The Division of Cornea & Anterior Segment, Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, MD 21231, USA.
Exp Eye Res. 2011 Dec;93(6):880-8. doi: 10.1016/j.exer.2011.10.001. Epub 2011 Oct 18.
Fuchs' endothelial corneal dystrophy is the most common corneal endotheliopathy, and a leading indication for corneal transplantation in the US. Relatively little is known about its underlying pathology. We created a cellular model of the disease focusing on collagen VIII alpha 2 (COL8A2), a collagen which is normally present in the cornea, but which is found in abnormal amounts and distribution in both early and late-onset forms of the disease. We performed cellular transfections using COL8A2 cDNAs including both wild-type and mutant alleles which are known to result in early-onset FECD. We used this cell model to explore the cellular production of wild-type and mutant monomeric and trimeric collagen VIII and measured production levels and patterns using Western blotting and immunofluorescence. We studied the thermal stability of the mutated collagen VIII helices using computer modeling, and further investigated these differences using collagen mimetic peptides. The Western blots demonstrated that similar amounts of wild-type and mutant collagen VIII monomers were produced in the cells. However, the levels of trimeric collagen peptide in the mutant-transfected cells were elevated. Intracellular accumulation of trimeric collagen VIII was confirmed on immunofluorescence studies. Both the computer model and the collagen mimetic peptides demonstrated that the L450W mutant was less thermally stable than either the Q455K or wild-type collagen VIII. Thus, although both mutant collagen VIII peptides were retained intracellularly, the biochemical reasons for the retention varied between genotypes. Collagen VIII mutations, which clinically result in Fuchs' dystrophy, are associated with abnormal cellular accumulation of collagen VIII. Different collagen VIII mutations may act via distinct biochemical mechanisms to produce the FECD phenotype.
福斯曼角膜内皮营养不良是最常见的角膜内皮病变，也是美国角膜移植的主要指征。对于其潜在的病理学，我们知之甚少。我们创建了一种疾病的细胞模型，重点关注胶原蛋白 VIII 阿尔法 2（COL8A2），这种胶原蛋白通常存在于角膜中，但在疾病的早发和晚发形式中，其含量和分布均异常。我们使用 COL8A2 cDNA 进行了细胞转染，包括已知导致早发性 FECD 的野生型和突变型等位基因。我们使用该细胞模型探索了野生型和突变型单体和三聚体胶原蛋白 VIII 的细胞产生，并使用 Western blot 和免疫荧光法测量了产生水平和模式。我们使用计算机建模研究了突变型胶原蛋白 VIII 螺旋的热稳定性，并使用胶原蛋白模拟肽进一步研究了这些差异。Western blot 表明，细胞中产生的野生型和突变型胶原蛋白 VIII 单体量相似。然而，突变转染细胞中三聚体胶原蛋白肽的水平升高。免疫荧光研究证实了三聚体胶原蛋白 VIII 的细胞内积累。计算机模型和胶原蛋白模拟肽均表明，L450W 突变型比 Q455K 或野生型胶原蛋白 VIII 的热稳定性差。因此，尽管两种突变型胶原蛋白 VIII 肽都被保留在细胞内，但基因型之间的保留的生化原因有所不同。临床上导致福斯曼营养不良的胶原蛋白 VIII 突变与胶原蛋白 VIII 的异常细胞积累有关。不同的胶原蛋白 VIII 突变可能通过不同的生化机制产生 FECD 表型。
