Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA.
Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA; Harvard Eye Associates, Laguna Hills, CA 92660, USA.
Free Radic Biol Med. 2018 Feb 20;116:19-30. doi: 10.1016/j.freeradbiomed.2017.12.036. Epub 2017 Dec 30.
Fuchs endothelial corneal dystrophy (FECD) is a genetic and oxidative stress disorder of post-mitotic human corneal endothelial cells (HCEnCs), which normally exhibit hexagonal shape and form a compact monolayer compatible with normal corneal functioning and clear vision. FECD is associated with increased DNA damage, which in turn leads to HCEnC loss, resulting in the formation rosettes and aberrant extracellular matrix (ECM) deposition in the form of pro-fibrotic guttae. Since the mechanism of ECM deposition in FECD is currently unknown, we aimed to investigate the role of endothelial-mesenchymal transition (EMT) in FECD using a previously established cellular in vitro model that recapitulates the characteristic rosette formation, by employing menadione (MN)-induced oxidative stress. We demonstrate that MN treatment alone, or a combination of MN and TGF-β1 induces reactive oxygen species (ROS), cell death, and EMT in HCEnCs during rosette formation, resulting in upregulation of EMT- and FECD-associated markers such as Snail1, N-cadherin, ZEB1, and transforming growth factor-beta-induced (TGFβI), respectively. Additionally, FECD ex vivo specimens displayed a loss of organized junctional staining of plasma membrane-bound N-cadherin, with corresponding increase in fibronectin and Snail1 compared to ex vivo controls. Addition of N-acetylcysteine (NAC) downregulated all EMT markers and abolished rosette formation. Loss of NQO1, a metabolizing enzyme of MN, led to greater increase in intracellular ROS levels as well as a significant upregulation of Snail1, fibronectin, and N-cadherin compared to normal cells, indicating that NQO1 regulates Snail1-mediated EMT. This study provides first line evidence that MN-induced oxidative stress leads to EMT in corneal endothelial cells, and the effect of which is further potentiated when redox cycling activity of MN is enhanced by the absence of NQO1. Given that NAC inhibits Snail-mediated EMT, this may be a potential therapeutic intervention for FECD.
弗氏角膜内皮营养不良（FECD）是一种后天性人眼角膜内皮细胞（HCEnC）的遗传和氧化应激疾病，这些细胞通常呈六边形形状，并形成一个紧凑的单层，与正常角膜功能和清晰视力相兼容。FECD 与 DNA 损伤增加有关，而 DNA 损伤反过来又导致 HCEnC 丧失，导致角膜内皮细胞形成玫瑰花结，并以纤维母细胞前质的形式出现异常细胞外基质（ECM）沉积。由于目前尚不清楚 FECD 中 ECM 沉积的机制，我们旨在使用先前建立的体外细胞模型来研究内皮-间充质转化（EMT）在 FECD 中的作用，该模型通过使用间苯二酚（MN）诱导的氧化应激来再现特征性玫瑰花结形成。我们证明，MN 单独处理或 MN 和 TGF-β1 的组合处理在玫瑰花结形成过程中诱导 HCEnC 中产生活性氧（ROS）、细胞死亡和 EMT，导致 EMT 和 FECD 相关标志物如 Snail1、N-钙粘蛋白、ZEB1 和转化生长因子-β诱导（TGFβI）的上调。此外，与体外对照相比，FECD 体外标本显示细胞膜结合型 N-钙粘蛋白的组织化连接染色丢失，相应的纤维连接蛋白和 Snail1 增加。添加 N-乙酰半胱氨酸（NAC）可下调所有 EMT 标志物并消除玫瑰花结形成。MN 代谢酶 NQO1 的缺失导致细胞内 ROS 水平显著增加，并且与正常细胞相比，Snail1、纤维连接蛋白和 N-钙粘蛋白的显著上调表明 NQO1 调节 Snail1 介导的 EMT。这项研究首次提供证据表明，MN 诱导的氧化应激导致角膜内皮细胞发生 EMT，并且当 MN 的氧化还原循环活性因缺乏 NQO1 而增强时，其作用会进一步增强。鉴于 NAC 抑制 Snail 介导的 EMT，这可能是 FECD 的一种潜在治疗干预措施。
