The Mechanism Of VEGF-Induced Cardiac Stem Cells Mobilization And Myocardial Repair In The Infracted Heart

Chapter 1 the effect of VEGF on cardiac stem cells (CSCs) migration and its mechanismAIM:To explore the effect of VEGF on cardiac stem cells(CSCs) migration and its probable signal transduction mechanism. Method:c-kit+CSCs were isolated from the hearts of 3-day-old Sprague-Dawley rats with immunomagnetic microbeads method. The expression of Nkx2.5, GATA4, MEF2C, CXCR4, Flk-1, Flt-1 and neuropilin-1 (NRP-1) mRNA in CSCs was detected by RT-PCR and immunofluorescence staining. And the expression of CXCR4, Flk-1, Flt-1 and NRP-1 was measured by flow cytometry (FCM). Migration assays, in vitro, were performed using transwell, 1×105 CSCs treated or untreated with VEGF isoforms (0.1、1、10、20、50、100 ng/mL) in 200μL of medium were seeded into the upper chamber (poresize,8μm); cells were collected from both chambers after 24 h of migration at 37℃. For the inhibition experiment, CSCs were preincubated with a VEGF receptor inhibitor or a PI3K/Akt inhibitor wortmannin (100 nM) for 30 min before seeding. pAkt expressions in CSCs with treatment of 20 ng/ml VEGF165 were performed with Western Blotting. Results: Using immunomagnetic microbeads, we isolated c-kit+ cells from the rat hearts with a purity of 91% by flow cytometry. Under light microscope, the cells were small, round, phase-bright and suspended in the medium. Nkx2.5, GATA4, MEF2C, CXCR4, Flk-1, Flt-1 and neuropilin-1 (NRP-1) mRNA in CSCs were detectable by RT-PCR. Immunofluorescence staining demonstrated the characteristic expressions of Flk-1, Flt-1, Nkx2.5 and GATA4 in CSCs. Furthermore, the CSCs spontaneously differentiated into myogenic and endothelial cells. By using tanswell in vitro migration assay system, in comparison with the control group, the migration index of CSCs increased with VEGF isoforms in a dosage-dependent manner, and reached a peak at 20ng/ml. And CSCs migration induced by VEGF165 or VEGF189 was greater than VEGF121. VEGF-induced CSCs migration could be inhibited by pretreatment of the CSCs with both VEGF-antibody and VEGFR blocker and PI3K-Akt inhibitor. We stern blot analysis showed that although the expression of total Akt protein was not markedly altered, the levels of pAkt were significantly increased in VEGF165-treated CSCs. The pAkt reached a peak at 30 min after incubation with 20 ng/mL VEGF165 and returned to the basal level at 60 min. Conclusion:The isolated c-kit+ CSCs showed the characteristic expressions of Flk-1, Flt-1, Nkx2.5 and GATA4, and the c-kit+ cells spontaneously differentiated into myogenic and endothelial cells. These results confirmed that the isolated c-kit+ cells were CSCs. The CSCs migration was mediated by VEGF through its receptors, and the PI3K/Akt signal pathway was involved in VEGF-induced CSCs migration.Chapter 2 the effect of VEGF on cardiac stem cells (CSCs) repair the infarcted heartAIM:To explore the effect of VEGF on cardiac stem cells(CSCs) repair the infarcted heart. Method:MSC culture was performed with the classical adhering method; characteristic of MSC was identified through multi-differentiation and surface marker assay (CD90, CD29, CD34, CD45, c-Kit). Myocardium infarction (MI) was achieved by the ligation of the left anterior descending coronary artery. Successful ligation of coronary was verified by color change in the ischemic area and ECG lead I and aVL S-T segment elevations after occlusion. One week after MI, the animals received an injection of 5.0×109 pfu Ad-VEGF or Ad-LacZ (0.2 ml) at four sites (0.05 ml per site) in each infarcted heart. Two injection sites were in the peri-ischemic area, and two, in the ischemic area. A total of 2×105 PKH26-labeled cells (50μL) were injected into the myocardium at the atrioventricular (AV) groove of the infarcted hearts. For the inhibition experiment, the cells were treated with 5μM of the VEGF receptor inhibitor SU5416 before injection. In the sham group, the animals underwent the identical surgery without ligation of the coronary artery, and in the control group, the animals underwent the ligation of the coronary artery, but were injected with an equivalent volume injection of cell-free PBS. One week after Ad-hVEGF injection, the expression of hVEGF in the myocardium were detected by immunohistochemistry, ELISA and WesternBlotting. c-Kit and Flk-1 in the myocardium were detected by RT-PCR. One week after Ad-hVEGF and PKH26-labeled CSCs injection, the number of CSCs in the infraction area and peri-infraction area were measured under fluorescent microscope. And the differentiation of CSCs into cardiomyocytes and endothelial cells invitro and in vivo was detected by immunostaining for aMHC、cTnt and CD31. One month after treatment, hemodynamic measurements were performed. Masson’s trichrome staining was performed to delineate collagen content as a percentage of the infarcted and peri-infarction area. Infarcted myocardium size was performed by TTC staining. The LV wall thickness was measured at three widely spaced locations within the scar segment as well as the non-infarcted region. Blood vessel density in the infarcted regions of the myocardium was measured by immunostaining for vWF-Ⅷ. Result:Cultured MSCs had the ability of multi-differentiation and highly expressed CD29 and CD90. hVEGF expression in the myocardium was shown by immunohistochemistry, the level of hVEGF protein expression as shown by ELISA analysis was significantly increased in the cardiac tissues and serum. At the same time, the serum level of rVEGF and the endogenous expression of VEGF, SDF-1 and HGF in the heart with or without injection of Ad-LacZ did not differ but Ad-hVEGF induced a higher level of these protein expressions in cardiac tissue. More importantly, the increased hVEGF was matched by increased levels of c-Kit and Flk-1 mRNA expression in the infarcted hearts. The assessment of CSC migration in vivo was performed using PKH26-labeled CSCs, which were injected into the AV groove. The increased VEGF was accompanied by a greater accumulation of CSCs, and the administration of SU5416 resulted in a significant reduction in accumulation of CSCs in the infarcted hearts at day 7 after treatment. And the CSCs could differentiate into cardiomyocytes and endothelial cells in vitro and in vivo was detected by immunostaining for aMHC、cTnt and CD31, and blood vessels density per field were higher in infarcted regions of the Ad-VEGF-CSCs group compared with those of the CSCs, MSCs, Ad-VEGF and Ad-Lac-Z groups, and SU5416 treatment clearly inhibited the angiogenesis induced by VEGF gene-transfection and/or CSCs ransplantation. In vivo hemodynamic measurements showed that the LV function was not significantly different between Ad-Lac-Z and control groups, but was significantly improved in Ad-VEGF+CSCs, CSCs, MSCs and Ad-VEGF groups compared to Ad-Lac-Z and control groups. Furthermore, Ad-VEGF+CSCs group showed a less decline in±dp/dt max and LVSP, and a lower increase in LVEDP than in CSCs, MSCs and Ad-VEGF groups. More importantly, LV function was significantly poorer in SU5416 group than in Ad-VEGF+CSCs group. TTC staining showed that Ad-VEGF-CSCs group had a significantly reduced infarct size in comparison to the other groups. Masson staining showed that more collagen accumulation in the infarcted and peri-infarction areas one month post-MI in the SU5416, control and Ad-LacZ groups compared to the CSCs, MSCs, Ad-VEGF-CSCs and Ad-VEGF groups. Semi-quantitative analysis indicated that the collagen volume fraction in the infarcted area was significantly decreased in the Ad-VEGF-CSCs group compared to the others groups. A marked increase in the average LV wall thickness and decrease in ventricular dilation was also observed in the Ad-VEGF-CSCs hearts. Conclusion:The greater accumulation of CSCs mediated by VEGF in the infarcted region could participate in the repair of ischemic myocardium through the beneficial effects of myogenesis and angiogenesis, which resulted in the decrease of infarction size and collagen content, increase of LV wall thickness and blood vessel density in the infracted hearts, accompanied by an improvement in cardiac function.Chapter 3. The effect of VEGF isoforms on CSCs adhesion in vitro and its mechanismAIM:To explore the effect of VEGF on CSCs adhesion in vitro and its probable signal transduction mechanism. Method:Adhesion assays, in vitro, were performed using 24-well on plates which CECs were plated, reaching confluence and labeled with DiO,3,3’-dioctadecyloxacarbocyanine perchlorate. All experiments were performed with 1×105 CSCs in 200μl. CSCs labeled with PKH26. After incubation at 37℃(24 hours) cells were washed 3 times with PBS, and adherent cells were counted in 25 fields per well using fluorescence microscopy (200×). The number of adherent cells was normalized to the total number of added cells. In some experiments, HUVECs and CSCs were pretreated for adhesion studies with one of the following substances VEGF (0.1、1、10、20、50、100ng/mL), VEGF-R inhibitor AMG706、SU5416、VEGFR2-I、PKC inhibitor GO6976、anti-VCAM-1, or anti-ICAM-1 one hour before and during the adhesion assay. ICAM-1 and VCAM-1 expressions in CSCs were performed with FCM after 20 ng/ml VEGF, VEGFR inhibitor or PKC inhibitor GO6976. Result:By using in vitro adhesion assays system, compared with the control group, the adhesion index of CSCs increased with VEGF isoforms in a dosage-dependent manner, and reached a peak at 20 ng/mL. And CSCs adhesion induced by VEGF165 was greater than VEGF121 or VEGF189. VEGF-induced CSCs adhesion could be inhibited by pretreatment of the CSCs with VEGFR blocker, and VCAM-1-antibody and PKC inhibitor. VCAM-1 expression in CSCs was enhanced by VEGF165. The increased expression of VCAM-1 in CSCs was blocked by VEGFR1, VEGFR-2, and PKC inhibitor. Conclusion:The CSCs adhesion was mediated by VEGF through its receptors, and PKC/VCAM-1 pathway was involved in VEGF-induced CSCs adhesion.Chapter 4 the effect of VEGF on adhesion of CSCs in the infarcted heart and its mechanismAIM:To explore the effect of VEGF on CSCs adhesion in the infarcted heart, and its probable signal transduction mechanism. Method:Adhesion assays, in vivo, were performed using infarcted heart in rats which CSCs labeled with PKH26 were injected into the infarcted area of heart. Three days after treatment, the number of CSCs in the infraction area and peri-infraction area were measured under fluorescent microscope. Result:The assessment of CSCs adhesion assays in vivo was performed using PKH26-labeled CSCs, which were injected into the infarcted area of heart. The VEGF-treated CSCs was accompanied by a greater adhesion and accumulation of CSCs in the infarcted heart, and the administration of VCAM-1-antibody and PKC inhibitor resulted in a significant reduction in adhesion and accumulation of CSCs in the infarcted hearts at day 3 after treatment. Conclusion:The CSCs adhesion mediated by VEGF involved in PKC/VCAM-1 pathway in the infarcted heart.