Inhibition of Connective Tissue Growth Factor prevents increase in extracellular matrix molecules in a rodent model of Diabetic Retinopathy.

Abstract

Connective tissue growth factor (CTGF) is a profibrotic factor that induces extracellular matrix (ECM) production and angiogenesis, two processes involved in diabetic retinopathy (DR). In this study, we examined whether insulin therapy or a CTGF specific siRNA administered to diabetic rats decreased the levels of CTGF and of selected putative downstream genes in retina. Rats with streptozotocin-induced diabetes were used for these studies. Animals received either no treatment or were administered constant insulin therapy for 12 weeks. mRNA and protein levels of CTGF and select ECM genes were determined using Real-Time PCR and western blotting of retina. Localization of CTGF, GFAP (Glial Fibrillary Acidic Protein), a marker of astrocytes and activated Müller cells, and Vascular Endothelial Growth Factor, (VEGF), a well studied growth factor in the pathologies of DR, were visualized in the retina using immunohistochemistry. CTGF mRNA and protein significantly increased in retina of diabetic rats at 8 and 12 weeks of hyperglycemia. There was also an increase CTGF, GFAP and VEGF immunostaining during hyperglycemia. Double label immunohistochemistry indicated that retinal Müller cells of diabetic rats expressed CTGF. Hyperglycemia also upregulated mRNA levels of fibronectin, laminin ß1, collagen IVa3 and VEGF, and this increase was prevented by insulin therapy. Treatment of diabetic rats with CTGF siRNA decreased laminin ß1, collagen IVa3 mRNAs and CTGF mRNA and protein but did not affect fibronectin or VEGF mRNA levels. Since insulin therapy is associated with oscillations in blood glucose levels, we sought to develop a supplementary gene therapy to inhibit CTGF in DR. Prior to its use in vivo, we sought to determine whether a gene therapy tool could inhibit CTGF in a rat cell culture model that expresses CTGF. First, we tested the efficiency of an adenovirus encoding for a CTGF antisense oligonucleotide and three CTGF siRNA's in vitro to decrease the levels of Transforming Growth Factor-β (TGF-β)-induced CTGF expression in Rat 2 fibroblasts. The adenovirus and siRNA both decreased CTGF in Rat 2 fibroblasts. To circumvent any possible immune responses caused by the adenovirus we chose to use the siRNA for our in vivo studies to inhibit CTGF in the diabetic retina. A subset of diabetic rats received a single intravitreal injection of CTGF siRNA in one eye and a scrambled, control, siRNA in the contralateral eye. Retinas were examined three and ten days after the injection. CTGF siRNA significantly inhibited CTGF mRNA and protein 3 days after injection. CTGF siRNA also induced a significant decrease in laminin β1, collagen IV α3 and GFAP mRNA but had no effect on fibronectin mRNA levels. The level of CTGF mRNA was similar in retina of control and CTGF siRNA injected animals 10 days after injection In conclusion, these studies indicate that CTGF and ECM genes are regulated with insulin therapy. Importantly, CTGF siRNA regulates changes in ECM molecules and reduced GFAP expression suggesting that the treatment decreased the level of glial cell activation and reactivity in DR. Taken together, our results indicate that CTGF is an important target for the treatment of DR.