Carvedilol may attenuate liver cirrhosis by inhibiting angiogenesis through the VEGF-Src-ERK signaling pathway
Carvedilol may attenuate liver cirrhosis by inhibiting angiogenesis through the VEGF-Src-ERK signaling pathway
复制标题
卡维地洛可能通过 VEGF-Src-ERK 信号通路抑制血管生成，从而减轻肝硬化
DOI：
10.3748/wjg.v21.i32.9566
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发表时间：
2015-08-28
影响因子：
4.3
通讯作者：
Zhang, Chun-Qing
中科院分区：
文献类型：
--
作者：
Ding, Qian;Tian, Xiang-Guo;Zhang, Chun-Qing
目的：探讨卡维地洛对血管生成的影响及其信号通路。方法：采用人脐血管内皮细胞（HUVEC）模型，观察卡维地洛对血管生成的影响。CCK8法测定卡维地洛对细胞活力的影响。流式细胞术评价卡维地洛对细胞周期进展的影响。通过细胞迁移、跨井迁移和成管实验分析卡维地洛对HUVEC功能的影响。血管内皮生长因子（VEGF）诱导HUVECs活化，分别用不同的卡维地洛浓度或不加卡维地洛预处理。Western blot分析检测了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来评估Src在vegf诱导的血管生成途径中的作用。结果：卡维地洛抑制HUVEC增殖呈剂量依赖性（IC50 = 38.5 mmol/L）。1、5和10 μ mol/L卡维地洛作用24 h后，S期细胞的分布分别从43.6%下降到37.2%、35.6%和17.8%。卡维地洛（10 μ mol/L）使vegf诱导的HUVEC迁移量从67.54 +/- 7.83降低至37.11 +/- 3.533 （P < 0.001）。卡维地洛浓度为5 μ mol/L和10 μ mol/L时，细胞侵袭率分别从196.3% +/- 18.76%降低至114.0% +/- 12.20%和51.68% +/- 8.28%。5 μ mol/L卡维地洛和10 μ mol/L卡维地洛对vegf诱导的试管形成也有显著抑制作用，分别从286.0 +/- 36.72降低到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水平，以确定这些减少的原因。10 μ mol/L卡维地洛使vegf诱导的VEGFR-2酪氨酸磷酸化从175.5% +/- 8.54%降低到52.67% +/- 5.33% （P < 0.01）。此外，10 μ mol/L卡维地洛使vegf诱导的ERK 1/2磷酸化从181.9% β 18.61%降低至56.45% β 7.64% （P < 0.01）。卡维地洛缓解了vegf诱导的Src激酶活性升高[在浓度为5和10 μ mol/L时，分别从141.8% β 15.37%降至53.57 β 7.18% （P < 0.01）和47.04% β 9.74% (P < 0.01)]。Src激酶抑制剂预处理HUVECs几乎完全阻止了vegf诱导的ERK上调[从213.2% β 27.68%下降到90.96% β 17.16% (P < 0.01)]。结论：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。
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.