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

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

Figures

Figure 1

Effect of carvedilol on human umbilical vascular endothelial cell proliferation. Subconfluent cultures of human umbilical vascular endothelial cells were exposed to increasing concentrations of carvedilol (0.01-40 μmol/L), and the extent of cell proliferation was measured by cholecystokinin-8 assay. The values are the means of three independent experiments.

Figure 2

Effects of carvedilol on cell cycle progression in human umbilical vascular endothelial cells. Cells were exposed to either control medium (containing 5% FBS) or medium containing the indicated concentrations of carvedilol and were treated as indicated in the Materials and Methods section. Representatives cytometric profiles (upper) and the percentage of cells at G0/G1, S and G2/M phases (%) (lower). The DNA content was determined by measuring the fluorescence intensity of incorporated propidium iodide.

Figure 3

Effect of carvedilol on cell migration. A: HUVECs were scraped and then treated with or without VEGF (50 ng/mL) and various concentrations of carvedilol. Images were taken at 0 and 24 h after scraping; B: Cell migration across the scraped area was determined using ImageJ software. (n = 12, aP < 0.05 vs control, bP < 0.01 vs VEGF alone). HUVEC: Human umbilical vascular endothelial cell; VEGF: Vascular endothelial growth factor.

Figure 4

Effect of carvedilol on cell invasion. A: HUVECs were seeded in the upper chamber of a Transwell and treated with various concentrations of carvedilol. The bottom chamber was filled with VEGF-enriched media. After 12 h, the HUVECs that migrated through the membrane were stained by hematoxylin and eosin; B: Cell numbers were quantified (n = 12, aP < 0.05 vs control, cP < 0.05, bP < 0.01 vs VEGF alone).

Figure 5

Carvedilol inhibited the vascular endothelial growth factor-induced tube formation of human umbilical vascular endothelial cells. A: HUVECs were placed in Matrigel with or without VEGF (50 ng/mL) in the presence or absence of different concentrations of carvedilol. After 8 h, tubular structures were imaged (magnification, × 100); B: Quantification of the area of cell alignment was determined using ImageJ software (n = 3, aP < 0.05, bP < 0.01 vs VEGF alone).

Figure 6

Effect of carvedilol on vascular endothelial growth factor angiogenic signaling. HUVECs were treated with VEGF (50 ng/mL) for 2 h in the presence or absence of different concentrations of carvedilol. Cell lysates were processed as indicated in the Materials and Methods. p-VEGFR2/VEGFR2 (A) and p-ERK/ERK (B) levels (n = 3, aP < 0.05 vs control, cP < 0.05, bP < 0.01 vs VEGF alone). ERK: Extracellular signal-regulated kinase.

Figure 7

Role of Src in angiogenic signaling. A: HUVECs were treated with VEGF (50 ng/mL) for 2 h in the presence or absence of different concentrations of PP2. Cell lysates were subjected to SDS-PAGE for Src phosphorylation; B: HUVECs were treated with VEGF (50 ng/mL) for 2 h in the presence or absence of different concentrations of PP2. Cell lysates were processed as indicated in the Materials and Methods (n = 3, aP < 0.05 vs control, bP < 0.01 vs VEGF alone).