Whittles C E, Pocock T M, Wedge S R, Kendrew J, Hennequin L F, Harper S J, Bates D O
Microvascular Research Laboratories, Department of Physiology, Preclinical Vet School, University of Bristol, Southwell Street, Bristol, UK.
Microcirculation. 2002 Dec;9(6):513-22. doi: 10.1038/sj.mn.7800164.
Vascular endothelial growth factor (VEGF) increases vascular permeability and angiogenesis in many pathological conditions including cancer, arthritis, and diabetes. VEGF activates VEGF-Receptor 1(VEGF-R1) and VEGF-Receptor 2 (VEGF-R2), which autophosphorylate to initiate a signaling cascade resulting in angiogenesis and increased microvascular permeability. Here we describe a novel VEGF-R2 selective inhibitor, ZM323881 (5-[[7-(benzyloxy) quinazolin-4-yl]amino]-4-fluoro-2-methylphenol), that is a potent and selective inhibitor of VEGF-R2 tyrosine kinase in vitro (IC(50) < 2 nM), compared with other receptor tyrosine kinases, including VEGF-R1 (IC(50) > 50 microM).
Endothelial cell proliferation was assayed by (3)H-thymidine incorporation in response to VEGF-A +/- ZM323881. The effect of ZM323881 on VEGF-mediated permeability was measured in frog microvessels using the Landis Michel technique. To ensure that ZM323881 was effective in frogs, western analysis was performed on protein extracted from frog lungs incubated in the presence or absence of VEGF-A or VEGF-A with ZM323881.
ZM323881 inhibits VEGF-A-induced endothelial cell proliferation (IC(50) = 8 nM) and VEGF-R2 tyrosine phosphorylation in vitro. VEGF-A-mediated increases in vascular permeability in perfused mesenteric microvessels in vivo were reversibly abolished by both ZM323881 and the class III receptor tyrosine kinase inhibitor PTK787/ZK222584.
These data suggest that VEGF-R2 phosphorylation is necessary for VEGF-A-mediated increases in microvascular permeability in vivo.
血管内皮生长因子（VEGF）在包括癌症、关节炎和糖尿病在内的许多病理状况下会增加血管通透性并促进血管生成。VEGF激活VEGF受体1（VEGF-R1）和VEGF受体2（VEGF-R2），它们会自动磷酸化以启动信号级联反应，从而导致血管生成和微血管通透性增加。在此，我们描述了一种新型的VEGF-R2选择性抑制剂ZM323881（5-[[7-(苄氧基)喹唑啉-4-基]氨基]-4-氟-2-甲基苯酚），与其他受体酪氨酸激酶（包括VEGF-R1，IC50>50μM）相比，它在体外是一种强效且选择性的VEGF-R2酪氨酸激酶抑制剂（IC50<2 nM）。
通过³H-胸腺嘧啶核苷掺入法检测内皮细胞对VEGF-A±ZM323881的增殖反应。使用兰迪斯·米歇尔技术在蛙微血管中测量ZM323881对VEGF介导的通透性的影响。为确保ZM323881在蛙体内有效，对在有或无VEGF-A或VEGF-A与ZM323881存在的情况下孵育的蛙肺中提取的蛋白质进行蛋白质印迹分析。
ZM323881在体外抑制VEGF-A诱导的内皮细胞增殖（IC50 = 8 nM）和VEGF-R2酪氨酸磷酸化。在体内灌注的肠系膜微血管中，ZM323881和III类受体酪氨酸激酶抑制剂PTK787/ZK222584均可可逆地消除VEGF-A介导的血管通透性增加。
这些数据表明，VEGF-R2磷酸化对于VEGF-A介导的体内微血管通透性增加是必需的。
