| BackgroundIschemic heart disease(IHD)is one of the most threatening diseases,accounting for 16%of the mortality rate and causing a huge hazard to human health.Clinical therapies,such as surgical bypass,percutaneous coronary intervention(PCI),and drug therapy,can effectively improve cardiac function and slow down the progression of IHD,but the incidence rate of heart failure after myocardial infarction in patients is still high and the quality of patient’s life is poor.Therefore,how to delay or prevent the occurrence and development of heart failure after MI is still a key problem to be solved.Stem cell therapy is a potential treatment strategy for IHD,which can reduce apoptosis,alleviate myocardial fibrosis,and prevent myocardial remodeling.Induced pluripotent stem cells(iPSCs),embryonic stem cells(ESCs),and mesenchymal stromal cells(MSCs)are used to treat IHD.Compared with other stem cell types,MSCs have the advantages of easy availability,low immunogenicity,no ethical problems,and no tumorigenicity.therefore,MSCs are expected to become a hopeful cell types for the treatment of IHD.There are three main types of stem cell injection,intra-coronary injection,intravenous injection,and intramyocardial injection,respectively.Compared with other infusion methods,intravenous injection has the outstanding advantages of being repeatable,non-invasive,and easy to operate.Some clinical trials have evaluated that MSCs transplantation for IHD is safe and feasible,and slight improvements in cardiac function,quality of life,and ventricular remodeling can be observed in patients.However,there is no significant decrease in mortality and incidence of heart failure of patients.The main problems are the less homing of heart after transplantation of MSCs,the low survival rate,the lack of cardiomyocyte differentiation ability,and the insufficient paracrine.Therefore,the application of new therapies to improve the quantity and survival of MSCs after transplantation is important.Colony stimulating factor 2(CSF2),also known as granulocyte-macrophage colony stimulating factor(GM-CSF),is a growth factor that can promote the differentiation of bone marrow progenitor cells into monocytes and macrophages in vitro.Our previous studies showed that the expression level of chemokine CSF2 in the infarcted myocardium significantly increased,which can act on the CSF2 receptor beta subunit(CSF2RB)on adipose-derived mesenchymal stem cell(ADSC),to promote the homing of intravenous ADSC to the heart.However,the expression level of CSF2RB in ADSC is extremely low,and the effect of the drug to improve the expression of CSF2RB in ADSC is limited.To solve this problem,we constructed adenovirus vectors CSF2RB and transfected ADSC,which successfully improved the expression level of CSF2RB in ADSC.Then we verified the effects of overexpression of CSF2RB in adipose-derived mesenchymal stem cell(ADSC)on cardiac homing and myocardial repair and explored its cytological and molecular mechanisms.Objective1.To verify the effect of multiple intravenous ADSC on myocardial ischemia injury in mice;2.Plasma was collected from non-coronary volunteers or acute myocardial infarction(AMI)patients who underwent PCI,to detect the expression of the CSF2 protein.In the same way,plasma and myocardial tissue were collected from myocardial ischemia/reperfusion(MI/R)mice or Sham mice,to detect the expression of CSF2 protein in plasma and myocardial tissue;3.To elucidate the effects of overexpression of CSF2RB in adipose-derived mesenchymal stem cell(ADSC)on cardiac homing and myocardial repair;4.To investigate the role and mechanism of overexpression of CSF2RB in ADSC on migration,apoptosis,and paracrine.Methods1.To verify the effect of intravenous ADSC injections on myocardial ischemic injury in mice,MI/R mouse model was first established by coronary artery ligation and randomly divided into three groups:Sham group,intravenous injections of cell-free vector after ischemia reperfusion(MI/R+Vehicle group)and intravenous injections of ADSC after ischemia reperfusion(MI/R+ADSC group).At 3 days after MI/R operation,MI/R+ADSC group received intravenous injections of td Tomato immunofluorescent protein labeled ADSC 7 times within 3 weeks,and MI/R+Vehicle group received intravenous injections of the same volume of cell-free vector 7 times within 3 weeks.At 3 weeks after MI/R operation,the immunofluorescence technique was used to detect the quantity of immunofluorescent protein labeled ADSC of td Tomato in the heart,lung,liver,and spleen of mice and the density of myocardial neovascularization.Cardiac function was detected by echocardiography and myocardial cell apoptosis was detected by TUNEL staining.After 6 weeks of MI/R injury,echocardiography was used to detect cardiac function,and Masson staining was used to detect the myocardial fibrosis area.We measured the gene levels of atrial natriuretic peptide(ANP)and brain natriuretic peptide(BNP)by using realtime quantitative polymerase chain reaction(PCR).2.Plasma was collected from 27 patients with AMI who underwent PCI and 23 noncoronary volunteers.Then,to detect the expression of the CSF2 protein in plasma by western blot assay and ELISA kit.In the same way,plasma and myocardial tissue were collected from MI/R mice and Sham mice.Then to detect the expression of CSF2 protein in plasma and myocardial tissue by western blot assay and ELISA kit.3.To elucidate the effects of overexpression of CSF2RB in ADSC on cardiac homing and cardiac function.MI/R models were first constructed by coronary ligation and randomized as follows:Sham group,MI/R+ADSC-Vehicle group,MI/R+ADSCNC group,and MI/R+ADSC-CSF2RB group.ADSC were labeled with DiR or EGFP to detect.At 3 days after MI/R,the mice were administered either ADSC infected with adenovirus-CSF2RB(ADSC-CSF2RB,intravenous injections of 5×105 cells every three days after the MI/R operation for a total of 7 times)or ADSC infected with adenovirus-control(ADSC-NC).MI/R+Vehicle group received intravenous injections of the same volume of cell-free vector 7 times within 3 weeks.At 3 weeks after MI/R,ADSC labeling and ex vivo fluorescence imaging were used to analyze the ADSC distribution in the heart,lung,liver,and spleen,cardiac function was detected by echocardiography,the density of myocardial neovascularization was detected by CD31 immunofluorescence staining,and myocardial cell apoptosis was detected by TUNEL staining.At 6 weeks after MI/R injury,echocardiography was used to detect cardiac function,and hemodynamic index such as:heart rate,systolic blood pressures,diastolic blood pressure,dP/dtmax and-dP/dtmax were collected.Masson staining was used to detect the myocardial fibrosis area,and ANP and BNP gene levels were detected by PCR.4.To investigate the role and mechanism of overexpression of CSF2RB in ADSC on migration,apoptosis,and paracrine.We verified the effects of overexpression of CSF2RB in ADSC on migration ability by Transwell assay and wound healing assay,and verified its anti-apoptotic ability by western blot assay and TUNEL staining.To investigate its paracrine effect,we collected supernatant from ADSC-NC cells and ADSC-CSF2RB cells to detect by western blot assay,capillary-like tube formation,transmission electron microscopy(TEM),and exosome extraction kit.Then the relevant regulatory factors were screened through RNA sequencing techniques,and the specific regulatory mechanisms were verified by siRNA,small molecule inhibitors,and other tools.Results1.Intravenous ADSC significantly improved cardiac function and reduced ventricular remodeling in MI/R mice.And we found that ADSC which were injected intravenously were mainly retained in lung tissue,with little distribution in myocardial tissue,spleen tissue and liver tissue.2.Compared with non-coronary volunteers,the expression level of CSF2 in plasmawith patients who underwent PCI of AMI was significantly increased.The expression level of CSF2 in plasma and myocardial tissue of MI/R mice was significantly increased after MI/R treatment than mice in the Sham group.3.(1)Compared with MI/R+ADSC-NC group,DiR signals and EGFP-positive cells in MI/R+ADSC-CSF2RB group were significantly increased in myocardial tissue,and it is no difference in lung,liver,and spleen.(2)Three weeks after MI/R,compared with MI/R+Vehicle group,the intravenous delivery of ADSC-NC did not significantly increase the cardiac function.In contrast,ADSC-CSF2RB treatment significantly increased cardiac function compared with those in the MI/R+Vehicle group.When we re-evaluated echocardiography at 6 weeks after MI/R(3 weeks after the last cell injections),we found that both ADSCNC and ADSC-CSF2RB significantly increased cardiac function.Importantly,the mice in the ADSC-CSF2RB group showed further improvement in cardiac function compared to those in the MI/R+ADSC-NC group.(3)Three weeks after MI/R,the capillary density in the infarcted heart areas was markedly decreased compared with that in sham hearts,as determined by CD31 immunofluorescence staining.After 7 ADSC-NC administrations,the capillary density was significantly increased in the ADSC-NC group and was further increased in the ADSC-CSF2RB group.TUNEL staining was performed to assess apoptosis in the peri-infarct regions of the hearts.ADSC-NC treatment significantly decreased the number of TUNEL positive cardiomyocytes compared to vehicle treatment,while ADSC-CSF2RB treatment further reduced the number of TUNEL positive cardiomyocytes.At 6 weeks after MI/R,Masson trichrome staining revealed that ADSC-CSF2RB group were more efficient in reducing the myocardial fibrotic area than ADSC-NC group.Moreover,cardiac remodeling was assessed at the molecular level.PCR showed that the mRNA levels of ANP and BNP were markedly downregulated by ADSC-NC treatment.Compared to ADSC-NC group,ADSC-CSF2RB group further decreased ANP and BNP mRNA expression.4.(1)Cell experiments showed that overexpression of CSF2RB could improve the migration capacity of ADSC and reduce the apoptosis induced by hypoxia/reoxygenation(H/R),but had no significant effect on the paracrine effect of ADSC.(2)Molecular biology experiments showed that the phosphorylation of signal transducer and activator of transcription 5(STAT5)was the key mechanism of CSF2RB in promoting ADSC migration and inhibiting ADSC apoptosis.STAT5-IN1,a STAT5 inhibitor,significantly weakened the migration promotion and antiapoptosis effect of overexpression of CSF2RB on ADSC.(3)RNA sequencing followed by PCR and western blots results showed that overexpression of CSF2RB increased the expression of ubiquitin-ligase RING finger protein 4(RNF4).Knockdown of RNF4 significantly reduced the phosphorylation level of STAT5 and weakened the migration promotion and antiapoptosis effect of overexpression of CSF2RB on ADSC.STAT5-IN-1 also attenuated RNF4 upregulation in ADSC with CSF2RB overexpression.Immunoprecipitation and immunofluorescence co-localization showed that RNF4 binds to and maintains STAT5 phosphorylation.Conclusions:1.Intravenous ADSC alleviate myocardial ischemia injury and enhance the antiremodeling effects.2.The expression level of CSF2 in plasma with patients who underwent PCI of AMI is significantly increased.The expression level of CSF2 in plasma and myocardial tissue of MI/R mice is significantly increased after MI/R treatment.3.Intravenously delivered CSF2RB-overexpressing ADSC enhance ADSC-mediated improvements in cardiac function and anti-remodeling effects in a long-term MI/R mouse model.4.CSF2RB overexpression increases the migration and anti-apoptosis of ADSC by increasing the response of the MSCs to a CSF2 gradient and CSF2RB-dependent STAT5/RNF4 activation without affecting their paracrine function.In conclusion,we demonstrate for the first time that CSF2RB overexpression optimizes the efficacy of intravenously delivered MSCs in the treatment of ischemic heart injury by increasing the response of the MSCs to a CSF2 gradient and CSF2RB-dependent STAT5/RNF4 activation. |
PDF Full Text Request