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

描述(申请人提供)：剥脱综合征(XFS)是一种与年龄相关的疾病，构成了开角型和闭角型青光眼最常见的可识别原因，其特征是异常纤维物质(XFM)在许多眼内组织中积聚，特别是那些排列在眼前段水浴表面的结构。XFM的沉淀物是由细胞外基质糖蛋白和蛋白多糖组成的复杂混合物，它们组装在一个超分子纤维结构中。由于XF纤维的生物化学成分差异很大，目前尚不清楚哪些是XF纤维形成的主要成分，也不清楚某些沉积成分或其直接前体是否从周围液体(如水)中吸收到病变中。因此，尽管有所有可用的信息，但仍然没有数据指出支持动物模型生成的特定候选基因。一种研究不足的替代方法是开发一种在体外产生XF样纤维的培养模型，这是一种潜在的有价值的范例，将允许搜索导致疾病的致病途径(S)，进行纤维组装的时程动力学，为详细的生化分析提供纤维，并最终适用于药物干预。在这个意义上，正如在初步研究部分中广泛说明的那样，我们从三维系统中培养XFS患者的Tenon囊成纤维细胞获得了令人鼓舞的数据，证明了XF纤维的产生在超微结构上与体内观察到的相似，如果不是相同的话。根据我们的结果，我们推测，在培养中使用选定的XFS眼细胞将为研究XF纤维组装提供一个非常必要的模型。为此，我们将利用尖端蛋白质组学方法、手术标本的可用性来建立来自患有和不患有XFS的个体的不同原代培养物、从相同病例获得房水体液的可获得性、新开发的Tenon成纤维细胞三维培养以及研究组在培养虹膜着色上皮(IPE)细胞方面先前获得的技术经验，以：(A)对XFS病例Tenon‘s成纤维细胞体外产生的XF样纤维进行蛋白质组学分析；(B)验证从XFS患者在不同条件下培养的IPE获得的细胞是否产生与Tenon成纤维细胞和/或体内产生的XF纤维具有相同EM特征和生化组成的XF样纤维；(C)识别XFS与非XFS对照不同培养上清液组成中的共同蛋白质组差异，并通过对相同XFS和非XFS对照水液的平行分析，验证所观察到的差异是否可作为该病的潜在特征标记。拟议的实验产生的数据将构成未来R01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，构成了全球青光眼最常见的可识别原因。到目前为止，构成XF损伤的分子的极端多样性排除了建立这种疾病亟需的动物模型的可能性。我们建议在生物化学和超微结构上描述一种在体外产生XF样纤维的有前景的细胞培养范例，这可能被证明是研究疾病的分子基础和探索新的药物干预的有用的替代方法。

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.