Genomic And Proteomic Characterization Of Non-proliferative Retinopathy In A Mouse Model

Abstract

Purpose: : Diabetic retinopathy is the leading cause of loss of visual acuity and blindness in adulthood. Transgenic (Tg) mice overexpressing Insulin-like Growth Factor type I (IGF-I) in the retina have retinal alterations characteristic of non-proliferative retinopathy and, with age, mice develop alterations that mimic the proliferative stage of the disease. The aim of this study was to perform genomic and proteomic analyses in IGF-I Tg eyes to identify key molecular markers in early stages of the disease

Methods: : Aqueous humor (AH) samples and retinas from 4-month-old Tg and wild-type (WT) animals were collected. Retinas were homogenized and total RNA and protein extracts were obtained. We compared gene expression profiles in Tg and WT retinas. The expression of some altered genes was confirmed by quantitative PCR (qPCR). Retinal protein extracts and AH samples were separated in bidimentional electrophoresis and proteins were identified by tandem mass-spectrometry

Results: : Gene profile analysis detected 37 genes differentially expressed, 25 of which up-regulated and 12 genes down-regulated more than 1.5-fold in Tg retinas. Most of the up-regulated genes were classified in three main categories: gliosis, retinal stress and angiogenesis, whereas down-regulated genes were related with CNS development and angiogenesis. By qPCR we found that Tg retinas already overexpressed gliosis-related genes at an early age (1.5 months), at which Tg mice do not present morphological alterations yet. This overexpression was maintained during the animal’s life. The same pattern was observed with retinal-stress-related genes. Retinal proteomic studies showed 45 proteins differentially produced in Tg retinas, 21 of which increased and 24 decreased. In AH 14 proteins were detected: 9 increased, 1 decreased and 4 presented no change. The majority of the identified proteins were involved in metabolic processes while their localization in the cell was mainly cytoplasmatic and mitochondrial

Conclusions: : Most of the alterations found in gene profile and proteomic analysis in Tg retinas and AH samples have also been reported in diabetic animal models and in human diabetic retinas and vitreous fluid, suggesting that the activation of glial and stress-response genes play a key role in initiating the pathology. Overall, these results also validate the IGF-I transgenic mouse model as an excellent tool to find therapeutic targets for early stages of retinopathy and to assay new therapies

Keywords: diabetic retinopathy • gene microarray • gene/expression