Regulation Of Subconjunctival Fibrotic Reaction By Blocking P38 MAPK Signaling Pathway

Glaucoma filtration surgery is the most frequently used procedure applied to decrease the intraocular pressure in cases of severe glaucoma. Fibrosis of filtering channel potentially causes the rate of surgical failure up to 15-25% following trabeculectomy in two years. There is a variety of risk factors in scarring of filtration channels, including youth, filtration surgery history, preoperatively long-term use of eye drops with preservatives, accompanying with uveitis, anterior segment neovascularization and so on. Characteristic of all failure causes in common is over proliferation of fibroblasts in filtering channels, leading to excessive fibrosis and scar formation. The cellular basis is myofibroblasts transdifferentiation in human Tenon's fibroblasts and excessive synthesis of extracellular matrix, which is mainly mediated by TGF-beta. Recent studies had found that p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway plays an important role in fibroblast phenotype transition. In this study, we observed the fibrosis suppression after trabeculectomy in rabbits by p38 MAPK chemical inhibitor, proving that p38 MAPK signaling pathway can be used as new targets of the anti-scarring. We cultured and characterized the human Tenon's fibroblasts (HTF) in vitro. The p38 MAPK signaling pathway was confirmed to involve in TGF-beta induced transdifferentiation of human Tenon's fibroblasts to myofibroblasts. We designed and synthesized the specificity siRNA to silence the expression of p38 MAPK and investigated if RNA interference the p38 MAPK signaling pathway suppressed the fibroblast proliferation and differentiation induced by TGF-beta.Part I. Fibrosis suppression after trabeculectomy in rabbits by inhibitoring p38 MAPK signal pathway Purpose:To investigate the fibrosis suppression after trabeculectomy in rabbits by a p38 MAPK signal pathway chemical inhibitor SB203580.Methods:24 eyes of 12 rabbits were randomly divided into 3 groups:A group (trabeculectomy alone group), B group (trabeculectomy+SB203580 group), C group (trabeculectomy+MMC group). Bleb appearance and intraocular pressure were observed during a 14-d period. Histological and transmission electron microscopy examinations, immunohistochemistry observations were evaluated. The mRNA expressions of a-SMA, type-I collagen and connective tissue growth factor (CTGF) were evaluated by real time-reverse transcriptase-polymerase chain reaction (Real-time RT-PCR). The protein expressions of a-SMA and fibronetin were evaluated by Elisa.Results:SB203580 enhanced the surgical outcome and inhibited fibroblast differentiation when compared with PBS. Bleb appearance in the SB203580-treated eyes were much diffuse and less neovascularization than those in the trabeculectomy alone eyes on d7 and 14. No significant differences in IOPs were observed between groups. Histological and ultrastructure analysis showed that conjunctival epithelium cells, fibroblasts and fibrous tissue differed between groups. Real-time RT-PCR and Elisa revealed the differences of a-SMA, type-I collagen, fibronectin, and CTGF expressions between groups.Conclusion:Fibrotic reaction after trabeculectomy in rabbits was suppressed by inhibitng p38 MAPK signal pathway. It proved that p38 MAPK signaling pathway can be used as new targets of the anti-scarring.PartⅡ. Culture of human Tenon's fibroblasts and inducing myofibrobasts transdifferentiation in vitroPurpose:To culture the human Tenon's fibroblasts (HTF) in vitro and characterize the myofibroblasts transdifferentiation induced by TGF-beta.Methods:The tissues were taken from human Tenon's capsule and placed in culture bottles containing Dulbecco's modified Eagle's medium (DMEM) for incubating. Inverted microscope was used to observe the morphology of fibroblast. The cell origin was identified by immunohistochemistry. The cell number was counted and the cell growth curve was drawn. TGF-beta dependent activation of p38 MAPK, Smad2 was examined by Western blot analysis. TGF-beta-induced mRNA expression of collagen Iα2, CTGF and the myofibroblast transdifferentiation marker alpha smooth muscle actin (α-SMA) was analyzed by real-time RT-PCR.α-SMA protein expression and fibronectin were determined by Elisa. Apoptosis was detected by flow cytometry.Results:Most cells could adhere to the wall of bottle. They were fusiform or polygon. The cells were negatively stained with Cytokeratin and positively with Vimentin by immunohistochemistry. Cell curve showed that the cells growed exponentially during day 2-8. TGF-beta stimulation of HTF induced activation of Smad2 and p38 MAPK. After 48 hours of TGF-beta stimulation, increased levels of collagen Iα2, CTGF andα-SMA transcripts were detected. And protein levels ofα-SMA and fibronectin were increased. TGF-beta also decreased HTF apoptosis.Conclusion:The cultured HTF provided a convenient source for preventing and treating the scarring after glaucoma filtration surgery in vitro. p38 MAPK signaling pathway participate in TGF-beta-induced myofibroblastic transdifferentiation and extracellular matrix expression.PartⅢ. Effects of siRNA mediated p38 MAPK gene silence on TGF-beta-induced human Tenon's fibroblasts transdifferentiation to myofibroblastPurpose:To explorer the influence of siRNA mediated p38 MAPK gene silence on transdifferentiation of hunman Tenon's fibroblasts to myofibroblast induced by TGF-beta.Methods:Three siRNA sequences targeted on special sequence of p38 MAPK gene were designed. HTFs were transfected by lipofectamine 2000. Western blot, flow cytometry and MTT were employed to evaluate the changes in the TGF-beta-induced p38 MAPK expression, the cell apoptosis and viability for screening the most effective siRNA. The p38 MAPK siRNA which was selected out was transfected into the cultured human Tenon's fibroblasts, and a nontargeted siRNA was transfected as a negative control. TGF-beta-induced p38 MAPK and Smad2 expression was analyzed with Western blot. TGF-beta-induced mRNA expression of collagen Iα2, CTGF andα-SMA was analyzed by real-time RT-PCR. Protein expression of a-SMA and fibronectin were tested by Elisa. Cell viability was determined with MTT assay. Morphology and ultrastructure of the fibroblasts was observed by atomic force microscopy.Results:The inhibitory rates of siRNA-1,2, and 3 were 71.01%,68.65% and 86.87% respectively, among which of siRNA-3 being the highest. The apoptosis differences compared with TGF-beta stimulated alone were 2.01%,1.72%,3.59% respectively. There is no significant difference in cell viability. Blocking p38 MAPK signal in the fibroblasts by siRNA, TGF-beta-induced p38 MAPK signaling was decreased rapidly, while Smad3 pathway was decreased slightly and increased lately. Both mRNA expression of collagen Iα2, CTGF and a-SMA and protein levels ofα-SMA and fibronectin were decreased. After siRNA transfection, TGF-beta stimulating time was interacted withα-SMA, CTGF, COL1A2 mRNA expression and a-SMA, FN protein levels. And the interactions were linear or quadratic trend. TGF-beta-induced cell viability was significantly inhibited after transfection. RNA interference p38 MAPK had a significant effect on the morphology and ultrastructure of fibroblasts.Conclusion:All three types of siRNA showed gene silencing effect on p38 MAPK, among which siRNA-3 was the most effective. siRNA targeted on p38 MAPK gene silence inhibited transdifferentiation of hunman Tenon's fibroblasts to myofibroblast induced by TGF-beta.