The Fundamental And Application Reaserch Of Thymosin β4 Optimizing The Function Of Endothelial Progenitor Cells

The morbility and mortality of cardiovascular desease are increasing nowdays, which poses threat to human health. The mechanism of cardiovascular desease is complicated, one of which is the dysfunction of endothelial cells. Although endothelial cells are capable of repairing impaired endothelium, their ability to regenerate is limited. Endothelial progenitor cells (EPCs) are the progenitor of endothelial cells that have the potential to differentiate into mature endothelial cells. Accumulating evidence indicates the impact of EPCs in cardiovascular repair and neovascularization. However, EPCs transplantation confronts multiple issues in clinical application. Various risk factors for coronary artery disease, such as aging, hypertension, hypercholesterolemia, and diabetes may affect the number and function of EPCs and restrict the application of EPCs-based cell therapy. Hence, improving the function of EPCs would be an important strategy for clinical therapy.Thymosin β4 (Tβ4), is a 43-amino acid polypeptide modulating various biological effects, such as angiogenesis, wound healing and inflammation control.Our previous studies have implicated Tβ4 could promoted proliferation, migration, suppress apoptosis and senescence of EPCs. However, wheather Tβ4 could improve angiogenesis, paracrine effects and effectiveness of transplantation of EPCs remains unclear.On the basis of these considerations, we first investigated the angiogenic capacity of EPCs exposed to Tβ4 and signal transduction pathways involved in this process. Then we studied the effects of Tβ4 on EPCs paracrine effects. Moreover, we tested wheather Tβ4 could improve effectiveness of transplantation of EPCs in vivo.Part 1:Effects of thy mo sin β4 on angiogenesis of endothelial progenitor cells and signal transduction pathways involvedThe aim of this study is to investigate the effects of thymosin 04 (Tβ4) on angiogenesis of endothelial progenitor cells (EPCs) and signal transduction pathways involved in this process. Peripheral blood mononuclear cells were isolated by density gradient centrifugation with Ficoll separating solution and were plated on culture dishes coated with fibronectin. Under a laser scanning confocal microscope, EPCs were characterized as cells double positive for lectin binding and DiI-acLDL-uptake by fluorescent staining after 7 days in culture. Attached cells were treated with Tβ4 (10ng/mL, 100ng/mL, 1000ng/mL) or vehicle control. EPCs angiogenesis were tested with Matrigel tube formation assay. We revealed that Tβ4 significantly promoted the angiogenic capacities of EPCs, maximum at 1000ng/mL. Using western blot analysis, we revealed that treatment of EPCs with thymosin β4 resulted in concentration-dependent phosphorylation of Akt, and endothelial nitric oxide synthase (eNOS). Functional analysis showed that thymosin β4-induced EPCs angiogenesis were blunted by Akt siRNA or eNOS siRNA. These results suggest that Tβ4 can enhance the angiogenic capacities of EPCs via Akt/eNOS signal transduction pathway.Part 2:Effects of thymosin β4 on paracrine effects of endothelial progenitor cellsOur previous study demonstrated that thymosin β4 (T04) can enhance the angiogenic capacities of endothelial progenitor cells (EPCs). In present study, we aimed to investigate the roles of T04 on EPCs paracrine effects. EPCs were isolated from peripheral blood and characterized as described previously.EPCs were treated with Tβ4 (10ng/mL, 100ng/mL, 1000ng/mL) or vehicle control. EPCs-deived conditioned medium (EPCs-CM) was obtained from EPCs subjected to trophic deprvation for 24 hours. RT-PCR and ELISA test were used to analyse the epression of paracrine factor in EPCs and secretion of them in EPCs-CM. HUVECs migration and in vitro angiogenesis were assayed with transwell migration assay and Matrigel tube formation assay. Tpβ4 increased the expression of some paracrine factors (VEGF, IL-8) within EPCs and in EPCs-CM, which can be reversed by Akt siRNA or eNOS siRNA. EPCs-CM significantly promoted the migration and angiogenesis in vitro, which can be further enhanced by treatment with Tβ4, and bocked by neutralizing antibody of VEGF and IL-8. In conclusion, Tβ4 can significantly enhanced the paracrine effects of EPCs on endothelial cells and increase the secretion of paracrine factors (VEGF, IL-8), which was inlvolved, a least in part, with Akt/eNOS signal transduction pathway.Part 3:Effects of thymosin β4 on the effectiveness of EPCs transplantation in vivoWe previously demonstrated that thymosin β4 (Tβ4) can increase the angiogenic capacities and paracrine effects of EPCs in vitro. In present study, we aimed to investigate the roles of Tβ4 in EPCs transplataion in vivo. EPCs were isolated from peripheral blood as described previously. We establish myocardial infarction model of rats by left coronary artery ligation.150 u L vehicle,1×106 EPCs and same amount of EPCs pre-treated with Tβ for 24 hours were injected in ischemic area of infarcted heart.Four weeks after myocardial infarction, we measured cadiac function by ultrasound. Using Dil-labeling and immunofluorescence, we detected capillary and arterioles density in ischemic area. We further investigated the vascular endothelial growth factor (VEGF) expression and fibrosis by immuohistochemisty study and western blots analysis as well. We observed that Tβ4 promoted the retention and vascular incorporation of EPCs in ischemic area. Tβ4 pre-treated EPCs transplantation could significantly increase capillary and arterioles density, augment VEGF expression and attenuated fibrosis in ischemic area, which contributed to the improved cardiac function of the post-MI rats. In conclusion, Tβ4 optimizes the effectiveness of EPCs transplantation in vivo.