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

Abstract

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 therefore 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.

Irisin Attenuates Pathological Neovascularization in Oxygen-Induced Retinopathy Mice

“…Furthermore, irisin decreased the expression of NF-κB and pNF-κB, which are classical regulators in angiogenesis, inflammation, and neuron apoptosis. 16 , 49 …”

Mechanism of thymosin β4 in ameliorating liver fibrosis via the MAPK/NF‐κB pathway

“…After cell infection with Tβ4 overexpression vectors for 6 h, the lentiviruses‐contained medium was displaced with complete medium, followed by 48‐h culture at 37°C for subsequent experimentation. [ 12 ]…”

“…After cell infection with Tβ4 overexpression vectors for 6 h, the lentiviruses-contained medium was displaced with complete medium, followed by 48-h culture at 37°C for subsequent experimentation. [12] LX-2 cells were allocated to the following 8 groups: blank group (normal LX-2 cells), TGF-β1 group (LX-2 cells stimulated with 5 ng/mL TGF-β1 for 24 h), TGF-β1 + oe-NC group (based on the TGF-β1 group, LX-2 cells treated with NC lentiviral infection for 6 h), TGF-β1 + oe-Tβ4 group (based on the TGF-β1 group, LX-2 cells treated with Tβ4 lentiviral infection for 6 h), TGF-β1 + MI group (treated with 10 μM MAPK inhibitor SB2035805 for 1 h, followed by stimulation with 5 ng/mL TGF-β1 for 24 h), TGF-β1 + dimethyl sulfoxide (DMSO) group (cultured with DMSO solvent for 1 h, followed by stimulation with 5 ng/mL TGF-β1 for 24 h), TGF-β1 + oe-Tβ4 + DMSO group (based on the TGF-β1 + oe-Tβ4 group, LX-2 cells supplemented with DMSO solvent for 1 h), TGF-β1 + oe-Tβ4 + MA group (based on the TGF-β1 + oe-Tβ4 group, LX-2 cells treated with 10 nmol/L MAPK activator U-46619 for 1 h). MAPK inhibitor SB203580 and MAPK activator U-46619 were provided by Calbiochem.…”

“…Thymosin β4 (Tβ4), a small 43-amino-acid polypeptide in all cells, regulates actin polymerization and participates in various cellular response processes, such as cell migration, cell survival, protease production, inflammation, wound healing, angiogenesis, and hair follicle growth. [11][12][13][14] Tβ4 is regarded as the most abundant thymosin peptide in mammalian tissues and can be detected in hepatocytes of adult normal livers. [15] Under-expressed serum Tβ4 level is documented in patients with acute-on-chronic liver failure and chronic liver failure.…”

Progress on the Function and Application of Thymosin β4

“…Moreover, Tb4 inhibited the proliferation and activation of hepatic stellate cells (HSCs), attenuated liver fibrosis by inhibiting Notch signaling, and markedly reduced expression levels of Notch2 and Notch3, which were increased in the liver cells (42). Furthermore, Tb4 enhanced HUVEC viability, angiogenesis, and migration, as well as promoted the expression of angiopoietin 2, VEGF A, Notch3, and other cytokines in HUVECs in a mouse model of critical limb ischemia (43). In addition, Takeshitak et al (44) reported that endothelial-specific Notch1 knockdown mice had impaired neovascularization after hindlimb ischemia, and Notch1 induced angiogenesis without VEGF involvement (45).…”