Effects And Mechanisms Of Thymosin β4 On Activity Of Endothelial Progenitor Cells From Peripheral Blood

Endothelial progenitor cells (EPCs) are a specific cell population that have the potential to differentiate into mature endothelial cells, but have not acquired characteristic markers of mature endothelial cells. EPCs express various combinations of antigens traditionally associated with hematopoietic stem cells as well as endothelial cells, such as CD34, CD133 and vascular endothelial growth factor receptor (VEGFR) -2. Accumulating evidence indicates the impact of EPCs in cardiovascular repair since these cells have been shown to contribute to angiogenesis in response to ischemia, reendothelialization after vascular injury, and reversal of endothelial dysfunction. However, various risk factors for coronary artery disease, such as aging, smoking, hypertension, hypercholesterolemia, and diabetes may affect the activity of circulating EPCs and restrict the application of EPCs-based cell therapy. Therefore, it is important to improve activity of EPCs.Thymosins comprise a family of polypeptides originally isolated from calf thynius, and are divided into three groups (α,βandγ) based on their isoelectric point. The most abundant member ofβ-thymosins is thymosinβ4 (Tβ4), a 43 amino acid polypeptide. Tβ4 has been found highly expressed in a number of tissues and cells, suggesting various functions. Previous studies have implicated Tβ4 in a number of physiological and pathological events, such as wound healing and angiogenesis. Recently, it showed that Tβ4 may play roles in various stem/progenitor cells proliferation, migration and differentiation to promote angiogenesis and cardiac repair. However, little is known about the effects of Tβ4 on circulating EPCs.On the basis of these considerations, we hypothesized that Tβ4 enhanced EPCs functional activity, thus promoted endothelial repair process and maintained the integrity of endothelium to perform its cardiovascular protection. To test this hypothesis, we measured the proliferative, migratory and adhesive capacities of EPCs exposed to Tβ4 and investigated the signal transduction pathways involved in these processes, and then we studied the effects of Tβ4 on EPCs apoptosis and senescence. Part 1: Effects of thymosinβ4 on activity of endothelial progenitorcells from peripheral bloodThe aim of this study is to investigate the effects of thymosinβ4 (Tβ4) on functional activity of endothelial progenitor cells (EPCs) from healthy volunteers. Peripheral blood mononuclear cells (PBMNCs) were isolated by density gradient centrifugation with Ficoll separating solution, and then the cells were plated on culture dishes coated with fibronectin. Under a laser scanning confocal microscope, EPCs were characterized as adherent cells double positive for lectin binding and DiI-acLDL-uptake by direct fluorescent staining after 7 days in culture. EPCs were further documented by showing the expression of VEGFR-2, CD34 and CD133 with flow cytometry. Attached cells were stimulated with TβM (1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL) or vehicle control. EPCs proliferation, colony-formation, migration and adhesion were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony-forming assay, transwell migration assay and adhesion assay respectively. We showed that Tβ4 significantly enhanced the proliferative, colony-forming, migratory and adhesive capacities of EPCs, maximum at 1000ng/ml (to compare with that of control subjects, proliferative capacities 0.409±0.020 vs 0.257±0.015, P＜0.05; colony-forming capacities 16.7±2.6 vs 6.0±1.0, P＜0.05; migratory capacities 79.6±5.6 vs 31.5±3.4, P＜0.05; adhesive capacities 45.3±4.7 vs 18.8±3.0, P＜0.05). These results suggest that Tβ4 can increase the proliferative, colony-forming, migratory and adhesive capacities of EPCs. Part 2: Signal transduction pathways involved in effects of thymosinβ4 on activity of endothelial progenitor cells from peripheral bloodOur previous study demonstrated that thymosinβ4 (Tβ4) can enhance the proliferative, migratory and adhesive capacities of endothelial progenitor cells (EPCs). In present study, we aimed to determine the possible signal transduction pathways involved in effects of Tβ4 on functional activity of EPCs. EPCs were isolated from peripheral blood and characterized as described previously. Using western blot analysis, we showed that treatment of EPCs with thymosinβ4 resulted in time and concentration-dependent phosphorylation of Akt, endothelial nitric oxide synthase (eNOS), and extracellular signal-regulated kinase (ERK)1/2. Functional analysis showed that thymosinβ4-induced EPCs proliferation, migration and adhesion were blocked by phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) or eNOS inhibitor (Nω-Nitro-L-arginine methyl ester), but were not significantly attenuated by ERK1/2 inhibitor (PD98059). These findings suggest that thymosinβ4 stimulates EPCs proliferation, migration and adhesion via PI3K/Akt/eNOS, rather than via ERK1/2 signal transduction pathway. Part 3: Effects of thymosinβ4 on apoptosis and senescence of endothelial progenitor cells from peripheral bloodAccumulating evidence has showed that reduced activity of endothelial progenitor cells (EPCs) was associated with EPCs apoptosis and senescence. We previously demonstrated that thymosinβ4 (Tβ4) can increase the proliferative, migratory and adhesive capacities of EPCs. In present study, we aimed to investigate the roles of Tβ4 in EPCs apoptosis and senescence. EPCs were isolated from peripheral blood and characterized as described previously. Using an apoptosis assay, we showed that Tβ4 decreased EPCs apoptosis induced by serum deprivation in a dose-dependent manner. After ex-vivo prolonged cultivation, EPCs became senescence as determined by acidicβ-galactosidase staining. Tβ4 dose-dependently inhibited the onset of EPCs senescence in culture. In conclusion, Tβ4 can significantly inhibit EPCs apoptosis and senescence, which may lead to the enhancement of EPCs activity.