Niclosamide inhibits TGF-ß1-induced fibrosis of human Tenon's fibroblasts by regulating the MAPK-ERK1/2 pathway.

Preventing postoperative bleb scar formation is an effective way of improving glaucoma filtration surgery (GFS) outcome. Use of more effective antifibrotic drugs with fewer adverse effects may be a good way to address the problem. In the present study, we use a primary cell model, consisting of Tenon's fibroblasts obtained from patients with glaucoma, which were stimulated with TGF-ß1 to induce the fibrotic phenotype. We explored the effects of niclosamide on TGF-ß1-induced fibrosis in these cells and examined its underlying mechanism of action. A transcriptome sequencing assay was used to explore possible signaling pathways involved. Niclosamide inhibited cell proliferation and migration, and decreased the levels of alpha-smooth muscle actin, type I and type III collagen in human Tenon's fibroblasts induced by TGF-ß1. Niclosamide also induced apoptosis and counteracted TGF-ß1-induced cytoskeletal changes and extracellular matrix accumulation. Moreover, niclosamide decreased TGF-ß1-induced phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) protein expression in human Tenon's fibroblasts. The results indicate that niclosamide inhibits TGF-ß1-induced fibrosis in human Tenon's fibroblasts by blocking the MAPK-ERK1/2 signaling pathway. Thus, niclosamide is a potentially promising antifibrotic drug that could improve glaucoma filtration surgery success rate.

Effects of atorvastatin on the function of Tenon's capsule fibroblasts in human eyes.

PURPOSE: This study aimed to explore the role of atorvastatin (ATO) in the prevention and treatment of the scarring of filtration channels after glaucoma surgery. METHODS: Human Tenon's capsule fibroblasts (HTFs) were co-cultured with various concentrations of ATO. First, Cell Counting Kit-8 assay was performed to evaluate the effects of various concentrations of ATO on the viability of HTFs. Then, after the ATO stimulated the HTFs for 24 h, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to evaluate the apoptosis of HTFs. Transwell assay was also performed to evaluate the migration of HTFs. Moreover, enzyme-linked immunosorbent assay (ELISA) was performed to detect the protein expression levels of transforming growth factor-ß1 (TGF-ß1) and TGF-ß2 in the cell culture supernatant of HTFs. Western blot was carried out to detect the protein expression levels of smooth muscle actin (SMA), p38, Smad3, fibronectin, collagen I and collagen III in different groups. RESULTS: The results revealed that ATO could inhibit the proliferation and migration of HTFs. Based on the TUNEL assay, 100 µM and 150 µM ATO could induce cell apoptosis. The ELISA results indicated that ATO could down-regulate the expression level of TGF-ß2, and western blot analysis revealed that the protein expression levels of SMA, p38, Smad3, fibronectin, collagen I and collagen III in the TGF-ß2 group were all up-regulated compared with the control group, whereas the addition of ATO could reverse this up-regulation. CONCLUSIONS: ATO could inhibit the proliferation and migration of HTFs and induce their apoptosis. It was preliminary proven that ATO could inhibit the signaling pathway induced by TGF-ß. It is suggested that ATO could be a basis for the treatment of the scarring of filtration channels after glaucoma surgery.

Differential effects of acetylsalicylic acid and mitomycin C on cytokine-induced Tenon's capsule myofibroblast transdifferentiation and activity: Implications for glaucoma surgery.

Inflammation-driven scarring is a major contributor to surgical failure after subconjunctival bleb forming glaucoma surgery. The current gold standard anti-scarring adjuvant mitomycin C (MMC) has variable effectiveness and is associated with significant risks. Acetylsalicylic acid (ASA), when delivered locally, repurposes the typically pro-inflammatory cyclooxygenase (COX-2) signaling for the resolution of inflammation and mitigating inflammation-mediated fibrosis. The aim of this study is to compare the effects of ASA and MMC in an in vitro model of subconjunctival scarring. Glaucoma patient-derived Tenon's capsule fibroblasts (HTCFs) were treated with TGFß1 (2 ng/mL) plus or minus ASA (1600 µg/ml), or MMC (0.05, 0.1, 0.2 mg/mL). In vitro collagen contraction, MTT, LDH, immunofluorescence, and Western blot assays were performed. To elucidate the mechanistic effects of ASA in TGFß1-induced HTCFs, liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to identify and measure pro-inflammatory and pro-resolving lipid mediator secretion. ASA was at least as effective as MMC in reducing TGFß1-induced HTCF-mediated collagen contraction, metabolic activity, and pro-fibrotic protein expression, with less cytotoxicity. Within cytokine-activated HTCFs, ASA significantly impaired secretion of pro-inflammatory lipid mediators prostaglandin E2 and 6-keto-prostaglandin F1α and significantly increased secretion of the pro-resolving mediators 5-hydroxyeicosatetraenoic acid (HETE), 15-HETE and 18-hydroxyeicosapentaenoic acid (HEPE). ASA reduces cytokine-induced myofibroblast transdifferentiation in HTCFs, being non-inferior to MMC in vitro. ASA's effects are associated with a unique lipid mediator expression profile, suggesting that the ASA-induced resolution of inflammation may be a promising strategy to mitigate inflammation-mediated scarring and could offer a novel alternative as a surgical adjuvant.

Involvement of METTL3/m6Adenosine and TGFß/Smad3 signaling on Tenon's fibroblasts and in a rabbit model of glaucoma surgery.

Glaucoma filtration surgery (GFS) is a classic operation for the treatment of glaucoma, which is the second leading cause of blindness, and scar formation caused by excessive human Tenon's capsule fibroblasts (HTFs) activation is responsible for surgery failure. However, the mechanism underlying excessive HTFs activation is largely unknown. Studies have revealed that N6-methyladenosine (m6A), which is one of the most common posttranscriptional modifications, plays an important role in multiple types of cellular processes. First, we isolated and identified primary HTFs and found that transforming growth factor-ß1 (TGF-ß1) enhanced cell viability and promoted cell proliferation and extracellular matrix (ECM) deposition in HTFs. We subsequently found that TGF-ß1 elevated the quantity of m6A and promoted the expression of m6A "writers", in the process from DNA to RNA, adenylate was methylated at the sixth N position by methylases methyltransferase-like 3 (METTL3). Furthermore, we demonstrated that METTL3 repression inhibited the promotion of cell viability, proliferation and ECM deposition in HTFs treated with TGF-ß1. We then illustrated that increased METTL3 played a role by promoting Smad3 in TGF-ß1-induced HTFs. We subsequently demonstrated that the METTL3/Smad3 regulatory axis was aberrantly expressed in the rabbit model of GFS. Thus, our study reveals that METTL3 indeed plays a role in modulating Smad3 in TGF-ß1-induced HTFs and further provides novel theoretical strategies based on METTL3 for the inhibition of scar formation after GFS.

AGTR1 blocker attenuates activation of Tenon's capsule fibroblasts after glaucoma filtration surgery via the NF-κB signaling pathway.

Activation of Tenon's capsule fibroblasts limits the success rate of glaucoma filtration surgery (GFS), the most efficacious therapy for patients with glaucoma. Angiotensin type 1 receptor (AGTR1) is involved in tissues remodeling and fibrogenesis. However, whether AGTR1 is involved in the progress of fibrogenesis after GFS is not fully elucidated. The aim of this study was to investigate the role of an AGTR1 in scar formation after GFS and the potential anti-fibrosis effect of AGTR1 blocker. AGTR1 expression level was increased in subconjunctival tissues in a rat model of GFS and transforming growth factor-beta 2 (TGF-ß2)-induced human Tenon's capsule fibroblasts (HTFs). AGTR1 blocker treatment suppressed TGF-ß2-induced HTF migration and α-smooth muscle actin (α-SMA) and fibronectin (FN) expression. AGTR1 blocker treatment also attenuated collagen deposition and α-SMA and FN expression in subconjunctival tissues of the rat model after GFS. Moreover, AGTR1 blocker decreased TGF-ß2-induced P65 phosphorylation, P65 nuclear translocation, and nuclear factor kappa B (NF-κB) luciferase activity. Additionally, BAY 11-7082 (an NF-κB inhibitor) significantly suppressed HTF fibrosis. In conclusion, our results indicate that AGTR1 is involved in scar formation after GFS. The AGTR1 blocker attenuates subconjunctival fibrosis after GFS by inhibiting the NF-κB signaling pathway. These findings indicate that targeting AGTR1 is a potential approach to attenuate fibrosis after GFS.

Studies on the mechanism of inhibitory effect of lithium chloride on the proliferation of human Tenon capsule fibroblasts / 中华实验眼科杂志

Objective:To investigate the effect of lithium chloride (LiCl) on the gap junctional intercellular communication (GJIC) in human Tenon capsule fibroblasts (HTFs) and its underlying mechanism.Methods:The Tenon capsule tissue of a patient who underwent strabismus surgery in Dezhou People's Hospital in April 2019 was collected and cut into tissue blocks of dimensions 1 mm×1 mm×1 mm.Primary culture and subculture were carried out, and the 4th-generation HTFs were taken for experiment.HTFs were divided into the control group and LiCl treatment group and were cultured with cell medium without or with 80 mmol/L LiCl for another 48 hours according to grouping.The cell scratch and dye labeling technique were used to label the coupling index and evaluate the GJIC function.The expression and localization of Cx43 in HTFs were detected by immunofluorescence staining.The expression levels of Cx43 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot, respectively.The study protocol was approved by an Ethics Committee of Dezhou People's Hospital (No.2019-023). Written informed consent was obtained from the subject.Results:The cultured spindle-shaped HTFs grew adhering to the wall showing radial monolayer or vortexlike, and the cytoplasm was vimentin positive.Results of dye tracer experiment of cell scratch showed that the cell coupling index of LiCl treatment group was 9.04±0.53, which was significantly higher than 4.94±0.39 of the control group ( t=-18.79, P<0.01). Immunofluorescence staining showed that the Cx43 fluorescence was dotted in the cell membrane between adjacent cells in the control group, and Cx43 staining was obviously enhanced in the LiCl treatment group.The results of real-time fluorescence quantitative PCR showed that with relative expression level of Cx43 mRNA in the control group set to 1, the relative expression level of Cx43 in the LiCl treatment group was significantly increased to 1.97±0.23, showing a statistical significance between them ( t=-14.426, P<0.01). Western blot showed that the relative expression level of Cx43 protein was 0.871±0.057 in the LiCl treatment group, which was significantly higher than 0.446±0.028 in the control group ( t=-11.682, P<0.01). Conclusions:LiCl can enhance the GJIC function between HTFs by upregulating the expression levels of Cx43 mRNA and protein, suggesting that the enhanced GJIC function by LiCl may be one of the mechanisms of its inhibition on HTFs proliferation.

Effects of HSP47 siRNA on biological behaviors of HTCF cells cultured <i>in vitro</i> and the expression level of TGF-β1 / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To explore the effects of heat shock protein 47(HSP47)siRNA on biological behaviors of human Tenon capsule fibroblasts(HTCF)cells cultured <i>in vitro</i> and the expression level of transforming growth factor-β1(TGF-β1).<p>METHODS: HTCF were cultured <i>in vitro</i> and divided into blank control group, empty vector group and transfection group. In transfection group, interfering siRNA sequences were designed and synthesized based on the HSP47 gene sequences, vectors were constructed and introduced into HTCF. The empty vector group was introduced with empty vectors. The expressions of HSP47 mRNA and protein in cells were detected by RT-PCR and Western blot. The proliferation, apoptosis, invasion and migration of cells were detected by clone formation assay, flow cytometry, Transwell method and scratch test. The expressions of proliferation, apoptosis, invasion and migration proteins, and TGF-β1 were detected by Western blot.<p>RESULTS: Compared with empty vector group, expression of HSP47 mRNA and protein, clone formation rate, cell healing rate, number of invasive cells, relative expression levels of Ki67, N-cadherin and TGF-β1 were significantly decreased in transfection group(<i>P</i><0.05), relative expression level of E-cadherin protein was significantly increased(<i>P</i><0.05), but there was no difference in apoptosis rate, and relative expression levels of Bcl-2 and Bax(<i>P</i>>0.05).<p>CONCLUSION: HSP47 siRNA can reduce proliferation, invasion and migration abilities of HTCF cells by inhibiting the expression of TGF-β1 protein, without significant effects on the apoptosis of HTCF cells.

The Pro-Fibrotic Behavior of Human Tenon's Capsule Fibroblasts in Medically Treated Glaucoma Patients.

PURPOSE: The aim of this study was to compare human Tenon's capsule fibroblasts (HTCFs) obtained from patients who received medical therapy for glaucoma (glaucomatous patients) and patients not treated for glaucoma (non-glaucomatous patients) in terms of wound healing and fibrosis. PATIENTS AND METHODS: Bioartificial tissues (BATs) were generated using primary HTCF-populated collagen lattices. Pro-fibrotic gene expression within HTCFs was compared between glaucomatous patients and non-glaucomatous patients after BAT culture. The BATs were also assessed regarding fibroblast-myofibroblast transition, collagen remodeling and collagen contraction using alpha-smooth muscle actin immunohistochemistry, picrosirius red staining and collagen contraction assays, respectively. RESULTS: Pro-fibrotic gene expression in BAT-cultured HTCFs derived from glaucomatous patients was significantly increased compared to non-glaucomatous patients. BATs imbued with HTCFs collected from glaucomatous patients exhibited a greater proportion of myofibroblasts as well as increased collagen contraction/remodeling compared to HTCFs isolated from non-glaucomatous patients. CONCLUSION: HTCFs from glaucomatous and non-glaucomatous patients differ in the expression of genes involved in fibrosis, proportion of fibroblasts undergoing transdifferentiation into myofibroblasts, contractile properties and collagen remodeling. These results suggest that for any number of reasons, at a cellular level, patients who received medical therapy for glaucoma have eyes primed for fibrosis.

Role of the long noncoding RNA H19 in TGF-ß1-induced Tenon's capsule fibroblast proliferation and extracellular matrix deposition.

Glaucoma filtration surgery (GFS) is a classic surgical method used to treat glaucoma, the second leading cause of blindness. Scar formation caused by excessive Tenon's capsule fibroblast activation leads to surgical failure. However, the mechanism underlying this activation is largely unknown. In this study, we first isolated primary human Tenon's capsule fibroblasts (HTFs) and found that TGF-ß promoted the viability, proliferation and extracellular matrix (ECM) deposition of HTFs. Then, we showed that TGF-ß promoted the expression of H19 in HTFs and that suppression of H19 inhibited the effect of TGF-ß on HTFs. Furthermore, we revealed that H19 exerted its effects by interacting with miR-200a in TGF-ß-treated HTFs. Additionally, we showed that ß-catenin was a target of miR-200a in TGF-ß-treated HTFs. We also demonstrated that H19 acted by modulating the H19/miR-200a/ß-catenin regulatory axis in TGF-ß-treated HTFs. Ultimately, we found that the components of the H19/miR-200a/ß-catenin regulatory axis were aberrantly expressed in a rat model of GFS. Our results show that H19 indeed acts by modulating ß-catenin expression via miR-200a in TGF-ß-treated HTFs, thus providing a novel rationale for the development of H19-based strategies to attenuate scar formation after GFS.

Effects of matrine on human Tenon capsule fibroblast in vitro / 中华实验眼科杂志

Objective To investigate the effects of matrine on proliferation and apoptosis of human Tenon capsule fibroblasts (HTFs) in.vitro.Methods After treated with 0,0.3,0.6 and 0.9 g/L matrine in vitro,cell counting kit-8 (CCK-8) method was used to assay the proliferation of HTFs at 24,48 and 72 hours,Western blot and PCR were performed to evaluate the expression of apoptosis-associated factor caspase-3 on both protein and RNA levels.Results The activity of human Tenon capsule fibroblast at 48 hours and 72 hours after treated with 0.3,0.6,0.9 g/L matrine was significantly inhibited when compared with the 0 g/L matrine group,and the inhibitory effect was dose-dependent and time-dependent (F ion =1 019.51,P =0.00;Ftime =5 848.66,P =0.00;Fi ion =147.45,P=0.00).After treated with 0,0.3,0.6 and 0.9 g/L matrine,the early apoptosis rate of HTFs was (2.68±0.30)％,(5.08±0.47)％,(6.97±0.69)％ and (10.30±1.20)％,the grey value ofcaspase-3 protein was 1.00±0.13,1.90±0.19,2.50±0.30 and 2.67±0.30,the relative expression of caspase-3 mRNA was 0.98 ±0.12,2.01 ±0.34,6.15 ± 0.60 and 11.40 ± 1.12,respectively,with significant differences among them (F =55.74,66.01,154.50;all at P＜0.01),the early apoptosis rate of HTFs,the grey value of caspase-3 protein and the relative expression of caspase-3 mRNA were all increased significantly as the concentration of matrine increased,with significant differences between any two groups (all at P＜0.05).Conclusions Matrine can inhibit the proliferation of HTFs and induce the apoptosis of HTFs in a time-and dose-depended manner.

The Anti-fibrotic Effect of Nilotinib on Tenon's Capsule Fibroblasts in Vitro

PURPOSE: To evaluate the anti-fibrotic effects of nilotinib on the survival of cultured human Tenon's capsule fibroblasts (HTFs). METHODS: HTF primary cultures were obtained from samples following glaucoma surgery. Primarily cultured HTFs were exposed to 1, 5, 10, and 20 µM nilotinib for 24 hours. The effects of nilotinib on HTF proliferation and cell viability were determined using the 3-(4,5-dimethylthiazone-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, and apoptosis was determined by flow cytometry using annexin-V/propidium iodide (PI) double staining. Apoptosis-related proteins were detected by western blotting. RESULTS: The MTT assay showed that nilotinib induced an inhibition of HTF proliferation at concentrations of 10 and 20 µM (p < 0.001 and p < 0.001, respectively). Annexin V/PI double staining showed significantly increased apoptosis in cells treated with nilotinib. Nilotinib activated caspase-3, -9, and poly adenosine diphosphate ribose polymerase cleavage, and downregulated the expression of B-cell lymphoma-extra large and Bax, which indicated that nilotinib-induced apoptosis was partly mediated through the mitochondrial pathway. In addition, treatment with nilotinib decreased the expression of α-smooth muscle actin and transforming growth factor-β. CONCLUSIONS: Nilotinib decreased cell survival of cultured HTFs and induced mitochondria-mediated apoptosis. The results suggested that nilotinib may exert antiproliferative effects on HTFs, making it a possible agent to control postoperative fibrosis in patients undergoing glaucoma surgery.

Antiproliferative effect of double suicide gene delivery mediated by polyamidoamine dendrimers in human Tenon's capsule fibroblasts.

The aim of the present study was to investigate the therapeutic potential of a double suicide gene, thymidine kinase (TK) combined with cytosine deaminase (CD), mediated by generation of 5-polyamidoamine dendrimers (G5-PAMAM-D) on human Tenon's capsule fibroblasts (HTFs) as an anti-scarring agent. The pAcGFP1-Hyg-TK-CD plasmid was transfected into HTFs, and reverse-transcription polymerase chain reaction (RT-PCR) was used to detect TK-CD expression. MTT cell proliferation assay was used to evaluate the cytotoxic effects of ganciclovir (GCV) and 5-flurocytosine (5-FC) on HTFs. The optimal concentration of GCV and 5-FC in TK-CD transfected HTFs (HTF-TK-CD) was selected by accessing the lowest and highest cytotoxicity caused, respectively. The morphological changes of transfected HTFs following treatment with GCV and 5-FC were observed by light and transmission electron microscopy. Results demonstrated that the double suicide gene TK-CD mediated by the G5-PAMAM-D delivery system was successfully expressed in HTFs as determined by RT-PCR. A concentration of 3 µg/ml GCV and 200 µg/ml 5-FC was identified as optimal for these prodrugs. The growth rate and number of HTF-TK-CD cells decreased following treatment with GCV and 5-FC as revealed by light microscopy. Additionally, the prodrugs GCV and 5-FC not only demonstrated toxicity on transfected HTFs but also exerted a 'bystander effect'. The present study illustrated that the double suicide gene TK-CD delivery mediated by G5-PAMAM-D was effective in reducing HTF proliferation and inducing cell apoptosis. Furthermore, TK-CD delivery mediated by G5-PAMAM-D may be used as an anti-scarring agent and provide a therapeutic potential for patients requiring glaucoma filtration surgery.

Overexpression of ALK5 Induces Human Tenon's Capsule Fibroblasts Transdifferentiation and Fibrosis In Vitro.

PURPOSE: To investigate the involvement of activin receptor-like kinase 5 (ALK5) in human Tenon's capsule fibroblasts (HTFs) transdifferentiation and fibrosis. METHODS: (1) Cultured HTFs were treated with transforming growth factor beta 1 (TGF-ß1) at different concentrations for different durations, mRNA expression of ALK5 and plasminogen activator inhibitor-1 (PAI-1) was measured by quantitative polymerase chain reaction (PCR) while protein expression of ALK5, α-smooth muscle actin (α-SMA), and extracellular matrix deposition including fibronectin (FN) and collagen I (Col1) was assessed by western blot. HTFs with or without TGF-ß1 were also treated with an ALK5 activity inhibitor, SB-431542, and fibrosis-related genes were assessed. (2) HTFs were transduced with ALK5 lentivirus (ALK5-OE group) or empty lentivirus (NC-OE) with or without the treatment of SB-431542. Protein expression of ALK5, α-SMA, FN, and Col1 was evaluated. (3) HTFs in the ALK5-OE group and NC-OE group were subjected to a scratch-wound assay and their migratory activities assessed. RESULTS: (1) TGF-ß1, in a concentration-dependent manner, upregulated ALK5 and PAI-1 expressions in the HTFs, which peaked between 24 and 36 h. These changes were associated with increases in protein levels of FN, Col1, and α-SMA. These TGF-ß1 effects were blocked by the ALK5 inhibitor SB-431542. (2) Similarly, overexpression of ALK5 by lentiviral vector significantly increased protein expression of α-SMA, FN, and Col1. Addition of TGF-ß1 to the ALK5-OE cells did not produce additional expression of any of the marker proteins. The upregulation of extracellular matrix and α-SMA can be reduced by SB-431542. (3) In ALK5-OE group, HTFs migration was significantly increased compared with normal control and TGF-ß1 could still promote ALK5-OE cells migration. CONCLUSIONS: Our findings suggest that ALK5 is an important mediator of HTFs fibrosis. ALK5 is a potential therapeutic target to suppress scar formation after filtration surgery.

Effects of artesunate on cell proliferation and apoptosis of human Tenon's capsule fibroblasts / 眼科新进展

Objective To investigate the effects of artesunate (Art) on cell proliferation and apoptosis of human Tenon's capsule fibroblasts (HTFs),and discuss the countermeasures of bleb scarfing in glaucoma.Methods In vitro,HTFs were cultivated and applicated by different concentrations (50 μg · mL-1,100 μg · mL-1,150 μg ·mL-1,200 μg · mL-1) of Art for 48 hours.The effect of Art on cell proliferation was assessed by MTT method.The rate of apoptosis induced by Art was determined by flow cytometry.Western Blot was performed to detect the relative expression levels of Bax and Bcl-2 after Art was treated.Results After treated with Art for 48 hours,compared with blank control group,Art (50 μg · mL-1,100 μg · mL-1,150 μg · mL-1,200 μg · mL-1) group exhibited notable anti-proliferative effect on HTFs with concentration-dependence (all P ＜ 0.05).The results of flow cytometry showed that the apoptosis rates (8.80％ ±0.88％,11.60％ ±0.56％,16.30％ ±1.03％,23.40％ ±1.62％) of HTFs were significantly enhanced with the increase of Art concentration (all P ＜ 0.05).The relative expression levels of Bax were obviously high with the increase of Art concentration,while Bcl-2 levels were significantly low with the increase of Art concentration (all P ＜ 0.05).Conclusion Art can inhibit the proliferation and induce cell apoptosis of HTFs possibly by enhancing the expression of Bax and reducing the expression of Bcl2.Art may be a potential drug in preventing fibrous scar formation after glaucoma filtration surgery.

Inducing effect of hydroxycamptothecin on autophagy of human Tenon capsule fibroblasts in vitro / 中华实验眼科杂志

Background The fibrosis of filtering area caused by proliferation of human Tenon fibroblasts (HTFs) is one of failure causes following glaucoma surgery.Researches revealed that hydroxycamptothecin can induce the apoptosis of HTFs,but its influence on autophagy of HTFs is unclear.Objective This study attempted to investigate whether hydroxycamptothecin can cause an alteration of autophagic activity in HTFs.Methods Human Tenon capsular tissue was obtained from 3 patients during strabismus correction surgery under the informed consent of patients and their parents for the primary culture and passaged of HTFs in DMEM containing 10％ fetal bovine serum.The generation 3 to 6 cells then were incubated with 0.0,0.5,1.0,4.0,10.0 mg/L hydroxycamptothecin for 24 hours,respectively.A cell counting kit-8 (CCK-8) was used to detect the cell viability in different treated groups.The autophagic activity of HTFs was evaluated by a Cyto-ID autophagy detection kit,and then the autophagic flux was evaluated by counting the Cyto-ID positive cells under a fluorescence microscope,and the green fluorescence intensity was determined by flow cytometry.Quantitative reverse transcriptase PCR (qRT-PCR) and Western blot analysis were employed to assay the relative expressions of autophagic-associated genes and their proteins in HTFs,including Beclin-1,autophagy related gene 5 (ATG-5) and light chain 3 (LC-3).Results The cell viability of HTFs in the 0.0,0.5,1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were (100.00 ± 6.44) ％,(91.70 ± 6.36) ％,(81.47 ± 6.00) ％,(68.43 ± 6.69) ％ and (59.97 ± 6.98) ％ respectively,showing a gradually declining trend with the increase of hydroxycamptothecin doses,with a significant difference among them (F=19.040,P＜0.001),and the viability of HTFs in the 1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were significantly decreased than the control group (P＜0.05,P＜0.01,P＜0.01).qRT-PCR analysis revealed that the relative expression levels of Beclin-1 mRNA,ATG-5 mRNA and LC-3 mRNA in 4.0 mg/L hydroxycamptothecin group were (3.225 ±0.346),(2.839 ±0.418) and (3.761±0.224) folds higher than those of the control group.The expressions of Beclin-1 and ATG-5 proteins were significantly increased in the 4.0 mg/L hydroxycamptothecin group in comparison with the control group,and the expression intensity ratio of LC-3-Ⅱ/Ⅰ was 0.965±0.159 in the hydroxycamptothecin group,which was significantly higher than 0.275 ±0.860 of the control group (P =0.003).Cyto-ID staining showed that the percentage of autophagic cells increased dramatically from (11.333±4.010) ％ to (55.000±9.013) ％ upon the exposure of HTFs to 4.0 mg/L hydroxycamptothecin (P=0.002).Flow cytometry analysis showed that the green fluorescence intensity in the 4.0 mg/L hydroxycamptothecin group was (3.037 ±0.513) fold relative to that in the control group,showing a significant difference between the two groups (P =0.003).Conclusions Hydroxycamptothecin can induce autophagy in HTFs in vitro.

lnhibition effects of Bevacizumab on migration of human Tenon capsule fibroblasts / 国际眼科杂志(Guoji Yanke Zazhi)

Abstract? AlM: To investigate the inhibition effect of bevacizumab on human Tenon capsule fibroblasts ( HTFs ) and discuss the countermeasures of bleb scarringscarring in glaucoma surgery countermeasures.? METHODS: Adopted cell recovery method and followed the aseptic principles, we performed the culture of HTFs which came from the Central Laboratory of Shaanxi People's Hospital cell library. Wound Healing assay:We scraped a cell-free zone on the cell surface when the cells reached confluence at 80%. The control group was added to serum-free DMEM medium. The HTFs of bevacizumab group were stimulated with 1mg/mL concentrations without DEME for 0, 24, 48, and 72h. The scratch width was observed and measured.? RESULTS: HTFs were long fusiform shape under microscope, the nucleus is in the center of the cell with larger nucleus, abundant cytoplasm, were arranged in a whorled growth out of shape, strong ability to proliferate, conform to general forms of fibroblast. The morphological and biological characteristics of cells after cryopreservation and resuscitation remain unchanged. Wound healing assay: 0h, equal to the initial width of the two groups, 24h when the migration distance of the two groups of cells are basically the same, 48h when the control group cell migration distance is greater than that in the bevacizumab processing group, 72h when the control group scratches basichealing, bevacizumab treated cells migrate closer than 48h no significant change, and a lot of cells died.?CONCLUSlON:Cell recovery method can successfully cultured HTFs, which was stability on morphology and biological properties, laying the cellular basis for experimental research. Fibroblast itself has a strong ability to migrate, outside - derived bevacizumab can inhibit HTFs migration evidently and it will cause excessive cell death when. Bevacizumab has certain extent inhibitory effect on HTFs migration, and it is likely to become one of the important drugs for creating bleb scarring after glaucoma surgery in the future.

Dexamethasone modifies mitomycin C-triggered interleukin-8 secretion in isolated human Tenon's capsule fibroblasts.

Intraoperative mitomycin C (MMC) is widely used to prevent pterygium recurrence and glaucoma filtering bleb failure, but it has been shown to induce corneal inflammation and cell death. Postoperative dexamethasone (DEX) is advocated to reduce MMC-related inflammation and cell death in corneal fibroblasts. Nevertheless, its long-term regulation mechanism in Tenon's capsule remains to be explored. The purpose of this study was to investigate how DEX modifies MMC's effects in human Tenon's capsule fibroblasts (HTFs). HTFs isolated from the pterygium surgical patients (n = 6) were treated with MMC at 0, 0.1, 0.2, and 0.4 mg/ml for 5 min and incubated in DEX at 10 µM for 0, 1, 2, and 3 days. Recombinant interleukin-8 (IL-8) was used to verify the effect of MMC-related IL-8 secretion. Cell proliferation of all the treated cells was analyzed by WST-1 assay. The amount of IL-8 secretion in HTFs was determined by enzyme-linked immunosorbent assay. Immunoblotting assay was used to analyze the expression of peroxisome-proliferator-activated receptor gamma (PPARγ) and B-cell lymphoma-extra large (Bcl-xL). Our results revealed that MMC significantly reduced the HTF cell proliferation rate. Additionally, MMC significantly upregulated IL-8 secretion in HTFs concentration-dependently. At 3 days post treatment (dpt), 5-min exposures to 0.1, 0.2, and 0.4 mg/ml MMC resulted in 1.4-fold (p = 0.012), 1.6-fold (p = 0.012), and 2.5-fold (p = 0.001) increases of IL-8 secretion. In contrast, DEX reversed the MMC-retarded cell proliferation rate (p = 0.036) and repressed MMC-related IL-8 secretion by 33.5% at 3 dpt (p = 0.003). Addition of recombinant IL-8 noticeably suppressed HTF cell proliferation in a concentration-dependent manner. DEX stimulated upregulation of both PPARγ and Bcl-xL at 1 dpt in normal HTFs and at 2 dpt in MMC-treated HTFs. PPARγ silencing reduced expression of PPARγ and Bcl-xL, but enhanced IL-8 secretion (p < 0.001). On the other hand, Bcl-xL silencing enhanced IL-8 secretion (p < 0.001), but did not affect PPARγ expression. These revealed that IL-8 secretion in HTFs is modulated by PPARγ-dependent Bcl-xL signaling. We conclude that DEX reversed the MMC-inhibited HTF cell proliferation via diminishing the MMC-induced IL-8 secretion, which resulted from a late-phase upregulation of the PPARγ and Bcl-xL. These long-term effects suggest a beneficial postoperative DEX treatment following intraoperative MMC application.

MicroRNA-216b/Beclin 1 axis regulates autophagy and apoptosis in human Tenon's capsule fibroblasts upon hydroxycamptothecin exposure.

Proliferation and fibrosis of human Tenon's fibroblasts (HTFs) have significantly challenged the outcome of glaucoma filtration surgery. Hydroxycamptothecin (HCPT) is considered as a potential chemical to overcome this issue as it was previously shown that HCPT inhibited cell proliferation and induced apoptosis in fibroblasts. Here, we further dissected the molecular pathway, through which the HCPT inhibit the proliferation of HTFs. We showed that HCPT induced significant autophagy as well as apoptosis, two self-destructive processes, and down-regulated the expression of miR-216b in HTFs. Overexpression of miR-216b in HTFs suppressed the autophagy and apoptosis induced by HCPT, whereas silence of miR-216b led to effects that were similar to those caused by the treatment with HCPT. Further, we showed that miR-216b could directly target a specific fragment in the 3' untranslated region of Beclin 1 as demonstrated by luciferase assay, and consequently decreased the expression of Beclin 1. Consistently, knocking down Beclin 1 significantly decreased HCPT-triggered autophagy and apoptosis, and increased the viability of HTFs treated with HCPT, thus implicating that Beclin 1 functions as a pro-apoptotic molecule in this circumstance. Altogether, we concluded that miR-216b regulated both autophagy and apoptosis by modulating Beclin 1 in HTFs treated with HCPT. We also demonstrated that HCPT-induced autophagy is one of the agent's anti-proliferative effects.

TGF-ß1 stimulates human Tenon's capsule fibroblast proliferation by miR-200b and its targeting of p27/kip1 and RND3.

PURPOSE: To evaluate the role of miR-200b expression in the proliferation of human Tenon's capsule fibroblasts (HTFs) induced by transforming growth factor-beta 1 (TGF-ß1). METHODS: Human Tenon's capsule fibroblasts were treated with various doses of TGF-ß1 for 24 hours. Cell proliferation was quantified by the cell counting kit-8 (cck-8) assay, cell cycle analysis, 5-ethynyl-2-deoxyuridine (EdU) assay, and analysis of cyclin E, cyclin D1, and proliferating cell nuclear antigen (PCNA) expression. MicroRNA-200b (miR-200b) was detected by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and its potential target genes were validated by the luciferase assay and Western blot analysis. The effect of miR-200b on the proliferation of HTFs was analyzed using both miR200b-mimic and inhibitor-transfected HTFs and confirmed in p27/kip1 and RND3 (the target of miR-200b) knockdown cells. RESULTS: The proliferation of the TGF-ß1-treated HTFs increased significantly in a dose- and time-dependent manner. Treatment with 5 ng/mL TGF-ß1 caused an upregulation of miR-200b. The luciferase assay identified p27/kip1 and RND3 as target genes for miRNA-200b, which was confirmed by the expression of p27/kip1 and RND3 and their downstream products (cyclinE and cyclinD1) in the TGF-ß1-treated cells. Transforming growth factor-ß1 and miR-200b mimics enhanced the proliferation of HTFs; suppressed the expression of p27/kip1 and RND3; and subsequently stimulated the expression of cyclinE, cyclinD1, and PCNA. The miR-200b inhibitor attenuated the effects of TGF-ß1 on HTFs. Furthermore, knockdown of p27/kip1 and RND3 resulted in an increase in cell proliferation and expression of the proliferation-related genes. CONCLUSIONS: MicroRNA-200b acts as a stimulant for the proliferation of HTFs by targeting p27/kip1 and RND3.

Effect of hydroxycamptothecin combined with etoposide on apoptosis of human Tenon capsule fibroblast and its mechanism / 中华实验眼科杂志

Background Previous study showed that both hydroxycamptothecin (HCPT) and etoposide (VP-16) can induce the apoptosis of human Tenon capsule fibroblasts (HTFs).However,whether the combination of HCPT with VP-16 enhance the efficacy of drugs is unknown.Olbjeetive This study was to investigate the synergistic effect and its mechanism of HCPT combined with VP-16 on apoptosis of HTFs.Methods Human Tenon capsule tissue was obtained from the eye bank of Jiangsu Province People's Hospital.HTFs were cultured in vitro using explant method and identified by immunofluorescence with vimentin.The fourth generation of cells were incubated in 96-well plate,and different concentrations of HCPT (1,5,10,50,100 mg/L),VP-16 (0.6,2.5,5.0,25.0,50.0 mg/L) and HCPT+VP-16 (2:1,final concentrations 0.80,3.75,7.50,37.50,75.00 mg/L) were added for 24 hours.The inhibiting rate of drugs to HTFs growth was detected using CCK-8 kit.The HTFs were divided into blank control group,HCPT (50 ng/L) treated group,VP-16 (25 mg/L) treated group and HCPT+ VP-16 (37.5 mg/L) treated group,and the apoptosis rates of HTFs in various groups were assayed by flow cytometry.The expressions of caspase-3,cleaved caspase-3,bax,bcl-2,JNK,p-JNK,Akt,p-Akt in the cells were detected by Western blot assay.Results Cultured cells grew well with the polygon shape and positive response for vimentin.The inhibiting rate was elevated with the increase of drug dosage 24 hours after addition of drugs (HCPT:F=41.34,P=0.00 ; VP-16:F =62.60,P =0.00 ; HCPT+VP-16:F =46.77,P =0.00).The half maximal inhibitory concentrations (IC50) of HCPT,VP-16,HCPT+VP-16 were 80.99,27.93,19.81 mg/L,respectively,and the combined index (CI) of HCPT with VP-16 was 0.399,showing a stronger synergistic action.The apoptotic rates of HTFs were (4.87±0.78) ％,(11.20± 1.94)％,(12.67±1.51)％ and (19.77±2.01)％ in the blank control group,HCPT treated group,VP-16 treated group and HCPT+VP-16 treated group,respectively,with a significant difference among them (F=18.23,P ＜ 0.01),and the apoptotic rate was significantly raised in the HCPT + VP-16 treated group,HCPT treated group and VP-16 treated group compared with the blank control group (q'=15.67,16.32,26.88,all at P＜0.01).Compared with the blank control group,the grey scale values of cleaved caspase-3,bax,p-JNK in the cells of HCPT+VP-16 treated group,HCPT treated group and VP-16 treated group were significantly increased (all at P＜0.01),and those in the HCPT+VP-16 treated group significant ascent in comparison with the HCPT treated group and VP-16 treated group (all at P＜0.01).However,the changes of caspase-3,JNK and Akt expression were insignificant.The grey scale values of bcl-2 and p-Akt in the HTFs of the HCPT,VP-16 and HCPT+VP-16 treated groups were significantly lower than those of the blank control group,with a dominant reducing in the HCPT+VP-16 treated group (all at P＜0.01).Conclusions HCPT and VP-16 induce the apoptosis of HTFs in vitro at a dose-dependent manner.The combination of HCPT with VP-16 has a stronger synergistic efficacy.The up-regulation of p-JNK and bax as well as the down-regulation of p-Akt and bcl-2 in HTFs are involved in the coaction of HCPT and VP-16.