Ding Qian, Tian Xiang-Guo, Li Yan, Wang Qi-Zhi, Zhang Chun-Qing
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.
World J Gastroenterol. 2015 Aug 28;21(32):9566-76. doi: 10.3748/wjg.v21.i32.9566.
To investigate the effect of carvedilol on angiogenesis and the underlying signaling pathways.
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.
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)].
Carvedilol has an anti-angiogenic effect on HUVECs. This inhibitory effect is mediated by VEGF-induced Src-ERK signaling pathways.
研究卡维地洛对血管生成的影响及其潜在的信号通路。
使用人脐静脉血管内皮细胞（HUVEC）模型检测卡维地洛对血管生成的影响。采用CCK8法检测卡维地洛对细胞活力的影响。运用流式细胞术评估卡维地洛对细胞周期进程的影响。进行细胞迁移、Transwell迁移和管腔形成实验，以分析卡维地洛对HUVEC功能的影响。用不同浓度卡维地洛或不用卡维地洛预处理人脐静脉血管内皮细胞后，检测血管内皮生长因子（VEGF）诱导的细胞激活情况。蛋白质免疫印迹分析检测三种细胞信号通路蛋白VEGFR-2、Src和细胞外信号调节激酶（ERK）的磷酸化水平。使用特异性Src抑制剂PP2评估Src在VEGF诱导的血管生成途径中的作用。
卡维地洛以剂量依赖性方式抑制HUVEC增殖（IC50 = 38.5 mmol/L）。1、5和10 μmol/L卡维地洛处理24小时后，S期细胞分布分别从43.6%降至37.2%、35.6%和17.8%。卡维地洛（10 μmol/L）使VEGF诱导的HUVEC迁移从67.54±7.83降至37.11±3.533（P < 0.001）。5 μmol/L和10 μmol/L卡维地洛浓度分别使细胞侵袭从196.3%±18.76%降至114.0%±12.20%和51.68%±8.28%。5 μmol/L和10 μmol/L卡维地洛也显著减少VEGF诱导的管腔形成，分别从286.0±36.72降至135.7±18.13（P < 0.05）和80.27±11.16（P < 0.01）。我们研究了几种细胞内蛋白质水平以确定这些降低的原因。10 μmol/L卡维地洛处理使VEGF诱导的VEGFR-2酪氨酸磷酸化从175.5%±8.54%降至52.67%±5.33%（P < 0.01）。此外，10 μmol/L卡维地洛使VEGF诱导的ERK 1/2磷酸化从181.9%±18.61%降至56.45%±7.64%（P < 0.01）。卡维地洛减轻了VEGF诱导的Src激酶活性增加[5和10 μmol/L浓度下分别从141.8%±15.37%降至53.57±7.18%（P < 0.01）和47.04%±9.74%（P < 0.01）]。用Src激酶抑制剂预处理HUVEC几乎完全阻止了VEGF诱导的ERK上调[从213.2%±27.68%降至90.96%±17.16%（P < 0.01）]。
卡维地洛对HUVEC具有抗血管生成作用。这种抑制作用是由VEGF诱导的Src-ERK信号通路介导的。
