Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase
Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase
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DOI：
10.1074/jbc.m301253200
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发表时间：
2003-09-19
影响因子：
4.8
通讯作者：
Avraham, S
中科院分区：
文献类型：
--
作者：
Avraham, HK;Lee, TH;Avraham, S
血管内皮生长因子（VEGF）在脑损伤后血脑屏障破坏和血管生成中发挥重要作用。 VEGF 诱导的内皮细胞迁移是血管生成反应的关键步骤，由粘着斑复合物组装和分解速度加快介导。在这项研究中，我们确定了 VEGF 调节人脑微血管内皮细胞 (HBMEC) 完整性和粘着斑组装的信号传导机制，粘着斑是由支架和信号蛋白组成的复合物，通过粘附到细胞外基质而组织。我们发现，对铺在层粘连蛋白或纤连蛋白上的 HBMEC 进行 VEGF 处理可刺激细胞骨架组织并增加粘着斑位点。用 VEGF 抗体或抑制 Flk-1/KDR 受体磷酸化的特异性抑制剂 SU-1498 预处理细胞，阻断 VEGF 刺激粘着斑组装的能力。 VEGF 诱导粘着斑激酶 (FAK) 与整合素 α(v)β(5) 偶联以及细胞骨架成分桩蛋白和 p130(cas) 的酪氨酸磷酸化。此外，FAK 和相关的粘着斑酪氨酸激酶 (RAFTK)/Pyk2 激酶被 VEGF 酪氨酸磷酸化，并且被发现对粘着斑位点很重要。野生型RAFTK/Pyk2的过表达增加了HBMEC的细胞扩散和迁移，而催化失活突变体RAFTK/Pyk2的过表达显着抑制了HBMEC的扩散（类似于70%）、粘附（类似于82%）和迁移（类似于65%）。此外，显性干扰突变体 FRNK（FAK 相关非激酶）对 FAK 的阻断显着抑制了 HBMEC 的扩散和迁移，并且还破坏了粘着斑。因此，这些研究定义了 VEGF 通过激活 FAK 和 RAFTK/Pyk2 在 HBMEC 中粘着斑复合物组装中发挥调节作用的机制。
Vascular endothelial growth factor ( VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury. VEGF-induced endothelial cell migration is a key step in the angiogenic response and is mediated by an accelerated rate of focal adhesion complex assembly and disassembly. In this study, we identified the signaling mechanisms by which VEGF regulates human brain microvascular endothelial cell (HBMEC) integrity and assembly of focal adhesions, complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. We found that VEGF treatment of HBMECs plated on laminin or fibronectin stimulated cytoskeletal organization and increased focal adhesion sites. Pretreating cells with VEGF antibodies or with the specific inhibitor SU-1498, which inhibits Flk-1/KDR receptor phosphorylation, blocked the ability of VEGF to stimulate focal adhesion assembly. VEGF induced the coupling of focal adhesion kinase (FAK) to integrin alpha(v)beta(5) and tyrosine phosphorylation of the cytoskeletal components paxillin and p130(cas). Additionally, FAK and related adhesion focal tyrosine kinase (RAFTK)/Pyk2 kinases were tyrosine-phosphorylated by VEGF and found to be important for focal adhesion sites. Overexpression of wild type RAFTK/Pyk2 increased cell spreading and the migration of HBMECs, whereas overexpression of catalytically inactive mutant RAFTK/Pyk2 markedly suppressed HBMEC spreading (similar to70%), adhesion (similar to82%), and migration (similar to65%). Furthermore, blocking of FAK by the dominant-interfering mutant FRNK (FAK-related non-kinase) significantly inhibited HBMEC spreading and migration and also disrupted focal adhesions. Thus, these studies define a mechanism for the regulatory role of VEGF in focal adhesion complex assembly in HBMECs via activation of FAK and RAFTK/Pyk2.