Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Aug 28, 2015; 21(32): 9566-9576
Published online Aug 28, 2015. doi: 10.3748/wjg.v21.i32.9566
Carvedilol may attenuate liver cirrhosis by inhibiting angiogenesis through the VEGF-Src-ERK signaling pathway
Qian Ding, Xiang-Guo Tian, Yan Li, Qi-Zhi Wang, Chun-Qing Zhang
Qian Ding, Xiang-Guo Tian, Yan Li, Qi-Zhi Wang, Chun-Qing Zhang, Department of Gastroenterology, Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China
Author contributions: Ding Q performed the majority of the experiments; Tian XG, Li Y and Wang QZ provided vital reagents and analytical tools and edited the manuscript; Ding Q and Zhang CQ designed the study and wrote the manuscript.
Supported by National Natural Science Foundation of China, No. 81370590.
Conflict-of-interest statement: The authors have no conflict of interest related to the manuscript.
Data sharing statement: No additional unpublished data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Chun-Qing Zhang, PhD, Department of Gastroenterology, Provincial Hospital Affiliated to Shandong University, 324 Jingwu Weiqi Road, Jinan 250021, Shandong Province, China. zhchqing@medmail.com.cn
Telephone: +86-531-66953227 Fax: +86-531-87906348
Received: March 30, 2015
Peer-review started: March 31, 2015
First decision: April 23, 2015
Revised: May 15, 2015
Accepted: July 8, 2015
Article in press: July 9, 2015
Published online: August 28, 2015
Processing time: 151 Days and 9 Hours
Abstract

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 μmol/L carvedilol for 24 h, respectively. Carvedilol (10 μmol/L) reduced VEGF-induced HUVEC migration from 67.54 ± 7.83 to 37.11 ± 3.533 (P < 0.001). Carvedilol concentrations of 5 μmol/L and 10 μmol/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 μmol/L and 10 μmol/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 μmol/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 μmol/L carvedilol reduced VEGF-induced ERK 1/2 phosphorylation from 181.9% ± 18.61% to 56.45% ± 7.64% (P < 0.01). The VEGF-induced increase in Src kinase activity was alleviated by carvedilol [decreased from 141.8% ± 15.37% to 53.57 ± 7.18% (P < 0.01) and 47.04% ± 9.74% (P < 0.01) at concentrations of 5 and 10 μmol/L, respectively]. Pretreatment of HUVECs with Src kinase inhibitor almost completely prevented the VEGF-induced ERK upregulation [decreased from 213.2% ± 27.68% to 90.96% ± 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.

Keywords: Carvedilol; Adrenergic β-antagonists; Angiogenesis; Liver cirrhosis; Drug utilization

Core tip: Carvedilol has been used for the treatment of portal hypertension for many years. In this study, carvedilol directly inhibited the proliferation and tube formation of cultured human umbilical vascular endothelial cells. Moreover, this study is the first to investigate the mechanism of the anti-angiogenic effect of carvedilol, which functions by inhibiting vascular endothelial growth factor-induced mitogen-activated protein kinase signaling pathways. These novel activities of carvedilol provide insight into the anti-angiogenic mechanisms involved, and highlight its potential therapeutic application against angiogenesis-dependent liver fibrosis.