| Backgrounds:Myocardial infarction (MI) is a sort of common but serious ischemic heart disease (IHD), which does great harm to people’s health. In recent years, the rapid development of percutaneous transluminal coronary angioplasty and coronary artery bypass graft surgery procedures helps to improve the myocardial re-perfusion, but is hard to stimulate angiogenesis and regenerate the myocytes. A large number of basic and clinical researches have shown that bone marrow-derived mesenchymal stem cells (MSCs) transplantation had the potential to regenerate cardiac myocytes, accelerate neovascularization, and improve the cardiac function after MI, which given a new hope for the treatment of IHD. But the studies also found that MSCs transplantation assisted improvement on cardiac function was limited, one important reason is their inability to reach the target tissues with high efficiency. The targeted homing ability of implanted cells is also need to be improved. How to promote the migration and homing of MSCs to the ischemic myocardium safely and effectively is the problem that needs to be solved urgently at present.Multiple studies have shown that the biological effects generated by ultrasound-targeted microbubble destruction (UTMD) could help to improve the homing ability and transplantation efficiency of the MSCs, and further improve the treating effect following MI. As far as the mechanism of MSCs migration and homing promoted by UTMD, the present research mainly focuses on the morphological and microenvironmental changes of the ischemic myocardial tissues in vivo. Some studies showed that UTMD could increase the permeability of myocardial capillary, and may help to promote the transmigration of MSCs from the blood vessels to the target tissues. Some other studies found that the local inflammatory response resulted from UTMD could change the microenvironment within tissues, and offer the target tissues as the cell niches that are more suitable for MSCs homing. However, studies about the effects of UTMD on MSCs themselves and the molecular mechanisms are comparatively few.It is known that SDF-1/CXCR4 axis is one of the important molecular pathways for targeted homing of stem cells. SDF-1 and its specific receptor, CXCR4, play a critical role in MSCs mobilization, migration, homing, and engraftment in the process of repairing infarcted hearts. This study intends to explore the potential molecular mechanism of UTMD prompted MSCs homing following MI via SDF-1/CXCR4 axis, as well as the effectiveness of ultrasound (US) combined with SDF-1-loaded microbubbles (MBs) on the improvement of MSCs homing and cardiac function after myocardial ischemia. Elucidation of this mechanism would provide a theoretical reference for UTMD-assisted MSCs homing and repairing capability on ischemic myocardium.Objectives:1. To explore the effects and the potential molecular mechanisms of UTMD on the migration and homing of MSCs following MI whereby regulating the SDF-1/CXCR4 axis, providing theoretical reference for UTMD-assisted MSCs homing to ischemic myocardium.2. To study the effectiveness of intravenous MSCs transplantation under diagnostic US combined with SDF-1-loaded MBs on the improvement of MSCs homing and cardiac function after rat myocardial infarction, and explore the preliminary mechanisms of the improvement on cardiac function.Methods:1. Isolation, cultivation, identification, and labeling of human and rat bone marrow derived MSCsDensity gradient centrifugation method and adherent culture method were performed in the isolation and cultivation of human MSCs and rat MSCs, respectively. The morphological, growth curve, cell cycle, and ultrastructure were detected for the biological characteristics. The expression of surface marker molecules on the MSCs was detected by flow cytometry (FCM). Adipogenesis induction and osteogenic induction of cultured cells were performed as well. In order to track the exogenous MSCs in hearts, rat MSCs were transfected with lentiviral vectors carrying eGFP. The expression of green fluorescent and cell toxicity reaction were observed after transfection.2. Establishment and evaluation of rat MI modelSurgical MI model was developed by ligation of the left anterior descending coronary artery. The successful establishment of MI was assessed with ultrasonic cardiogram (UCG) and pathology (HE staining and masson staining).3. Mechanism on UTMD promoting the migration and homing of MSCs after MI by up-regulating SDF-1/CXCR4(1) Optimization of UTMD parameters in vitro. Each experiment was arranged based on the uniform design. After detected the concentration of SDF-1 and the cell viability, the results of the optimized irradiation parameters (the irradiation time, acoustic intensity, and the dosage of MBs) were analyzed by regression analysis ultimately.(2) In vitro study. Human MSCs was used in this part of the experiment. To fully validate the assumptions, two types of conditional culture medium were prepared for simulating a normal or ischemic/hypoxic environment in vitro: ① Normal or hypoxia cultured medium of human cardiac myocytes (HCM) and human cardiac microvascular endothelial cells (HCMEC); ② Myocardial tissue extracts (MTE) obtained from normal rats or MI rats. The in vitro grouping was divided into two corresponding parts, namely "supernatant groups" and "MTE groups". Using the optimized irradiation parameters, the effects of UTMD on the MSCs cultured in different conditional medium were investigated. Evaluation indicators were list as followed:CCK-8 was used to test the cell viability of MSCs; ELISA was applied to detect the concentration of SDF-1, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1); FCM was used to detect the expression of surface CXCR4 on MSCs; Real-time quantitative PCR (qPCR) and Western blot were applied to test the expression of CXCR4 mRNA and intracellular proteins, respectively; and Transwell migration assay was performed to evaluate the migration ability of MSCs in vitro.(3) In vivo study. Rat MSCs was used in this part of the experiment. Fourty-eight hours after implantation of GFP-labeled MSCs in MI rats, the fluorescence distribution within ischemic myocardium in the MSCs infusion group and the US+MB+MSCs group was observed, and the number of GFP-positive cells was counted respectively for statistical analysis. In addition,7d after implantation of unlabeled MSCs in MI rats, both immunohistochemistry (IHC) and western blot were used to detect the expression of SDF-1 and CXCR4 in the control group, the MSCs infusion group, the US+MB group, and the US+MB+MSCs group.4. Study on diagnostic US combined with SDF-1-loaded MBs improving MSCs homing and cardiac function after rat MI(1) Preparation and evaluation of SDF-1-loaded MBs. On the basis of the common lipid MBs made by our department, covalent binding method was used to prepare SDF-1-loaded MBs. Light microscope was used to observe their morphology and distribution; particle counter was applied to analyze the concentration and mean diameter of MBs; FCM used to test the carriage rate of SDF-1; and ELISA performed to detect the SDF-1 loading amount and the SDF-1 encapsulation efficiency.(2) Effect of UTMD on the hypoxic preconditioned MSCs. Human MSCs was used in this part of the experiment. MSCs were cultured in hypoxic environment (1%O2,94% N2, 5% CO2) for 24h for hypoxic preconditioning. After treatment with UTMD, CCK-8 was used to test the cell viability of MSCs; FCM was used to detect the expression of surface CXCR4 on MSCs; and Transwell migration assay was performed to evaluate the migration ability of MSCs in vitro.(3) Animal experiments. Hypoxic preconditioned rat MSCs were used in cell transplantation in all animal experiment groups. Fourty-eight hours after implantation of GFP-labeled MSCs in MI rats, the number of GFP-positive cells within ischemic myocardium in the MSCs infusion group, the MSC+UM group (common MBs), and the MSC+UMSDF-1 group (SDF-1-loaded MBs) was counted, respectively.In addition,7d after implantation of unlabeled MSCs in MI rats, IHC was used to detect the expression of hepatocyte growth factor (HGF) in the control group, the MSCs infusion group, the MSC+UM group and the MSC+UMSDF-1 group; Western blot were used to analyze the expression of SDF-1 and vascular endothelial growth factor (VEGF); HE staining worked to detect the capillary density (CD) within ischemic myocardium; and the UCG used to evaluate the systolic function of left ventricular (LV).Results:1. Isolation, cultivation, identification, and labeling of human and rat bone marrow derived MSCsThe human MSCs and rat MSCs grew with adherence. Most of them were spindle shaped, and became more uniform after several passages. The results of FCM indicated that the human MSCs were positive expressed CD44 (95.25%) and CD105 (99.98%), while not expressed CD34 (0.44%) and CD45 (4.86%); the rat MSCs were positive expressed CD29 (94.88%), CD44 (99.96%), and CD90 (99.86%), while not expressed CD34 (0.48%), CD45 (4.17%), and CD11b (4.63%). Adipogenic and osteocyte differentiation was successfully induced after 14d and 21d of treatment respectively both in human MSCs and rat MSCs. Choosing multiplicity of infection (MOI)=10 for GFP lentiviral transduction. Fourty-eight hours after transfection, bright green fluorescence could be observed in the cytoplasm and nucleus of almost all MSCs. The transfected MSCs still maintained spindle shape and good proliferation ability. The stable expression of GFP was also observed in the subcultured MSCs.2. Establishment and evaluation of rat MI modelMI models were successfully built by ligation of LAD. Masson staining showed that a blue dye was located around the MI areas. The results of UCG showed that the LV ejection fraction (EF) and fractional shortening (FS) were significantly lower than normal rats, indicating that LV systolic function was attenuated greatly after MI.3. Molecular Mechanism exploration on UTMD promoting the migration and homing of MSCs after MI by up-regulating SDF-1/CXCR4(1) Optimization of UTMD parameters in vitro:The best match of parameters is as follows:the frequency= 1MHz, the irradiation time= 30s, acoustic intensity= 0.6W/cm2 and the dosage of MBs=106/mL. The concentration of SDF-1 (551.67±40.88 pg/mL) and the cell viability (88.51±4.03%) both reached a high level while using these optimal parameters.(2) In vitro study.Using human MSCs and two types of conditioned medium, statistical differences of various indexes between groups in "supernatant part" or "MTE part" are basically identical. The specific results are list as follows: ① ELISA results of SDF-1:Take the "supernatant part" as an example, the hypoxic supernatants contained a certain amount of SDF-1 (344.89±74.93 pg/mL), when the MSCs were co-cultured with this hypoxia medium, the level of SDF-1 was increased by about 34%(462.51±101.07 pg/mL). After UTMD treatment, the concentration of SDF-1 was further increased by approximately 22% (563.75±76.22 pg/mL), while the viability of MSCs only decreased by approximately 7%-9% as compared to the control group; ② FCM results of surface CXCR4 expression: UTMD could up-regulate the expression of surface CXCR4 on MSCs in vitro. Especially, after treatment with UTMD on MSCs when cultured with infarcted MTE, the percentage of MSCs expressing surface CXCR4 was increased to (12.45±.73)%, which was 1.49-fold higher than the untreated MSCs (8.34±1.33%), and 22.23-fold higher than the control group (0.56±0.19%). ③ qPCR and Western blot results of CXCR4:Whether in the normal or hypoxia conditioned medium, UTMD could elevate the expression of CXCR4 mRNA significantly. The results of Western blot were similar to that of qPCR. The expression of CXCR4 protein was the highest in the group which UTMD acted on the MSCs cultured with hypoxia conditioning medium as compared to the other groups, P<0.01. ④ ELISA results of VCAM-1 and ICAM-1:After treatment with UTMD, the concentration of VCAM-1 and ICAM-1 was increased by 34% and 40.2%, respectively, in the groups which MSCs cultured with the hypoxia supernatant; and was increased by 53.9% and 51.7%, respectively, in the groups which MSCs cultured with the infarcted MTE. ⑤ Results of Transwell migration assay:UTMD could improve the migration ability of MSCs in vitro, But after MSCs pre-treated with AMD310O, a blocker of SDF-1/CXCR4 axis, the number of migrated MSCs was rapidly decreased, and there was no differences when compared to the control group (P>0.05).(3) In vivo study.① Quantitative analysis of homing GFP-MSCs:The GFP-positive MSCs were located in the ischemic myocardium and the border areas. The average number of GFP-MSCs in the MSC+US+MB group (41.27±6.34) was significantly increased compared to the MSCs infusion group (29.23±4.08),P<0.01. ② IHC results of SDF-1 and CXCR4:SDF-1 and CXCR4 were predominantly localized to the MI area and border zones. The number of SDF-1-or CXCR4-positive cells was smaller in the control group, relatively more in the MSC group and the UM group, and largest in the MSC+UM group. ③ Western blot results of SDF-1 and CXCR4:The level of SDF-1 was higher in the MSC group (0.65±0.05) and the UM group (0.54±0.05) than the control group (0.45±0.02), P<0.01. The MSC+UM group (0.86±0.03) had the highest level compared with all the other groups, P<0.01. The level of CXCR4 in the MSC+UM group was also significantly higher than the other three groups.4. Study on diagnostic US combined with SDF-1-loaded MBs improving MSCs homing and cardiac function after rat MI(1) Preparation and evaluation of SDF-1-loaded MBs.The MBsdf-1 presented as round, homogeneously distributed under the light microscope. The average mean size and concentration of them were 1.92 μm and 2～6×109/mL, respectively. The carriage rate of SDF-1 was 79.74%. The SDF-1 encapsulation efficiency was 79%, and the SDF-1 loading amount was 15.8μg/mL.(2) Effect of UTMD on the hypoxic preconditioned MSCs.① FCM results of surface CXCR4 expression:After hypoxic preconditiong, the proportion of MSCs expressed membrane CXCR4 was increased from (0.86±0.21)% to (8.52±2.17)%, while the cell viability had no significant change. After UTMD treatment, the proportion was further increased to (15.62±3.65)%, while the cell viability only decreased by about 9%-10%. ② Results of Transwell migration assay:After treatment with SDF-1-loaded MBs mediated UTMD, the number of successfully migrated cells in MSC+UMSDF-1 group (184.57±19.52) was significantly more than the MSC+UM group (using common MBs) (95.25±9.21) and the control group (43.13±6.86). But the number of migrated cells was sharply reduced to (62.34±8.95) after pre-treatment MSCs with AMD3100,all P<0.01.(3) Animal experiments.① Quantitative analysis of homing GFP-MSCs:The number of GFP-positive MSCs was 58.33±13.51 in the MSC+UMSDF-1 group, which was 1.27-flod higher than the MSC+UM group (45.92±8.08) and 2.12-flod greater than the MSC group (27.46±5.21). ② Results of capillary density (CD):The number of capillaries stained by HE in the MSC+UMSDF-1 group was much greater than that of the MSC+UM group, the MSC group, and the control group. ③ Semi-quantitative analysis of HGF by IHC:The integral optical density (IOD) tested by Pro-Image in the MSC+UMSDf-1 group (45014.66±6230.07) was higher than the MSC+UM group (32879.28±3851.45), the MSC group (18786.90±1229.33), and the control group (5791.51±834.12), all P<0.01. ④ Western blot results of SDF-1 and VEGF:The expression of both SDF-1 and VEGF in MI area was highest in the MSC+UMSDF-1 group, followed by the MSC+UM group, the MSC group, and the control group. ⑤ Results of LV systolic function:There was significant difference of LVEF between the MSC+UMSDF-1 group (67.83±5.78%) and the MSC+UM group (58.83±5.50%), the MSC group (48.74±3.13%) and the control group (39.80±4.51%), all P<0.01. So was the difference of FS between the MSC+UMSDF-1 group (43.58±4.94%) and the MSC+UM group (39.84±3.68%, P<0.05), the MSC group (31.01±3.94%, P,0.01) and the control group (20.73±2.83%, P<0.01), respectively.Conclusions:1. Successfully isolated, purified, cultured and identified human bone marrow-derived MSCs and rat bone marrow-derived MSCs. Stable expression of green fluorescent could be observed in the eGFP transfected MSCs. GFP-MSCs could track the distribution of MSCs in heart effectively.2. Using the optimized parameters (frequency=1MHz, T=30s, Q=0.6W/cm2, MB=106/mL), on the premise of maintaining relatively high level of cell viability, UTMD can promote the secretion of SDF-1, VCAM-1 and ICAM-1. UTMD can also up-regulate the expression of CXCR4 on the surface of MSCs, it may benefit from the "sonoporation" and the increased intracellular CXCR4 storage due to UTMD.3. MSCs co-cultured with MTE or supernatant of HCM and HCMEC. The results get from two types of hypoxic/ischemic model in vitro confirmed the molecular mechanism that UTMD could promote the migration of MSCs by up-regulating SDF-1/CXCR4.4. Intravenous transplantation of MSCs under diagnostic US combined with MBs can promote the migration and homing of MSCs to ischemic myocardium, and improve the expression of SDF-1 and CXCR4 in MI area. The increased SDF-1 level may result from biological effect of UTMD in target tissues and the up-regulated paracrine effect of MSCs by UTMD.5. An increased expression of SDF-1 in target tissue and the surface levels of CXCR4 on exogenous MSCs may be one of the important molecular mechanisms that UTMD can improve the migration and homing of MSCs via up-regulating SDF-1/CXCR4.6. SDF-1-loaded MBs were successfully prepared with small particle size, high concentration, uniform distribution, stable connection, good loading amount and encapsulation efficiency by covalent binding method.7. Hypoxic preconditioning can up-regulate the expression of surface CXCR4 on MSCs, UTMD can further improve this functional expression of CXCR4, both of them promote the migration and homing of MSCs.8. The elevated concentration of SDF-1 in MI areas due to its release from the SDF-1-loaded MBs destruction by UTMD, and the up-regulated surface levels of CXCR4 on MSCs caused by hypoxic preconditioning and UTMD, the two factors are interacted for promoting exogenous MSCs homing to ischemic myocardium.9. Under the effect of diagnostic US irradiated on SDF-1-loaded MBs, the number of the homing MSCs was increased. The VEGF and HGF in MI areas was elevated by UTMD associated with the improved paracrine effect of exogenous MSCs, in which resulted in an improvement of LV perfusion and cardiac function following MI. |
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