| Cardiovascular disease is a leading cause of human death worldwide. The prevalence ofcardiovascular disease is in a rising phase in China[1]. The adult myocardium has very limitedpotential of self-regeneration[2]. Current drugs or interventional treatments can only delay theprogression of heart failure but can not re-build the myocardium[3]. Stem cell based therapyemerges as a new strategy for cardiac repair[4]. Direct differentiation and paracrine effect arethe two main mechanisms for stem cell mediated therapeutic effect. Studies in recent yearshave shown that stem cell therapy can significantly limit ventricular remodeling and improvecardiac function (including left ventricular systolic function, diastolic function and coronaryflow reserve)[5,6]. Some studies suggest mesenchymal stem cells (MSC) can differentiate intocardiomyocytes and vascular cells both in vitro and in vivo, leading tomyocardialregeneration to some degree. However, other studies suggest that mesenchymal stem cells cannot differentiate into cardiomyocytes and vascular cells, and the therapeutic effect is due tothe indirect paracrine effect[7,8], which includes protection of ischemic myocardium,promotion of angiogenesis, and mobilization of endogenous stem cell. However, the relativecontribution of direct differentiation and paracrine in the stem cell mediated therapeutic effectis unclear.[9,10]Adipose-derived mesenchymal stem cell (ADSC) emerged as a preferable cellsource to treat myocardial infarction (MI)[8], due to its advantages over other MSC types:easy accessibility, minimal morbidity upon harvest, clinically relevant abundance withoutexpansion[11]. ADSC therapeutic effect of MI was significantly better than bonemarrow-derived stem cells[8]. The role of direct differentiation and paracrine mechanism inthe the ADSC treatment for myocardial infarction has not been reported. Recent studiesshowed that ADSC conditioned medium (CM) promotes endothelial cell survival andmigration in vitro[12]. However, Whether ADSC-CM and ADSC exert comparativetherapeutic effect for MI has not been defined. whether ADSC-CM protects the injuredcardiomyocytes and promotes angiogenesis is not reported Therefore, our study compared the therapeutic effects of ADSC and ADSC-CM for MI, explored the mechanism of ADSCparacrine anti-apoptotic effect on cardiomyocytes, and evaluated the relative contribution ofdifferentiation and paracrine effect on ADSC mediated therapeutic function.Methods:Primarily cultured human ADSCs (passage3-5) were characterized and used in theexperiments.1.8-10week-old male C57B/L mice(20-24g) were subjected to MI injury by ligation ofleft descending artery. The experimental groups include sham, MI+DMEM, MI+ADSC-CMand MI+ADSC(N=12). DMEM, ADSC-CM or ADSC were intramyocardially injected intoinfarct border zone after MI injury. To determine the therapeutic effect, the animal survival ratewas observed, myocardial infarcte size was measured by TTC staining, cardiac function wasevaluated by echocardiography, cardiomyocyte apoptosis was determined by TUNEL staining.2. An in vitro cell injury model was established by using neonatal rat ventricularmyocytes (NRVMs) with H2O2treatment, and the cardioprotective effect of ADSC-CM wasexamined,The experimental groups include H2O2, H2O2+DMEM and H2O2+ADSC-CM(N=5). The cardiomyocyte apoptosis was determined by caspase-3with western bloting andTUNEL staining.Result:1.The survival rate of DMEM groups was66.7%, the ADSC-CM groups was70.0%.The ADSC groups was72.7%, It’s has no significant difference between the three groupssurvival group (P>0.05), suggesting that the injection of ADSC-CM or ADSC can’timprove the survival rate of MI mice.2. Compared with the control group, ADSC and ADSC-CM can reduce infarct size.(DMEM:41.9±3.0%; ADSC:32.2±2.8%; ADSC-CM:35.7±2.5%; p <0.05), but nostatistically significant difference between the ADSC and ADSC-CM3. Four weeks after MI,the ultrasound results show no significant change before andafter the sham-operated mice heart function after MI. the control mice after myocardialinfarction (EF:43.8±3.7%, FS:19.0±3.8%) compared with the sham group (EF:90.8±5.4%FS:45.6±6.8%, p <0.05) was significantly reduced. Intramyocardial injection ofADSC-CM improved the cardiac function compared with the control group(EF:60.4±5.8%, FS:27.6±3.5%). ADSC injection further improved the cardiac function compared withADSC-CM injection (EF:69.2±4.3%; FS:34.3±2.5%).4. Angiogenesis in the the infarct border zone4weeks after MI. The vascular density ofADSC and ADSC-CM treated hearts have greater angiogenesis compared with the controlgroup, but there is no significant difference between the two treating groups.5. Compared with the control group, H2O2damage increased caspase-3expression levelsin cardiomyocytes. ADSC-CM pretreatment significantly blocked the increased caspase-3expression. TUNEL staining also showed that: ADSC-CM pretreatment significantly reducedthe TUNEL-positive rate induced by H2O2.6. The cardiomyocyte apoptosis in the infarct border zone2days after MI. The numberof TUNEL-positive cardiomyocytes is significantly reduced by ADSC or ADSC-CMtreatment compared with the control group, but no significant difference between the twotreating groups.7. The survived ADSCs were found4weeks after cell transplantation GFP tracking.ADSCs do not express tropomyosin or α-actinin. Some ADSCs were demonstrated to expressvWF or SMA, indicating that some injected cells differentiated into endothelial cells andsmooth muscle cells.8. ADSC-CM or ADSC promote angiogenesis in the infarct border zone4weeks afterMI. ADSC increased the angiogenesis density by64.3%compared with control group.Neovascularization CD31and GFP double positive cells account for3.4±0.7%. of the totalangiogenesis. Direct differentiation plays a minor role in ADSC mediated therapeutic effect(<10%), and paracrine effect dominate the therapeutic effect (>90%).Conclusion:ADSC-CM can reduce infact size and promote cardiac function, similar to the effectinduced by ADSC transplantation. ADSC can differentiate into vessel cells thereby promoteangiogenesis by forming new cells, but this effect is minor and paracrine effect dominate theADSC mediated therapeutic effect. |
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