Recombinant RGD-disintegrin DisBa-01 blocks integrin αvβ3 and impairs VEGF signaling in endothelial cells
Recombinant RGD-disintegrin DisBa-01 blocks integrin αvβ3 and impairs VEGF signaling in endothelial cells
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DOI：
10.1186/s12964-019-0339-1
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发表时间：
2019-03-20
影响因子：
8.4
通讯作者：
Selistre-de-Araujo, Heloisa S.
中科院分区：
文献类型：
--
作者：
Danilucci, Tais M.;Santos, Patty K.;Selistre-de-Araujo, Heloisa S.
背景整合素通过连接细胞内的机械和周围的细胞外基质来调节细胞的黏附、迁移和存活。以往的研究表明，(V3)整合素与血管内皮生长因子2型受体(VEGFR2)在血管生成中存在相互作用。Disba-01是松墨天牛蛇毒重组His-Tag融合RGD-去整合素，通过纳米分子亲和力与(V3)整合素结合，阻断细胞与细胞外基质的黏附。方法人脐静脉内皮细胞(HUVECs)在纤维连接蛋白(FN)或玻璃体连接蛋白(VN)包被的平板上培养，同时加入血管内皮生长因子(VEGF)、血管内皮细胞生长因子(Disba-01)(1000 Nm)或血管内皮生长因子(VEGF)和血管内皮细胞生长因子(Disba-01)进行迁移、侵袭和增殖实验。Western blotting分析(V3)/VEGFR2受体的磷酸化和细胞内信号通路的激活。结果Disba-01处理内皮细胞可抑制血管内皮细胞的迁移、侵袭和小管生成等血管生成的关键步骤。该去整合素阻断(V3)/VEGFR2信号通路，减少VEGFR2和(3)整合素亚基的蛋白表达和磷酸化，调节FAK/SRC/paxlin下游信号，抑制ERK1/2和PI3K信号通路。这些事件导致肌动蛋白重组并抑制HUVEC的迁移和黏附。结论去整合素抑制(V3)整合素抑制VEGFR2信号转导通路，即使在有血管内皮生长因子存在的情况下，也能阻断VEGFR2信号通路，从而影响血管生成机制。这些结果加深了我们对药物抑制血管生成机制的理解。
BackgroundIntegrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the interaction between (v3) 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 (v3) 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 (v3)/VEGFR2 cross-talk and its downstream pathways.MethodsHuman 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 (1000nM) or VEGF and DisBa-01 simultaneously. Phosphorylation of (v3)/VEGFR2 receptors and the activation of intracellular signaling pathways were analyzed by western blotting. Morphological alterations were observed and quantified by fluorescence confocal microscopy.ResultsDisBa-01 treatment of endothelial cells inhibited critical steps of VEGF-mediated angiogenesis such as migration, invasion and tubulogenesis. The blockage of (v3)/VEGFR2 cross-talk by this disintegrin decreases protein expression and phosphorylation of VEGFR2 and (3) 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 (v3) integrin and VEGFR2 in treated cells.ConclusionsDisintegrin inhibition of (v3) 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.