Birth and Death of Choriocapillaris.

项目总结： 脉络膜毛细血管(CC)是一个小叶的有窗的血管系统，提供所有的氧气和血清 RPE和光感受器的营养物质。我们从血管生成的角度阐述了人类CC的诞生。 在最后一次授权期内，CC在湿润和干燥AMD中死亡。在干性AMD中，CC细胞死亡与丢失有关 相邻RPE的。在湿性AMD中，CC的丢失先于脉络膜新生血管膜(CNV) 形成，这一点到目前为止还无法解释。我们观察到CC周围有高浓度的血清蛋白 在干燥和潮湿的AMD中。这种积累的原因和这些蛋白对CC的影响尚不清楚。 虽然假设CC为RPE和光感受器提供营养，并清除废物， 确切的传输机制尚不清楚。拟议的研究将描述CC的正常运输 通过开窗、凹陷和涂层凹坑的机制。这些机制将使用Gold进行评估 规定大小的纳米颗粒、标记的血清蛋白和标记的血清脂质。我们将确定是否 CC转运的失调通过使用缺乏小窝的小鼠影响蛋白质积累(敲除Cav-1， 小窝系统的主要成分)，或开窗(RPE产生的血管内皮生长因子被敲除或 中和)，或视网膜色素上皮细胞过度表达血管内皮生长因子，或产生基底板沉积。建议数 研究将确定Bruchs膜沉积对CC转运的影响以及CC的影响 输运缺陷对矿床形成的影响。 我们假设CC转运功能障碍导致血清蛋白在脉络膜积聚。 我们观察到，这对CC是有毒的，这可能是CC死于AMD的原因。我们将对其毒性进行评估 脉络膜内皮细胞上的血清蛋白(白蛋白、C反应蛋白和α-2巨球蛋白糖基化前后) 细胞(CEC)，并确定这些血清蛋白是否导致紧密连接的丢失，数量的变化 在CEC中形成小窝或开窗。这也被认为是CC功能障碍与AMD有关。我们的预赛 研究表明，窗孔的丢失与Bruchs膜沉积和RPE丢失有关。在……里面 格雷格·哈格曼为透射电子显微镜准备的大量AMD眼睛，我们将确定CC 人类AMD的开窗、凹陷、包被凹坑和/或紧密连接的变化及其与基底动脉的关联 随着这些变化，沉积物、玻璃体和RPE损失。 总而言之，这项提案将定义CC用于向 视网膜。我们还将确定这些传输过程在AMD中是否发生了变化。我们将确定血清是否 随着年龄的增长，脉络膜中积累的蛋白质导致CC转运功能障碍和CC WE死亡 在AMD中观察到。我们将研究血管内皮生长因子的降低或升高如何改变CC的转运。这是一项新的 了解CC转运及其在AMD中的变化对开发新的系统将是非常有价值的 用于预防AMD视网膜变性和CNV的治疗性纳米颗粒。

Project Summary: The choriocapillaris (CC) is a lobular, fenestrated vasculature that provides all of the oxygen and serum nutrients to the RPE and photoreceptors. We have elaborated the birth of human CC by hemo-vasculogenesis and the death of CC in wet and dry AMD in the last grant period. In dry AMD, CC cell death is related to loss of adjacent RPE. In wet AMD, loss of CC occurs in advance of choroidal neovascular membrane (CNV) formation, which is unexplained to date. We have observed high concentrations of serum proteins around CC in dry and wet AMD. The reason for this accumulation and influence of these proteins on CC is unknown. Although it is assumed that CC provides the nutrients for RPE and photoreceptors and removes waste, the exact mechanisms of transport are unknown. The proposed studies will characterize CC's normal transport mechanisms via fenestrations, caveolae, and coated pits. These mechanisms will be evaluated using gold nanoparticles of defined sizes, tagged serum proteins, and tagged serum lipids. We will determine if dysregulation in CC transport affects protein accumulation by using mice lacking caveolae (knock out cav-1, the major component in the caveolae system), or fenestrations (RPE-produced VEGF knocked out or neutralized), or mice with RPE over expressing VEGF, or producing basal laminar deposits. The proposed studies will determine the effects of Bruchs membrane deposits on CC transport and the effects of CC transport defects on deposit formation. We hypothesize that dysfunction in CC transport results in the serum protein accumulation in choroid we observe, which is toxic to CC and may be the reason that CC die in AMD. We will evaluate the toxicity of serum proteins (albumin, CRP, and alpha-2 macroglobulin with and without glycation) on choroidal endothelial cells (CEC) in vitro and determine if those serum proteins cause loss of tight junctions, changes in numbers of caveolae or fenestrations in CEC. It is also assumed that CC dysfunction is involved in AMD. Our preliminary studies demonstrated that loss in fenestrations is associated with Bruchs membrane deposits and RPE loss. In a large collection of AMD eyes that Greg Hageman has prepared for TEM, we will determine if CC fenestrations, caveolae, coated pits and/or tight junctions change in human AMD and the association of basal deposits, drusen, and RPE loss with these changes. In conclusion, this proposal will define the normal transport processes used by CC to supply nutrients to retina. We will also determine if these transport processes are altered in AMD. We will determine if serum proteins that accumulate with age in choroid contribute to dysfunction in CC transport and the death of CC we have observed in AMD. We will investigate how reduced or elevated VEGF changes CC transport. This new knowledge of CC transport and how it changes in AMD will be invaluable in developing new systemic therapeutic nanoparticles for preventing retinal degeneration and CNV that occurs in AMD.