thapsigargin and Fuchs--Endothelial-Dystrophy

The present study evaluated survival effects of N-acetylcysteine (NAC) on cultured corneal endothelial cells exposed to oxidative and endoplasmic reticulum (ER) stress and in a mouse model of early-onset Fuchs endothelial corneal dystrophy (FECD). Cultured bovine corneal endothelial cell viability against oxidative and ER stress was determined by CellTiter-Glo(®) luminescent reagent. Two-month-old homozygous knock-in Col8a2(L450W/L450W) mutant (L450W) and C57/Bl6 wild-type (WT) animals were divided into two groups of 15 mice. Group I received 7 mg/mL NAC in drinking water and Group II received control water for 7 months. Endothelial cell density and morphology were evaluated with confocal microscopy. Antioxidant gene (iNos) and ER stress/unfolded protein response gene (Grp78 and Chop) mRNA levels and protein expression were measured in corneal endothelium by real time PCR and Western blotting. Cell viability of H2O2 and thapsigargin exposed cells pre-treated with NAC was significantly increased compared to untreated controls (p < 0.01). Corneal endothelial cell density (CD) was higher (p = 0.001) and percent polymegathism was lower (p = 0.04) in NAC treated L450W mice than in untreated L450W mice. NAC treated L450W endothelium showed significant upregulation of iNos, whereas Grp78 and Chop were downregulated compared to untreated L450W endothelium by real time PCR and Western blotting. NAC increases survival in cultured corneal endothelial cells exposed against ER and oxidative stress. Systemic NAC ingestion increases corneal endothelial cell survival which is associated with increased antioxidant and decreased ER stress markers in a mouse model of early-onset FECD. Our study presents in vivo evidence of a novel potential medical treatment for FECD.

Topics: Acetylcysteine; Animals; Blotting, Western; Cell Count; Cell Survival; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Endothelium, Corneal; Free Radical Scavengers; Fuchs' Endothelial Dystrophy; Heat-Shock Proteins; Hydrogen Peroxide; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Nitric Oxide Synthase Type II; Oxidative Stress; Polymerase Chain Reaction; RNA, Messenger; Thapsigargin; Transcription Factor CHOP

Lithium previously has been shown to reduce both endoplasmic reticulum (ER) and oxidative stress in other in vitro and in vivo model systems. We investigated lithium's effects on cultured corneal endothelial cells (CECs) exposed to these types of stress and in a mouse model of Fuchs endothelial corneal dystrophy (FECD).. Viability of cultured bovine CECs was determined by CellTiter-Glo. 2-month-old Col8a2(Q455K/Q455K) mutant (Q455K) and C57/Bl6 wild type animals were divided into two groups of 15 mice. Group I received 0.2% lithium carbonate-containing chow and Group II received control chow for 7 months. Confocal microscopy, transmission electron microscopy, real-time PCR (RT-PCR) and western blot were performed.. Pretreatment with lithium increased viability of cultured CECs after H2O2 and thapsigargin exposure compared with untreated controls (p<0.05). In vivo analysis of mouse corneal endothelium showed the following: endothelial cell density of lithium treated Q455K was higher than for untreated Q455K (p<0.01). transmission electron microscopy of lithium treated Q455K showed normal endothelium with enlarged autophagosomes, but untreated Q455K showed dilated ER and guttae. Compared with untreated Q455K endothelium, lithium treated Q455K showed significant upregulation of P62, Tmem74, Tm9sf1 and Tmem166 by RT-PCR and of Atg5-12 conjugate by western blotting indicating that lithium treatment increased autophagy. Although RT-PCR unexpectedly showed increased levels of lithium response genes, caspase 12, Gsk3β, Arrβ2 and Impa1, western blotting showed the expected downregulation of Arrβ2 and Impa1 proteins in response to lithium treatment.. Lithium increases cultured CEC survival against ER and oxidative stress. Increased autophagy in lithium treated endothelium in a mouse model of FECD suggests autophagy may contribute to increased endothelial cell survival.

Topics: Animals; Apoptosis; Blotting, Western; Cattle; Cell Count; Cell Survival; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Endothelium, Corneal; Fuchs' Endothelial Dystrophy; Gene Expression Regulation; Heat-Shock Proteins; Hydrogen Peroxide; Lithium Carbonate; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Microscopy, Electron, Transmission; Oxidative Stress; Real-Time Polymerase Chain Reaction; Thapsigargin; Transcription Factor CHOP