| Background and Objective:Myocardial infarction (MI) is one of the leading causes of death and the ischemicinjury is usually irreversible despite aggressive medical and revascularization treatment.Cardiac myocytes have been traditionally regarded as terminally differentiated cells thatadapt to increased work and compensate for diseases exclusively through hypertrophy.Recent studies have shown that the resident cardiac stem cells (CSCs) exist in heart and candifferentiate into cardiomyocytes, smooth muscle cells or endothelial cells， andtransplantation of CSCs can reduce infarcted size and enhance heart function, representinga new strategy for cardiac repair. But CSCs are scattered throughout the myocardium, andconcentrated relatively in the atria and apex and are less numerous at the base andmid-portion of the left ventricle. It is still not fully understood how these CSCs arerecruited into the injured myocardium after MI.Up to now, stromal cell-derived factor-1α (SDF-1α) and its cellular receptor CXCR4（C-X-C chemokine receptor type4） are known as the most prominent stem cellchemotaxis. SDF-1α has been shown to be significantly upregulated in myocardialinfarction and attract the CXCR4+stem cells towards SDF-1α gradient. Thus, upregulationof SDF-1α expression in ischemial myocardium represents a promising therapeutic strategyto improve post-infarction therapy. However, the role of SDF-1α/CXCR4pathway in CSCsmigration needs to be further clarified.Phosphatidylinositol3-kinase (PI3K) is widely expressed and plays crucial roles inregulating multiple cell processes, such as cell migration, proliferation, differentiation,motility, survival and angiogenesis. Previous studies have demonstrated PI3K is alsoinvolved in SDF-1α/CXCR4-mediated chemotaxis and multiple stem/progenitor cellsmigration.The purpose of this study was to address the following questions:1) whetheroverexpression of SDF-1α resulted in CSCs migration and accumulation in the infarcted region, and2) whether SDF-1α-induced CSCs migration was mediated via CXCR4/PI3Ksignaling pathway.Materials and Methods：1. rAAV1-SDF1α-eGFP vector was contructed through three plasmids co-transfectionsystem.First, SDF-1α cDNA fragments combining with3bp stop codons were synthesizedand inserted into the cloning sites between Sal Ⅰ and Bgl Ⅱ in plasmidpSNAV2.0-mCMV-eGFP to construct plasmid pSNAV2.0-SDF1α-eGFP. The recombinantplasmid was determined by PCR, restriction enzyme digestion and gene sequencing. Andthen, the recombinant plasmid pSNAV2.0-SDF1α-eGFP was co-transfected into HEK293cells with the control plasmid pAAV-R2C1and pHelper in AAV Helper-Free System forpackaging recombinant AAV. The recombinant AAV was determined by detecting GFP andSDF-1α expression in infected cardiomyocytes (at a MOI of105) by immunoflorescence.2. Neonatal mouse heart fragments were cultured in complete explants medium andc-kit+CSCs were isolated from it by magnetic activiated cell sorting (MACS) andamplified in cardiosphere-growing medium. Cells phenotypes were analyzed byfluorescence-activated cell sorting (FACS) by co-stained with phycoerythrin(PE)-conjugated antibodies against CXCR4, Sca-1or CD45, and FITC-conjugatedantibodies against c-kit. The purified c-kit+CSCs were determined by immunofluorescenceusing a c-kit antibody. The multiple differentiation potency of c-kit+cells were determinedby immunofluorescence with specific antibodies for cardiac troponin I （cTn-I）,cardiomyocyte specific protein, α smooth muscle actin (α-SMA), smooth muscle cellspecific marker, and von Willebrand factor（vWF）, the endothelial specific marker afterconditioned culture.3. The in vitro chemotaxis effect of SDF-1α on CSCs was assessed using Transwellchambers. Cardiac myocytes were seeded on lower chambers and infected withrAAV1-SDF1α-eGFP at a MOI of105. c-kit+CSCs were added into the upper chambers.For inhibition experiments, CSCs were pre-incubated with AMD3100(10μg/ml, CXCR4antagonist) or LY294002(20μmol/L, PI3K inhibitor) for1h before seeding. The migratedcells were stained by DAPI and counted under five random high-power scopes.The SDF-1αprotein concentration in lower chamber supernatant was determined by ELISA assay4. For in vivo CSCs homing assays, rAAV1-SDF1α-eGFP and DAPI-labeled c-kit+ CSCs were injected into infarcted zone and infarcted border individually. Mouse MI modelwas established by ligation of left anterior descending coronary artery (LAD). The micewere divided into6groups:1. Sham group: the mice chest was opened but without LADligation.2. MI group: LAD ligation but without other treatment.3. Control (Empty) group:50μl empty vector rAAV1-eGFP and1×105DAPI-labeled c-kit+CSCs in50μl saline wereinjected.4. SDF-1α group:50μl rAAV1-SDF1α-eGFP and1×105DAPI-labeled c-kit+CSCs were injected.5. AMD3100and LY294002group: rAAV1-SDF1α-eGFP injection butCSCs were pre-incubated with AMD3100(10μg/ml) or LY294002(20μmol/L) for1hbefore injection. Three weeks later, immunofluorence was performed in infarcted regions todetect the engraftment of DAPI-labeled cells. Ten confocal images were counted forengraftment assay. The expression of SDF-1α in infected zone was determined by westernblot in3weeks. The ratio of OD value of each group with sham group served as theintensity of SDF-1α expression.5. In vivo differentiation of transplanted CSCs was tested by immunohistochemistryusing cTn-I antibody after21days. The differentiated CSCs were double positive for DAPIand cTn-I. Ten confocal images were counted for differentiation assay. The differentiationpercentage of migrated c-kit+CSCs into infracted zone was caculated as the percentage ofcTn-I-positive nuclei vs total labelled nuclei.6. At day21post-MI, the infarcted size was measured by triphenyltetrazoliumchloride (TTC) staining. The infarcted myocardium and the non-ischemic zones weredivided and weighed. The infarction proportion was counted by ratio of infarcted mass/totalheart.7. For proliferation analysis of transplanted c-kit+CSCs, CSCs were incubated withBrdU for24hours before being transplanted. rAAV1-SDF1α-eGFP injection and CSCstransplantation were operated as above. Three weeks later, BrdU was detected byimmunofluorescent using a monoclonal antibody in border zones and infarcted region andthe tissuse was stained by DAPI. Proliferation of transplanted CSCs was determined byBrdu/DAPI double staining. The proliferation percentage of transplanted c-kit+CSCs wascaculated as the percentage of BrdU-positive nuclei vs total DAPI-stained nuclei.Result：1. The PCR and restriction enzyme digestion could amplify an about270bps band, consistent with the cDNA length of SDF-1α. Gene sequencing showed that the insertedsequence was fully consistent with the SDF-1α cDNA. In cardiomyocytes infected withrAAV1-SDF1α-eGFP, almost all cells expressed GFP. Furthermore, SDF-1α could bedetected in infected cardiomycytes by an anti-SDF-1α antibody and TRITC-conjugatedsecond antibody under fluorescent microscope. These results manifested that rAAVconstruction was successful and infection was effective.2. The immunofluorescence results indicated that the purified c-kit+cells by MACSisolation were c-kit+positive and expresse cTn-I, α-SMA, and vWF after differentiationinduction. The FACS results indicated c-kit+cells accounted for8.3%of the total unsortedcells. A few cells were double positive for c-kit/sca-1(1.05%±0.16%), c-kit/CD45(0.87%±0.12%) and c-kit/CXCR4(5.62%±0.54%).However,97%cells were c-kit positiveafter MACS isolation, and most c-kit+cells expressed CXCR4(91.47%±7.73%) and a fewc-kit+cells expressed CD45(4.54±1.79%) and Sca-1(2.58±1.16%)（n=3）.3. The in vitro migration results showed that the cardiomyocytes infected withrAAV1-SDF1α-eGFP significantly increased SDF-1α concentration5-fold more insupernatant than that in the control group (10.27±1.8ng/ml vs.2.23±0.67ng/ml, p<0.01, n=3)by ELISA assays and subsequently attracted more CSCs migration(48.3±7.6vs.21.3±6.1,p<0.01, n=3). This effect was diminished by administration of AMD3100(29.7±6.0vs.48.3±7.6, p<0.05) or LY294002(26.0±5.6vs.48.3±7.6, p<0.05).4. Western blot revealed that there was no difference of SDF-1α expression ininfarcted zone between MI and Empty group (1.20±0.19vs.1.30±0.17, p>0.05). However,infection with rAAV1-SDF1α-eGFP significantly increased SDF-1α expression in infarctedmyocardium but there was no difference among SDF-1α, AMD3100and LY294002groups(2.22±0.38vs.2.08±0.25and2.02±0.31, p>0.05, n=3).In myocardial infarction mice,overexpression of SDF-1α in the infarcted area resulted in more CSCs retention to theinfarcted myocardium (SDF-1α vs. Empty,30.3±6.5vs.10.3±2.5, p<0.01n=6), which wasattenuated by AMD3100(8.7±1.5, p<0.01vs. SDF-1α) or ly294002(8.0±2.0, p<0.01vs.SDF-1α) pretreatment.5. In vivo differentiation assay of transplanted CSCs in infarcted zone indicated thatsome DAPI-labeled CSCs were positive for cTn-I, suggestting differentiation of homingCSCs into cardiomyocytes. However, we found that there was no difference of CSCs differentiation percentage among4groups in infarcted zone.6. TTC staining revealed empty vector infection didn’t improve infarcted size (Emptyvs. MI group,30.0±4.0%vs.27.3±3.2%, p>0.05, n=6). However, rAAV1-SDF1α-eGFPinjection resulted in reduced infarcted size (17.3±2.5%, p<0.05vs. MI group, n=6). Thiseffect was abolished by AMD3100pretreatment (24.0±3.0%, p<0.05vs. SDF-1α group,n=6) or LY294002pretreatment (25.3±3.1%, p<0.05vs. SDF-1α group, n=6).7. Immunofluorence staining showed more BrdU positive cells in the infarctedregions in SDF-1α group than Empty group (SDF-1α vs. Empty,28.8±1.5vs.8.9±1.8,p<0.01n=6)(Fig.4). However, the cells number decreased in AMD3100(7.7±1.7, p<0.01vs. SDF-1α) and LY294002group (7.9±0.9, p<0.01vs. SDF-1α). There was no differencein border zone among all the groups.Conclusions：1. rAAV1-SDF1α-eGFP vector was conctructed successfully.2. c-kit+CSCs were isolated successfully and displayed multiple differentiationpotential after differentiation induction.3. In vitro rAAV1-SDF1α-eGFP infection increased SDF-1α concentration insupernatant and attracted more CSCs migration which was diminished by administration ofAMD3100or LY294002, suggesting CXCR4/PI3K involving in SDF-1α-promoted CSCsmigration.4. Overexpression of SDF-1α in the infarcted area resulted in more CSCs retention tothe infarcted myocardium, a higher percentage of proliferation and reduced infarcted areawhich was attenuated by AMD3100or ly294002pretreatment, indicating that SDF-1αenhanced engraftment of transplanted CSCs and reduced infarcted size via CXCR4/PI3Kpathway. |
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