Birth and Death of Choriocapillaris.

描述（由申请人提供）：绒毛膜毛细血管（CC）是一个小叶状、开孔的脉管系统，为RPE和光感受器提供所有的氧气和血清营养。我们在最后一期拨款中详细阐述了人类CC通过血液血管发生的诞生以及湿性和干性AMD中CC的死亡。在干性AMD中，CC细胞死亡与邻近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，这一点迄今尚未得到解释。我们观察到干性和湿性AMD患者CC周围有高浓度的血清蛋白。这种积累的原因和这些蛋白质对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，但运输的确切机制尚不清楚。拟议的研究将通过开窗、小泡和涂层坑来描述CC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进行评估。我们将通过使用缺乏小泡（敲除cav-1，小泡系统的主要成分）或开窗（RPE产生的VEGF敲除或中和）的小鼠，或RPE过度表达VEGF或产生基底层沉积的小鼠，来确定CC转运失调是否会影响蛋白质积累。提出的研究将确定布鲁克斯膜沉积对CC运输的影响以及CC运输缺陷对沉积形成的影响。我们假设CC转运功能障碍导致我们观察到的脉络膜中血清蛋白积累，这对CC是有毒的，可能是AMD中CC死亡的原因。我们将在体外评估血清蛋白（白蛋白、CRP和α -2巨球蛋白）对脉络膜内皮细胞（CEC）的毒性，并确定这些血清蛋白是否会导致CEC中紧密连接的丧失、小泡或开孔数量的变化。也有人认为，CC功能障碍与AMD有关。我们的初步研究表明，开窗的损失与布鲁氏膜沉积和RPE损失有关。在Greg Hageman为TEM准备的大量AMD眼睛中，我们将确定人类AMD的CC开孔、小泡、涂层凹坑和/或紧密连接是否发生变化，以及基底沉积物、水肿和RPE损失与这些变化的关系。总之，该提案将定义CC向视网膜提供营养物质的正常运输过程。我们还将确定这些转运过程是否在AMD中发生改变。我们将确定随着年龄在脉络膜中积累的血清蛋白是否有助于我们在AMD中观察到的CC运输功能障碍和CC死亡。我们将研究VEGF降低或升高如何改变CC转运。这一关于CC转运及其在AMD中如何变化的新知识将对开发新的系统性治疗纳米颗粒预防AMD中发生的视网膜变性和CNV具有宝贵的价值。

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.