Carvedilol may attenuate liver cirrhosis by inhibiting angiogenesis through the VEGF-Src-ERK signaling pathway

AIM: To investigate the effect of carvedilol on angiogenesis and the underlying signaling pathways. METHODS: The effect of carvedilol on angiogenesis was examined using a human umbilical vascular endothelial cell (HUVEC) model. The effect of carvedilol on cell viability was measured by CCK8 assay. Flow cytometry was used to assess the effect of carvedilol on cell cycle progression. Cell migration, transwell migration and tube formation assays were performed to analyze the effect of carvedilol on HUVEC function. Vascular endothelial growth factor (VEGF) induced activation of HUVECs, which were pretreated with different carvedilol concentrations or none. Western blot analysis detected the phosphorylation levels of three cell signaling pathway proteins, VEGFR-2, Src, and extracellular signal-regulated kinase (ERK). The specific Src inhibitor PP2 was used to assess the role of Src in the VEGF-induced angiogenic pathway. RESULTS: Carvedilol inhibited HUVEC proliferation in a dose-dependent manner (IC50 = 38.5 mmol/L). The distribution of cells in the S phase decreased from 43.6% to 37.2%, 35.6% and 17.8% by 1, 5 and 10 mu mol/L carvedilol for 24 h, respectively. Carvedilol (10 mu mol/L) reduced VEGF-induced HUVEC migration from 67.54 +/- 7.83 to 37.11 +/- 3.533 (P < 0.001). Carvedilol concentrations of 5 mu mol/L and 10 mu mol/L reduced cell invasion from 196.3% +/- 18.76% to 114.0% +/- 12.20% and 51.68% +/- 8.28%, respectively. VEGF-induced tube formation was also reduced significantly by 5 mu mol/L and 10 mu mol/L carvedilol from 286.0 +/- 36.72 to 135.7 +/- 18.13 (P < 0.05) and 80.27 +/- 11.16 (P < 0.01) respectively. We investigated several intracellular protein levels to determine the reason for these reductions. Treatment with 10 mu mol/L carvedilol reduced VEGF-induced tyrosine phosphorylation of VEGFR-2 from 175.5% +/- 8.54% to 52.67% +/- 5.33% (P < 0.01). Additionally, 10 mu mol/L carvedilol reduced VEGF-induced ERK 1/2 phosphorylation from 181.9% beta 18.61% to 56.45% beta 7.64% (P < 0.01). The VEGF-induced increase in Src kinase activity was alleviated by carvedilol [decreased from 141.8% beta 15.37% to 53.57 beta 7.18% (P < 0.01) and 47.04% beta 9.74% (P < 0.01) at concentrations of 5 and 10 mu mol/L, respectively]. Pretreatment of HUVECs with Src kinase inhibitor almost completely prevented the VEGF-induced ERK upregulation [decreased from 213.2% beta 27.68% to 90.96% beta 17.16% (P < 0.01)]. CONCLUSION: Carvedilol has an anti-angiogenic effect on HUVECs. This inhibitory effect is mediated by VEGF-induced Src-ERK signaling pathways.