Effects And Mechanisms Of Thymosin β4 On Circulating Endothelial Progenitor Cells

The essential role of vascular endothelium in cardiovascular disorders is increasingly recognized. Mature endothelial cells contribute to the repair of endothelial injury, whereas they possess limited regenerative capacities. This has led to growing interest in circulating endothelial progenitor cells (EPCs) among scientific researchers, especially into their roles in vascular repair, remodeling and postnatal neovascularization. Although the exact origin and functional definition of EPCs remains rather controversial, emerging evidence demonstrated that EPCs contribute to neoangiogenesis after tissue ischemia. However, these positive effects after autologous transfusion or mobilization of EPCs are significantly limited by low survival rate, insufficient cell number, and impaired funcition in the patients with cardiovascular risk factors and cardiovascular diseases. Indeed, there is increasing evidence for reduced availability and impaired EPCs function in the presence of cardiovascular risk factors as well as various cardiovascular disease states, such as aging, hypertension, hypercholesterolemia, smoking, and diabetes. Since allogeneic transfusion of EPCs from healthy donors bears the problem of immunologic incompatibilities, it is quite reasonable to regard functional enhancement (such as enhancing mobilization, homing, survival, and secretion of growth factors in a paracrine/autocrine manner) of autologous EPCs as the future strategy for EPCs-based therapy in cardiovascular diseases.Thymosinβ4 (Tβ4), a small ubiquitous protein containing 43 amino acids, as the major actin-sequestering molecule in eukaryotic cells, plays pleiotropic roles in tissue development, maintenance, repair, and pathology. Previous study found that Tβ4 can stimulate migration of cardiomyocytes and endothelial cells and promote survival of cardiomyocytes. Despite the extensive identification of multiple biological activities for Tβ4, little progress is actually available for identifying the effects of Tβ4 on circulating EPCs. Our recent study demonstrated that Tβ4 can induce EPCs migration via PI3K/Akt/eNOS signal transduction pathway, but the effect of T(34 on circulating EPCs apoptosis, proliferation and senescence remains unexplored.Based on these considerations, we hypothesized that Tβ4 enhanced EPCs functional activity, subsequently promoted endothelial repair process and maintained the integrity of endothelium to perform its cardiovascular protection. To test this hypothesis, we examined the cell viability, proliferation activity, colonies formation, adhesive capacities and senescence of EPCs exposed to Tβ4, and then we studied the effect of Tβ4 on EPCs apoptosis and the signal transduction pathways involved in this process. Finally, we investigated the effects of Tβ4 on paracrine effects of EPCs.Part 1:Effects of exogenous thymosinβ4 on activity of circulating endothelial progenitor cellsEmerging evidence indicates that circulating endothelial progenitor cells (EPCs) contribute to neoangiogenesis after tissue ischemia. Here we aimed to investigate the effects of exogenous thymosin (34 (TβM) on circulating EPCs from healthy volunteers. EPCs, isolated from peripheral blood, were cultured on fibronectin-coated dishes. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding under a laser scanning confocal microscope. They were further documented by demonstrating the expression of VE-cadherin, KDR, CD34 and AC133 by flow cytometry. Cells were treated with Tββ4 (lng/mL, 10ng/mL, 100ng/mL and 1000ng/mL) or vehicle control. Cell viability, proliferation, adhesion, colony-formation and sensence of EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Brdu incorporation, adhesion assay, colony-forming assay and acidicβ-galactosidase staining respectively. Incubation of EPCs with Tβ4 significantly increased the cell viability, proliferation, adhesion, and colony-formation of EPCs, reached a maximum at 1000ng/mL. In addition, Tβ4 significantly inhibited EPCs senescence in a dose dependent manner. Together, these results revealed that T04 can dose-dependently improve cell activities of EPCs including cell viability, proliferation, adhesive capacities and colony-formation of EPCs, meanwhile inhibit EPCs senescence in a dose dependent manner.Part 2:Effects and mechanisms of thymosinβ4 on circulating endothelial progenitor cells apoptosisThymosinβ4 (Tβ4) has been suggested to regulate multiple cell signal pathways and a variety of cellular functions such as cell migration, proliferation, survival and angiogenesis. Here, we investigated the effect of Tβ4 on endothelial progenitor cells (EPCs) apoptosis induced by serum deprivation and the corresponding signal transduction pathways involved in this process. Circulating EPCs, isolated from healthy volunteers, were cultured in the absence or presence of T04 and various signal cascade inhibitors. Apoptosis was evaluated with Annexin V immunostaining and cytosolic cytochrome c expression. Incubation of EPCs with Tβ4 caused a concentration dependent increase in cell viability and proliferation activity. It also caused an inhibitory effect on EPCs apoptosis, which was abolished by PI3K inhibitors (either LY294002 or Wortmannin) or JNK MAPK inhibitor SP600125. In addition, the expression and activity of caspase-3 and-9 were decreased by T(34, which markedly increased the Bcl-2/Bax ratio within EPCs. Furthermore, Tβ4 was immunoprecipitated with integrin-linked kinase (ILK), accompanied by augmentation of ILK activity. Transfection of EPCs with ILK-siRNA resulted in abolishment of the activation of ILK-Akt and the ameliorative effect on apoptosis by Tβ4. Together, T(34 mediated inhibitory effect on EPCs apoptosis under serum deprivation can be attributed, at least in part, to ILK-Akt activation. The activation of JNK MAPK might also be involved in this process.Part 3:Effects of thymosinβ4 on paracrine effects of circulating endothelial progenitor cellsEmerging evidence suggests that paracrine effects of EPCs play a pivotal role in various processes of tissue repair. We previously demonstrated that thymosinβ4 (Tβ4) can increase the cell viability, proliferation, adhesive capacities and colony formation of EPCs. In present study, we aimed to investigate the roles of Tβ4 on EPCs paracrine effects. EPCs, isolated from healthy volunteers, were cultured and characterized as described previously. EPCs-derived conditioned medium (EPCs-CM) was obtained from culture EPCs subjected to trophic deprivation 24 h. HUVECs proliferation, migration and in vitro angiogenesis were assayed with Ki-67 immunofluorescence assay, trans-well migration assay and capillary-like tube formation on Matrigel gel respectively. EPCs-CM significantly improved endothelial function in vitro including proliferation, migration and capillary-like tube formation, which can be further enhanced by treatment with exogenous Tβ4. Furthermore, Tβ4 increased the expression of pro-angiogenic factors (such as VEGF, FGF-2 and IL-8) within EPCs. Taken together, Tβ4 can significantly enhace the paracrine effects of EPCs on endothelial cells, increased secretion of pro-mitogenic cytokines (such as VEGF, FGF-2 and IL-8) from EPCs may involve in this process.