Birth and Death of Choriocapillaris.

描述(申请人提供)：脉络膜毛细血管(CC)是一种有窗的小叶血管系统，为RPE和光感受器提供所有的氧气和血清营养物质。我们详细阐述了在上一次资助期间，人类CC是通过血管生成而诞生的，而CC在湿性和干性AMD中死亡。在干性AMD中，CC细胞的死亡与邻近RPE的丢失有关。在湿性AMD中，CC的丢失发生在脉络膜新生血管膜(CNV)形成之前，这一点至今无法解释。我们观察到干性和湿性AMD患者CC周围的血清蛋白浓度较高。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物质，并清除废物，但确切的运输机制尚不清楚。拟议的研究将描述CC通过开窗、洞穴和涂层坑的正常运输机制。这些机制将使用确定大小的金纳米颗粒、标记的血清蛋白和标记的血清脂类进行评估。我们将通过使用缺乏小窝(敲除小窝系统中的主要成分Cav-1)或开窗(RPE产生的VEGF被敲除或中和)或RPE过度表达VEGF的小鼠，或产生基底层沉积的小鼠，来确定CC转运的失调是否影响蛋白质的积累。拟议的研究将确定Bruchs膜沉积对CC传输的影响以及CC传输缺陷对沉积形成的影响。我们假设CC转运功能障碍导致了我们观察到的脉络膜中血清蛋白的积聚，这对CC是有毒的，可能是CC在AMD中死亡的原因。我们将评估血清蛋白(白蛋白、C反应蛋白和α-2巨球蛋白)在体外对脉络膜内皮细胞(CEC)的毒性作用，并确定这些血清蛋白是否会导致CEC紧密连接的丧失、小凹数量的变化或开窗。这也被认为是CC功能障碍与AMD有关。我们的初步研究表明，窗孔的丢失与Bruchs膜沉积和RPE丢失有关。在Greg Hageman为透射电子显微镜准备的一大批AMD眼睛中，我们将确定人类AMD中的CC窗口、凹陷、涂层凹坑和/或紧密连接是否发生变化，以及基底沉积、玻璃体和RPE丢失与这些变化的关联。总而言之，这项提议将定义CC向视网膜供应营养物质所使用的正常运输过程。我们还将确定这些传输过程在AMD中是否发生了变化。我们将确定脉络膜中随年龄积累的血清蛋白是否与我们在AMD中观察到的CC转运功能障碍和CC死亡有关。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。这一关于CC转运及其在AMD中的变化的新知识将对开发新的系统治疗纳米粒以预防AMD的视网膜变性和CNV具有非常宝贵的价值。 公共卫生相关性：这项研究将确定脉络膜毛细血管(CC)如何将营养物质运输到视网膜色素上皮(RPE)和光感受器，以及在存在太少或太多VEGF的情况下，CC运输系统是否受到失调。这将在小鼠身上完成，使用统一的大小和带电的金纳米颗粒，并标记白蛋白和脂蛋白来评估凹陷、开窗、涂层凹陷和紧密连接。CC转运也将在没有开窗和凹陷的小鼠身上进行评估，或者在Bruchs膜上有沉积物。这些结果将与一组具有良好特征的AMD和老年受试者的透射电子显微镜进行比较。这些研究将确定CC转运的机制以及它们在AMD中是如何受到影响的，这两项都是未知的。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: This study will determine how the choriocapillaris (CC) transports nutrients to retinal pigment epithelium (RPE) and photoreceptors and if the CC transport systems are dysregulated in the presence of too little or too much VEGF. This will be accomplished in mice using uniform size and charge gold nanoparticles, and labeled albumin and lipoprotein to evaluate caveolae, fenestrations, coated pits, and tight junction. CC transport will also be evaluated in mice lacking fenestrations and caveolae, or with deposits on Bruch's membrane. These results will be compared to transmission electron microscopy of a well-characterized cohort of AMD and aged subjects. These studies will determine mechanisms of CC transport and how they are affected in AMD, both of which are unknown.