The Role And Mechanism Of STAT3 In Subconjunctival Fibrosis After Glaucoma Filtration Surgery

BackgroundGlaucoma,a chronic progressive optic neuropathy,is a major cause of blindness worldwide.Elevation of intraocular pressure(IOP)is a major risk factor for glaucoma and a permanent loss of vision.Currently,the only evidence-based treatment available is to lower IOP by medical and surgical methods.However,surgical failure can occur due to the fibrotic responses induced by glaucoma filtration surgery(GFS)injury at the subconjunctival area.Although antimetabolic agents,such as mitomycin C(MMC)and5-fluorouracil,have been used to inhibit scar formation at the filtering site,some patients continue to have poor prognoses after GFS.Furthermore,the current antifibrotic therapies have a large side-effect profile.Therefore,the scarring after GFS remains a pressing issue that needs to be resolved by glaucoma doctors.Wound healing after GFS involves a series of complex and dynamic cascade processes,including hemostasis,inflammation,proliferation and tissue remodeling,and a variety of cytokines and growth factors participate in the process.Interleukin 6(IL-6),as an important inflammatory cytokine,can be released during the inflammatory phase.Accumulated evidence has shown that IL-6 can induce fibroblasts activation.In the proliferative phase of wound healing,transforming growth factor β(TGF-β)can stimulate the migration and proliferation of human Tenon’s capsule fibroblasts(HTFs),as well as the synthesis of extracellular matrix(ECM)and the phenotypic differentiation to myofibroblasts.The phosphorylated signal transduction and activator of transcription 3(STAT3)can be activated by a variety of cytokines and growth factors.Subsequently,the phosphorylated STAT3 dimerizes and translocates to the nucleus,causing the transcription of target genes including proliferation,differentiation,survival and immunosuppression.In normal conditions,negative feedback regulated by the suppressor of cytokine signaling 3(SOCS3)controls the signal transduction of STAT3.It was reported that fibrosis can be induced by various cytokines and growth factors,and the biological processes regulated by STAT3,such as proliferation,differentiation,survival and inflammation,are involved in the formation of fibrosis.Therefore,we speculate that STAT3 activation may integrate common profibrotic pathways to promote fibrosis.Currently,no publications have studied the role of STAT3 in scar formation following GFS.Therefore,the purpose of this study is to collect subconjunctival tissues from glaucoma patients and detect the expression and activation of STAT3 in human subconjunctival tissues.Additionally,we aim to examine the role and mechanism of STAT3 in HTFs fibrosis using two complementary fibrosis models of HTFs: one involving inflammation-dependent pathways induced by IL-6,and the other involving fibroblast activation devoid of inflammation induced by TGF-β1.Finally,we will investigate the effect of STAT3 inhibitor S3I-201 on subconjunctival fibrosis around the filtration area after GFS in Sprague-Dawley rats.PurposeTo explore whether STAT3 is activated and involved in the formation of subconjunctival scar after GFS and to understand its molecular mechanism.Methods1.Human subconjunctival Tenon’s capsules were collected from patients undergoing GFS.Normal tissues were obtained from patients who had no history of ocular surgery,and scarred tissues were collected from patients who had undergone GFS with uncontrolled IOP.The obtained tissues were used for frozen sectioning and immunofluorescence staining to identify the expression of P-STAT3 and STAT3.2.HTFs were isolated from human subconjunctival tissues.The expressions of PSTAT3 and STAT3 in HTFs treated with TGF-β1 and IL-6/s IL-6R were detected by western blot and immunofluorescence.3.The HTFs were cultured with S3I-201,a small molecular inhibitor of STAT3,and the gene expression level of STAT3 was further reduced by utilizing STAT3 si RNA.Afterward,the proliferation ability of the cells was tested using the CCK-8 assay,the migration ability of the cells was examined through wound healing and transwell chamber assays,and the expression of fibronectin,collagen-I and α-SMA was detected by western blot and immunofluorescence.4.TGF-β1 and S3I-201 were used simultaneously,while IL-6/s IL-6R and S3I-201 were also used to culture the HTFs.The proliferation ability of the cells was tested using the CCK-8 assay,the migration ability of the cells was examined through wound healing and transwell chamber assays,and the expression of fibronectin,collagen-I andα-SMA was detected by western blot and immunofluorescence.5.HTFs were treated with TGF-β1 and IL-6/s IL-6R respectively,and the expression level of SOCS3 was detected by western blot.Lentivirus was used to overexpress SOCS3 in HTFs,and the infected cells were treated with TGF-β1 and IL-6/s IL-6R respectively.The proliferation ability of cells was tested by the CCK-8,the migration ability of cells was detected by wounding healing assay and transwell chamber assay,and the expression of P-STAT3,STAT3,fibronectin,collagen-I and α-SMA was detected by western blot.6.GFS was performed on Sprague-Dawley rats,which were divided into four groups: control,vehicle,MMC,and S3I-201.The IOP of the rats was measured before and after surgery on days 3,7,14,21,and 28.The morphology of filtering blebs was observed and recorded on days 3,7,14,21,and 28 after surgery.The rats were killed on day 28 after GFS and their eyes were used for the frozen section and paraffin section.TUNEL assay was used to evaluate the drug toxicity,while HE and Masson’s staining was used to observe the pathological changes and collagen deposition of the subconjunctival tissues.The expression of collagen-Ⅰ and fibronectin in the subconjunctival tissues was detected using immunofluorescence.Results1.Immunofluorescence staining of tissue sections showed a positive expression of STAT3 in the subconjunctival tissues.Moreover,the expression level of P-STAT3 was found to be increased in scarred subconjunctival tissues compared to non-scarred subconjunctival tissues.2.Western blot and immunofluorescence results revealed that STAT3 was expressed in HTFs.Additionally,the expression level of P-STAT3 was found to be increased in HTFs that had been treated with TGF-β1 and IL-6/s IL-6R.The activated P-STAT3 was located in the nucleus based on the immunofluorescence results.3.The results of CCK-8 demonstrated that the proliferation ability of HTFs was reduced in both the S3I-201 group and STAT3 si RNA group when compared to the vehicle group.Wounding healing and transwell chamber assays showed that the migration ability of HTFs was also significantly reduced in both the S3I-201 group and STAT3 si RNA group when compared to the vehicle group.Western blot and immunofluorescence evidenced that the expression level of fibronectin,collagen-I andα-SMA was decreased in the S3I-201 group and STAT3 si RNA group when compared to the vehicle group.4.The results of CCK-8 indicated that the proliferation ability of HTFs increased in the TGF-β1 and IL-6/s IL-6R treatment groups compared to the vehicle group.However,S3I-201 could inhibit the proliferation of HTFs induced by TGF-β1 and IL-6/s IL-6R.Wounding healing assay and transwell chamber assay showed that the migration ability of HTFs was higher in the TGF-β1 and IL-6/s IL-6R treatment groups than in the vehicle group.However,S3I-201 could inhibit the migration of HTFs induced by TGF-β1 and IL-6/s IL-6R.Western blot and immunofluorescence demonstrated that the expression level of fibronectin,collagen-I,and α-SMA increased in the TGF-β1 and IL-6/s IL-6R treatment groups compared to the vehicle group.However,S3I-201 could inhibit the expression level of fibronectin,collagen-I,and α-SMA induced by TGF-β1 and IL-6/s IL-6R.5.The expression of SOCS3 was decreased in HTFs cells treated with TGF-β1 or IL-6/s IL-6R.HTFs overexpressing SOCS3 were treated with TGF-β1 or IL-6/SIL-6R,respectively.The overexpression of SOCS3 in HTFs inhibited cell proliferation and migration in response to TGF-β1 or IL-6/s IL-6R.This was evident from the CCK-8assay,wounding healing assay,and transwell chamber assay.Additionally,the expression of P-STAT3,fibronectin,collagen-I,and α-SMA was significantly reduced in the LV-SOCS3 group compared to the LV-NC group in response to TGF-β1 or IL-6/s IL-6R.6.The results of the TUNEL assay showed that there were a lot of TUNEL-positive cells in the subconjunctival area of the MMC group.On the other hand,there were few TUNEL-positive cells found in the S3I-201 group.7.After GFS,a drastic decrease in IOP was observed at postoperative day 3 with animals in all three surgical groups.Animals in the vehicle group showed a complete rebound IOP elevation to the level equivalent to baseline at days 21 to 28.Animals in S3I-201 and MMC treated groups also displayed a rebound IOP elevation.However,the rebound occurred at a slower rate than that of the vehicle group.Of which,S3I-201 treated rats showed the slowest rate of rebound IOP elevation in a period of 28 days after GFS.8.Bleb survival analysis demonstrated that rats in the vehicle group displayed a rapid loss of filtering blebs after GFS and the filtering blebs were completely lost in this group on day 28.Compared with the vehicle group,filtering blebs in MMC and S3I-201 groups survived a significantly longer period.Loss of filtering blebs in the MMC group started on day 14,and the survival rate of filtering blebs was 50% on day28.Loss of blebs in the S3I-201 group occurred on day 21,and the survival rate of filtering blebs was 62.5% on day 28.9.HE and Masson staining revealed that the subconjunctival tissue structure was loose,with a small number of fibroblasts and less collagen deposition in the control group.The vehicle group demonstrated a significantly thickened subconjunctival tissue with marked hyperplasia of fibrous connective tissue.Inflammatory cells and fibroblasts were present in high densities,grew in clumps,and collagen deposition was increased.In the MMC group,the subconjunctival fibrous layer appeared looser and thinner with the formation of cavities.Sparse cells and less collagen deposition were found.The S3I-201 group showed a loose structure of fibrous connective tissue,forming voids.Sparse cells and reduced collagen deposition were observed.Immunofluorescence staining of tissue sections revealed that the expression of collagen-I and fibronectin in the subconjunctival tissue of the surgical area was higher in the vehicle group compared to the control group.However,expression of collagen-I and fibronectin was lower in the MMC group and S3I-201 group as compared to the vehicle group.Conclusions1.The expression of STAT3 was observed in human subconjunctival tissues and HTFs,and STAT3 activation was found to be upregulated in the scarred subconjunctival tissues and fibrosis HTFs induced by TGF-β1 and IL-6/s IL-6R.2.STAT3 was demonstrated to promote HTFs proliferation,migration,ECM synthesis,and phenotype transformation to myofibroblasts.3.STAT3 was found to mediate HTFs fibrosis induced by TGF-β1,which lacked inflammation,as well as IL-6,which involved an inflammatory-dependent pathway.STAT3 is a common target of both TGF-β1 and IL-6 in promoting fibrosis.4.TGF-β1 and IL-6 were observed to reduce the expression level of SOCS3 which,in turn,promoted STAT3 signal activation and HTFs fibrosis.5.Administration of S3I-201,a STAT3 inhibitor,was found to alleviate subconjunctival scar formation and benefit the stabilization of IOP reduction,prolonging survival of filtering blebs after GFS in rat models.Additionally,S3I-201 was observed to have a less toxic effect on eye tissues than MMC.