Thymosin β4 promotes glucose-impaired endothelial progenitor cell function via Akt/endothelial nitric oxide synthesis signaling pathway

Abstract

Circulating endothelial progenitor cells (EPCs) are a subtype of hematopoietic stem cells, which can differentiate into endothelial cells and restore endothelial function. However, high glucose decreases the number and impairs the function of EPCs. A previous study showed that thymosin β4 (Tβ4), a pleiotropic peptide beneficial for multiple functions of various types of cells, could promote EPC migration and dose-dependently upregulate the phosphorylation of Akt and endothelial nitric oxide synthesis signaling (eNOS). In present study, the hypothesis that Tβ4 can improve glucose-suppressed EPC functions via the Akt/eNOS signaling pathway and restores the production of nitric oxide (NO) is investigated. EPCs were isolated from the peripheral blood of healthy volunteers and formed a cobblestone shape after 3-4 weeks of cultivation. Then, EPCs were treated with high concentrations of glucose (25 mM) for 4 days and administrated with Tβ4 for further study. Transwell migration and tube formation assays were performed to access the migratory and angiogenic ability of EPCs. In addition, the quantity of Akt, eNOS and the concentration of nitric oxide (NO) was investigated. Functional studies showed that high concentrations of glucose significantly suppressed EPC function, while this adverse effect was reversed by the administration of Tβ4. In addition, Akt small interfering (si)RNA and eNOS siRNA were demonstrated to reduce the protective effect of Tβ4 against glucose-impaired EPC functions. These findings suggest that Tβ4 improves glucose-impaired EPC functions via the Akt/eNOS signaling pathway.

Keywords: angiogenesis; endothelial progenitor cells; hyperglycemia; migration; thymosin β4.

Figure 1.

Tβ4 restores glucose-suppressed EPC migration and angiogenesis. Following treatment of EPCs with various concentrations of Tβ4 (10 ng/ml, 100 ng/ml and 1000 ng/ml) for 24 h, cultured EPCs were evaluated by using functional assays following incubation with high glucose medium for 4 days. (A) A transwell migration assay was used to assess the effect of Tβ4 on EPC migration in high concentrations of Glu. 4′6-diamidino-2-phenylindole staining was performed to determine the number of migrated EPCs (magnification, ×400). (B) An in vitro tube formation assay was performed to investigate the effect of Tβ4 on EPC neovascularization in high concentrations of Glu. Number of branch points was counted to evaluate the effect of Tβ4 on EPC angiogenesis in high-glucose medium. (n=4). *P<0.05 and **P<0.01 vs. the control; ^#P<0.05 and ^##P<0.01 vs. the high-glucose group. Tβ4, thymosin β4; EPC, endothelial progenitor cell; Glu, glucose.

Figure 2.

Tβ4 recovers glucose-inhibited EPC Akt/eNOS activation and NO production. (A) Effects of Tβ4 on the expression and phosphorylation of Akt and eNOS, examined in EPCs by western blotting. (B) Nitrate production was measured by Griess reagent. (n=4). *P<0.05 and **P<0.01 vs. the control group; ^#P<0.05 and ^##P<0.01 vs. the high-glucose group. Tβ4, thymosin β4; EPC, endothelial progenitor cell; Glu, glucose; NO, nitric oxide; eNOS, endothelial NO synthesis; p-eNOS, phosphorylated eNOS t-eNOS, total eNOS; p-Akt, phosphorylated-Akt.

Figure 3.

Tβ4 improves glucose-impaired EPC functions via the Akt/eNOS signaling pathway. (A) Tβ4 (1000 ng/ml) was administrated to EPCs 4 days after incubation with high concentrations of glucose. EPCs were pretreated with or without Akt and eNOS siRNA (50 nM) 24 h prior to the administration of Tβ4 (magnification, ×400). (B) An in vitro angiogenesis assay was performed to determine the effect of Tβ4 on EPC angiogenesis in high concentrations of glucose following the ablation of Akt and eNOS activity by Akt and eNOS siRNA. (n=4; magnification, ×100). *P<0.05 and **P<0.01 vs. the control group; ^#P<0.05 and ^##P<0.01 vs. the high-glucose group; ^&P<0.05 vs. the high-glucose + Tβ4 group. Tβ4, thymosin β4; EPC, endothelial progenitor cell; Glu, glucose; eNOS, endothelial NO synthesis; SC, scramble; siRNA, small interfering RNA.