| Background:With the development of stem cell regenerative medicine, transplantation of bone morrow-derived mesenchymal stem cells (MSCs) into the infarcted heart after acute myocardial infarction (AMI) has emerged as a promising therapy for myocardial repair. However, poor engraftment of donor MSCs limits reparative capability of the therapy. Mobilization and migration of MSCs are mainly controlled by stromal cell-derived factor1(SDF-1) and its receptor CXC chemokine receptor4(CXCR4). Statins can increase the survival of MSCs. However, whether statins could enhance MSCs migration and engraftment is still unknown. Therefore, we designed the study to investigate whether atorvastatin (ATV) could enhance CXCR4expression of MSCs and promote them homing toward the injured myocardium.Methods:In vitro, MSCs from the Sprague-Dawley rats bone marrow (60-80g, male) were treated with different concentrations of ATV (0.01μM,0.1μM,1μM,10μM) different ATV culture durations (1h,3h,6h,12h,24h,36h,48h). For inhibitory studies, MSCs were incubated with CXCR4neutralizing antibody. Expression of CXCR4was evaluated using flow cytometry and real time PCR. A transwell system was used to assess MSCs migration. In vivo, female Sprague-Dawley rats were randomized into the following groups:SDF-1dynamic groups (AMI30min,1h,12h,24h,48h,72h,5d,7d), Sham group, AMI group, MSCs group, ATV-pretreated-MSCs group (ATV-MSCs). AMI was created by ligating the left anterior descending coronary artery.24h after AMI,2.0×106CM-Dil-labeled MSCs, CM-Dil-labeled ATV-MSCs, or PBS were injected in a total volume of500μL through the tail vein. Cardiac function, histology, inflammation cytokines were examined at3days and30days after MSCs transplantation.Results:Cell surface expression of CXCR4assessed by flow cytometry showed that ATV enhanced CXCR4expression in a dose-dependent manner, especially in the1μM ATV group (14.76±3.05%vs.1.98±0.40%, P<0.001). Then the time-course experiments at1μM ATV concentration revealed that, compared with the control group, CXCR4expression was significantly increased with ATV treatment (1to48h), peaking at12h (22.77±2.03%vs.2.20±0.18%, P<0.001) and maintaining at a high level within 24h (20.34±4.13%vs.2.20±0.18%, P<0.001). CXCR4mRNA expression showed the same tendency as the cell surface expression in each group. As expected, MSCs pretreated with ATV showed enhanced migration ability demonstrated by the increased number of cells migrating toward SDF-1compared with untreated MSCs (24.65±5.57vs.12.70±2.40, P<0.001). However, the effect was largely abolished by CXCR4neutralizing antibody, indicating that the benefit was mediated by CXCR4expression.In AMI model of Sprague-Dawley rats, we found much more ATV-pretreated MSCs homing toward the infarcted myocardium than non-treated cells (41.68±10.80vs.65.30±13.37, P<0.05). In addition, almost no MSCs were detected in the non-infarcted myocardium. At30days after cell injection, the survival rate in both ATV-MSCs and MSCs groups were low, fewer cells can be seen in the MSCs group. Compared with AMI group, left ventricular end-diastolic diameter and left ventricular ejection fraction were slightly improved in MSCs group. Cardiac performance was further improved in ATV-MSCs group than MSCs group, evidenced by increased left ventricular ejection fraction (62.28±3.27%vs.52.77±7.05%, P=0.014) and left ventricular fractional shortening (27.80±2.16%vs.22.27±3.84%, P=0.015). Histology analysis showed that compared with AMI and MSCs groups, the left ventricular fibrotic area was markedly reduced in ATV-MSCs group, and the inflammation was obviously attenuated. Western Blot analysis demonstrated that the expression of inflammation cytokines IL-6and TNF-a were decreased in ATV-MSCs group at3days and30days.Conclusions:ATV increases MSCs migration ability and improves cardiac performance due to up-regulated expression of CXCR4. These results suggest that ATV pretreatment of donor MSCs is an effective way to promote cell therapeutic potential for AMI. Background:Bone morrow-derived mesenchymal stem cells (MSCs) are the optimal candidate cells for acute myocardial infarction. However, the poor survival of the implanted cells because of the harsh environment hampered the therapeutic potential of the treatment. Tongxinluo (TXL), a traditional Chinese medicine, is widely used to treat cardiovascular diseases in China. Our previous study has demonstrated the pro-survival role of TXL on mesenchymal stem cells (MSCs) in vivo. But whether TXL could decrease apoptosis of MSCs in vitro and the underlying mechanism are still unknown. Moreover, AMPK/eNOS pathway is crucial in regulating cell apoptosis. Therefore, we designed the study to investigate whether TXL could decrease MSCs apoptosis under hypoxia and serum deprivation (H/SD) conditions and to determine the role of AMPK/eNOS pathway.Methods:MSCs from the Sprague-Dawley rats bone marrow (60-80g, male) were treated with TXL (50μg/mL,100μg/mL,200μg/mL,400μg/mL) for6h under H/SD conditions. For inhibitor studies, the cells were preincubated with AMPK inhibitor compound C (10μM) prior to the addition of TXL (400μg/mL). Cell apoptosis was assessed using Hocchst33342and TUNEL by Fluorescence microscope, Annexin V/PI by flow cytometry, mitochondrial membrane potential using fluorescent dye JC-1by flow cytometry, and apoptosis related protein cytochrome C, bax and bcl-2by western blot. The expression of AMPK and eNOS were measured by western blot.Results:Cell apoptosis was significantly upregulated under H/SD conditions compared with the normal (Annexin V+/PI-cells:21.91±3.28%vs.2.13±0.33%, P<0.001; JC-1red/green signal ratio:5.40±0.43vs.2.34±0.25, P<0.001)。TXL decreased the apoptosis level in a dose-dependent manner especially in the400μg/mL group, demonstrated by reduced Annexin V+/PI-cells (3.47±0.69%vs.21.91±3.28%in H/SD, P<0.001), increased ratio of JC-1red/green signal (4.78±0.37vs.2.34±0.25, P<0.001), decreased expression of bax(1.06±0.24vs.2.92±0.29, P<0.001) and cytochrome C, and incerased expression of bcl-2(2.59±0.15vs1.14±0.09, P<0.001). Further, TXL upregulated the phosphorylation of AMPK and eNOS. While, treatment with compound C decreased the phosphorylation of AMPK and eNOS and was accompanied by attenuated anti-apoptotic effect of TXL.Conclusions:TXL protected MSCs against H/SD-induced injury at least in part through the AMPK/eNOS signal pathway, which provides a novel explanation for the multi-effect of TXL on cardiovascular system. And TXL administration may be a useful method to improve MSCs survival for myocardial infarction patients. |
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