| Ischemic heart disease is the leading cause of death in developed countries and carries significant morbidity. After an acute myocardial infarction (MI), the heart has limited capacity for self-renewal and undergoes remodeling with resulting depressed left ventricular function. Attempts have been made to restore myocardial infarction using several different cell types to determine the best cell type for cardiac regeneration. Mesenchymal stem cells (MSCs) were first identified and isolated from bone marrow more than40years ago and have emerged as one of the leading candidates in cellular cardiomyoplasty. The unique properties of MSCs (easily isolated and amplified from the bone marrow, immunologically tolerated as an allogeneic transplant, and their multilineage potential) have led to their intense investigation as a cell-based therapeutic strategy for cardiac repair.The promising therapeutic effect of MSCs is dependent on their capacity to survive and engraft in the target tissue. However, the transplantation of as many as6×107of these putative MSCs into infarcted porcine hearts yielded only marginal improvement in cardiac function. Toma et al. further reported that less than0.44%MSCs survive by day4after engraftment in an immunodeficient mouse heart model. Hence, it is imperative to reinforce the stem cells to withstand the rigors of the microenvironment of the infarcted heart incurred from ischemia, inflammatory responses, and pro-apoptotic factors in order to develop an effective therapeutic modality.Plasma levels of high density lipoprotein cholesterol (HDL-C) are a well known negative risk factor for the development of cardiovascular disease (CVD). A widely accepted basis for the inverse relationship between human plasma HDL-C and CVD is the ability of HDL, and its major protein constituent apolipoprotein (apo)A-I, to mediate reverse cholesterol transport (RCT). However, recent studies have provided tantalizing clues that HDL might be more than it appears. The application of mass spectrometry (MS) based proteomic approaches has revealed unexpected diversity in the HDL proteome. Interestingly, only about one-third of HDL proteins are known to mediate lipid transport. The rest play roles in such areas as protease inhibition, complement regulation and acute phase response. This suggests that HDL has other broader functions, including anti-inflammatory, anti-oxidant, anti-apoptotic properties. It suggests that HDL might be a platform that mediates protection from a host of disease states ranging from CVD to diabetes, and to other diseases concerning with apoptosis.Based on the above knowledgement, we wonder whether HDL plays a critical role in the cardiac repair through influencing the biological properties of MSCs. In our study, we will investigate the role and mechanism of HDL in the proliferation, migration, paracrine and anti-apoptotic capacity of MSCs, and will further examine the efficacy of the transplantation of MSCs preconditioned with HDL on the myocardial infarction model of rats. The results of this study are expected to provide clinical practice, particularly for those suffer from ischemic heart diseases, with experimental evidences. Objectives To investigate the effect of HDL on the proliferation and the migration of mesenchymal stem cells (MSCs), and to elucidate the molecular mechanisms involved.Methods MSCs derived from rats was treated with HDL in different concentrations or for different periods. The proliferation of MSCs was measured with MTT and BrdU assay, and the migration rate was detected by scarification test. The expressions of p21and phosphorylation of Akt, ERK1/2were evaluated by Western-blot. The activities of pathways were down-regulated by the respective specific inhibitors, and the gene of Scavenger Receptor-B Type Ⅰ (SR-BI) was knocked down by RNA interference to identify their roles in the proliferation of MSCs induced by HDL.Results We found that HDL did not influence the migration of MSCs, while promoted MSCs proliferation in a time-and concentration-dependent manner, in which the phosphorylation of Akt, ERK1/2were up-regulated and the level of p21was down-regulated. When MSCs was preconditioned with the specific inhibitor to respective pathways, the decrease of p21induced by HDL was significantly attenuated compared with that without preconditioning (LY294002:1.03±0.16vs.0.691±0.13,p<0.05; U0126:1.68±0.17vs.0.76±0.15,p<0.05). SR-BI contributed to HDL-induced proliferation of MSCs, which was effectively abolished by the knock-down of SR-BI. Compared with that in respective PBS-treatment group, the MSCs transfected with mock siRNA displayed a notable higher BrdU incorporation rate in HDL-treatment group (0.98±0.16vs.1.57±0.23,p<0.05), while the MSCs transfected with SR-BI siRNA in HDL-treatment group showed no change (1.08±0.15vs.1.08±0.14,p>0.05).Conclusions HDL improves the proliferation of MSCs in a time-and concentration-dependent manner through the activation of PI3K/Akt, MAPK/ERK1/2pathwavs and binding SR-BI receeptor. Objectives The effect of HDL on the secretome of MSCs has not been well elucidated yet. The aim of this study was to examine the hypothesis that preconditioning with HDL promoted MSCs secretome, subsequently protecting cardiomyocytes from apoptosis induced by hypoxia.Methods MSCs derived from bone marrow of rats were preconditioned with HDL in various concentrations or for different periods. Afterwards, the mRNA of some soluble factors (including VEGF, bFGF, HGF, TGF-β2) in MSCs was analyzed by real-time PCR, and the expression levels of above factors in supernatant were analyzed by ELISA. Cardiomyocytes of neonatal rats were exposed to hypoxia for12hours, incubated in culture medium which were respectively added with the supernatant filtered from MSCs and HDL cultured system (MSCs+HDL), from MSCs alone cultured medium (MSCs), or the same terminal concentration of HDL (HDL) or PBS (Positive Control). Apoptosis of cardiomyocytes was evaluated by TUNEL assay and Western-blot. RNA interference was administrated to confirm the effect of soluble factors on the cardiomyocytes apoptosis induced by hypoxia, in which flow cytometry was addressed to evaluate apoptosis rate.Results When MSCs were exposed to HDL, the expression of TGF-β2, both mRNA level in MSCs and protein level in supernatant, increased significantly in a time-and concentration-dependent manner. Group(MSCs+HDL) exhibited remarkable lower apoptosis rate compared with Group(Positive Control), Group(HDL) and Group(MSCs)[TUNEL:(10.23±5.68)%vs.(28.36±4.98)%, vs.(16.38±5.38)%, vs.(17.82±6.24)%, p all<0.05; bcl-2/bax:(0.846±0.16) vs.(0.156±0.03), vs.(0.411±0.10), vs.(0.346±0.08), p all<0.05]. Knocked down the TGF-β2gene, Group (siRNA) showed a significant higher apoptosis rate compared with Group(MSCs+HDL)[(10.28±1.33)%vs.(5.34±0.67)%,p<0.05].Conclusions TGF-β2released by mesenchymal stem cells preconditioned with high density lipoprotein protects cardiomyocyte from injury of hypoxia. Objectives The aim was to explore the effect of HDL on the anti-apoptotic character of MSCs, and to clarify the molecular mechanism involved.Methods Hydrogen peroxide (H2O2) was used to induce apoptosis of MSCs to develop the oxidative stress model in vitro. MSCs was treated with H2O2in different concentrations (0,20μM,50μM,100μM and200μM) for1hour. A Hochest33342assay was addressed to observe the apoptosis of MSCs. Afterwards, MSCs was randomly divided into four groups:preconditioned with HDL (100μg/ml) for24hours followed by injury with H2O2(100μM) for1hour (H2O2+HDL), only preconditioning without injury(HDL), only injury without preconditioning(H2O2), and neither preconditioning nor injury(Control). The accumulation of reactive oxygen species (ROS) in MSCs was detected by ROS probe, and the apoptosis of MSCs was analyzed by TUNEL assay and Western-blot. A Bio-Plex chip was applied to screen the changes of the pathways concerned with apoptosis.Results The treatment of H2O2(100μM) significantly induced the apoptosis of MSCs. Group(H2O2+HDL) demonstrated a significant low accumulation of ROS compared with Group(H202)[(1.56±0.38) vs.(5.68±0.56), p<0.05], a remarkable decrease in apoptosis rate [(28.77±6.91)%vs.(47.37±7.53)%,p<0.05],a statistical increase of bcl-2/bax (1.36±0.44vs.0.44±0.17,p<0.05). Compared with that in Group(H2O2), the change of phosphorylation of MAPK/JNK and MAPK/P38pathways in Group(H2O2+HDL) were not significant [MAPK/JNK:(70.56±5.91) vs.(51.03±4.56), p>0.05; MAPK/P38:(10.70±3.46) vs.(3.34±1.28), p>0.05], while the phosphorylation of ERK in Group(H2O2+HDL) significantly decreased[(51.03±5.73) vs.(17.73±3.22), p<0.05].Conclusions The preconditioning with HDL protects MSCs from the apoptosis induced by oxidative stress, in which the inhibition of activation in MAPK/ERK nathwav may be an underlying molecular mechanism Objectives The aim was to examine the effect of transplantation of MSCs preconditioned with HDL on cardiac function in a rat model of acute myocardial infarction.Methods MSCs harvested from male SD rat bone marrow were transfected with GFP, followed by preconditioning with HDL. A model of acute myocardial infarction was developed in female rats which were given an intramyocardial injection of PBS (Control), MSCs(MSCs) or HDL-preconditioned MSCs(MSCs+HDL) in the border zone of infarcted myocardium. The survival rate of MSCs transplanted was compared on day4after transplantation between Group(MSCs) and Group(MSCs+HDL) through detecting fluorescence image of GFP+cells and measuring expression of sry gene by real-time PCR. After4weeks, cardiac functions were examined using transthoracic echocardiography. Afterwards, rats were sacrificed and hearts were excised to calculate infarcted size using Masson’s staining, and to examine neovascularization using an immunohistochemical staining for CD31.Results Compared with Group(MSCs), Group(MSCs+HDL) demonstrated a significantly more GFP+cells in the border zone of infarcted myocardium, significantly higher expression of sry gene (1.00±0.22vs.2.83±0.74,p<0.05); showed a notable decrease in infarcted size [(31.58±4.36)%vs.(20.25±5.36)%, p<0.05] and statistically higher capillary density in the border zone of infarcted myocardium[(10.68±3.22)/HP vs.(15.17±3.83)/HP,p<0.05); indicated a remarkable improvement of cardiac function [LVEF:(35.32±3.6)%vs.(42.33±5.2)%; LVFS:(20.35±1.56)%vs.(25.35±3.22)%; LVIDs:(7.29±1.6)mm vs.(5.79±0.8)mm; LVIDd:(8.35±1.9)mm vs.(7.95±1.6)mm;p all<0.05].Conclusions The preconditioning with HDL enhances MSCs’ survival in infracted myocardium, consequently leading to an improvement of cardiac function. |
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