Endothelial Cell Whole Genome Expression Analysis in a Mouse Model of Early-Onset Fuchs' Endothelial Corneal Dystrophy

Abstract

PURPOSE. To investigate the endothelial gene expression profile in a Col8a2 Q455K mutant knock-in mouse model of earlyonset Fuchs' endothelial corneal dystrophy (FECD) and identify potential targets that can be correlated to human late-onset FECD.METHODS. Diseased or normal endothelial phenotypes were verified in 12-month-old homozygous Col8a2 Q455K/Q455K mutant and wild-type mice by clinical confocal microscopy. An endothelial whole genome expression profile was generated by microarray-based analysis. Result validation was performed by real-time PCR. Endothelial COX2 and JUN expression was further studied in human late-onset FECD compared to normal samples.RESULTS. Microarray analysis demonstrated endothelial expression of 24,538 genes (162 up-regulated and 172 down-regulated targets) and identified affected gene ontology terms including Response to Stress, Protein Metabolic Process, Protein Folding, Regulation of Apoptosis, and Transporter Activity. Real-time PCR assessment confirmed increased Cox2 (P ¼ 0.001) and Jun mRNA (P ¼ 0.03) levels in Col8a2 Q455K/Q455K mutant compared to wild-type mice. In human FECD samples, real-time PCR demonstrated a statistically significant increase in COX2 mRNA (P < 0.0001) and JUN mRNA (P ¼ 0.002) and tissue microarray analysis showed increased endothelial COX2 (P ¼ 0.02) and JUN protein (P ¼ 0.04).CONCLUSIONS. The present study provides the first endothelial whole genome expression analysis in an animal model of FECD and represents a useful resource for future studies of the disease. In particular endothelial COX2 up-regulation warrants further investigation of its role in FECD. (Invest Ophthalmol Vis Sci.

Identification of Circulating Fibrocytes and Dendritic Derivatives in Corneal Endothelium of Patients With Fuchs' Dystrophy

“…Apart from FOSB, another activator protein 1 transcription factor subunit, namely JUN, was also significantly upregulated in our data, consistent with the current literature. 50,51 With respect to unfolded protein response, our data confirm a significant downregulation of prefoldin subunit 5, as reported by Jalimarada et al 50 As a last remark, differences in gene expression levels between studies can also be a result of the use of different reference genes for normalization. GAPDH, a frequently used reference gene, 39,41 is unstably expressed in FECD versus normal (significantly upregulated in our study and significantly downregulated in other studies 48,49 ) and is therefore not the best reference gene to study gene expression in FECD.…”

Autophagy in the eye: Implications for ocular cell health

“…The primary component of the basement membrane is collagen type VIII, thus it is not surprising that the Col8a2 Q455K knockin mouse exhibits a FECD-like endothelial morphology and is used as a model of early onset disease (Matthaei et al, 2013). Morphological and molecular analysis of the Col8a2 Q455K mouse reveals an increase in damage-regulated autophagy modulator Dram1, a p53 target gene encoding a lysosomal protein that induces macroautophagy (Meng et al, 2013).…”

GRIK5 Genetically Regulated Expression Associated with Eye and Vascular Phenomes: Discovery through Iteration among Biobanks, Electronic Health Records, and Zebrafish

“…The zebrafish studies provided key information that the zebrafish grik5 ortholog was highly expressed during early embryonic development (day 3) of the eye, ear, and brain; these results have also been observed in the mouse eye. 34,53,54 Complete or substantial reduction of grik5 by morpholino-or CRISPR-based editing at this time results in notably reduced numbers of blood vessels and in increased vascular permeability. The fact that many of the comorbid vascular phenotypes are very early onset (congenital anomalies of the vasculature or Mendelian diseases present from birth) and that top SNP signals from the large meta-analysis of macular degeneration had such strong enrichment in vascular diseases that substantially predate macular degeneration are both consistent with the hypotheses that early developmental and/or subtle, lifelong deficits in vascular function can increase the risk of late onset eye diseases.…”

“…Although we are confident that the vascular consequences of reduced expression of GRIK5 contribute to an increased risk for eye disease, we cannot be sure that this is the only mechanism by which reduced expression of GRIK5 might contribute to eye disease. GRIK5 is expressed in endothelial cells ( Figure S3C) 38,53,54 but has also been shown to be expressed in mice in the photoreceptor cells of the retina. 56 We observed GRIK5 to be highly expressed in the eye of 3-day-old zebrafish embryos ( Figure S3).…”