重组 RGD-整联蛋白 disintegrin DisBa-01 阻断整合素 αβ 并损害内皮细胞中的 VEGF 信号通路。

Recombinant RGD-disintegrin DisBa-01 blocks integrin αβ and impairs VEGF signaling in endothelial cells.

机构信息

Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, Rod. Washington Luis, km 235 - SP-310 - São Carlos, São Paulo, CEP 13565-905, Brazil.

出版信息

Cell Commun Signal. 2019 Mar 20;17(1):27. doi: 10.1186/s12964-019-0339-1.

DOI:10.1186/s12964-019-0339-1

PMID:30894182

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6425665/

Abstract

BACKGROUND

Integrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the interaction between αβ integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. DisBa-01, a recombinant His-tag fusion, RGD-disintegrin from Bothrops alternatus snake venom, binds to αβ integrin with nanomolar affinity blocking cell adhesion to the extracellular matrix. Here we present in vitro evidence of a direct interference of DisBa-01 with αβ/VEGFR2 cross-talk and its downstream pathways.

METHODS

Human umbilical vein (HUVECs) were cultured in plates coated with fibronectin (FN) or vitronectin (VN) and tested for migration, invasion and proliferation assays in the presence of VEGF, DisBa-01 (1000 nM) or VEGF and DisBa-01 simultaneously. Phosphorylation of αβ/VEGFR2 receptors and the activation of intracellular signaling pathways were analyzed by western blotting. Morphological alterations were observed and quantified by fluorescence confocal microscopy.

RESULTS

DisBa-01 treatment of endothelial cells inhibited critical steps of VEGF-mediated angiogenesis such as migration, invasion and tubulogenesis. The blockage of αβ/VEGFR2 cross-talk by this disintegrin decreases protein expression and phosphorylation of VEGFR2 and β integrin subunit, regulates FAK/SrC/Paxillin downstream signals, and inhibits ERK1/2 and PI3K pathways. These events result in actin re-organization and inhibition of HUVEC migration and adhesion. Labelled-DisBa-01 colocalizes with αβ integrin and VEGFR2 in treated cells.

CONCLUSIONS

Disintegrin inhibition of αβ integrin blocks VEGFR2 signalling, even in the presence of VEGF, which impairs the angiogenic mechanism. These results improve our understanding concerning the mechanisms of pharmacological inhibition of angiogenesis.

摘要

背景

整合素通过将细胞内机制与周围细胞外基质连接起来，介导细胞黏附、迁移和存活。先前的研究表明，在 VEGF 诱导的血管生成中，αβ 整合素与 VEGF 型 2 受体（VEGFR2）之间存在相互作用。DisBa-01 是一种重组 His 标签融合物，是来自矛头蝮蛇蛇毒的 RGD 去整合素，以纳摩尔亲和力与 αβ 整合素结合，阻断细胞黏附到细胞外基质。在这里，我们提供了 DisBa-01 与 αβ/VEGFR2 串扰及其下游途径直接干扰的体外证据。

方法

将人脐静脉内皮细胞（HUVECs）培养在涂有纤维连接蛋白（FN）或 vitronectin（VN）的平板上，并在 VEGF、DisBa-01（1000 nM）或 VEGF 和 DisBa-01 同时存在的情况下进行迁移、侵袭和增殖测定。通过 Western blot 分析 αβ/VEGFR2 受体的磷酸化和细胞内信号通路的激活。通过荧光共聚焦显微镜观察和量化形态改变。

结果

DisBa-01 处理内皮细胞抑制了 VEGF 介导的血管生成的关键步骤，如迁移、侵袭和小管形成。这种去整合素阻断 αβ/VEGFR2 串扰，降低 VEGFR2 和β 整合素亚基的蛋白表达和磷酸化，调节 FAK/SrC/Paxillin 下游信号，并抑制 ERK1/2 和 PI3K 通路。这些事件导致肌动蛋白重排和 HUVEC 迁移和黏附的抑制。标记的 DisBa-01 与处理细胞中的 αβ 整合素和 VEGFR2 共定位。