Dataset Information

L450W and Q455K Col8a2 knock-in mouse models of Fuchs endothelial corneal dystrophy show distinct phenotypes and evidence for altered autophagy.

ABSTRACT:

Purpose

We compared the cellular phenotypes and studied the role of autophagy in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD) using two ?2 collagen VIII (Col8a2) knock-in mouse models and human FECD tissues.

Methods

In vivo corneal endothelial cell (CEC) counts and morphology were analyzed by clinical confocal microscopy. Ultrastructural analysis of CECs was performed by transmission electron microscopy. Real-time PCR and Western blotting were performed using total RNA, and protein extracted from mouse CECs and human CECs obtained from FECD and autopsy patients.

Results

Both Col8a2 mouse models exhibited hallmarks of FECD; however, the Col8a2(L450W/L450W) mice exhibited a milder phenotype compared to the Col8a2(Q455K/Q455K) mice. Both models exhibited upregulation of the unfolded protein response (UPR) as evidenced by dilated rough endoplasmic reticulum (RER), and upregulation of UPR-associated genes and proteins. Real-time PCR of Col8a2(L450W/L450W) and Col8a2(Q455K/Q455K) CECs at 40 weeks revealed a 2.1-fold (P < 0.05) and a 5.2-fold (P < 0.01) upregulation of the autophagy marker Dram1, respectively. Real-time PCR of human FECD endothelium revealed a 10.4-fold upregulation of DRAM1 (P < 0.0001) compared to autopsy controls.

Conclusions

The Col8a2(L450W/L450W) and Col8a2(Q455K/Q455K) mouse models of FECD showed distinct endothelial cell phenotypes. Dram1 was associated with activation of the UPR and increased autophagy. Overexpression of this gene in mouse and human FECD endothelial cells suggested a role for altered autophagy in this disease.

SUBMITTER: Meng H

PROVIDER: S-EPMC3626522 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Publications

L450W and Q455K Col8a2 knock-in mouse models of Fuchs endothelial corneal dystrophy show distinct phenotypes and evidence for altered autophagy.

Meng Huan H Matthaei Mario M Ramanan Narendrakumar N Grebe Rhonda R Chakravarti Shukti S Speck Caroline L CL Kimos Martha M Vij Neeraj N Eberhart Charles G CG Jun Albert S AS

Investigative ophthalmology & visual science 20130328 3

<h4>Purpose</h4>We compared the cellular phenotypes and studied the role of autophagy in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD) using two α2 collagen VIII (Col8a2) knock-in mouse models and human FECD tissues.<h4>Methods</h4>In vivo corneal endothelial cell (CEC) counts and morphology were analyzed by clinical confocal microscopy. Ultrastructural analysis of CECs was performed by transmission electron microscopy. Real-time PCR and Western blotting were performed using tot ...[more]

PMID: 23422828

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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

L450W and Q455K Col8a2 knock-in mouse models of Fuchs endothelial corneal dystrophy show distinct phenotypes and evidence for altered autophagy.

Purpose

Methods

Results

Conclusions

Publications

L450W and Q455K Col8a2 knock-in mouse models of Fuchs endothelial corneal dystrophy show distinct phenotypes and evidence for altered autophagy.

Similar Datasets

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