Effect Of Connective Tissue Growth Factor On Posterior Capsule Opacification

Psterior capsule opacification (PCO) is the the most common complication of catraractsurgery. At present the only means of treating cataract is by surgical intervention, and thisinitially restores high visual quality. Unfortunately, PCO develops in a significantproportion of patients to such an extent that a secondary loss of vision occurs, whichconsequently required further corrective laser surgery which is not without risk.After cataract surgery, residual LECs transdifferentiate into myofibroblast, and oftenmigrate and proliferate on the posterior capsule, induce capsule wrinkling. Thetransdifferentiated cells expressα-smooth muscle actin (α-SMA) which is the mark ofmyofibroblast; abnormal extracellular materials (ECM), including collagen-â… (CA-â… )and fibronectin (FN), accumulate around the transdifferentiated cells, that will induce PCO.Connective tissue growth factor (CTGF) is a 36 to 38kDa cysteine-rich peptide, and isfirst identified from human umbilical endothelial cells. As the downstream regulatory factorof transforming growth factor-β(TGF-β), CTGF could promote cells proliferation,migration, adhesion, and ECM production. As reported by previous research, CTGFupregulates the expression CA-â… and FN in human lung fibroblasts and vascular smoothmuscle cells. Overproduction of CTGF therefore has been implicated as an importantpathway leading to lung fibrosis and kidney fibrosis. It has been reported that CTGF playsan important role in pathogenesis of PCO. In order to research the effect and possible mechanism of the action about the CTGF on the PCO, we are going to this reseach project,which includes four sections.Partâ… The research of migration and transdifferentiation effects ofconnective tissue growth factor in bovine lens epithelial cellsObjective To observe the migration and transdifferentiation effects of connective tissuegrowth factor (CTGF) in bovine lens epithelial cells (BLECs).Methods Cultured 2～3 passage BLECs were divided into control group and experimentalgroup with 0.1×10⁶ng·L^-1, 0.5×10⁶ng·L^-1, 1.0×10⁶ng·L^-1 CTGF for 24h, the mRNA andprotein ofα-smooth muscle actin (α-SMA) in BLECs were examined respectively bysemiquantitative RT-PCR and Western blotting. In migration experiment, transwell insertswere used to evaluate migration ability of BLECs.Results In comparison with control group, experimental group could promote theexpression ofα-SMA at mRNA and protein level with dose dependent obviously inBLECs (P＜0.01). In migration experiment, CTGF could significantly promote BLECsmigration (P＜0.01).Conclusion As the downstream regulatory factor of TGF-β, CTGF play an important rolein lens epithelial cells migration, transdifferentiation and deposition of ECM in posteriorcapsule opacification (PCO).Partâ…¡Effect of connective tissue growth factor SiRNA inbovine lens epithelial cellsObjective To observe the effects of connective tissue growth factor (CTGF) SiRNA on the expression of CTGF,α-smooth muscle actin (α-SMA) and fibronectin(FN) in bovine lensepithelial cells (BLECs).Methods Cultured 2～3 passage BLECs were divided into control group,TGF-β₁+negative SiRNA group, TGF-β₁ group, TGF-β₁+CTGF SiRNA₁ group andTGF-β₁+CTGF SiRNA₂ group. The experimental group was treated by 50nm·L^-1 SiRNA₁and SiRNA₂, and then incubation with 10.0×10³ng·L^-1 TGF-β₁. The mRNA and proteinexpression ofα-SMA, FN and CTGF in BLECs were examined respectively by Real-TimePCR and Western blotting.Results Real-Time PCR indicated that in comparison with TGF-β₁ group, mRNAexpression of FN,α-SMA and CTGF decreased in tranfection group (P＜0.05). Westernblotting indicated that in comparison with TGF-β₁ group, CTGF SiRNA could inhibitedthe expression of FN,α-SMA and CTGF notablely (P＜0.05).Conclusion The data suggested that CTGF SiRNA could inhibit the expression of CTGF,α-SMA and ECM. Because CTGF play an essential role in lens epithelial cells migration,transdifferentiation and deposition of ECM in posterior capsule opacification (PCO), thatinterferring of CTGF could prevent PCO.Partâ…¢The research of mechanism at connective tissue growth factorinduced fibronectin expression in bovine lens epithelial cellsObjective To observe the effect of JNK and PI3K/Akt on connective tissue growth factor(CTGF)-induced fibronectin (FN) expression in bovine lens epithelial cells (BLECs).Methods The Cultured 2～3 passage BLECs were respectively treated by JNK inhibitorSP-600125, Akt inhibitor LY-294002, JNK SiRNA and Akt SiRNA, and then incubationwith 1μg/ml CTGF for 24h. The protein of FN, p-JNK and p-Akt were examined by western blotting.Results In the presence of JNK inhibitor (SP-600125) or transfected by JNK SiRNA,CTGF-induced protein expression of FN increase was completely inhibited (P＜0.05). Aktinhibitor (LY-294002) and Akt SiRNA had no effect on CTGF-stimulated proteinexpression of FN (P＞0.05).Conclusion FN expression which induced by CTGF is mediated through the JNK pathway,but not affected by PI3K/Akt pathway. From these, we could know more about theCTGF-induced PCO.Partâ…£The expression of connective tissue growth factor andα-smoothmuscle actin in rat posterior capsule opacification modelsObjective Posterior capsule opacification (PCO), the major complication of cataractsurgery, would induce visual loss. In this study, the expression of connective tissue growthfactor (CTGF) andα-smooth muscle actin (α-SMA) in rat posterior capsule opacificationmodels has been studied.Methods An extracapsular lens extraction (ECLE), was performed in 50 Sprague-Dawley(SD) adult male rats. The pathological characteristics of PCO were observed in rat modelswith slit lamp at each time point. The posterior capsule was taken out for examining theexpression of CTGF andα-SMA.Results A classical pathological characteristics of posterior capsule opacification could beobsrved in PCO models with slit lamp. In this process, we detected the expression ofα-SMA and CTGF in the posterior capsules of models,α-SMA, a special hallmark formyofibroblast, increased significantly at 3 day, and reached the peak level at 7 day aftersurgery. However, the expression decreased at 14 day after surgery, and further decreased at28 day. It demonstrated that the lens epithelial cells transdifferentiated into mesenchyme-like cells. CTGF, a major downstream regulatory factor of transforminggrowth factor-β(TGF-β), also showed a significant increase after surgery. The regressionanalysis strongly indicated thatα-SMA expression was associated with CTGF expression(R²=0.893, P＜0.05).Conclusions These results suggest that CTGF may be involved in regulatingα-SMAexpression, which is the important marker of transdifferentiation of lens epithelial cells inrat PCO models.