| Objectives:Mesenchymal stem cells (MSCs) have been the optimal candidate cells for cardiovascular regeneration in recent years; however, the poor survival of the implanted cells because of the harsh environment, hampered the progress of the treatment. Our previous studies have proven that Statins could enhance the survival of implanted bone marrow MSCs, but the exact mechanism remained to be investigated. There has been evidence demonstrating that Statins could protect stem cells from apoptosis. AMP-activated protein kinase (AMPK), Phosphatidylinositol-3kinase/Protein kinase B (PI3K/Akt) pathway or endothelial nitric oxide synthase (eNOS) may play a role in such process. Based on these, we hypothesized that Atorvastatin could protect MSCs from H/SF induced apoptosis and investigated the internal mechanism and pathway.Methods:Chinese mini-swine's bone marrow derived MSCs cultured in vitro exposed to hypoxia and serum-free (H/SF), different Atorvastatin concentrations (0.001μM-10μM), Compound C (10mM), Atorvastatin+Compound C, LY294002(50μM) or Atorvastatin+LY294002. Cell apoptosis was assessed using Annexin V/Propidine Iodine kit by flow cytometry. Bax, Bcl-2, phosphorylation of AMPK, Akt and eNOS was tested with Western blot. Real time-PCR was performed to analyze the gene expression of AMPK, Akt and eNOS.Results:Flow cytometry showed that Atorvastatin (000O1μM-10μM) inhibited apoptosis of e bone marrow derived MSCs cultured in H/SF condition; which could be blocked by compound C, an inhibitor of AMPK, but not the PI3K inhibitor-LY294002. The similar trend was observed as to Bax protein expression by Western blot; whereas the Bcl-2protein, an anti-apoptosis protein, increased in Atorvastatin group. The gene expression of AMPK, Akt and eNOS increased in Atorvastatin. Meanwhile, phosphorylation of AMPK and eNOS increased in MSCs treated with Atorvastatin, Phosphorylation of eNOS correlated with AMPK phosphorylation (r=0.599, P=0.004), which could be inhibited by compound C as well. Conclusions:Atorvastatin can protect MSCs from H/SF induced apoptosis through AMPK pathway, which resulting in activation of eNOS. Meanwhile, the role of P13K/Akt was still not confirmed. Objectives:To study whether Atorvastatin combined with Mesenchymal stem cells (MSCs) transplantation can improve cardiac function recovery and survival of implanted cells in swine acute myocardial Infarction (AMI) model. Using NOS inhibitor-L-NNA to evaluate the role of NOS/NO system in the process.Methods:42Chinese mini-swine were divided into6groups (n=7), including:Sham operation (Sham), AMI control (AMI), Atorvastatin alone (Ator), MSCs transplantation alone (MSCs), Atorvastatin+MSCs transplantation (Ator+MSCs) and Atorvastatin+MSCs transplantation+L-NNA (Ator+MSCs+L-NNA). Ator (0.25mg/kg-d) or L-NNA (5mg/kg-day) was fed from3days before to4weeks post-transplantation. Serum lipids, High sensitivity C-reactive protein (Hs-CRP), NO concentration and constructive NOS (cNOS) activity were detected before drug administration (baseline) and animal sacrificed (end point).AMI/Reperfusion model was created by ligating the left anterior descending artery90minutes. Then DAPI/DiI labeled autologous bone marrow MSCs (3×107cells per animal,1mL) were injected into peri-infarct myocardium30minutes after reperfusion. At the first week (baseline) and fourth week (end point), the data of infarction size, viable myocardium and cardiac function were obtained by Single photon emission computed tomography (SPECT), Positron emission tomography-computed tomography (PET-CT) and Magnetic resonance imaging (MRI). At the end point, performing pathological tissue staining to evaluate the infarct size, inflammatory cell infiltration and myocardial fibrosis. The cellular apoptosis in the peri-infarction region was detected with TUNEL assay. The survival and the expression of cardiac specific proteins such as Cardiac troponine-T (CTn-T) and Connexin-43(Cx-43) of implanted cells in vivo were detected by immunofluorescent staining.Results:Serum lipid level were not significantly different at both baseline and end point (P>0.05). In Ator+MSCs group, Hs-CRP decreased significantly (-0.23±0.007μg/L, P=0.0002),and cNOS activity significantly increased at endpoint(18.32±3.89U/mL, P=0.001).Baseline SPECT and PET-CT scan showed that the area of myocardial infarction was not significantly different among all groups (P>0.05). At endpoint, Atorvastatin+MSCs had the minimum infarction size and proportion (-7.00±3.16cm3, P=0.0001;-3.00±1.14%,P=0.0004), with increased viable myocardium (SUV)(3.42±1.16, P=0.043),which were not different in MSCs or Ator group compared with AMI control(P>0.05). Baseline MRI indicated that there were no significant differences in cardiac function among groups (P>0.05). At end point, MSCs or Ator group was not significantly different with AMI control group (P>0.05). The LVEF, LVCO and LVCI remarkably increased in the Ator+MSCs group compared with AMI control group (14.22±12.85%, P=0.019;0.70±0.45L/min, P=0.036;0.80±0.49L/min·m2, P=0.013). However, the effect disappeared in Ator+MSCs+L-NNA group. H&E and Masson's Trichrome staining showed that there were extensive fibrosis and serious inflammatory cell infiltration with poor surviving myocardium within infracted regions in AMI control group. Fibrosis and inflammation were slighter in MSCs and Ator group. Furthermore, inflammation and fibrosis were significantly decreased in the Ator+MSCs group. At end point, the apoptotic index was remarkably decreased in Ator+MSCs group (2.8±0.8, P<0.05). The survival of implanted MSCs in the group was significantly more than that in MSCs or Ator+MSCs+L-NNA group (88.1±8.6/HPF, P=0.000). Some of the implanted cells expressed cardiac specific proteins, such as CTn-T and Cx-43, there was no significant difference among groups.Conclusion:1.Intramyocardial injection of MSCs after AMI and reperfusion resulted in limited survival of implanted cells, without significant benefits in cardiac function;2.Atorvastatin could significantly improved survival of implanted cells, accompanying by significant decrease of infarction size and benefits in cardiac function;3. The effect of Atorvastatin improving the quality of local microenvironments, facilitate the survival of implanted cells and improving the results of stem cell therapy is associated with NOS/NO system.
|
PDF Full Text Request