Therapeutic Effect Of Drugs On Myocardial Stem Cells In The Treatment Of Acute Myocardial Infarction

Background:The therapeutic potential of stem cell therapy for cardiac repair is hampered partly due to their low survival rate and low engraftment rate within the ischemic myocardial.The interaction between stromal cell-derived factor 1(SDF-1)and its receptor chemokine CXC motif receptor 4(CXCR4)plays a vital role in the migration of cardiac stem cells(CSCs)in the injured myocardium.While PODXL(podocalyxin-like protein 1,podocalyxin)is a marker on the cell membrane of CSCs and was confirmed to play an important role in cell adhesion.This study aims 1)to investigate the effect of Atorvastatin(ATV)on the apoptosis of cardiac stem cells,and to determine the underlying pathway;2)to investigate whether the combination of loading dose of ATV and ATV-pretreated CSCs can promote the migration,adhesion and transendothelial engraftment of CSCs and enhance the cardiac performance in acute myocardial infarction(AMI)through improving the expression of CXCR4 and PODXL on the surface of the CSCs.Methods:Mouse CSCs were separated and cultured with the treatment of ATV for 12 h under hypoxia and serum deprivation(H/SD)conditions.Apoptosis was detected by flow cytometry and Hoechst 33342 staining.Western blotting was used to detect the expression of the phosphorylation of AMPK and mTOR.Then CSCs were pretreated with different concentrations of ATV for a series of lengths of time.Expression of CXCR4 and PODXL was evaluated by flow cytometry.A transwell system was used to assess CSCs migration ability.Cell adhesion assay and transendothelial migration assay were used to detect the adhesion and transendothelial migration ability of CSCs.In vivo,320 female C57 mouse were randomly assigned to sham,AMI,ATV,CSCs,ATV-CSCs,ATV+ATV-CSCs,ATV-PODXL-SiRNA-CSCs,ATV-CXCR4-SiRNA-CSCs groups.CSCs or PBS were injected through the jugular vein 1 week after AMI.Cardiac functions were assessed using echocardiography at baseline(3 days after AMI)and endpoint(4 weeks after CSCs injection).Recruitment of CSCs to the infarcted heart,inflammatory factors and fibrosis were evaluated with histopathology and immunofluorescence.Results:ATV decreased the apoptosis of CSCs concentration-dependently.Furthermore,ATV significantly enhanced the phosphorylation of AMPK,while the anti-apoptotic effect was attenuated by the AMPK inhibitor compound C.Cell surface expression of CXCR4 and PODXL assessed by flow cytometry showed that ATV enhanced CXCR4 and PODXL expression in a dose-dependent manner,especially in the 1?M ATV group,peaking at 12 h.As expected,CSCs pretreated with ATV showed enhanced migration ability demonstrated by the increased number of cells migrating toward SDF-1 compared with untreated CSCs.However,the effect was largely abolished by CXCR4 neutralizing antibody,indicating that the benefit was mediated by CXCR4 expression.Also,CSCs pretreated with ATV showed improved adhesion and transendothelial migration ability,which was remarkably inhibited with the addition of PODXL-siRNA,indicating that these effects were mediated by PODXL.In vivo,administration of loading dose of ATV combined with ATV-pretreated CSCs can increase the recruitment of CSCs and improve the cardiac performance.This may attribute to the inhibition of inflammation,apoptosis and fibrosis,the upregulation of c-kit+ stem cells and the promotion of arteriogenesis and angiogenesis.Conclusions:ATV inhibits H/SD-induced apoptosis in CSCs in concentration dependent manner,possibly linked to the activation of AMPK/mTOR pathway.Administration of ATV-pretreated CSCs can increase the recruitment of CSCs and improve the cardiac performance by targeting CXCR4 and PODXL.These results suggest that combined use of loading dose of ATV and ATV pretreatment of donor CSCs is an effective way to promote cell therapeutic potential for AMI.Background:Atorvastatin(ATV)has an important pro-survival role in cardiomyocytes after acute myocardial infarction(AMI).AMP-activated protein kinase(AMPK)has an important effect on mediating energy metabolism and cell survival.It could suppress mammalian target of rapamycin(mTOR)activation,which could regulate apoptosis and autophagy.The objectives of this study were to:1)determine whether ATV could affect autophagy of cardiomyocytes via the AMPK/mTOR pathway,and 2)investigate the balance between autophagy and apoptosis pathways.Methods:Male Wistar rats(n=100)weighing 200-220g were randomly divided into sham,control,ATV,compound C,and ATV+compound C groups.The MI model was established in all rats except the sham group by ligating the left anterior descending coronary artery(LAD).Sham operations were performed by passing a suture around the LAD without ligation.ATV treatment(10mg/kg/d)was given orally from induction of MI by surgical ligation till 4 weeks after AMI,and compound C(20mg/kg/w)was intraperitoneal injected from induction of MI till 4 weeks after AMI.Transthoracic echocardiography measurements were taken 3 days and 4 weeks after AMI induction to detect the change of cardiac function.The fibrosis,infarcted area,and inflammatory level were determined by pathological and histological studies.The expression of apoptotic,autophagic,and AMPK pathway proteins was detected by immunohistochemical staining and Western blot.Results:Transthoracic echocardiography showed that the ejection fraction in the ATV group increased over the control group 4 weeks after AMI.Histological analysis was conducted 4 weeks after AMI.There was more surviving myocardium,less fibrotic area,and accumulation of extracellular matrix in the peri-infarct heart in the ATV group than in the other groups.Also,ATV treatment increased the expression of autophagic protein LC3 in infarcted myocardium and inhibited the expression of autophagic protein Bax,thus promoting autophagy and inhibiting apoptosis.AMPK inhibitor compound C reversed these beneficial effects.The phosphorylation of AMPK was upregulated and the phosphorylation of p53 and mTOR was down-regulated in ATV-treated rats compared to sham-treated rats.In contrast,the levels of total AMPK and mTOR were not different between the five groups.Compound C reduced the phosphorylation of AMPK dramatically and abrogated the effects of ATV on the down-regulation of p53 and mTOR phosphorylation significantly.Conclusions:ATV improves survival of cardiomyocytes and decreases alterations in morphology and function of infarcted hearts 4 weeks after AMI by inducing autophagy and inhibiting apoptosis through the activation of AMPK/mTOR pathway.Background:Cardiac microvascular endothelial cells(CMECs)regulate the microvascular function,modulate autocrine and paracrine function and barrier protection of cardiomyocytes in ischemia/reperfusion injury(I/R).In contrast to cardiomyocytes,CMECs are more sensitive to I/R.Tongxinluo(TXL)is a Traditional Chinese Medicine compound which is constituted of 12 kinds of natural products.In this study,we used the TMT-labeled proteomics to detect the various proteins in an in vitro model of cardiac microvascular endothelial cells(CMECs)suffering ischemia/reperfusion injury.Our aims are to investigate whether TXL could modulate protein expression of CMECs,and to synthetically analysis the underlying mechanism of the regulation.Methods:Human CMECs were subjected to 2 h of hypoxia followed by 2 h of reoxygenation with different concentrations of TXL.Apoptotic rates of the cells were measured to determine the optimal concentration.Protein expression profiles of CMECs were determined using tandem mass spectrometry.We evaluated several proteins with altered expression in I/R injury and summarized some reported proteins related to I/R injury through GO analysis.Also,We chose the differential proteins including HMOX1 and ANGPT2 to further verify the cell migration,tube formation and cell proliferation functions of differential proteins through inhibition or overexpression,respectively.Results:TXL concentration-dependently decreased apoptosis of CMECs.The optimal concentration of TXL was 800?g/mL.We found 50 differential proteins in CMECs were connected with ischemia reperfusion injury and described involved functions and pathways.I/R significantly changed the cytokines level of CMECs,meanwhile TXL treatment modulated the cytokines secretion ulteriorly.30 differential proteins between group N and group HR were detected.TXL treatment up-regulated 6 and down-regulated 5 in ischemia/reperfusion injury.These kind of protein mainly had vital functions including cell proliferation,stress response,regulation of multicelluler organismal metabolic process.We evaluated several proteins played important roles in ischemia/reperfusion injury including human heme oxygenase 1(HMOX1),angiopoietin 2(ANGPT2).TXL significantly inhibited the apoprosis of CMECs and promoted the cell proliferation,cell migration and tube formation under I/R.However,these effects were largely blocked by HMOXl inhibitor TiiPPIX or overexprssion of ANGPT2.Conclusion:TMT-labeled proteomic provides a way to study ischemia reperfusion injury comprehensively at molecular level.We identified 50 differential proteins associated with I/R and analysis their functions and pathways.It suggested that TXL may be protective for microvascular endothelial cells against ischemia reperfusion injury through inhibition of inflammatory chemokines secretion,reducing oxidative stress factors,and regulation of multicellular organismal metabolic process.