Microarray Analysis of Retinal Gene Expression in the VLDLR Knockout Mouse Model of Retinal Neovascularization

Abstract

Purpose: : Juxafoveal telangiectasia is a progressive blinding disease in which retinal blood vessels become leaky and subretinal neovascularization occurs. The biochemical mechanisms responsible for these vascular abnormalities are unknown. Mice lacking the very low density lipoprotein receptor (VLDLR -/-) are characterized by abnormal intraretinal and subretinal neovascularization, starting as early as postnatal day 15, and therefore provide a good animal model to study the factors involved in abnormal retinal angiogenesis. The purpose of this study was to examine gene expression in the outer nuclear layer of VLDLR -/- mice right before the onset of the phenotype using microarray. We aim to identify the genes/pathways involved in triggering the pathological neovascularization.

Methods: : The outer nuclear layer of flash-frozen retinas of P15 VLDLR -/- and control mice was laser-captured and RNA was extracted. High-quality RNA was amplified and the resulting cDNAs were labeled. The samples were then hybridized to an Illumina MouseWG-6 BeadChip. Microarray data analysis was performed using the SAM software.Hybridization signals were calculated using rank invariant normalization. Genes were identified as differentially expressed if they showed a fold-change of at least 3 and a p-value < 0.05. Additionally, we performed genes cluster analysis to isolate cellular pathways specifically affected.

Results: : We found a significant increase in the expression levels of several genes of the PGC-1 pathway, which has been involved in the regulation and maintenance of lipids and energy metabolism. The Wnt pathway is one of the major pathways found to be downregulated, in particular the wnt5B and frizzled-6 genes. This pathway has been recently linked to angiogenesis regulation in the retina. Additionally fatty acids and PPAR pathways are found to be downregulated, indicating a disruption of the lipid metabolism. Surprisingly, VEGF expression level in the ONL of VLDLR KO mice remained low and within range of what is observed in the age-matched controls.

Conclusions: : We have identified several pathways and genes differentially expressed in the photoreceptors of the VLDLR KO mouse before the onset of the abnormal vessel growth. These findings will be important for the identification of early biochemical alterations leading to neovascularization as well as for the understanding of the pathological process.

Keywords: retinal neovascularization • gene/expression • gene microarray