The Role Of CTGF, VEGF And TGF-β₂ In Retina Of The Diabetic Rats Elucidated By Treatment With CTGFsiRNA

Background and ObjectiveDiabetic retinopathy is a leading cause of blindness,ultimately resulting in an advanced stage of proliferative retinopathy with neovascularization,fibrovascular proliferation,and retinal detachment.Recently the incidence of diabetes increased significantly,which is higher in developing countries than others as 38%～90%.It has been attracted extensive attention.The pathogenesis of DR is very complex and has not yet been fully clarified.Moreover cells are lost during this early stage of retinopathy in diabetes.Cytokines have been implicated in diabetic retinopathy,the most notable being vascular endothelial growth factor(VEGF) and transforming growth factor-beta2 (TGF-β2).Connective tissue growth factor(CTGF) is a cysteine-rich matricellular protein belonging to the CCN family of proteins,which have many diverse functions concerned with angiogenesis,fibrosis,and apoptosis etc.CTGF,accompanied by VEGF and TGF-β2 are increasingly recognized as playing important roles in the pathogenesis of diabetic retinopathy.Recently,a novel method of post-transcriptional silencing of gene expression,called RNA interference(RNAi),was discovered.RNAi is a conserved cellular mechanism that silences the expression of a protein in a specific and potent fashion.Thus small interference RNA(siRNA) is a potent method for investigating the function of gene products in tissues.Although much progress has been made recently,the interaction between these factors is not very clear and further research is needed,as the disease remains neither preventable nor curable.In this study, we measure the gene and protein expression and the distribution of CTGF,VEGF and TGF-β2 in the retina of diabetic and normal rats,and analyze the correlation between expression of these three factors and the relationship between the degree of apoptosis and cytokine production using siRNA targeting CTGF(CTGFsiRNA) to silence the CTGF gene. MethodsDiabetes was induced in rats by the 1%β-cell toxin streptozotocin(STZ).The blood glucose above 16.7mmol/L and urine glaucose+++ is the standard of setting up the model.Animals were divided to control,diabetic rats in 4w,8w,12w,16w,24 weeks and similar animals diabetic 16 weeks treated with CTGFsiRNA by intravitreal injection.The vessels of retina were detected by ADPase stained mRNA level and protein expression of CTGF,VEGF and TGF-β₂ was measured by RT-PCR and Western-blotting,and located by immunohistochemistry.Retinal apoptosis was detected by TUNEL staining.The results were statistically tested by SPSS10.0 with t test and correlations analysis.ResultsThe model of diabetes in rat was set up successfully.At early stage of diabetic retina before 16 weeks,there were similar to the control.Till 24 weeks diabetes,the vessels became straight and narrow,and the layers of the retina became unregular and the inner layer membrane edema.The levels of CTGF,VEGF and TGF-β₂ and TUNEL-positive nuclei number in the diabetic retinas were significantly higher than the control(P＜0.05).CTGF rose at 8 weeks,earlier than VEGF and TGF-β₂ at 12 weeks after the onset of diabetes.The difference was significant(P＜0.05). siRNA-mediated inhibition of CTGF mRNA inhibited retinal VEGF and TGF-β₂ and also resulted in a significant decrease in apoptosis.Significant correlations were found between CTGF and VEGF(P=0.04),CTGF and TGF-β₂(P=0.05),VEGF and TGF-β₂ (P＜0.01),apoptosis and these three cytokines(P＜0.01) in the rat retina early in diabetes.ConclusionThese results suggest that the diabetes-mediated increase in CTGF up-regulates VEGF and TGF-β₂ expression and induces apoptosis in retina.And the elevation could be inhibited by treatment with CTGFsiRNA.CTGF,VEGF and TGF-β₂ play critical roles in the early stage of diabetic retina.CTGF could serve as a potential target for the prevention and treatment of diabetic retinopathy.