Alternative Pathway of Complement Activation in Age-Related Macular Degeneration

长期目标是了解眼睛中补体信号传导的基本基础，以及如何在眼睛中激活补体。 在这个过程中的失调导致病理学，最终有助于治疗方法的发展- 毁灭性的致盲疾病。视网膜相关性黄斑变性（AMD）以两种形式发生，干性和 湿了干性AMD的特征在于玻璃疣、RPE损伤和感光细胞损失。在一些患者中， 干性形式可转变为湿性AMD。湿性AMD，本身表现为脉络膜新生血管（CNV），莱亚- 这些新血管的老化和感光细胞的快速丧失。最近的遗传学证据表明 在补体抑制蛋白因子H（CFH）以及补体因子B（CF B）的基因中， C2和C3是疾病的潜在危险因素。AMD中常见的环境应激源是氧化应激 应力三种途径激活补体系统：经典途径（CP）、替代途径（AP）和甘露糖途径。 结合凝集素途径（LP）;所有三个汇聚在相同的下游级联。我们的实验 自己的实验室以及其他实验室，使用CNV的激光损伤模型，已经表明AP信号传导 是CNV发展所必需的;而在涉及CNV的研究中， 其他途径。AP活性被发现控制促血管生成因子血管内皮细胞的生成。 内皮生长因子（VEGF），触发新血管生长所需。最后，我们证明了AP 激活参与氧化应激介导的RPE功能障碍，其特征在于VEGF和MMP重新激活。 从RPE单层租赁。氧化应激被发现通过再灌注使RPE对补体攻击敏感。 从而降低膜结合内源性补体抑制剂的水平。对于这一提议，我们将 根据我们的总体假设，即AP的病理激活对RPE有直接影响， 为AMD病理学提供一个宽松的细胞环境。这一假设将在三个目标进行测试， 在体内以及在RPE培养物（原代人和小鼠RPE细胞）中。使用不同的小鼠， 补体级联途径被破坏和补体耗尽的血清，我们将检查 补体激活机制的相对作用，并确定AP激活是否是必需的或不足的。 很好。为了鉴定AP蛋白的来源（即，肝或眼），分析组织特异性转基因小鼠。 接下来，我们将测试补体激活是否对CNV具有特异性，或者AMD病理是否与CNV相关。 Sod 1-/-小鼠中的氧化应激需要过度活跃的补体级联反应来发展。费用 将进行试验以测试VEGF是否参与补体介导的损伤机制。 最后，将在体内对这一假设进行检验。我们将使用补体抑制策略使用焦油- 获得了在不同水平阻断补体级联反应的抑制剂，以干扰CNV。检测仪器- 这些药物不仅可以确定其治疗价值，而且还可以阐明其在CNV小鼠中的作用机制 将研究不同补体成分在CNV中的作用和贡献。

The long-term goal is to understand the fundamental basis of complement signaling in the eye, and how mi- sregulation in this process leads to pathology, to ultimately aid in the development of therapeutic approach- es for devastating blinding diseases. Age-related macular degeneration (AMD) occurs in two forms, dry and wet. Dry AMD is characterized by drusen, RPE damage, and photoreceptor cell loss. In some patients, the dry form can transition to wet AMD. Wet AMD, presents itself with chroidal neovascularization (CNV), lea- kage of these new vessels, and rapid photoreceptor loss. Recent genetic evidence has implicated variations in the complement inhibitory protein factor H (CFH), as well as in the genes for complement factor B (CFB), C2 and C3, as potential risk factors for the disease. A common environmental stressor in AMD is oxidative stress. Three pathways activate the complement system: the classical (CP), alternative (AP), and mannose- binding lectin pathway (LP); all three converge on the same down-stream cascade. Experiments from our own laboratory as well as others, using the laser-damage model of CNV, have suggested that AP signaling is required for CNV development; whereas conflicting evidence has been published in the involvement of the other pathways. AP activity was found to control the generation of the proangiogenic factor vascular en- dothelial growth factor (VEGF), required for triggering new vessel growth. Finally, we have shown that AP activation is involved in oxidative stress-mediated RPE dysfunction characterized by VEGF and MMP re- lease from RPE monolayers. Oxidative stress was found to sensitize the RPE to complement attack by re- ducing the levels of membrane-bound endogenous complement inhibitors. For this proposal we will be guided by our overall hypothesis that pathologic activation of the AP has direct effects on the RPE, generat- ing a permissive cellular environment for AMD pathology. This hypothesis will be tested in three aims, both in vivo as well as in RPE cultures (primary human and mouse RPE cells). Using mice in which different pathways of the complement cascade are disrupted and complement-depleted serum, we will examine the relative roles of complement activation mechanisms and determine whether AP activation is required or suf- ficient. To identify the source of AP proteins (i.e., liver or eye), tissue-specific transgenic mice are analyzed. Next, we will test whether complement activation is specific for CNV, or whether AMD pathologies related to oxidative stress in the Sod1-/- mouse require a hyperactive complement cascade for them to develop. Expe- riments will be performed to test whether VEGF is involved in mechanisms of complement-mediated injury. And finally, the hypothesis will be put to test in vivo. We will use complement inhibitory strategies using tar- geted inhibitors that block the complement cascade at different levels to interfere with CNV. Testing inhibi- tors will not only establish their therapeutic value, but in addition, elucidating their mechanisms in CNV mice will investigate the roles and contributions of the different complement components in CNV.