Targeting gamma-secretase activation for anti-angiogenesis

靶向伽玛分泌酶激活以抗血管生成

• 批准号: 7917776
• 负责人: Michael Edwin Boulton
• 金额: $ 38.33万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7917776
• 关键词: Adhesions; Animal Model; Binding; C-terminal; COS Cells; Cell Nucleus; Cell membrane; Cells; Chimeric Proteins; Choroidal Neovascularization; Cleaved cell; Complex; Cytoplasm; Data; Developed Countries; Developing Countries; Endothelial Cells; Eye diseases; Family; Family member; Image; Impairment; In Vitro; Intracellular translocation; Label; Lasers; Membrane; Modeling; Molecular; Oxygen; Permeability; Phosphorylation; Play; Process; Proteins; Regulation; Research Personnel; Retinal; Retinal Diseases; Retinal Neovascularization; Role; Signal Transduction; Site; Small Interfering RNA; Testing; Time; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Vascular Permeabilities; Vision; angiogenesis; antiangiogenesis therapy; base; c-ets1 transcription factor; cadherin 5; gamma secretase; in vitro Model; in vivo; in vivo Model; mouse model; neovascular; neovascularization; occludin; ocular neovascularization; programs; response; secretase; trafficking

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.

描述（由适用提供）：眼睛的新血管疾病是发达国家视力障碍的主要原因。集体证据表明，血管内皮生长因子（VEGF）家族对眼部血管生成至关重要。在VEGF受体中，长期以来一直认为VEGFR2是眼部新血管化的主要效应因子，而VEGFR1的贡献直到最近才被认识到。我们的数据支持VEGFR1在VEGFR-2诱导的血管生成中的关键作用，并表明这取决于 - 分泌酶调节的裂解和VEGFR1的细胞内易位（Cai等人2002; 2006）。我们可以证明？ - 分泌酶是体外和体内血管渗透性和血管生成的潜在调节剂，并且这种作用取决于VEGFR1的C末端域的靶向易位及其与» - 钙宁和转录因子ETETS-1的关联。基于这些观察结果，我们提出了以下假设：“ VEGFR2驱动的新血管形成受VEGFR1的细胞内结构域的易位，以及将vegfr1的动员，募集和结合到这一过程中，我们进一步验证了VEGFR1的bigalline gromince，我们进一步验证了bine to and vegfr1。 - 蛋白酶。我们认为 - 分泌酶配合物和/或VEGFR1体内的操纵会降低血管渗透性，并抑制异常的残留残留新血管新生血管。 Using cultured residual 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 translation 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 residual and choriodal microvascular endothelial cells and e) characterize the ？ - 分泌酶在确定VEGFR1与连接蛋白的结合中的作用。我们将使用三种视网膜新血管形成的动物模型（即氧诱导的视网膜病模型，视网膜新生血管化模型，激光诱导的脉络性新血管化模型的ADCAS模型，与对生物的贡献相结合的贡献？我们认为， - 分泌酶复合物与VEGFR1之间的相互作用以及随后的血管生成反应将确定确定血管渗透性和抑制异常的新策略。