在斑马鱼胚胎发育过程中，基础Flt1酪氨酸激酶活性是内皮细胞存活和血管形成的正向调节因子。
Basal Flt1 tyrosine kinase activity is a positive regulator of endothelial survival and vascularization during zebrafish embryogenesis.
作者信息
Li Shang, Zhou Xue Lin, Dang Yuan Ye, Kwan Yiu Wa, Chan Shun Wan, Leung George Pak Heng, Lee Simon Ming-Yuen, Hoi Maggie Pui Man
机构信息
State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao, China.
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China.
出版信息
Biochim Biophys Acta. 2015 Feb;1850(2):373-84. doi: 10.1016/j.bbagen.2014.10.023. Epub 2014 Oct 29.
BACKGROUND
The role of Kdr (VEGFR-2/Flk-1) in vascular formation has been well described, but the role of Flt1 (VEGFR-1) is not well studied and is generally considered as a decoy receptor for trapping VEGF.
METHODS
The effects of VEGFR1/2 kinase inhibitor (VRI) and calycosin on Flt1 tyrosine kinase (TK) activity were evaluated by molecular docking, enzymatic inhibition assay, protein co-immunoprecipitation and siRNA gene knock-down analysis in HUVECs. Toxicities of the chemicals were examined using HUVECs viability. Their effects on angiogenesis and vessel formation were furthered studied in HUVECs in vitro and Tg(fli-1:EGFP) zebrafish in vivo. The gene and protein expression of VEGF and VEGF receptors were investigated by quantitative RT-PCR and Western blot.
RESULTS
VRI strongly inhibited physiological functions of both VEGF receptors and suppressed endothelial cell survival. This resulted in blood vessel loss in zebrafish embryos. Interestingly, calycosin co-treatment impeded VRI-induced blood vessel loss. Docking and kinase inhibition assay revealed that calycosin competed with VRI for the tyrosine kinase domain of Flt1 without affecting ATP binding. On the contrary, calycosin did not affect the interaction between VRI and Kdr-TK. Consistent with these results, calycosin counteracted the inhibition of Flt1-TK and PI3K phosphorylation induced by VRI in HUVECs. Further studies in vitro and in vivo showed that the minimizing effect of calycosin on VRI-mediated endothelial cytotoxicity was blocked by wortmannin (a PI3K inhibitor). The impeding effect of calycosin on VRI-induced blood vessel loss was absent in zebrafish embryos injected with Flt1 MO.
CONCLUSIONS
Flt1-tyrosine kinase (TK) activity contributed significantly in endothelial cells survival and vascular development during embryo angiogenesis in zebrafish by engaging PI3K/Akt pathway.
GENERAL SIGNIFICANCE
The roles of Flt1 activity in endothelial cell survival in physiological vascular formation may have been previously under-appreciated.
背景
Kdr（血管内皮生长因子受体-2/Flk-1）在血管形成中的作用已得到充分描述，但Flt1（血管内皮生长因子受体-1）的作用尚未得到充分研究，通常被认为是一种捕获血管内皮生长因子的诱饵受体。
方法
通过分子对接、酶抑制试验、蛋白质免疫共沉淀和siRNA基因敲除分析，在人脐静脉内皮细胞（HUVECs）中评估血管内皮生长因子受体1/2激酶抑制剂（VRI）和毛蕊异黄酮对Flt1酪氨酸激酶（TK）活性的影响。使用HUVECs活力检测化学物质的毒性。在体外HUVECs和体内Tg（fli-1：EGFP）斑马鱼中进一步研究它们对血管生成和血管形成的影响。通过定量逆转录聚合酶链反应和蛋白质免疫印迹法研究血管内皮生长因子（VEGF）和血管内皮生长因子受体的基因和蛋白质表达。
结果
VRI强烈抑制两种血管内皮生长因子受体的生理功能并抑制内皮细胞存活。这导致斑马鱼胚胎血管缺失。有趣的是，毛蕊异黄酮联合处理可阻止VRI诱导的血管缺失。对接和激酶抑制试验表明，毛蕊异黄酮与VRI竞争Flt1的酪氨酸激酶结构域，而不影响ATP结合。相反，毛蕊异黄酮不影响VRI与Kdr-TK之间的相互作用。与这些结果一致，毛蕊异黄酮抵消了VRI在HUVECs中诱导的Flt1-TK和磷脂酰肌醇-3-激酶（PI3K）磷酸化的抑制作用。体外和体内的进一步研究表明，渥曼青霉素（一种PI3K抑制剂）可阻断毛蕊异黄酮对VRI介导的内皮细胞毒性的最小化作用。在注射Flt1吗啉代寡核苷酸的斑马鱼胚胎中，毛蕊异黄酮对VRI诱导的血管缺失的阻碍作用不存在。
结论
Flt1酪氨酸激酶（TK）活性通过参与PI3K/蛋白激酶B（Akt）途径，在斑马鱼胚胎血管生成过程中的内皮细胞存活和血管发育中起重要作用。
普遍意义
Flt1活性在生理性血管形成中对内皮细胞存活的作用可能此前未得到充分认识。
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