Targeting gamma-secretase activation for anti-angiogenesis

描述（由申请人提供）：眼睛的新生血管性疾病是发达国家视力损害的主要原因。大量证据表明血管内皮生长因子（VEGF）家族在眼部血管生成中起着关键作用。在VEGF受体中，长期以来一直认为VEGFR 2是眼部新生血管形成的主要效应物，而VEGFR 1的作用只是最近才被认识到。我们的数据支持VEGFR 1在调节VEGFR-2诱导的血管生成中的关键作用，并表明这依赖于？分泌酶调节VEGFR 1的切割和细胞内易位（Cai et al 2002; 2006）。我们可以展示吗？-分泌酶是体外和体内血管通透性和血管生成的有效调节剂，并且这种作用依赖于VEGFR 1的C-末端结构域的靶向移位及其分别与β-连环蛋白和转录因子Ets-1的结合。基于这些观察结果，我们提出了以下假设：“VEGFR 2驱动的新血管形成受VEGFR 1细胞内结构域易位的负调控，并且？分泌酶转化为VEGFR 1对这一过程至关重要。我们进一步假设VEGFR 1的细胞内结构域通过与β-连环蛋白结合来调节血管通透性。我们认为操纵？-分泌酶复合物和/或VEGFR 1在体内将降低血管通透性并抑制异常视网膜新生血管形成。“使用培养的视网膜和脉络膜微血管细胞，以及适当的COS细胞，我们将a）确定参与？分泌酶在质膜中的组装和活化，并鉴定VEGF 1中的结合和切割位点，B）如何表征？分泌酶磷酸化VEGFR-1的细胞内结构域，以及这如何有助于VEGFR-1的易位和信号传导，c）使用由荧光标记的VEGFR 1组成的融合蛋白与真实的时间成像相结合，以表征在？分泌酶活性，d）使用siRNA研究来鉴定？在培养的视网膜和脉络膜微血管内皮细胞中调节渗透性和血管生成的分泌酶的形成和激活，以及e）表征？-分泌酶调节VEGFR 1与连接蛋白的结合。我们将证实？-使用三种视网膜新生血管形成的动物模型（即氧诱导的视网膜病变模型、视网膜新生血管形成的ADCaS模型和激光诱导的脉络膜新生血管形成模型）联合调节特异性？分泌酶亚基，以评估其贡献。我们认为，表征之间的相互作用？-分泌酶复合物和VEGFR 1，以及随后的血管生成反应，将确定新的策略，调节血管通透性和抑制异常视网膜新生血管在体内。

DESCRIPTION (provided by applicant): Neovascular diseases of the eye are the leading causes of vision impairment in developed nations. The collective evidence suggests that the vascular endothelial growth factor (VEGF) family is critical for ocular angiogensis. Of the VEGF receptors it has long been considered that VEGFR2 is the major effector of ocular neovascularization while the contribution of VEGFR1 has only recently been recognized. Our data support a critical role for VEGFR1 in the regulation of VEGFR-2 induced angiogenesis and show that this is dependent on ?-secretase regulated cleavage and intracellular translocation of VEGFR1 (Cai et al 2002; 2006). We can show that ?-secretase is a potent regulator of vascular permeability and angiogenesis both in vitro and in vivo and that this effect is dependent on the targeted translocation of the C-terminal domain of VEGFR1 and its association with ¿-catenin and transcription factor Ets-1 respectively. Based on these observations we put forward the following hypothesis:"VEGFR2 driven neovascularization is negatively regulated by the translocation of the intracellular domain of VEGFR1 and that the mobilization, recruitment and binding of ?-secretase to VEGFR1 is critical to this process. We further postulate that the intracellular domain of VEGFR1 regulates vascular permeability through binding to ¿-catenin. We believe that manipulation of the ?-secretase complex and/or VEGFR1 in vivo will reduce vascular permeability and inhibit aberrant retinal neovascularization." Using cultured retinal and choroidal microvascular cells, and where appropriate COS cells, we will a) determine the molecular mechanisms involved in ?-secretase assembly and activation in the plasma membrane and identify the binding and cleavage sites in VEGFR1, b) characterize how ?-secretase phosphorylates the intracellular domain of VEGFR-1 and how this contributes to VEGFR-1 translocation and signaling, c) use fusion proteins consisting of fluorescently labeled VEGR1 in combination with real time imaging to characterize the translocation and intracellular localization of VEGFR-1 following changes in ?-secretase activity, d) use siRNA studies to identify the steps in ?-secretase formation and activation that regulate permeability and angiogenesis in cultured retinal and choriodal microvascular endothelial cells and e) characterize the role of ?-secretase in regulating the binding of VEGFR1 to junctional proteins. We will substantiate the effects of ?-secretase on in vitro permeability and angiogenesis using three animal models of retinal neovascularization (i.e. oxygen-induced retinopathy model, ADCaS model of retinal neovascularization and the laser-induced choroidal neovascularization model) in conjunction with agents that regulate specific ?-secretase subunits to evaluate their contribution. We believe that characterization of the interaction between the ?-secretase complex and VEGFR1, and the subsequent angiogenic response will identify new strategies for regulating vascular permeability and inhibiting aberrant retinal neovascularization in vivo.