NQO1 downregulation potentiates menadione-induced endothelial-mesenchymal transition during rosette formation in Fuchs endothelial corneal dystrophy

Abstract

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.

Pathological molecular mechanism of symptomatic late-onset Fuchs endothelial corneal dystrophy by bioinformatic analysis

“…It may cause blurred vision or even blindness[ 24 , 25 ]. Previous studies have found that changes in cell function, such as ECM deposition[ 26 ], oxidative stress[ 27 ], apoptosis[ 9 ] and EMT[ 26 , 28 ], play a key role in the formation of FECD. However, FECD pathogenesis and the molecular mechanism are incomplete[ 24 – 26 , 29 , 30 ].…”

A Potential Link Between Oxidative Stress and Endothelial-to-Mesenchymal Transition in Systemic Sclerosis

“…EndMT accounts for the increased fibroproliferative vasculopathy and fibrosis in several diseases ( 16 ) and is considered a novel mechanism for the generation of activated myofibroblasts in SSc ( 17 – 20 ). On the other hand, increased ROS generation has been reported to mediate TGF-β-induced EndMT in several conditions including atherosclerosis, Fuchs endothelial corneal dystrophy, and diabetic nephropathy ( 21 – 23 ). TGF-β-mediated ROS generation also promotes cardiac fibroblast differentiation into myofibroblasts, which accounts for the increased production of ECM proteins such as type I and III collagen and the initiation of α-smooth muscle actin expression (α-SMA) during the EndMT process ( 24 ).…”

MitoROS due to loss ofSlc4a11in corneal endothelial cells induces ER stress, lysosomal dysfunction and impairs autophagy

“…Several studies have identified mitochondrial dysfunction, or oxidative stress as the trigger behind cell death in each of these diseases 3,4,[44][45][46] . Our study further characterizes the crosstalk between mitochondria, lysosomes and ER in the corneal endothelial cells, and reveal that at least two important physiological processes (autophagy and ER function) are disabled by increased mitochondrial ROS.…”

“…The energy needed for this process is derived from a high density of mitochondria. Corneal endothelial diseases, such as Fuchs Corneal Endothelial Dystrophy (FCED) and Congenital Hereditary Endothelial Dystrophy (CHED), are associated with dysfunctional mitochondria, increased reactive oxygen species (ROS), alterations in cell morphology, function, and eventually cell death [3][4][5][6] . Corneal edema is the main clinical manifestation of FCED and CHED 7,8 , and corneal transplantation is the only available treatment option.…”