Elsevier

Free Radical Biology and Medicine

Volume 116, 20 February 2018, Pages 19-30
Free Radical Biology and Medicine

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

https://doi.org/10.1016/j.freeradbiomed.2017.12.036Get rights and content

Highlights

  • Menadione-induced oxidative stress activates EMT in HCEnCs.

  • Menadione-induced EMT is potentiated by the absence of NQO1.

  • Menadione-induced EMT is inhibited with the addition of NAC.

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.

Introduction

Human corneal endothelial cells (HCEnCs) are a monolayer of regular hexagonal cells that form the most posterior layer of the cornea, facing the anterior chamber of the eye. These cells are arrested in post-mitotic state and have minimal proliferative capacity in vivo [1], [2], [3]. The post-mitotic arrest is believed to be attributed to contact inhibition [4], transforming growth factor (TGF)-β in the aqueous fluid, and lack of paracrine stimulation by cell cycle promoting growth factors [5]. Fuchs endothelial corneal dystrophy (FECD) is the most common degenerative condition of corneal endothelium, manifesting in gradual and age-related loss of HCEnCs and production of extracellular matrix (ECM) deposits in the form of guttae. These dome shaped excrescences protrude from the underlying Descemet's membrane (DM), a basement membrane located between the corneal stroma and endothelium, and are thought to be secreted by degenerating endothelial cells [6].

Although little is known about the exact composition of guttae, studies have shown abnormal wide-spaced collagen in the posterior collagenous layer of DM [7], [8], as well as increased collagen VIII, collagen IV, laminin [9], [10], collagen I [11], [12], [13], collagen XVI [12], and fibronectin [12], [13]. Similar ECM changes have been associated with endothelial-mesenchymal transition (EMT) occurring during tissue fibrosis and scar formation in other organ systems such as liver, intestine, kidney [14], [15], [16], lung [17], [18], [19], and heart [20], [21]. EMT is a repair-associated event that leads to organ fibrosis in response to pathological stress [20]. It generates activated mesenchymal-like cells that produce excessive amounts of collagen-rich ECM and results in upregulation of characteristic EMT markers such vimentin, Snail1, ZEB1, collagen 1A1, laminin, fibronectin, and N-cadherin [20], [21], [22]. Moreover, TGF-β, the main modulator of EMT, is abundant in aqueous humor [23] and has been shown to induce the production of TGFβI [24], which is known to be upregulated in FECD [25]. Increased levels of TGF-β1 and TGF-β2 have been noted in aqueous fluid of a subgroup of FECD patients [26]. Although components of EMT have been described in FECD, the etiology of purported EMT in endothelial cell degeneration needs further investigation. The mechanism of morphological and functional changes that accompany HCEnC loss and ECM deposition in the development of FECD is not yet known. Both the intracellular crosstalk and the microenvironmental triggers of EMT phenotypes are of particular interest in these mitotically incompetent cells.

One of the major inducers of EMT under pathologic conditions is oxidative stress [27], [28]. Previous studies have implicated oxidative stress and DNA damage in the pathogenesis of FECD [25], [29], [30], [31], [32], [33], [34] and have shown that generation of intracellular ROS with menadione (MN) leads to DNA damage during rosette formation in normal HCEnCs, modeling the changes seen in FECD [35]. Rosette formation is prominent feature seen in FECD ex vivo specimens and signifies a degenerative process, during which the endothelial cells form a ring or rosette around the bases of dome-shaped extracellular matrix deposits [36]. In this study, we sought to investigate whether oxidative DNA damage induced by MN causes EMT during rosette formation and leads to cellular remodeling involved in the pathogenesis of FECD. MN is a quinone that is reduced and metabolized by NQO1 to menadiol by 2-electron transfer, quenching ROS generation and macromolecular damage of un-metabolized quinones. NQO1, regulated by Nrf2 transcriptional activation, has a broad role in cellular protection from oxidative stress and has been shown to play a key role in oxidant-antioxidant imbalance seen in FECD [30], [37]. Furthermore, given that TGF-β is one of the main modulators of EMT, we aimed to evaluate the interplay between MN-induced oxidative stress and TGF-β in HCEnCs.

Section snippets

Human tissue

This study was conducted according to the tenets of the Declaration of Helsinki and approved by the Massachusetts Eye and Ear Institutional Review Board. Written and informed consent was obtained from patients undergoing surgical treatment for FECD. After surgical removal, the tissue was immediately placed in storage medium (Optisol-GS; Bausch & Lomb) at 4 °C. For western blot, human FECD endothelium was derived from post-keratoplasty specimens (n = 4, age range 57–87). Normal donor corneas (n =

MN and TGF-β induce ROS during rosette formation in HCEnCs

As FECD has been deemed as an oxidative stress disorder [29], [30], [39], it is important to investigate the mechanism of rosette formation in response to MN-induced intracellular ROS generation, and to assess the interplay between ROS and TGFβ. Our lab demonstrated that MN-induced DNA damage in HCEnCs leads to rosette formation, corresponding to findings in an ex vivo FECD specimen [35]. In order to investigate the role EMT in rosette formation, normal HCEnCs were grown in hexagonal monolayers

Discussion

Corneal endothelium consists of a post-mitotic cell type which is exposed to oxidative stress via chronic exposure to UV light, exuberant metabolic activity of pumping ions to maintain corneal deturgescence, and high mitochondrial respiratory demands. Loss of regular hexagonal mosaic has been the major cause of loss barrier and pumping functions of HCEnCs resulting in corneal edema in FECD [51]. In FECD, excessive deposition of ECM, in the form of guttae, leads to destruction of adjacent

Conclusions

By using an in vitro model that recapitulates characteristic rosette formation in HCEnCs, we detected that oxidative stress induces EMT, causing loss of cell–cell adhesion, acquisition of fibroblastic morphology, and upregulation of Snail1, ZEB1, fibronectin, and N-cadherin in human corneal endothelium. This effect was potentiated when redox cycling activity of MN was enhanced by the absence of NQO1. This data highlights the regulatory role of intracellular changes in inducing EMT in post

Acknowledgments

This work was supported by NIH/NEI Grant R01EY20581, Core Grant P30EY003790, Alcon Research Award, and New England Transplantation Fund.

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    Work performed at: Schepens Eye Research Institute, Massachusetts Eye and Ear, 20 Staniford Street, Boston, Massachusetts 02114, USA.

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    Authors contributed equally.

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