodulated by miR221.The anti-apoptotic effect of miRNA221 is exerted by inhibiting the expression of target gene PTEN.Part ? Confirmed that miR221-PTEN-PI3K/Akt pathway is involved in the cardioprotection of GATA4-Exo in vivoObjective:Explore the role of PTEN-PI3K/Akt pathway in miR221-mediated GATA4-Exo cardiac protection by mouse acute myocardial infarction model.Methods:1)In vivo,after myocardial infarction,PBS,GATA4-Exo,NC-Exo are injected into the myocardium around the infarct area,24h after LAD ligation,(1)RT-q PCR were used to detect the expression of miR221 in myocardial tissue.(2)Western-blot were used to detect the expression of PTEN and c-Caspase3 in myocardial tissue.2)After LAD ligation,PBS,NC-Exo,GATA4-Exo,GATA4-Exo+miR221 inhibitor and miR221 mimics were injected into the myocardium,24h after LAD ligation,(1)TUNEL staining was used to evaluate the apoptosis.(2)Western-blot was used to detect the expression of apoptosis-related proteins in myocardial tissue;28 days after LAD ligation,(1)Cardiac function was measured by echocardiography.(2)Masson staining was used to observe infarct size.Results:1)(1)RT-q PCR results showed that both NC-Exo and GATA4-Exo can up-regulate the expression of miRNA221 in myocardial tissue,and the increase in GATA4-Exo group was more significant.(2)compared with NC-Exo injection,GATA4-Exo significantly downregulated the protein expression of PTEN and c-caspase3 in the acute MI model.2)(1)TUNEL staining revealed that delivery of GATA4-Exo can reduce the number of apoptotic cells after myocardial infarction,while the administration of GATA4-Exo+miR221 inhibitor did not show a significant anti-apoptotic effect.(2)The results of western blot showed that GATA4-Exo+miR221 inhibitor can elimination the effect of GATA4-Exo to downregulate the protein expression of c-caspase3 in the acute MI model,while miR221 mimics can simulate the function of GATA4-Exo.(3)Echocardiography showed that GATA4-Exo treatment improved systolic function,which is similar to the miR221mimics treatment,while GATA4-Exo+miR221inhibitor injection neutralizes the protection.(4)Masson trichrome staining showed that the percentage of the fibrotic area in the entire LV cross-sectional area was distinctly reduced in the GATA4-Exo group compared with the NC-Exo and PBS groups.The effect of the miR221mimics is consistent with the GATA4-Exo delivery.However,the injection of GATA4-Exo+miR221 inhibitor does not significantly reduce the fibrotic area after myocardial infarction.Conclusion:Injection of GATA4-Exo can increase the expression of miR221 in the myocardium.By inhibiting the expression of the target gene PTEN,miRNA 221involved in the cardioprotection of GATA4-Exo.In this study,we used a genetic modification technique to promote the paracrine function of cCFU-Fs for repair of the ischemic myocardium.Exosomes derived from GATA4 overexpressed cCFU-Fs prevent cardiomyocyte apoptotic program,at least partly,via anti-apoptotic miRNAs contained therein.Our data provide compelling evidence of the role of GATA4-Exo in improving the healing response following injury.Therefore,exploring the active ingredients and functional mechanisms of GATA4-Exo is of great significance to developing a new biotherapy of myocardial ischemia based on exosomes.This study offered a new approach to alleviating myocardium ischemic injury.