| Objectives:To investigate the therapeutic efficacy of pre-treatment of bone marrow derived mesenchymal stem cells with growth factors in acute myocardial infarction (AMI) rat and to explore its mechanisms.Methods:1. Establishment of AMI rat 8-week-old female SD rats (n=30) were randomly divided into AMI group (n=15) and Sham group (n=15). AMI were induced in SD rats by left anterior descending coronary artery ligation. After 4 weeks:Measured the body weight of all groups, observed general state and mortality rate of rats; Cardiac structure and function were measured by echocardiography and hemodynamic examination; Sacrificed all the rats to remove the heart and weight each heart; Myocardial histopathology HE stain and Masson's trichrome stain were performed.2. Isolation and culture of BM-MSCs The MSCs were isolated from the femur of 4-week old male SD rats and expanded ex vivo; Cell surface markers of BM-MSCs were analyzed by flow cytometry; Ex vivo pre-treatment of MSCs with growth factors (GF-MSCs); GF-MSCs were identified by immunofluorescence, Tnl and Des mRNA expression level of GF-MSCs examined by reverse transcription polymerase chain reaction (RT-PCR); Cell cycle analysis of MSCs and GF-MSCs were performed by flow cytometry; Protein concentration of HGF and VEGF in cell and cell culture supernatant were measured by ELISA.3. Evaluation of the theraputic efficacy AMI-rat models were randomly assigned into the MSCs group, GF-MSCs group and NS group. Cultured cells were labeled with CM-Dil, and then suspended in PBS at a density of 5×106 cells per milliliter for transplantation. CM-Dil labeled cells or normal saline was transplanted into myocardium by direct myocardial injection. Finally,39 rats were enrolled in the study with 13 rats in each group. Four weeks after transplantation:(1) Rat weight, general state and mortality rate were observed in all of groups; (2) Echocardiography and hemodynamic exams were performed to evaluate the cardiac structure and function, respectively. (3) After hemodynamic exams sacrificed all the rats to remove the hearts and weight each heart. Myocardial samples were embedded by OCT compound, snap-frozen in 20?, and cut into sections. (4) HE stain and Masson trichromic stain were performed to evaluate the maocardial infarction. And fibrosis areas were calculated.4. The mechanism of present cell therapy (1) The presence of Y chromosome of male transplanted cells was analyzed by RT-PCR; (2) Immunofluorescence staining was performed with monoclonal mouse anti-cardiac troponin I and anti-desmin. FITC-conjugated antibody was used as a secondary antibody; (3) Myocardial MMP2, MMP9, TIMP1, SDF1-a, Tn1 and Des mRNA expression levels were examined by RT-PCR; (4) Concentration of myocardial VEGF, HGF and TNF-a were determined by ELISA. (5) To evaluate cardiomyocyte apoptosis, the TUNEL assay and Western Blot for Caspase-3 expression were performed; (6) To examine myocardial oxidative stress level, the enzymatic activities of superoxide dismutase (SOD) was measured through xanthine-oxidase method, the myocardial content of malondialdehyde (MDA) was determined by thiobarbituric acid-reactive substance method.Results:1. Cardiac structure LVEDD and LVESD were significantly greater and cardiac systolic function LVEF and FS were significantly lower in the AMI group than those in the sham group (P?0.01). Hemodynamic examination results showed that LVSP,+dP/dtmax,-dP/dtmax were significantly lower and LVEDP were significantly higher in the AMI group than those in the sham group (P?0.01). The HE histopathology study showed derangement of myofibrillar formation, loss of myofibrils, breaks in myofibril, hypertrophy myofibril; interstitial edema; partial loss of cardiomyocyte, myocyte vacuolar degeneration; myofibril scattered infiltration of lymphocytes in the AMI group. Masson's trichrome stain showed myocardial interstitial fibrosis in the AMI group. Both HE and Masson's trichrome staining showed normal myocardial tissue in the sham group.2. Cultured bone-marrow derived mesenchymal stem cells showed spindle shaped morphology, plastic adherent, and expressed CD 105, CD44, CD29. The majority of MSCs were negative for CD31, CD34 and CD45. Pretreated MSC with growth factors (GF-MSCs) demonstrated spindle shaped or synaptic morphology, similar to untreated MSCs. Immunofluorescence examination and RT-PCR showed GF-MSCs were positive for cardiac troponin?and desmin. Cell cycle analysis demonstrated that more than 90%MSCs were in G1 phase, significantly higher than GF-MSCs (P<0.05). In contrast, most of GF-MSCs were in proliferation G2 and S phase, significantly more than MSCs (P<0.05). Protein concentration of HGF and VEGF in cell and cell culture supernatant were significantly higher in the GF-MSCs than that in the MSCs (P?0.01).3. (1) Four weeks after cell transplantation, no rat died in all groups. General state of cell transplantation groups was better than the NS group. Heart weight-to-body weight ratios were significantly greater in the NS group than that in both MSCs group and GF-MSCs group (P<0.01). (2)Echocardiographic studies demonstrated LVEDD and LVESD were significantly smaller and systolic function parameters (LVEF and FS) were significantly higher in both MSCs group and GF-MSCs group than those in the NS group (P<0.01); LVEF and FS were significantly higher and LVEDD and LVESD were significantly smaller in the GF-MSCs group than those in the MSCs group (P<0.05). (3) Hemodynamics examination showed LVSP,+dP/dtmax and-dP/dtmax were significantly higher and LVEDP was significantly lower in both MSCs group and GF-MSCs group than those in the NS group (P<0.01); Compared with the MSCs group, the GF-MSCs group had higher LVSP,+dP/dtmax and-dP/dtmax and lower LVEDP (P?0.05). (4)The HE histopathology study showed derangement of myofibrillar formation, hypertrophy myofibril; interstitial edema; myofibril scattered infiltration of lymphocytes in the PBS group. Compared with NS group, both MSCs group and GF-MSCs group had similar myofibrillar morphology, but lesser infiltration of lymphocytes. Masson's trichrome staining demonstrated modest myocardial fibrosis in the NS group. However, MSCs or GF-MSCs transplantation significantly attenuated the development of myocardial fibrosis. Quantitative analysis also showed that mean fibrosis area was significantly larger in the NS group than that in the MSCs group and the GF-MSCs group (P?0.01). Mean fibrosis area in the GF-MSCs group was significantly smaller than that in the MSCs group (P<0.01).4. (1) PCR showed the presence of Y chromosome mRNA expression in female AMI rats in the MSCs group and GF-MSCs group. (2) Immunofluorescence demonstrated that transplanted red fluorescence-labeled MSCs and GF-MSCs were positive for the cardiac markers cardiac troponin I and desmin. Tnl and Des mRNA expression levels were significantly lower in the NS group than those in the MSCs group or the GF-MSCs group (P<0.01), and were significantly higher in the GF-MSCs group than those in the MSCs group (P?0.01). (3) Protein concentration of myocardial TNF-a were significantly higher in the NS group than those in the MSCs group and the GF-MSCs group (P<0.01), and were significantly lower in the GF-MSCs group than those in the MSCs group (P?0.01). (4) Myocardial MMP2 mRNA expression level was lower in the NS group than those in the MSCs group and the GF-MSCs group (P>0.05). MMP9 and TIMP1 mRNA expression levels were significantly higher in the NS group than those in the MSCs group and the GF-MSCs group (P?0.01). The increase in MMP9 and TIMP1 mRNA level was attenuated by GF-MSCs transplantation better than by MSCs transplantation (P?0.01). (5) Myocardial SDFl-a mRNA expression levels were significantly lower in the NS group than those in the MSCs group and the GF-MSCs group (P<0.01), and were significantly higher in the GF-MSCs group than those in the MSCs group (P?0.01). Protein concentration of myocardial VEGF and HGF was significantly lower in the NS group than that in the MSCs group and the GF-MSCs group (P?0.01). Transplantation of GF-MSCs increased the protein concentration of myocardial VEGF and HGF in AMI rats significantly more than MSCs transplantation (P<0.01). (6) Cardiomyocyte apoptotic index was significantly lower in the NS group than that in the MSCs group and GF-MSCs group (P<0.01). Compared with MSCs group, GF-MSCs group had lower apoptotic index (P?0.01). Western Blot analysis showed higher myocardial Caspase-3 protein expression in the NS group than that in the MSCs group and GF-MSCs group (P<0.01). However, the increase in Caspase-3 level was attenuated by GF-MSCs transplantation more than MSCs transplantation (P?0.01). (7) Myocardial T-SOD activity was significantly lower in the NS group than that in the MSC and the GF-MSC group (P<0.01); compared with the MSCs group, the GF-MSCs group had higher T-SOD activity (P?0.01). MDA content was significantly higher in the NS group than that in the MSCs and the GF-MSCs group (P?0.01); compared with the MSCs group, the GF-MSCs group had lower MDA content (P<0.01).Conclusions:1. Left anterior descending coronary artery ligation can successfully establish AMI-rat model.2. Ex vivo pretreatment rat BM-MSCs with growth factors induced cardiomyocyte like cell differentiation and enhanced the paracrine effect of MSCs.3. Pretreatment MSCs with growth factors can improve cardiac structure and function of AMI-rat, and its efficacy is superior to MSCs alone.4. The mechanisms may be related to:growth factors pretreatment promotes the differentiation of MSCs into cardiomyocyte-like cells; enhances paracrine effect of MSCs; increases mRNA expression of myocardial functional protein; enhances the anti-apoptotic capacity; inhibits myocardial inflammation and oxidative stress; reduces myocardial fibrosis as well as ameliorates myocardial remodeling. |
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