Human Tenon's Capsule Fibroblasts Research Articles

In vitro and in vivo studies on bioactive hydroxyapatite-coated magnesium for glaucoma drainage implant

Given the alarmingly increasing rates of glaucoma worldwide and the lack of satisfactory treatments, there is a dire need to explore more feasible treatment options. Magnesium (Mg) is an essential element in maintaining the functional and structural integrity of vital ocular tissues, but Mg and its alloys are rarely mentioned in ophthalmic applications. Our previous research found that hydroxyapatite-coated Mg (Mg@HA) shows the best biocompatibility and bioactivity, and exhibits the effect of inhibiting fibrosis after filtration surgery in the rabbit model, which is expected to be a promising material for glaucoma drainage device. In this study, we further demonstrated the anti-fibrosis effect of Mg@HA from the molecular signal level and the efficacy of implantation in the rabbit filtration surgery model. In vitro experiments showed the surface modification of Mg affects the adhesion behavior and the reorganization of cytoskeleton of Human Western blot analysis and immunofluorescence found that Mg@HA regulates the adhesion and motility of human Tenon's capsule fibroblasts mainly by down-regulating the phosphorylation of Smad2 and Smad3 in the canonical transforming growth factor-beta (TGF - β) signaling pathway. By observing and recording the condition of filtering blebs and intraocular pressure after surgery, the effectiveness of Mg@HA applied in the rabbit filtration surgery model was further evaluated. In conclusion, the application of hydroxyapatite-coated Mg in the eye has good biocompatibility and has the potential to resist postoperative glaucoma filtration fibrosis, which may be mediated by the regulation of the TGFβ/Smad signaling pathway.

Studying Monocyte-Myofibroblast Immunomodulation in a 3D Co-culture System.

Simple and reproducible 3D cell culture systems that mimic biological interactions within physiological tissues (biomimetics) can provide unique insight for scientific inquiries compared to 2D cell cultures. Fibroblast-populated collagen lattices (FPCLs) are commonly used for mimicking physiological collagen matrices, potentiating biomechanical stresses on embedded fibroblasts. Here, we describe a novel 3D co-culture model that incorporates human Tenon's capsule fibroblasts embedded in FPCLs co-cultured with THP-1 monocytes suspended in culture media. This method can be used for the assessment of cell-cell interactions in various stages of the wound healing process and can facilitate various types of immune cells in co-culture. This system can also be used to study pharmacological agents that may eventually improve clinical outcomes in patients affected by inflammatory disorders.

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

This study aimed to explore the role of atorvastatin (ATO) in the prevention and treatment of the scarring of filtration channels after glaucoma surgery. 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 24h, 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-β1 (TGF-β1) and TGF-β2 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. The results revealed that ATO could inhibit the proliferation and migration of HTFs. Based on the TUNEL assay, 100μM and 150μM ATO could induce cell apoptosis. The ELISA results indicated that ATO could down-regulate the expression level of TGF-β2, and western blot analysis revealed that the protein expression levels of SMA, p38, Smad3, fibronectin, collagen I and collagen III in the TGF-β2 group were all up-regulated compared with the control group, whereas the addition of ATO could reverse this up-regulation. 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-β. It is suggested that ATO could be a basis for the treatment of the scarring of filtration channels after glaucoma surgery.

ALK5 Inhibition of Subconjunctival Scarring From Glaucoma Surgery: Effects of SB-431542 Compared to Mitomycin C in Human Tenon's Capsule Fibroblasts.

The gold standard for managing postoperative ocular fibrosis in glaucoma surgery is the chemotherapeutic mitomycin C (MMC) despite its association with significant adverse effects. This study compares in vitro the antifibrotic efficacy and cytotoxicity of the small-molecule TGFβ1 inhibitor SB-431542 (SB) to MMC. To measure collagen contraction, human Tenon's capsule fibroblasts (HTCFs) embedded in a three-dimensional collagen lattice were exposed to 0.2 mg/mL MMC or 20 µM SB followed by incubation with 2 ng/mL TGFβ1. Total protein extracted from experimentally treated HTCFs underwent immunoblotting for α-smooth muscle actin (α-SMA), matrix metallopeptidase 9 (MMP-9), and EDA splice-variant fibronectin (EDA-FN) expression. Cytotoxicity and cell metabolism were assessed using LIVE/DEAD staining, lactate dehydrogenase (LDH) assay, and methylthiazole tetrazolium (MTT) assay. Collagen lattice contraction in TGFβ1-induced HTCFs was significantly lowered by SB and MMC. Pretreatment with SB and MMC significantly lowered protein expression of α-SMA, MMP-9, and EDA-FN in HTCFs relative to TGFβ1 alone. HTCF viability in collagen lattices was significantly reduced with MMC pretreatment but not SB pretreatment. MMC-pretreated HTCFs had a significant increase in LDH release after 3 hours and a decrease in MTT activity after 20 minutes, while SB-pretreated HTCFs showed no significant changes via MTT or LDH assay during the same treatment period. SB shows comparable efficacy to MMC in reducing expression of fibrosis-promoting proteins in HTCFs and in vitro scarring activity. SB distinguishes itself from MMC by exhibiting less cytotoxicity in both two-dimensional and three-dimensional in vitro assays. This study demonstrates in vitro the potential of SB as a safer alternative ocular antifibrotic agent.

Liensinine, an Isoquinoline-Type Alkaloid in Lotus Seeds, Suppressed TGF-beta1-Induced Proliferation and Migration in Human Tenon Capsule Fibroblast Cells Through MAP3K7 Gene

Liensinine, an Isoquinoline-Type Alkaloid in Lotus Seeds, Suppressed TGF-beta1-Induced Proliferation and Migration in Human Tenon Capsule Fibroblast Cells Through MAP3K7 Gene

TGF-β1 Induces Interlukin-11 Expression and Pro-Fibrotic Effect by DNA Demethylation in Subconjunctival Fibroblasts.

Objective To assess Interlukin-11 (IL11) expression in the tears of patients after filtration surgery and to find out its pro-transdifferentiational and pro-fibrotic functions and mechanisms on subconjunctival human Tenon's capsule fibroblasts (HTFs) induced by transforming growth factor beta1 (TGF-β1). Methods Tears were collected from glaucoma patients with or without filtration surgery. The expression of IL11 in tears was examined by enzyme-linked immunosorbent assay. Primary HTFs were prepared as an expansion culture of human Tenon's explants from patients undergoing cataract surgery. TGF-β1 and IL11 were used to stimulate the cultured HTFs. Quantitative RT-PCR and western blot analyzed the roles of TGF-β1 in IL11 and DNA methyltransferase (DNMT) expression and the effects of IL11 on collagen-1A1 and α-smooth muscle actin expression. The effects of IL11 on human HTFs' migration were tested via the scratch-wound assay. MassARRAY platform of Sequenom was applied for analyzing the quantitative methylation of the IL11 promoter region. Result Our data presented significantly high levels of IL11 in the tears of patients who underwent filtration surgery with uncontrolled intraocular pressure (IOP) compared with those who underwent filtration surgery with controlled IOP. The up-regulation of IL11 was related to TGF-β1. We also found that TGF-β induced IL11 up-regulation in the HTFs, which activates the HTFs and enhanced the translation of the pro-fibrotic protein expression. This is correlated with inhibiting the activity and expression of DNMTs and demethylating the IL11 promoter. Therefore, IL11 may be an ideal target to be regulated to control the filtering pathway scar formation.

LINC01605 knockdown induces apoptosis in human Tenon's capsule fibroblasts by inhibiting autophagy.

Glaucoma is an irreversible disease that causes blindness. Formation of a hypertrophic scar (HS) is the main cause of failure of glaucoma surgery. The long non-coding RNA LINC01605 is closely associated with the formation of HS; however, the function of LINC01605 in the formation and development of HS remains unclear. For this study, firstly, human Tenon's capsule fibroblasts (HTFs) and corneal epithelial cells (control cells) were collected from patients (n=5) with POAG who underwent glaucoma filtration surgery at Fuyang People's Hospital. Immunofluorescence analysis was performed to detect the expression levels of vimentin (one of the main components of medium fiber and plays an important role in the cytoskeleton and motility), keratin (the main component of cytoskeletal proteins) and LC3 (an autophagy marker). In addition, reverse transcription-quantitative PCR analysis was performed to detect LINC01605 expression. Besides, the Cell Counting Kit-8 assay was performed to assess the viability of human Tenon's capsule fibroblasts (HTFs). Next, flow cytometry was performed to detect HTF apoptosis. Furthermore, western blot analysis was performed for Bax, Bcl-2, Pro-caspase-3, cleaved caspase-3, phosphorylated (p-)Smad2, Smad2, α-SMA, MMP9, ATG7, p62, beclin 1, p-AMPK and AMPK in HTFs to determine the mechanism by which LINC01605 regulates the formation and development of HS. Moreover, a Transwell assay was performed to detect the migratory ability of HTFs. The results demonstrated that LINC01605 was significantly upregulated in HS tissues compared with that in normal (control/healthy) tissues. In addition, vimentin was highly expressed in HTFs, whereas keratin was expressed at a low level. Also, in HTFs, LINC01605 knockdown inhibited cell viability by inducing apoptosis, decreasing Smad2 activation and inhibiting autophagy. Furthermore, LINC01605 knockdown significantly inhibited the migratory ability of HTFs. Transfection with LINC01605 small interference RNAs significantly downregulated the expression levels of p-Smad2, α-SMA and MMP9 in HTFs. Furthermore, LINC01605 knockdown notably inhibited the viability and migration, and induced the apoptosis of HTFs, the effects of which were reversed following treatment with TGF-β. Taken together, the results of the present study suggested that LINC01605 knockdown may inhibit the viability of HTFs by inducing the apoptotic pathway. These findings may provide novel directions for the treatment of HS.

Collecting and deactivating TGF-β1 hydrogel for anti-scarring therapy in post-glaucoma filtration surgery.

Scar formation can lead to glaucoma filtration surgery (GFS) failure, wherein transforming growth factor (TGF)-β is the core regulator. To reducing scar formation, this paper presents our study on the design of hydrogels to deactivate TGF-β1. We hypothesized that excess TGF-β1 can be removed from aqueous humor through the addition of oxidized hyaluronic acid (O-HA) hydrogels that are seeded with decorin (O-HA ​+ ​D). Immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) were performed to demonstrate the adsorption properties of O-HA ​+ ​D hydrogel, thus reducing the TGF-β1 concentration in aqueous humor. In the light that collagen contraction in human Tenon's capsule fibroblasts (HTFs) and the angiogenesis of human umbilical vein endothelial cells (HUVECs) can be activated by TGF-β1 and β2, we performed the quantitative analysis of polymerase chain reaction to determine the effect of O-HA ​+ ​D on the type I collagen, fibronectin, and angiogenesis. Our results illustrate that O-HA ​+ ​D can inhibit the increase of α-SMA expression in HTF induced by TGF-β1 and that O-HA ​+ ​D can inhibit the production of collagen I and fibronectin in HTF treated with TGF-β1. Furthermore, we performed in vivo studies by employing a rabbit model, where rabbits were treated with hydrogels post GFS. Our results demonstrate that, as compared with other groups, the rabbits treated with O-HA ​+ ​D had the greatest reduction in inflammatory cells with reduced level of collagen in wounds. Taken together, the present study paves the way toward the treatment of post-glaucoma fibrosis following surgery.

Involvement of METTL3/m6Adenosine and TGFβ/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-β1 (TGF-β1) enhanced cell viability and promoted cell proliferation and extracellular matrix (ECM) deposition in HTFs. We subsequently found that TGF-β1 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-β1. We then illustrated that increased METTL3 played a role by promoting Smad3 in TGF-β1-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-β1-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-β2)-induced human Tenon's capsule fibroblasts (HTFs). AGTR1 blocker treatment suppressed TGF-β2-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-β2-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.

Long Noncoding RNA LINC01518 Modulates Proliferation and Migration in TGF-β1-Treated Human Tenon Capsule Fibroblast Cells Through the Regulation of hsa-miR-216b-5p.

In this study, we investigated the expression and functions of long noncoding RNAs (LncRNAs) of LINC01518 in an in vitro model of TGF-β1-treated human Tenon capsule fibroblast (HTF) cells. qRT-PCR was used to examine LINC01518 expression in in situ human glaucoma tissues, and in vitro HTF cells treated with TGF-β1. Lentivirus-mediated LINC01518 knockdown was performed in HTF cells to investigate its effect on TGF-β1-induced cell proliferation, migration and autophagy signaling pathway. The potential ceRNA candidate of LINC01518, hsa-miR-216b-5p, was probed by dual-luciferase assay and qRT-PCR. Hsa-miR-216b-5p was also knocked down in LINC01518-downregulated HTF cells to investigate the function of this lncRNA-miRNA epigenetic axis in TGF-β1-treated HTF cells. LINC01518 was upregulated in human glaucoma tissues and cultured HTF cells. LINC01518 downregulation significantly suppressed TGF-β1-induced cell proliferation, migration and autophagy signaling pathway in HTF cells. Hsa-miR-216b-5p was confirmed to be a ceRNA target of LINC01518. Knocking down hsa-miR-216b-5p reversed the suppressing effects of LINC01518 downregulation in TGF-β1-treated HTF cells. Our study demonstrated that LINC01518 is a functional factor in regulating proliferation and migration in TGF-β1-treated HTF cells, and hsa-miR-216b -5p may also be involved. Targeting the epigenetic axis of LINC01518/hsa-miR-216b-5p may provide new insight into the pathological development of human glaucoma.

Dihydroartemisinin Inhibits TGF-β-Induced Fibrosis in Human Tenon Fibroblasts via Inducing Autophagy.

BackgroundThe formation of hypertrophic scars (HS) can result in the failure of glaucoma surgery, and fibrosis is known to be closely associated with the progression of HS. Dihydroartemisinin (DHA) has been reported to inhibit the progression of fibrosis; however, whether DHA can alleviate the formation of HS remains unclear.MethodsIn the present study, in order to examine the effects of DHA on the progression of HS, human Tenon’s capsule fibroblasts (HTFs) were isolated from patients who underwent glaucoma surgery. In addition, Western blot analysis, microtubule associated protein 1 light chain 3 α staining and reverse transcription-quantitative PCR were performed to detect protein and mRNA expression levels in the HTFs, respectively. Cell proliferation was detected by Ki67 staining. Flow cytometry was used to examine apoptosis and reactive oxygen species (ROS) levels in the HTFs.ResultsThe results revealed that TGF-β promoted the proliferation and fibrosis of HTFs; however, DHA significantly reversed the effects of TGF-β by increasing cell autophagy. In addition, DHA notably induced the apoptosis of TGF-β-stimulated HTFs by increasing the ROS levels, while these increases were partially reversed by 3-methyladenine. Furthermore, DHA notably increased the expression of microRNA (miR)-145-5p in HTFs in a dose-dependent manner.ConclusionThe present study demonstrated that DHA inhibits the TGF-β-induced fibrosis of HTFs by inducing autophagy. These findings may aid in the development of novel agents for the prevention of the formation of HS following glaucoma surgery.

Bio-modulation of scaring Glaucoma Filtration Surgery using a novel application of coated magnesium

Filtration surgery still plays a mainstream role of treatment for glaucoma. Postoperative scarring is the main cause of surgical failure. This study evaluated the biocompatibility and anti-proliferative properties of pure magnesium with three different coatings, which are hydroxyapatite (HA), dicalcium phosphate dihydrate (DCPD) and DCPD + stearic acid (SA), in a primary culture of human tenon's capsule fibroblasts (HTCFs) and in rabbit Glaucoma Filtration Surgery. Titanium and glass were used as controls in vitro , and trabeculectomy was used as control in vivo . The results show the number and shape of HTCFs seeded on different coatings showed less quantity and poor cell morphology. Each type of coated magnesium demonstrated significantly decreased metabolic activity of HTCFs. DCPD + SA showed higher cytotoxicity than the other coatings. Significant inhibition of proliferation was observed with the DCPD + SA coating. The expression of α-SMA was decreased in the cells when seeded on all of the coated magnesium disks. In vivo , no obvious adverse effects were observed after operation. No significant difference existed for any of the different samples regarding different ion concentrations in the aqueous humor. The inflammatory response in the titanium, DCPD and DCPD + SA treated eyes was more intense than in the trabeculectomy alone and HA groups. Western Blot analysis showed that collagen-1 and α-SMA expression was significantly lower in the titanium, HA, DCPD and DCPD+SA groups compared with the control. Different coatings on magnesium were able to affect the corrosive properties, which in turn, influenced the morphology and function of HTCFs. HA coated magnesium may be considered a very promising biodegradable material for the next generation of glaucoma drainage devices.

Isoliquiritigenin protects against angiotensin II-induced fibrogenesis by inhibiting NF-κB/PPARγ inflammatory pathway in human Tenon's capsule fibroblasts

PurposeTo examine the protective effects of Isoliquiritigenin (ISL) in angiotensin II (ANG II)-induced inflammation and fibrosis on Human Tenon's capsule Fibroblasts (HTFs) and Mouse Peritoneal Macrophages (MPMs). This study also investigated the potential mechanism of action of ISL. MethodMethyl-thiazolyl tetrazolium (MTT) assay was used to test ISL toxicity. An ELISA and an RT-qPCR assay detected the inflammatory cytokines (TNF-α, IL-6, COX-2, and ICAM-1). A Western blot investigated the expression levels of inflammation-related signals [nuclear factor-κB (NF-κB), peroxisome proliferator-activated receptor γ (PPARγ)], and fibrogenesis, including fibronectin and alpha-smooth muscle actin (α-SMA)]. Protein expressions of α-SMA were measured by immunofluorescence. ResultsPre-treatment with ISL (10 or 20 μM) dose-dependently decreased the mRNA levels of TNF-α, IL-6, ICAM-1, and COX-2 induced by ANG II (1 μg/ml) in both MPMs and HTFs. ANG II remarkably increased the amount of P65 in the nuclei and decreased the amount of P65 in the cytoplasm. Additionally, ANG II reduced PPARγ expression levels in a time-dependent manner. Furthermore, these effects which were induced by ISL were remarkably neutralized by ISL pre-treatment. Finally, ANG II markedly elevated the expression of fibronectin and α-SMA. ConclusionISL could alleviate ANG II-induced fibrogenesis by inhibiting the NF-κB/PPARγ inflammatory pathway. In addition, ISL may be a potential agent for the treatment of conjunctival fibrosis. Most importantly, the NF-κB/PPARγ signaling pathway could be an effective therapeutic target for the prevention and treatment of conjunctival fibrosis after glaucoma surgery.

<p>The Pro-Fibrotic Behavior of Human Tenon’s Capsule Fibroblasts in Medically Treated Glaucoma Patients</p>

PurposeThe 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 MethodsBioartificial 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.ResultsPro-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.ConclusionHTCFs 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.

LINC00028 regulates the development of TGFβ1-treated human tenon capsule fibroblasts by targeting miR-204-5p

Glaucoma is a leading cause of blindness worldwide with complex pathogenesis. The excessive proliferation and fibrosis of human tenon capsule fibroblasts (HTFs) trigger the scar formation after glaucoma filtration surgery. The purpose was to investigate the role of long intergenic non-protein coding RNA 28 (LINC00028) and mechanism in transforming growth factor β1 (TGFβ1)-treated HTFs. The detection of LINC00028 and miR-204-5p expression was conducted using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed by cell counting kit-8 (CCK-8) assay. Cell migration and invasion were monitored by transwell assay. The expression of Epithelial-mesenchymal transition (EMT)-related markers, including E-cadherin, α-Smooth muscle actin (α-SMA), fibronectin and β-catenin, and autophagy-related markers, including Beclin 1 and light chain 3 (LC3-II and LC3-I) at the protein level was quantified using western blot. The prediction of the relationship between LINC00028 and miR-204-5p was performed by the online tool miRcode, and the verification of the relationship between them was conducted using dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay. The expression of LINC00028 was elevated in glaucoma tissues and TGFβ1-treated HTFs. LINC00028 downregulation blocked proliferation, migration, invasion, EMT, fibrosis and autophagy of TGFβ1-treated HTFs. MiR-204-5p was a target of LINC00028, and its reintroduction exerted a similar role of LINC00028 downregulation. The inhibition of miR-204-5p reversed the effects of LINC00028 downregulation in TGFβ1-treated HTFs. LINC00028 regulated proliferation, migration, invasion, EMT, fibrosis and autophagy to induce the development of HTFs by competitively targeting miR-204-5p, and LINC00028 was regarded as a promising biomarker for glaucoma filtration treatment.

Role of the long noncoding RNA H19 in TGF-β1-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-β promoted the viability, proliferation and extracellular matrix (ECM) deposition of HTFs. Then, we showed that TGF-β promoted the expression of H19 in HTFs and that suppression of H19 inhibited the effect of TGF-β on HTFs. Furthermore, we revealed that H19 exerted its effects by interacting with miR-200a in TGF-β-treated HTFs. Additionally, we showed that β-catenin was a target of miR-200a in TGF-β-treated HTFs. We also demonstrated that H19 acted by modulating the H19/miR-200a/β-catenin regulatory axis in TGF-β-treated HTFs. Ultimately, we found that the components of the H19/miR-200a/β-catenin regulatory axis were aberrantly expressed in a rat model of GFS. Our results show that H19 indeed acts by modulating β-catenin expression via miR-200a in TGF-β-treated HTFs, thus providing a novel rationale for the development of H19-based strategies to attenuate scar formation after GFS.

Differential effects of dexamethasone and indomethacin on Tenon's capsule fibroblasts: Implications for glaucoma surgery

Dysregulated wound healing and subsequent fibrosis represents the most common cause of failure in glaucoma filtration surgery. Primary means to prevent this outcome are the anti-metabolite surgical adjuvants, however, topical corticosteroids are commonly used postoperatively to permit further control of wound healing and development of the filtration bleb. Unfortunately, they carry important side effects such as raised intraocular pressure, cataract and increased infection risk. Non-steroidal anti-inflammatory drugs (NSAIDs) show promising results in clinical trials as an alternative wound modulatory drug. NSAIDs exhibit non-inferiority to steroids in terms of post-operative intraocular pressure control and secondary IOP lowering interventions, however there is little known about the differing effects these drugs exert on human Tenon's capsule fibroblast (HTCF) mediated wound healing. The purpose of this study was to assess the individual effects of dexamethasone and indomethacin on the extracellular matrix modifying actions of HTCFs in vitro. To this end, HTCFs were cultured in 3D collagen matrices as well as in 2D monolayers and exposed to clinically relevant concentrations of dexamethasone or indomethacin for up to seven days. HTCF-mediated wound healing functions were assayed through collagen matrix contraction, extracellular matrix morphology, estimation of HCTF proliferation and differentiation into myofibroblasts within the collagen matrices, as well as western blot. Both drugs significantly reduced HTCF-mediated collagen contraction relative to control however there was a significant trend towards greater inhibition with indomethacin exposure compared to dexamethasone. Indomethacin exposure significantly reduced HTCF-mediated collagen remodelling activity compared vehicle control, whereas dexamethasone was unable to reduce remodelling activity at any of the studied exposures. Both drugs reduced myofibroblast differentiation, however indomethacin alone demonstrated an inhibitory effect on final cell number relative to control whereas dexamethasone had no significant effect at any studied exposure. These findings demonstrate that both steroidal and NSAID treatment can mitigate HTCF-mediated collagen contraction and αSMA expression. However, NSAIDs may function to better impede HTCF proliferation and remodelling activity. Taken in the context of previous glaucoma surgical trials, NSAIDs appear to be a viable alternative to steroids for post-operative wound modulation.

The Smad2/3/4 complex binds miR-139 promoter to modulate TGFβ-induced proliferation and activation of human Tenon's capsule fibroblasts through the Wnt pathway.

The activation and proliferation of human Tenon's fibroblasts (HTFs) play a vital role in the fibrosis in the pathology of the scar formation after the glaucoma filtration surgery. Transforming growth factor β1 (TGFβ1)/Smads signaling has been reported to promote fibrosis. In our previous study, we revealed that TGFβ1-induced orbital fibroblast activation and proliferation through Wnt/β-catenin signaling. As microRNA (miR)-139 could target several factors in Wnt signaling to modulate fibrosis, here, the effect and mechanism of miR-139 in HTF activation and proliferation were investigated. miR-139 overexpression significantly reversed the TGFβ1-induced increase in collagen I and α-smooth muscle actin contents and proliferation in HTFs. CTNNB1 and CTNND1 were direct downstream of miR-139 and can significantly restore the suppressive effect of miR-139 on the activation and proliferation in HTFs under TGFβ1 stimulation. Smad2/3/4 complex inhibits the transcription activity of miR-139, most possibly by Smad4 binding to the miR-139 promoter. Taken together, we demonstrated a new mechanism of HTF activation and proliferation from the perspective of miRNA regulation, which may provide new strategies for improving the fibrosis after the glaucoma filtration surgery.

Inhibition of hepatocyte growth factor on proliferation and transdifferentiation of human Tenon capsule fibroblasts triggered by transforming growth factor-β1in vitro

Objective To investigate the effects of hepatocyte growth factor (HGF) on proliferation and transdifferentiation of human Tenon capsule fibroblasts induced by transforming growth factor -β1 (TGF-β1) in vitro. Methods Human Tenon capsule fibroblasts were cultured and divided into blank control group, TGF-β1 treated group and different concentrations HGF+ TGF-β1 groups.The TGF-β1 at 10 μg/L was added into culture medium of the TGF-β1 treated group, and different concentrations of HGF (25, 50, 100, 200 μg/L) and 10 μg/L TGF-β1 were added into culture medium of the HGF25 μg/L+ TGF-β1, HGF50 μg/L+ TGF-β1, HGF100 μg/L+ TGF-β1, HGF200 μg/L+ TGF-β1 group respectively, Methyl thiazolyl tetrazolium (MTT) was employed to measure the cell proliferation (absorbance at 560 nm). Immunofluorescence staining was used to evaluate and locate the expression of α-smooth muscle action (α-SMA) in the cells.The expression of α-SMA protein in the cells was detected by Western blot assay. Results Cultured cells showed fusiform in shape with the positive response for vimentin.The proliferation value of the cells was 0.203±0.025, 0.497±0.101, 0.426±0.062, 0.354±0.040, 0.272±0.084, 0.241±0.011 in the blank control group, TGF-β1 treated group, HGF25 μg/L+ TGF-β1 group, HGF50 μg/L+ TGF-β1 group, HGF100 μg/L+ TGF-β1 group and HGF200 μg/L+ TGF-β1 group, respectively, showing a significant difference among the groups (F=9.210, P=0.003). Compared with the TGF-β1 treated group, the proliferation values of the cells were significantly reduced in the blank control group and HGF+ TGF-β1 group (all at P<0.05). Immunofluorescence staining showed that α-SMA protein mainly located in cytoplasm with the strong red fluorescence in the cells of the TGF-β1 treated group and weak red fluorescence in HGF+ TGF-β1 group, and the expression of α-SMA was absent in the blank control group.The percentage of α-SMA-positive cells was (60.0±4.7)% in the TGF-β1 treated group and (14.3±3.1)% in the HGF+ TGF-β1 group, with significant difference between the two groups (t=19.856, P<0.001). The relative expression levels of the α-SMA protein in the cells were 0.642±0.032, 1.330±0.069 and 0.884±0.040 in the blank control group, TGF-β1 group and HGF100 μg/L+ TGF-β1 group, respectively, showing a significant difference among the groups (F=13.370, P<0.001), and relative expression levels of the α-SMA protein in the cells were significantly lower in the blank control group and HGF100 μg/L+ TGF-β1 group than that in the TGF-β1 treated group (all at P<0.05). Conclusions HGF can inhibit the proliferation of human Tenon capsule fibroblasts, down-regulate the expression of α-SMA protein induced by TGF-β1 and arrest the phenotype transformation of fibroblasts in vitro. Key words: Tenon capsule/cytology; Fibroblasts/metabolism; Hepatocyte growth factor/administration & dosage; Transforming growth factor beta type 1; Fibrosis/prevention & control; α-Smooth muscular actin; Humans; Cells, cultured