Differential Gene Expression of CCN–Family Members in Retinas of VEGF–Injected Rat Eyes

Abstract

Abstract: : Purpose: Proliferative diabetic retinopathy (PDR) and exudative age related macular degeneration (AMD) are the leading causes of blindness in Western countries. Progression of both diseases is characterized by neovascularization and fibrosis, which eventually leads to irreversible loss of vision. The major factor responsible for neovascularization was identified as vascular endothelial growth factor–A (VEGF). VEGF is suggested to act in concert with many other cytokines. A recently discovered growth factor, which also plays a role in extracellular matrix production and fibrosis, is connective tissue growth factor (CTGF, CCN2). In vitro studies have shown that VEGF can induce CCN2 transcription in retinal endothelial cells and pericytes, indicating a possible role of CCN2 in ocular neovascularization and fibrosis. In order to study possible VEGF–induced expression of CCN2 in vivo, we investigated mRNA expression patterns of CCN2 in the retina of VEGF–injected rat eyes. In addition, we investigated mRNA expression of other CCN–family members and extracellular matrix components. Methods: Recombinant rat VEGF164 (rrVegf164, 300ng) or PBS was injected intra–vitreally in male Wistar rats. Gene expression in whole retinas was investigated after 24 and 48 hours by real–time quantitative RT–PCR. A normalization factor based on the expression levels of three housekeeping genes was calculated by using the geometric mean of the absolute values. Results: We demonstrated a significant induction of CCN2 and CCN1 (CYR61) 24 hours after VEGF injection. 48 hours after injection, CCN2 transcription levels were on average higher, although no longer significantly, than in control retinas and CCN1 expression was back to normal. As for the remaining CCN–family members expression levels were unchanged for CCN4, while CCN3, –5 and –6 expression levels were undetectable. Fibronectin and transforming growth factor–ß2 (TGF–ß2) expression was elevated significantly 24 hours after injection and decreased 48 hours after injection to levels that were not significantly different from controls. This was in contrast to expression levels of collagen type IV (Col3a4), TGF–ß1 and tissue inhibitor of metalloproteinase 2 (TIMP–2), which remained comparable to control levels at both time points. Conclusions: VEGF–injection in rat eyes induces upregulation of retinal CCN1, CCN2 and TGF–ß2 genes and selected extracellular matrix genes. In vivo regulation of these genes by VEGF may underlie crucial mechanisms involved in fibrosis and neovascularization in the progression of DR and AMD.

Keywords: diabetic retinopathy • gene/expression • extracellular matrix