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


CONCLUSIONS
Carvedilol has an anti-angiogenic effect on HUVECs. This inhibitory effect is mediated by VEGF-induced Src-ERK signaling pathways.
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.
Qian
Q
Ding
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.
Xiang-Guo
XG
Tian
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.
Yan
Y
Li
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.
Qi-Zhi
QZ
Wang
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.
Chun-Qing
CQ
Zhang
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D002227:Carbazoles / Q000494:pharmacology*; D000077261:Carvedilol; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D008103:Liver Cirrhosis / Q000188:drug therapy* / Q000378:metabolism / Q000503:physiopathology; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010766:Phosphorylation; D011412:Propanolamines / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D059807:S Phase Cell Cycle Checkpoints / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D020533:Angiogenesis Inhibitors; D002227:Carbazoles; D011412:Propanolamines; D047428:Protein Kinase Inhibitors; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D000077261:Carvedilol; C000588866:KDR protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases
Adrenergic β-antagonists; Angiogenesis; Carvedilol; Drug utilization; Liver cirrhosis