Thymosin-β 4 induces angiogenesis in critical limb ischemia mice via regulating Notch/NF-κB pathway

Thymosin-β 4 (Tβ4) has been reported to exert a pro-angogenic effect on endothelial cells. However, little is known on the role and underlying mechanisms of Tβ4 on critical limb ischemia (CLI). The present study aimed there-fore to investigate the mechanisms and pro-angiogenic effects of Tβ4 in CLI mice. Tβ4 overexpression lentiviral vector was first transfected into HUVEC and CLI mice model, and inhibitors of Notch pathway (DAPT) and NF-κB pathway (BMS) were also applied to HUVEC and CLI mice. Subsequently, MTT, tube formation and wound healing assays were used to determine the cell viability, angiogenesis and migratory ablity of HUVEC, respectively. Western blotting, reverse transcription, quantitative PCR, immunofluorescence and immunohistochemistry were used to detect the expression of the angiogenesis-related factors angiopoietin-2 (Ang2), TEK receptor tyrosine kinase 2 (tie2), vascular endothelial growth factor A (VEGFA), CD31 and α-smooth muscle actin (α-SMA) and the Notch/NF-κB pathways-related factors NOTCH1 intracellular domain (N1ICD), Notch receptor 3 (Notch3), NF-κB and p65 in HUVEC or CLI mice muscle tissues. The results demonstrated that Tβ4 not only enhanced the cell viability, angiogenesis and migratory ability of HUVEC but also promoted the expression of Ang2, tie2, VEGFA, N1ICD, Notch3, NF-κB, and phosphorylated (p)-p65 in HUVEC. In addition, Tβ4 promoted the expression of CD31, α-SMA Ang2, tie2, VEGFA, N1ICD and p-p65 in CLI mice muscle tissues. Treatment with DAPT and BMS had opposite effects of Tβ4, whereas Tβ4 reversed the effect of DAPT and BMS. The findings from the present study suggested that Tβ4 may promote angiogenesis in CLI mice via regulation of Notch/NF-κB pathways.