AMD 相关变异 rs704 引起的蛋白功能改变将玻连蛋白与疾病病理联系起来。
Altered Protein Function Caused by AMD-associated Variant rs704 Links Vitronectin to Disease Pathology.
机构信息
Institute of Human Genetics, University of Regensburg, Regensburg, Germany.
Institute of Clinical Human Genetics, University Hospital Regensburg, Regensburg, Germany.
出版信息
Invest Ophthalmol Vis Sci. 2020 Dec 1;61(14):2. doi: 10.1167/iovs.61.14.2.
PURPOSE
Vitronectin, a cell adhesion and spreading factor, is suspected to play a role in the pathogenesis of age-related macular degeneration (AMD), as it is a major component of AMD-specific extracellular deposits (e.g., soft drusen, subretinal drusenoid deposits). The present study addressed the impact of AMD-associated non-synonymous variant rs704 in the vitronectin-encoding gene VTN on vitronectin functionality.
METHODS
Effects of rs704 on vitronectin expression and processing were analyzed by semi-quantitative sequencing of VTN transcripts from retinal pigment epithelium (RPE) cells generated from human induced pluripotent stem cells (hiPSCs) and from human neural retina, as well as by western blot analyses on heterologously expressed vitronectin isoforms. Binding of vitronectin isoforms to retinal and endothelial cells was analyzed by western blot. Immunofluorescence staining followed extracellular matrix (ECM) deposition in cultured RPE cells heterologously expressing the vitronectin isoforms. Adhesion of fluorescently labeled RPE or endothelial cells in dependence of recombinant vitronectin or vitronectin-containing ECM was investigated fluorometrically or microscopically. Tube formation and migration assays addressed effects of vitronectin on angiogenesis-related processes.
RESULTS
Variant rs704 affected expression, secretion, and processing but not oligomerization of vitronectin. Cell binding and influence on RPE-mediated ECM deposition differed between AMD-risk-associated and non-AMD-risk-associated protein isoforms. Finally, vitronectin affected adhesion and endothelial tube formation.
CONCLUSIONS
The AMD-risk-associated vitronectin isoform exhibits increased expression and altered functionality in cellular processes related to the sub-RPE aspects of AMD pathology. Although further research is required to address the subretinal disease aspects, this initial study supports an involvement of vitronectin in AMD pathogenesis.
目的
纤连蛋白是一种细胞黏附和铺展因子，它可能在年龄相关性黄斑变性（AMD）的发病机制中发挥作用，因为它是 AMD 特异性细胞外沉积物（如软性玻璃膜疣、视网膜下玻璃膜疣样沉积物）的主要成分。本研究探讨了纤连蛋白编码基因 VTN 中的 AMD 相关非同义变体 rs704 对纤连蛋白功能的影响。
方法
通过对源自人诱导多能干细胞（hiPSC）的视网膜色素上皮（RPE）细胞和人神经视网膜的 VTN 转录本进行半定量测序，以及对异源表达的纤连蛋白异构体进行 Western blot 分析，分析 rs704 对纤连蛋白表达和加工的影响。通过 Western blot 分析纤连蛋白异构体与视网膜和内皮细胞的结合。通过免疫荧光染色分析在异源表达纤连蛋白异构体的培养 RPE 细胞中细胞外基质（ECM）的沉积。通过荧光标记的 RPE 或内皮细胞的黏附实验，在依赖于重组纤连蛋白或含纤连蛋白的 ECM 的情况下进行荧光法或显微镜观察。管形成和迁移实验研究了纤连蛋白对血管生成相关过程的影响。
结果
变体 rs704 影响纤连蛋白的表达、分泌和加工，但不影响其寡聚化。细胞结合和对 RPE 介导的 ECM 沉积的影响在 AMD 风险相关和非 AMD 风险相关蛋白异构体之间存在差异。最后，纤连蛋白影响黏附和内皮管形成。
结论
AMD 风险相关的纤连蛋白异构体在与 AMD 病理的 SUB-RPE 相关的细胞过程中表现出增加的表达和改变的功能。尽管还需要进一步的研究来解决 SUBRETINAL 疾病方面的问题，但这项初步研究支持纤连蛋白在 AMD 发病机制中的作用。
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