Tenon's Fibroblasts Research Articles

Anti-glaucoma medications lowered decorin and altered profibrotic proteins in human tenon's fibroblasts

Long term exposure to anti-glaucoma medications (AGMs) leads to an increase in extracellular matrix (ECM) accumulation in primary glaucoma patients. This study aims to evaluate the effect of topical AGMs in primary human tenon's fibroblasts (HTFs) and analyze the expression of profibrotic and anti-fibrotic proteins. Primary HTFs were cultured from patients undergoing cataract (control) and trabeculectomy. The different types of AGMs in single/multiple combinations (BB, PG, AA, CAI, CH, combinations of 3- PG + AA + CAI, 4A- BB + PG + AA + CAI, 4B– BB + PG + CAI + CH and 5- BB + PG + AA + CAI + CH) on chronic exposure were tested for cell viability using MTT assay and morphological alterations. Profibrotic proteins mainly SPARC, LOXL2, COL1A1 and anti-fibrotic DCN were analyzed in treated HTFs using q-PCR and ELISA. Sirius red staining and collagen gel contraction (CGC) assay were performed to assess collagen synthesis and the contractility of HTFs, respectively. Except for AA and CH, the other AGMs at a higher concentration were found to decrease the cell viability of HTFs. The morphology of HTFs were altered on exposure to BB, CH and AA; Profibrotic proteins i.e., SPARC, LOXL2 and COL1A1 were significantly increased (p < 0.05) on exposure to a combination of AGMs with TGF-β1, whereas the anti-fibrotic DCN expression was significantly lowered (p < 0.05) in single/multiple AGM exposure. Sirius red staining showed increased collagen synthesis with combinations of AGMs with TGF-β1. Meanwhile, HTFs showed increased collagen gel contraction with TGF-β1, CAI and CH. This study reveals that altered profibrotic proteins, with significantly lowered DCN on chronic exposure of AGMs in HTFs.

Elucidating the Therapeutic Potential of Cell-Penetrating Peptides in Human Tenon Fibroblast Cells.

Cell-penetrating peptides (CPPs) have been widely used as vehicles for delivering therapeutic molecules to the site of action. Apart from their delivering potential, the biological effects of CPPs have not been explored in detail. JTS-1 is a CPP that has been reported to have gene delivery functions, although its biological role is yet to be determined. Hence, in this study, we revealed the biological mechanism such as its uptake mechanism and immunogenic potential and function using primary human tenon fibroblast (TF) cells collected from patients undergoing glaucoma trabeculectomy surgery. Our results showed that the JTS-1 peptide has an α-helical structure and is nontoxic up to 1 μM concentration. It was found to be colocalized with early endosome (Rab5), recycling endosome (Rab7), and Rab11 and interacted with major histocompatibility complex (MHC) class I and II. The peptide also affected actin polymerization, which is regulated by cofilin phosphorylation and ROCK1 localization. It also inhibited TF cell proliferation. Therefore, the JTS-1 peptide could be used as a possible therapeutic agent for modifying the fibrosis process, where TF proliferation is a key cause of surgery failure.

Understanding the cellular response of human tenon fibroblast on polycaprolactone-Aloe vera blend fiber.

The signalling response is determined by the cell's reaction to different biochemical and biophysical inputs such as stiffness, topological, and structural alignment. The surface patterns at the nano-scale can be an influential factor in cell signalling behaviour. It is important to understand the cellular response to the biophysical cues for biomedical applications. Biomaterials have an important role in regenerative tissue engineering. In this study, we have fabricated electrospun polycaprolactone (PCL) and PCL-Aloe vera (PCL-AV) nanofibrous matrix and studied its effect on the human tenon fibroblast (HTF) cellular and morphological changes. The electrospun fibers were characterized using Scanning Electron Microscope (SEM), Fourier Transform Infrared spectroscopy (FTIR), Atomic Force Microscopy (AFM) and Brunaur, Emette and Teller (BET) analysis for their morphology, composition, topography, surface area and porosity. The results revealed fiber size, roughness and porosity has been altered by addition of AV. The HTF cell viability, proliferation and expression of focal adhesion proteins, such as FAK, Ezrin, Vasp and Cofilin on the PCL-AV fiber matrix were examined. The results showed a change in cellular morphology and a significant change in the cofilin phosphorylation on PCL-AV nanofiber. The influence of Aloe vera composition on the nano-dimension of the PCL has made a significant impact on the cellular morphology at both gene and protein levels. This observation suggests that AV composition in the nanofiber can significantly influence the HTF cellular adhesions.

The Effect of Irradiated Riboflavin in Human Tenon’s Fibroblast – A Study on Cellular Viability

ABSTRACT Purpose/ Aim The main purpose of this work is to study the cellular viability effect of irradiated riboflavin in cultured human tenon fibroblasts. Materials and Methods The tenon tissue was harvested from a patient undergoing strabismus surgery. The human tenon fibroblast cell culture and isolation were performed according to the standard laboratory cell culturing protocol. The cells were divided into three groups: control, treatment with irradiated and non-irradiated riboflavin. There were five different concentrations (0.00156%, 0.003125%, 0.00625%, 0.0125%, 0.025%) in each group of riboflavin. The fibroblasts were treated with riboflavin and the cellular viability was assessed at 24-hour and 48-hour post treatment with MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide colorimetric assay. The absorbance values were analysed using Magellan microplate reader data analysis. A triplicate of readings was taken. The data were presented as mean ± standard deviation of the triplicates. Statistical analysis was performed with Statistical Package for Social Sciences (SPSS) analysis version 23. Results Irradiated riboflavin caused a concentration-dependent cell death in human tenon fibroblast cell culture (p < .05). The antiproliferative difference between irradiated and non-irradiated riboflavin was significant up to 48 hours (p < .05). Post hoc multiple comparisons showed higher concentrations of irradiated riboflavin (0.0125% and 0.025%) caused more reduction in cellular viability in human tenon fibroblast cells (p < .05). The duration of treatment is not a causative factor in this study. Conclusions This pilot experiment demonstrated that irradiated riboflavin induced cell death in human tenon fibroblast culture in a concentration-dependent manner, but is not time-dependent. Further exploratory investigations should be performed to determine the mechanism of cell death. We postulate that apoptosis occurred in these irradiated riboflavin-treated cells.

The Effect of CHIR 99021, a Glycogen Synthase Kinase-3β Inhibitor, on Transforming Growth Factor β-Induced Tenon Fibrosis.

PurposeThis study investigated the effect of glycogen synthase kinase-3β (GSK-3β) inhibition on the fibrosis of human Tenon's fibroblasts (HTFs) induced by transforming growth factor-β (TGF-β).MethodsQuantitative real-time PCR and Western blot analyses were performed to determine the expression levels of molecules associated with the fibrosis of HTFs by TGF-β (fibronectin, collagen Iα, and α-smooth muscle actin) and GSK-3β. The levels of phosphorylated Smad2 and Smad3 were also analyzed in the presence of the GSK-3β inhibitor CHIR 99021. The wound healing assay was performed to determine the effect of CHIR 99021 on the migration of HTFs. All experiments were conducted using primary cultured HTFs or human tenon tissues obtained from normal subjects and patients with glaucoma.ResultsTreatment with TGF-β resulted in an increase in the levels of molecules associated with the fibrosis of HTFs. The expression levels of these molecules were higher in the tenon tissues obtained from patients with glaucoma than those from normal subjects. When the HTFs were treated with TGF-β, a significant increase in the active form of GSK-3β (Y216) was observed. A significant decrease in the active form of GSK-3β and molecules associated with fibrosis by TGF-β was noted in HTFs treated with CHIR 99021. CHIR 99021 treatment reduced the phosphorylated Smad2/Smad2 and phosphorylated Smad3/Smad3 ratios in HTFs and attenuated HTF migration.ConclusionsOur results demonstrated the effect of GSK-3β inhibition on the regulation of TGF-β–mediated fibrosis of HTFs, suggesting GSK-3β to be a potential target for maintaining bleb function after glaucoma filtration surgery.

Pirfenidone inhibits proliferation of rabbit tenon fibroblasts by down-regulating TGF-β3 in the TGF-β/Smad pathway

To investigate the molecular mechanism by which pirfenidone inhibits scar formation through the TGF-β/Smad pathway. Cultured rabbit tenon fibroblasts (RTFs) were treated with different concentrations of pirfenidone to determine its initial active concentration and optimum concentration of pirfenidone for inhibiting RTF proliferation using CCK-8 assay. In RTFs treated with pirfenidone at the initial and optimal concentrations, expressions of TGF-β3, collagen I and collagen III were examined with both immunofluorescence assay and Western blotting, and their mRNA expression levels were detected using RT-PCR. The initial and optimal concentrations of pirfenidone for inhibiting RTF proliferation were 0.1 mg/mL and 0.27 mg/mL, respectively. In RTFs treated with pirfenidone at the two concentrations for 24 h, both immunofluorescence assay and Western blotting showed significantly lowered protein expressions of TGF-β3, collagen I and collagen III as compared with those in the control group (P < 0.05). The mRNA expressions of TGF-β3, collagen I and collagen III in the RTFs were also significantly lowered after treatment with pirfenidone at the initial and optimal concentrations (P < 0.05). Pirfenidone concentration-dependently inhibits the proliferation of RTFs possibly by down-regulating the expression of TGF-β3 in the TGF-β/Smad pathway.

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.

Hydrophilic Bile Acid, Ursodeoxycholic Acid Attenuates the Effect of TGF On Human Tenon Fibroblast

Excessive scarring of the filtering bleb was reported post-surgery and thus lead to failed trabeculectomy.

Benzalkonium chloride-induced myofibroblastic transdifferentiation of Tenon\u2019s capsule fibroblasts is inhibited by coculture with corneal epithelial cells or by interleukin-10

Benzalkonium chloride (BAC) is used as a preservative in eyedrops but induces subconjunctival fibrosis that can result in failure of glaucoma surgery. Tenon’s capsule fibroblasts in subconjunctival tissue interact with the corneal epithelium through tear fluid. With the use of a coculture system, we have now investigated the effect of human corneal epithelial (HCE) cells on myofibroblastic transdifferentiation of human Tenon fibroblasts (HTFs) induced by BAC (5 × 10−6%). Immunofluorescence and immunoblot analyses revealed that the BAC-induced expression of α smooth muscle actin (αSMA) in HTFs was suppressed by coculture of these cells with HCE cells (p < 0.01). The concentration of interleukin-10 (IL-10) in culture supernatants of BAC-treated HTFs was increased by coculture with HCE cells (17.26-fold, vs. coculure, p < 0.001). Immunofluorescence and immunoblot analyses also showed that exogenous IL-10 (300 pg/ml) suppressed the BAC-induced expression of αSMA by 43.65% (p < 0.05) as well as the nuclear translocation of myocardin-related transcription factor-A (MRTF-A) by 39.32% (p < 0.01) in HTFs cultured alone. Our findings suggest that corneal epithelial cells may protect against subconjunctival fibrosis by maintaining IL-10 levels and preventing the MRTF-A-dependent transdifferentiation of HTFs into myofibroblasts.

Forkhead domain inhibitory-6 attenuates subconjunctival fibrosis in rabbit model with trabeculectomy

Antiproliferative therapies are crucially important for improving the success rate of the glaucoma filtration surgeries. In this study, we investigated the potential efficacy of Forkhead Domain Inhibitory-6 (FDI-6) in inhibiting post-trabeculectomy subconjunctival fibrosis. In vitro, the effect of FDI-6 (10 μM) on fibrotic response and its underlying mechanism were investigated in rabbit tenon's fibroblasts (RTFs) treated with or without transforming growth factor-β1 (TGF-β1, 20 ng/mL). In vivo, FDI-6 (40 μM) was injected subconjunctivally to a rabbit trabeculectomy model. Intraocular pressure (IOP) changes were monitored within the 14-day period post-surgery. Bleb morphology and subepithelial fibrosis at the operating area were evaluated with slit lamp and confocal microscopic examinations and with histologic examinations. The results showed that, in cell culture studies, FDI-6 suppressed the proliferation, migration, collagen gel contraction and the expression levels of fibronectin (FN) and α-smooth muscle actin (α-SMA) in RTFs with TGF-β treatment by down-regulating the TGF-β1/Smad2/3 signaling pathway. In animal studies, the IOPs of the FDI-6-treated group were significantly lower than those of the saline-treated group after trabeculectomy. The FDI-6-treated eyes showed a better bleb appearance with fewer blood vessels compared to the saline-treated eyes. The analysis of confocal microscopy in vivo and histopathology revealed that subconjunctival fibrosis after trabeculectomy was significantly attenuated in the FDI-6-treated group compared to the controls. In conclusion, our studies indicate that FDI-6 exerts an inhibitory effect on subconjunctival fibrosis caused by trabeculectomy, holding potentials as a new antiproliferative agent used in anti-glaucoma filtration surgeries in the future.

Improved Treatment on Ocular Inflammation with Rationally Designed Thermoresponsive Nanocomposite Formulation

AbstractCataract is the principal cause of preventable blindness in older people globally. Intraocular surgeries stimulate multiple postoperative inflammatory responses. The current clinical treatment by dropping prednisolone acetate (PA) and levofloxacin (LF) suffers from drawbacks, such as low bioavailability and poor patient compliance. This study aims to develop a rationally designed thermoresponsive formulation combining PA and LF using nancomposites of mesoporous silica nanoparticle (MSN) and thermo‐nanogel (thermogel) (PA@MSN‐LF@Thermogel). The in vitro release profiles of PA and LF from PA@MSN‐LF@Thermogel are tailor‐made to adapt to wound healing by a sustained release of 28 days. PA@MSN‐LF@Thermogel exhibits little cytotoxicity to human Tenon's fibroblasts and human corneal epithelial cells, while presents excellent in vitro antibacterial effectiveness to Staphylococcus bacteria. After implanted into the rabbit eye by a subconjunctival injection, PA@MSN‐LF@Thermogel displays overwhelming anti‐inflammatory and antibacterial effectiveness up to 21 days using only one‐eighth of the total dose, compared with PA and LF eye drops. The formulation does not cause any ocular hypertension and noticeable histopathological abnormality which indicate good biosafety. This thermoresponsive nanocomposite formulation is thus a promising drug delivery alternative for treatment of postoperative inflammation after intraocular surgeries.

Effect of Vitamin D3 on Regulating Human Tenon's Fibroblasts Activity.

PurposeTo study the in vitro effect of vitamin D3 on the healing response of human Tenon's fibroblasts (HTF) and its possible role in preventing excessive postoperative subconjunctival fibrosis.MethodsEffect of vitamin D3 on cytotoxicity and cell survival of primary cultured HTF was measured by lactate dehydrogenase and PrestoBlue assays, respectively. Proliferation and migration of vitamin D3–treated HTF (D3-HTF) was determined by CyQUANT proliferation and scratch assay, respectively. The mRNA expression profiles of control-HTF and D3-HTF from six subjects (three with glaucoma and long-term use of topical medications, three with primary pterygium) were assessed by RNA sequencing analyses to identify potential biomarkers for the inhibitory effect on HTF by vitamin D3. Validation of these biomarkers and their potential pathways were performed by quantitative real-time polymerase chain reaction (qRT-PCR) detection.ResultsPure monolayers of HTF from controls (retinal detachment or squint surgeries), pterygium, and glaucoma subjects were successfully prepared and passaged. Proliferation and migration of pterygium and glaucoma HTF were inhibited by vitamin D3 in a dose-dependent manner, and without cytotoxicity or decrease in cellular viability with concentrations up to 10 µM. The qRT-PCR results were consistent with the transcriptome analyses, vitamin D3 appears to enhance CYP24A1, SHE, KRT16 but suppresses CILP expression in HTF.ConclusionsVitamin D3 can inhibit the in vitro activity of HTF without compromising cellular survivability at concentration up to 10 µM. This has potential clinical application for improving the outcome of pterygium and filtering surgeries.Translational RelevanceVitamin D3 can suppress the in vitro proliferation, migration, and transdifferentiation of human Tenon's fibroblasts, without the cytotoxicity of mitomycin-C, the current standard antifibrotic agent in clinical use.

Zinc oxide nanoparticle-triggered oxidative stress and autophagy activation in human tenon fibroblasts

Glaucoma is a leading cause of irreversible blindness worldwide due to elevated intraocular pressure, and filtering surgery can efficiently control intraocular pressure of glaucoma patients. However, failure of filtering surgery commonly results from scarring formation at the surgical site, in which fibroblast proliferation plays an essential role in the scarring process. Our previous study has demonstrated that zinc oxide (ZnO) nanoparticles could efficiently inhibit human tenon fibroblasts (HTFs) proliferation. The present study aimed to explore the underlying mechanism involved in oxidative stress and autophagy signaling in zinc oxide (ZnO) nanoparticles-induced inhibition of HTFs proliferation. In this study, we investigated the effect of ZnO nanoparticles on HTFs proliferation, mitochondrial function, ATP production and nuclear morphology. Moreover, we also explored the interactions between ZnO nanoparticles and HTFs, investigated the influence of ZnO nanoparticles on the autophagosome formation, the expression of autophagy-related 5 (Atg5), Atg12 and Becn1 (Beclin 1), and the level of light chain 3 (LC3). The results suggested that ZnO nanoparticles can efficiently inhibit HTFs proliferation, disrupt the mitochondrial function, attenuate the adenosine triphosphate (ATP) generation, and damage the nuclear morphology of HTFs. Exposure of HTFs to ZnO nanoparticles can also induce the shifted peak, elevate the expression of Atg5, Atg12 and Becn1, enhance the autophagosome formation, and promote the LC3 expression, and thus activate autophagy signaling. Overall, ZnO nanoparticles can apparently trigger oxidative stress and activate autophagy signaling in HTFs, and thus inhibit HTFs proliferation and mediate HTFs apoptosis.

Inhibiting corneal neovascularization by sustainably releasing anti-VEGF and anti-inflammation drugs from silica-thermogel nanohybrids

Corneal neovascularization (CNV) is one of the main factors that induce blindness worldwide. To effectively inhibit CNV, a novel nanohybrid has been developed by incorporating anti-VEGF bevacizumab (BEV)-loaded mesoporous silica nanoparticles (BEV@MSN) into the thermogel matrix with anti-inflammation cyclosporine A (CsA) (BEV@MSN-CsA@Thermogel). This nanohybrid regulates the in vitro release of both bevacizumab and cyclosporine A in a sustainable way for up to four weeks to enhance CNV inhibition through the synergistic anti-VEGF and anti-inflammation. The carrier materials (i.e. silica and thermogel) in this nanohybrid do not show any cytotoxicity to human Tenon's fibroblasts, corneal epithelial cells and corneal endothelial cells. BEV@MSN-CsA@Thermogel effectively prevents proliferation, migration, and tube-like structure formation of human umbilical vein endothelial cells. Moreover, subconjunctival injection of BEV@MSN-CsA@Thermogel significantly inhibits corneal neovascularization in terms of the CNV area, the new vessel length, the corneal opaque area, the corneal inflammation and abnormal fibrosis in a rabbit model. This nanohybrid is thus a promising alternative for effective CNV treatment.

Epigallocatechin-3-gallate increases autophagic activity attenuating TGF-β1-induced transformation of human Tenon's fibroblasts

We previously found that epigallocatechin-3-gallate (EGCG) could inhibit the myofibroblast transformation of human Tenon's fibroblasts, however, the underlying mechanism remained unclear. We therefore investigated whether the autophagic regulation involved in the anti-fibrotic function of EGCG. The fibroblasts were subjected to transforming growth factor beta-1 (TGF-β1) induction followed by EGCG treatments. The autophagic flux was examined by transmission electron microscopy and autophagic flux analysis. The levels of autophagy-related proteins (LC3β and p62) and alpha-smooth muscle actin (α-SMA) were measured by Western blot and immunofluorescence. Results showed that TGF-β1 partially inhibited the autophagic function of Tenon's fibroblasts. But this inhibition effect was rescued by LY2157299, a TGF-βR1 selective inhibitor. Compared with the cells treated with TGF-β1 alone, EGCG treatments increased the amount of autophagosomes and autolysosomes, evaluated the ratio of LC3-II to LC3-I and decreased p62 level. Our results indicated that EGCG could recover the activity of autophagy in the TGF-β1-treated cells. Moreover, treatments with EGCG significantly decreased the α-SMA expression. Taken together, these findings revealed that autophagic regulation involved in the action of EGCG against TGF-β1-induced transformation of Tenon's fibroblasts. Through increasing intracellular autophagy, EGCG could be a potential anti-fibrotic reagent for preventing subconjunctival fibrosis after glaucoma filtration surgery.

Positive-charge tuned gelatin hydrogel-siSPARC injectable for siRNA anti-scarring therapy in post glaucoma filtration surgery

Small interfering RNA (siRNA) therapy is a promising epigenetic silencing strategy. However, its widespread adoption has been severely impeded by its ineffective delivery into the cellular environment. Here, a biocompatible injectable gelatin-based hydrogel with positive-charge tuned surface charge is presented as an effective platform for siRNA protection and delivery. We demonstrate a two-step synthesis of a gelatin-tyramine (Gtn-Tyr) hydrogel with simultaneous charge tunability and crosslinking ability. We discuss how different physiochemical properties of the hydrogel interact with siSPARC (siRNA for secreted protein, acidic and rich in cysteine), and study the positive-charge tuned gelatin hydrogel as an effective delivery platform for siSPARC in anti-fibrotic treatment. Through in vitro studies using mouse tenon fibroblasts, the positive-charge tuned Gtn-Tyr hydrogel shows sustained siSPARC cellular internalization and effective SPARC silencing with excellent biocompatibility. Similarly, the same hydrogel platform delivering siSPARC in an in vivo assessment employing a rabbit model shows an effective reduction in subconjunctival scarring in post glaucoma filtration surgery, and is non-cytotoxic compared to a commonly used anti-scarring agent, mitomycin-C. Overall, the current siRNA delivery strategy involving the positive-charge tuned gelatin hydrogel shows effective delivery of gene silencing siSPARC for anti-fibrotic treatment. The current charge tunable hydrogel delivery system is simple to fabricate and highly scalable. We believe this delivery platform has strong translational potential for effective siRNA delivery and epigenetic silencing therapy.

Pathophysiological Role and Drug Modulation of Calcium Transport in Ocular Surface Cells.

The ocular surface structure and extraocular accessory organs constitute the ocular surface system, which includes the cornea, conjunctiva, eyelids, lacrimal organs, and lacrimal passages. This system is composed of, and stabilized by, the corneal epithelium, conjunctival cells, conjunctival goblet cells, lacrimal acinar cells and Tenon's fibroblasts, all of which maintain the healthy eyeball surface system. Ocular surface diseases are commonly referred to corneal and conjunctival disease and external ocular disease, resulting from damage to the ocular surface structure. A growing body of evidence has indicated that abnormal activation of the KCa3.1 channel and Ca2+/ calmodulin-dependent kinase initiates ocular injury. Signaling pathways downstream of the irregular Ca2+ influx induce cell progression and migration, and impair tight junctions, epithelial transport and secretory function. In this overview, we summarize the current knowledge regarding ocular surface disease in terms of physical and pathological alteration of the ocular system. We dissect in-depth, the mechanisms underlying disease progression, and we describe the current calcium transport therapeutics and the obstacles that remain to be solved. Finally, we summarize how to integrate the research results into clinical practice in the future.

Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon

Abstract Despite decades of research, fibrosis still remains a significant challenge for medicine in many different fields. Although there is a general model of fibrosis, the causes and characteristics of the various pathologies are as diverse as the variety of organs and tissues that can be affected by fibrosis. Moreover, fibrosis also impedes the long-term prospects of success in implantation surgery. One possibility to address this challenge is the development of biocompatible implants featuring drug delivery systems loaded with antifibrotic pharmaceuticals. Due to diverse regulatory mechanisms in organs, tissues and also cell types, these active substances must consequentially be designed for diverse specific applications. Compared to fibrosis in organs like lung or liver, these mechanisms were poorly addressed in ophthalmologic research, but it is known that transforming growth factor beta (TGF-β) plays a key role. This gene expression study revealed 30 genes being upregulated more than two fold in TGF-β1 treated human primary tenon fibroblasts (hTF). Furthermore, 15 genes were found to be downregulated more than two fold. Tumor necrosis factor (TNF), vascular endothelial growth factor A (VEGFA) and inhibin beta (INHBE) were particular strongly regulated in TGF-β1 treated hTFs.

Suppression of the TGF-β pathway by a macrolide antibiotic decreases fibrotic responses by ocular fibroblasts in vitro.

To elucidate and to inhibit post-surgical fibrotic processes after trabeculectomy in glaucoma therapy, we measured gene expression in a fibrotic cell culture model, based on transforming growth factor TGF-β induction in primary human tenon fibroblasts (hTFs), and used Connectivity Map (CMap) data for drug repositioning. We found that specific molecular mechanisms behind fibrosis are the upregulation of actins, the downregulation of CD34, and the upregulation of inflammatory cytokines such as IL6, IL11 and BMP6. The macrolide antibiotic Josamycin (JM) reverses these molecular mechanisms according to data from the CMap, and we thus tested JM as an inhibitor of fibrosis. JM was first tested for its toxic effects on hTFs, where it showed no influence on cell viability, but inhibited hTF proliferation in a concentration-dependent manner. We then demonstrated that JM suppresses the synthesis of extracellular matrix (ECM) components. In hTFs stimulated with TGF-β1, JM specifically inhibited α-smooth muslce actin expression, suggesting that it inhibits the transformation of fibroblasts into fibrotic myofibroblasts. In addition, a decrease of components of the ECM such as fibronectin, which is involved in in vivo scarring, was observed. We conclude that JM may be a promising candidate for the treatment of fibrosis after glaucoma filtration surgery or drainage device implantation in vivo.

Epigallocatechin-3-gallate (EGCG) inhibits myofibroblast transformation of human Tenon's fibroblasts

Myofibroblast transformation of human Tenon's fibroblasts severely challenges the outcome of glaucoma filtration surgery. epigallocatechin-3-gallate (EGCG) is considered as a potential reagent to overcome this issue for its anti-fibrosis effect on various human diseases, but it is unclear on the fibrosis of Tenon's fibroblasts. This study was conducted to investigate the effect of EGCG on TGF-β1-induced myofibroblast transformation of human Tenon's fibroblasts. The human Tenon's fibroblasts were incubated in the medium containing 10 ng/mL TGF-β1, and subsequently treated with EGCG or mitomycin C (MMC). The cell proliferation and migration were analyzed. The expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col-I), and p-Smad2/3 were also evaluated. It showed that EGCG and MMC strongly inhibited the elevation in cell number in tissue explants compared to the tissues treated with TGF-β1 alone. Scratch-Wound assay showed that 48 h after TGF-β1 induction, only 10% of the wound width remained. But cells treated with EGCG still showed over 93% wound width. Further, EGCG effectively inhibited TGF-β1-induced expression of α-SMA and Col-I as well as phosphorylation of Smad2/3 in Tenon's fibroblasts. Altogether, we concluded that EGCG suppressed the myofibroblast transformation in Tenon's fibroblasts through inactivating TGF-β1/Smad signaling. These findings demonstrate that EGCG can be considered as one of the possible antifibrotic reagents for preventing postoperative scarring in glaucoma filtration surgery.