Exfoliation Syndrome: Development of a cell culture model of fibril formation

描述（由申请人提供）：剥脱综合征（XFS）是一种年龄相关性疾病，构成开角型和闭角型青光眼最常见的可识别病因，其特征在于异常纤维状物质（XFM）在许多眼内组织中的积聚，特别是在眼前段的水浸表面的结构中。XFM的沉积物由组装在超分子纤维状结构中的细胞外基质糖蛋白和蛋白聚糖的复杂混合物组成。由于高度多样化的生物化学组成，仍然不清楚哪些是参与XF纤维形成的主要元素，以及一些沉积的组分或其直接前体是否从周围流体（例如水性流体）募集到病变中。因此，尽管有所有可用的信息，仍然没有数据指出特定的候选基因来支持动物模型的产生。一种未充分研究的替代方法是开发一种培养模型，该模型在体外产生XF样纤维，这是一种潜在的有价值的范例，可以搜索导致疾病的致病途径，进行原纤维组装的时程动力学，为详细的生化分析提供原纤维，并最终适用于药理学干预。从这个意义上说，并在初步研究部分中广泛说明，我们已经获得了令人鼓舞的数据，从培养Tenon囊成纤维细胞从XFS患者在三维系统中，记录XF原纤维的生产超微结构相似，如果不是相同的，在体内观察到的。基于我们的研究结果，我们假设在培养中使用所选择的XFS眼细胞将提供一个急需的模型来研究XF原纤维组装。为此，我们将利用尖端的蛋白质组学方法，手术标本的可用性，以建立来自有和没有XFS的个体的原代培养物的多样性，来自相同病例的房水的可获得性，新开发的Tenon成纤维细胞三维培养物以及研究小组在培养虹膜色素上皮（IPE）细胞方面获得的先前技术经验，以：（B）验证在各种条件下培养的从XFS患者的IPE获得的细胞是否产生具有与体内产生的Tenon成纤维细胞和/或XF原纤维相同的EM特征和生物化学组成的XF样原纤维;（c）鉴定XFS与非XFS对照的各种培养上清液的组成中的共同蛋白质组差异，并通过平行分析来自相同XFS和非XFS对照的水性流体来验证，观察到的差异是否可以作为疾病的预期特征标记。拟议实验产生的数据将构成未来R 01应用的基础。公共卫生相关性：剥脱（XF）综合征是一种与年龄相关的疾病，是全球青光眼最常见的可识别原因。到目前为止，构成XF病变的分子的极端多样性排除了建立急需的疾病动物模型的可能性。我们建议生物化学和超微结构特征的一个有前途的细胞培养模式，在体外产生XF样纤维，这可能被证明是一个有用的替代方法来研究疾病的分子基础，并探索新的药理学干预措施。

DESCRIPTION (provided by applicant): Exfoliation syndrome (XFS), an age-related disorder which constitutes the most common identifiable cause of open-angle and angle-closure glaucoma, is characterized by the accumulation of an abnormal fibrillar material (XFM) in many intraocular tissues, particularly those structures that line the aqueous-bathed surfaces of the anterior segment. Deposits of XFM are composed of a complex mixture of extracellular matrix glycoproteins and proteoglycans assembled in a supramolecular fibrillar structure. Due to the highly diverse biochemical composition it is still not clear which are the primary elements involved in the formation of XF fibers and whether some of the deposited components or their immediate precursors are recruited to the lesions from the surrounding fluids (e.g. aqueous). Thus, despite all available information, there is still no data pointing out to specific candidate genes to support the generation of animal models. An understudied alternative approach is the development of a culture model that produces XF-like fibers in vitro, a potentially valuable paradigm that would allow the search for the pathogenetic pathway(s) leading to disease, conduct time-course kinetics of fibril assembly, supply fibrils for detailed biochemical analysis and be eventually amenable for pharmacologic interventions. In this sense, and as extensively illustrated in the Preliminary Studies section, we have obtained encouraging data from culturing Tenon's capsule fibroblasts from XFS patients in tri-dimensional systems, documenting the production of XF fibrils ultrastructurally similar, if not identical, to those observed in vivo. Based on our results we hypothesize that the use of selected XFS ocular cells in culture will provide a much needed model to study XF fibril assembly. To this end, we will take advantage of cutting-edge proteomic approaches, the availability of surgical specimens to establish a diversity of primary cultures from individuals with and without XFS, the accessibility to aqueous humors from the same cases, the newly developed Tenon's fibroblasts tri-dimensional cultures as well as previous technical experience acquired by the research group in culturing iris pigmented epithelial (IPE) cells to: (a) conduct proteomic analysis of XF-like fibrils produced in vitro by Tenon's fibroblasts from XFS cases; (b) verify whether cells obtained form IPE of XFS patients, cultured under various conditions, produce XF-like fibrils with the same EM characteristics and biochemical composition than Tenon's fibroblasts and/or XF fibrils produced in vivo; (c) identify common proteomic differences in the composition of the various culture supernatants of XFS vs. non-XFS controls and validate, through the parallel analysis of aqueous fluids from the same XFS and non- XFS controls, whether the observed differences might serve as prospective signature markers of the disease. The data generated by the proposed experiments will constitute the basis of a future R01 application. PUBLIC HEALTH RELEVANCE: Exfoliation (XF) syndrome is an age-related disorder which constitutes the most common identifiable cause of glaucoma worldwide. The extreme diversity of the molecules composing the XF lesions has so far precluded the creation of a much needed animal model of the disease. We propose to biochemically and ultrastructurally characterize a promising cell culture paradigm that produces XF-like fibers in vitro, which may prove to be a useful alternative approach to study the molecular basis of the disease and explore novel pharmacologic interventions.