Connective tissue growth factor binds vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis
Connective tissue growth factor binds vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis
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DOI：
10.1096/fj.01-0332fje
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发表时间：
2001-12-01
期刊：
影响因子：
4.8
通讯作者：
Okada, Y
中科院分区：
文献类型：
--
作者：
Inoki, I;Shiomi, T;Okada, Y
血管内皮生长因子（VEGF）是一种强的促血管生成的有丝分裂原，在多种病理生理条件下的血管生成中起重要作用。分泌的VEGF的体内血管生成活性可由细胞外抑制剂调节，因为它也在无血管组织如软骨中产生。为了寻找VEGF的结合抑制剂，我们以VEGF（165）为诱饵，利用酵母双杂交系统从软骨细胞cDNA文库中筛选出结缔组织生长因子（CTGF）。VEGF（165）与CTGF的复合物形成首先通过来自过表达两种结合伴侣的细胞的免疫沉淀来建立。竞争性亲和力结合测定也证明CTGF以两类结合位点（Kd = 26 +/- 11 nM和125 +/- 38 nM）特异性结合VEGF（165）。使用CTGF缺失突变体的结合试验表明，CTGF的血小板反应蛋白1型重复（TSP-1）结构域与VEGF的外显子7编码区结合（165），COOH末端结构域保留了与VEGF（165）和VEGF（121）的亲和力。VEGF（165）与CTGF的相互作用抑制VEGF（165）与内皮细胞和固定化的KDR/IgG Fc的结合;即VEGF（165）受体的重组蛋白。通过内皮细胞的体外管形成测定，全长CTGF和具有TSP-1结构域的缺失突变体以复合物形式显著抑制VEGF（165）诱导的血管生成。CTGF的这种抗血管生成活性通过使用Matrigel注射模型在小鼠中的体内血管生成测定进一步证明。这些数据首次证明VEGF（165）通过蛋白质-蛋白质相互作用与CTGF结合，并表明VEGF（165）的血管生成活性在细胞外环境中受CTGF负调控。
Vascular endothelial growth factor (VEGF) is a strong angiogenic mitogen and plays important roles in angiogenesis under various pathophysiological conditions. The in vivo angiogenic activity of secreted VEGF may be regulated by extracellular inhibitors, because it is also produced in avascular tissues such as the cartilage. To seek the binding inhibitors against VEGF, we screened the chondrocyte cDNA library by a yeast two-hybrid system by using VEGF(165) as bait and identified connective tissue growth factor (CTGF) as a candidate. The complex formation of VEGF(165) with CTGF was first established by immunoprecipitation from the cells overexpressing both binding partners. A competitive affinity-binding assay also demonstrated that CTGF binds specifically to VEGF(165) with two classes of binding sites (Kd = 26 +/- 11 nM and 125 +/- 38 nM). Binding assay using deletion mutants of CTGF indicated that the thrombospondin type-1 repeat (TSP-1) domain of CTGF binds to the exon 7-coded region of VEGF(165) and that the COOH-terminal domain preserves the affinity to both VEGF(165) and VEGF(121). The interaction of VEGF(165) with CTGF inhibited the binding of VEGF(165) to the endothelial cells and the immobilized KDR/IgG Fc; that is, a recombinant protein for VEGF(165) receptor. By in vitro tube formation assay of endothelial cells, full-length CTGF and the deletion mutant possessing the TSP-1 domain inhibited VEGF(165)-induced angiogenesis significantly in the complex form. This antiangiogenic activity of CTGF was demonstrated further by in vivo angiogenesis assay by using Matrigel injection model in mice. These data demonstrate for the first time that VEGF(165) binds to CTGF through a protein-to-protein interaction and suggest that the angiogenic activity of VEGF(165) is regulated negatively by CTGF in the extracellular environment.