Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA, 95616, USA.
Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA, 95616, USA; Cleveland Clinic, Cleveland, OH, 44195, USA.
Exp Eye Res. 2023 Jan;226:109303. doi: 10.1016/j.exer.2022.109303. Epub 2022 Nov 4.
Fuchs endothelial corneal dystrophy (FECD) is a progressive corneal disease that impacts the structure and stiffness of the Descemet's membrane (DM), the substratum for corneal endothelial cells (CECs). These structural alterations of the DM could contribute to the loss of the CECs resulting in corneal edema and blindness. Oxidative stress and transforming growth factor-β (TGF-β) pathways have been implicated in endothelial cell loss and endothelial to mesenchymal transition of CECs in FECD. Ascorbic acid (AA) is found at high concentrations in FECD and its impact on CEC survival has been investigated. However, how TGF-β and AA effect the composition and rigidity of the CEC's matrix remains unknown.
In this study, we investigated the effect of AA, TGF-β1 and TGF-β3 on the deposition, ultrastructure, stiffness, and composition of the extracellular matrix (ECM) secreted by primary bovine corneal endothelial cells (BCECs).
Immunofluorescence and electron microscopy post-decellularization demonstrated a robust deposition and distinct structure of ECM in response to treatments. AFM measurements showed that the modulus of the matrix in BCECs treated with TGF-β1 and TGF-β3 was significantly lower than the controls. There was no difference in the stiffness of the matrix between the AA-treated cell and controls. Gene Ontology analysis of the proteomics results revealed that AA modulates the oxidative stress pathway in the matrix while TGF-β induces the expression of matrix proteins collagen IV, laminin, and lysyl oxidase homolog 1.
Molecular pathways identified in this study demonstrate the differential role of soluble factors in the pathogenesis of FECD.
Fuchs 内皮角膜营养不良（FECD）是一种进行性角膜疾病，会影响角膜内皮细胞（CEC）的基质——Descemet 膜（DM）的结构和硬度。DM 的这些结构改变可能导致 CEC 丧失，从而导致角膜水肿和失明。氧化应激和转化生长因子-β（TGF-β）途径已被牵涉到 FECD 中内皮细胞丧失和内皮向间充质转化的过程中。抗坏血酸（AA）在 FECD 中浓度较高，其对 CEC 存活的影响已得到研究。然而，TGF-β和 AA 如何影响 CEC 基质的组成和刚性尚不清楚。
在这项研究中，我们研究了 AA、TGF-β1 和 TGF-β3 对原代牛角膜内皮细胞（BCEC）分泌的细胞外基质（ECM）的沉积、超微结构、硬度和组成的影响。
脱细胞化后的免疫荧光和电子显微镜显示，ECM 的大量沉积和独特结构对处理有明显反应。原子力显微镜测量显示，用 TGF-β1 和 TGF-β3 处理的 BCEC 基质的模量明显低于对照组。用 AA 处理的细胞与对照组之间基质的刚度没有差异。蛋白质组学结果的基因本体分析表明，AA 调节了基质中的氧化应激途径，而 TGF-β则诱导了胶原蛋白 IV、层粘连蛋白和赖氨酰氧化酶同源物 1 等基质蛋白的表达。
本研究中确定的分子途径表明可溶性因子在 FECD 发病机制中具有不同的作用。
