Dataset Information

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.

ABSTRACT:

Aims

Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death.

Results

We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ??m and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs.

Innovation

This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue.

Conclusion

MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072-1083.

SUBMITTER: Halilovic A

PROVIDER: S-EPMC4931310 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Publications

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.

Halilovic Adna A Schmedt Thore T Benischke Anne-Sophie AS Hamill Cecily C Chen Yuming Y Santos Janine Hertzog JH Jurkunas Ula V UV

Antioxidants & redox signaling 20160411 18

<h4>Aims</h4>Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death.<h4>Results</h4>We modeled the pathognomonic rosette formation of postmitotic corneal cells by incr ...[more]

PMID: 26935406

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Project description: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-?1 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 (TGF?I), 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.

Project description:Fuchs’ endothelial corneal dystrophy (FECD) is a progressive vision impairing disease caused by thickening of Descemet’s membrane and gradual degeneration and loss of corneal endothelial cells. The aim of this study was to identify differentially expressed genes between FECD-affected and unaffected corneal endothelium to gain insight into the pathophysiological mechanisms underlying this disease. Microarray gene expression analysis was performed on total RNA from FECD-affected and unaffected corneal endothelium-Descemet’s membrane (CE-DM) specimens using the Illumina HumanHT-12 v3.0 expression array. RNA from pools of FECD-affected (n=3 per pool) and individual unaffected (n=3) specimens was used for comparison. Altered expression of a sub-set of differentially expressed genes was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in independent specimens. Bioinformatics analysis was performed using InnateDB to reveal functional relationships among the differentially expressed genes and molecular pathways involved in the disease. A total of 16,513 genes were found expressed in the corneal endothelium of which 142 genes were differentially expressed between FECD-affected and unaffected endothelium (log2 fold-change ≥1.5, corrected p-value ≤0.05). Most of the genes were up-regulated (126) and a small proportion down-regulated (16) in affected corneal endothelium. Of the twelve genes prioritised for validation, differential expression of 10 genes, including those ranked 57th and 81st by significance validated by qRT-PCR (8 up-regulated and 2 downregulated, corrected p ≤0.05), one gene showed a trend for up-regulation in affected endothelium, consistent with the microarray analysis and another was up-regulated in an independent study indicating robustness of the differential expression dataset. Bioinformatic analysis revealed significant over-representation of differentially expressed genes in extracellular matrix reorganisation, cellular remodelling, immune response, and inflammation. Network analysis showed functional inter-relatedness of the majority of the dysregulated genes and revealed known direct functional relationships between 20 of the genes; many of these genes have roles in macrophage differentiation, phagocytosis and inflammation. This is the second report of microarray gene expression analysis in FECD. This study revealed a set of highly dysregulated genes in the corneal endothelium in FECD. More than a third of the dysregulated genes in the disease have been discovered for the first time and thus are novel. The dysregulated genes strongly suggest the presence of phagocytic cells, most likely immune cells, and inflammation in corneal endothelium in the disease. This study provides a molecular framework for delineating the mechanisms underlying these cellular processes in FECD.

Dataset Information

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.

Aims

Results

Innovation

Conclusion

Publications

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.

Similar Datasets

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