Alternative Pathway of Complement Activation in Age-Related Macular Degeneration

我们的长期目标是了解补体信号在眼睛中的基本基础，以及补体信号是如何丢失的。 这一过程中的调节导致了病理，最终有助于治疗方法的发展- Es治疗毁灭性的致盲疾病。年龄相关性黄斑变性(AMD)以两种形式发生，干性和 湿的。干性AMD以玻璃体、RPE损伤和光感受器细胞丢失为特征。在一些患者中， 干型可过渡到湿型AMD。湿性AMD表现为嗜铬细胞新生血管(CNV)，Lea- 这些新血管的凯奇，和快速的光感受器丧失。最新的遗传证据表明变异 在补体抑制蛋白因子H(CFH)以及补体因子B(CFB)的基因中， C2和C3是该病的潜在危险因素。AMD常见的环境应激源是氧化 压力。有三条途径激活补体系统：经典(CP)、替代(AP)和甘露糖。 结合凝集素途径(LP)；所有三个都汇聚在同一个下游级联上。来自我们的实验 自己的实验室以及其他实验室，使用CNV的激光损伤模型，已经提出AP信号 是CNV发展所必需的；而在涉及 另一条路。AP活性可控制促血管生成因子-血管内皮细胞生长因子的生成。 血管内皮生长因子(VEGF)，触发新血管生长所必需的。最后，我们向您展示了AP 活化参与氧化应激介导的以血管内皮生长因子和基质金属蛋白酶为特征的RPE功能障碍 从RPE单层租赁。研究发现，氧化应激可使RPE对补体攻击变得敏感。 降低膜结合型内源性补体抑制物水平。对于这项提案，我们将 在我们关于AP的病理性激活对RPE有直接影响的总体假设的指导下，产生了- 为AMD病理提供了一个允许的细胞环境。这一假设将在三个目标上进行检验，两者都是 在体内以及在RPE培养(原代人和小鼠RPE细胞)中。使用不同的小鼠 补体级联的通路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，并决定AP激活是必需的还是超激活的。 足够了。为了确定AP蛋白的来源(即肝脏或眼睛)，分析了组织特异性转基因小鼠。 接下来，我们将测试补体激活是否是CNV的特异性，或者AMD的病理是否与 SOD1-/-小鼠的氧化应激需要一种高度活跃的补体级联反应才能发展。费用- 将进行试验，以测试血管内皮生长因子是否参与补体介导的损伤机制。 最后，这一假说将在体内进行检验。我们将使用补体抑制策略，使用TAR- Geted在不同水平上阻断补体级联以干扰CNV的抑制剂。测试抑制物- TORS不仅将确定其治疗价值，而且还将阐明其在CNV小鼠中的作用机制 将研究不同的补体成分在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.