Targeting gamma-secretase activation for anti-angiogenesis

描述(申请人提供)：眼部新生血管疾病是发达国家视力受损的主要原因。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重要作用。在血管内皮生长因子受体中，VEGFR2被认为是眼部新生血管的主要效应因子，而VEGFR1的作用则是最近才被认识到的。我们的数据支持VEGFR1在VEGFR-2诱导的血管生成调节中的关键作用，并表明这依赖于？-分泌酶调节的VEGFR1的裂解和细胞内转位(Cai等人，2002；2006)。我们可以证明，β-分泌酶在体外和体内都是一种有效的血管通透性和血管生成的调节因子，并且这种作用依赖于VEGFR1 C-末端结构域的靶向转位及其分别与β-连环蛋白和转录因子Ets-1的关系。基于这些观察，我们提出了以下假说：“VEGFR2驱动的新生血管是受VEGFR1胞内区移位的负性调节的，而β-分泌酶与VEGFR1的动员、募集和结合是这一过程的关键。我们进一步假设VEGFR1的胞内区通过与β-连环蛋白结合来调节血管通透性。我们相信在体内操纵β-分泌酶复合体和/或VEGFR1将降低血管通透性并抑制异常的视网膜新生血管。”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)确定涉及质膜中？分泌酶组装和激活的分子机制，并确定VEGFR1中的结合和切割位置；b)表征？分泌酶如何磷酸化VEGFR-1的细胞内区域，以及这如何促进VEGFR-1的转位和信号传递；c)使用由荧光标记的VEGR1组成的融合蛋白，结合实时成像来表征？分泌酶活性变化后VEGFR-1的移位和细胞内定位。D)利用siRNA研究确定调节培养的视网膜和脉络膜微血管内皮细胞通透性和血管生成的β-分泌酶形成和激活的步骤，以及e)表征β-分泌酶在调节VEGFR1与连接蛋白结合中的作用。我们将利用三种视网膜新生血管的动物模型(即氧诱导的视网膜病变模型、AdCAS视网膜新生血管模型和激光诱导的脉络膜新生血管模型)，结合调节特定的β-分泌酶亚基的药物来证实β-分泌酶对体外通透性和血管生成的影响，以评估它们的作用。我们相信，表征分泌酶复合体和VEGFR1之间的相互作用，以及随后的血管生成反应，将为体内调节血管通透性和抑制异常的视网膜新生血管确定新的策略。

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.