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The principle reason of high failure rate of glaucoma filtration surgery is the loss of filtration function caused by postoperative scar formation. We investigated the effects of 5-aza-2′-deoxycytidine (5-Aza-dc), a DNA methyltransferases inhibitor, on human Tenon's capsule fibroblasts (HTFs) differentiation and fibrosis and its mechanism of action, especially in relation to transforming growth factor (TGF)-β1 signaling. TGF-β1 was used to induce differentiation of cultured HTFs. 5-Aza-dc suppressed DNA methyltransferases (DNMTs) activity 6 h after treatment with a course corresponding to that of TGF-β1-induced reduction of DNMT activity without affecting cell viability as measured by Cell Counting Kit-8 assay. 5-Aza-dc also reduced DNMT1 and DNMT3a protein expression from 24 to 48 h. HTFs migration evaluated by scratch-wound assay were significantly increased 24 h after 5-Aza-dc treatment, a time course similar to that of TGF-β1. Treatment with 5-Aza-dc significantly increased the mRNA and protein levels of α-smooth muscle actin (α-SMA), collagen-1A1 (Col1A1), fibronectin (FN) and TGF-β type I receptor (TGFβRI). Furthermore, the effects of 5-Aza-dc on DNMT activity suppression, cell migration, and fibrosis were all reversed by a TGFβRI inhibitor- SB-431542. Meanwhile, knockdown of DNMT1 upregulated TGFβRI expression and had the same fibrosis-inducing effect in HTFs, which was also inhibited by SB-431542. Thus, the results indicate that DNA hypomethylation induces HTFs differentiation and fibrosis through up-regulation of TGFβRI. DNA methylation status plays an important role in subconjunctival wound healing.

To investigate the involvement of activin receptor-like kinase 5 (ALK5) in human Tenon's capsule fibroblasts (HTFs) transdifferentiation and fibrosis.(1) Cultured HTFs were treated with transforming growth factor beta 1 (TGF-β1) 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-β1 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.(1) TGF-β1, 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-β1 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-β1 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-β1 could still promote ALK5-OE cells migration.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.

PURPOSE: To examine the expression of Angiotensin II (Ang II) and its type I, and type II receptors (AT1R, AT2R) in rabbit Tenon's capsule fibroblasts after trabeculectomy, and to investigate the effects of Ang II on cultured human Tenon's capsule fibroblasts (HTFs) proliferation, migration, phenotype transition, and extracellular matrix (ECM) synthesis. METHODS: In the rabbit, expression of Ang II, AT1R, and AT2R in Tenon's capsule fibroblasts of eyes after trabeculectomy was evaluated by immunohistochemistry. Ang II levels in aqueous humor and plasma were assessed by ELISA. Cultured HTFs, obtained from patients undergoing cataract surgery, were treated with Ang II, TGF-β1, or vehicle control. Cell proliferation and migration were evaluated by Cell Counting Kit-8 and Transwell assay, and wound scratch assay, respectively. Protein expressions of α-smooth muscle actin (α-SMA) and fibronectin (FN) were measured by Western blot and immunofluorescence. Messenger RNA expressions of α-SMA and FN were measured by real-time PCR. RESULTS: In the rabbit, the expression of Ang II and AT1R increased from 1 day after surgery while AT2R increased from 7 days. In cultured HTFs, Ang II promoted cell proliferation and migration significantly (P < 0.05). Interestingly, the effect of 10(-7) M Ang II was more prominent than higher concentrations (10(-5) M; P < 0.05). Ang II also markedly induced the expression of α-SMA and FN, suggesting a phenotypic transition to myofibroblasts. CONCLUSIONS: Our results show that trabeculectomy alter the levels of Ang II and its receptors in Tenon's capsule fibroblasts, and that Ang II increase HTFs proliferation, migration, and phenotype transition, suggesting that Ang II may play a role in wound healing after trabeculectomy.

Primary open-angle glaucoma (POAG) with complex pathogenesis is one of the many major causes of blindness. It is widely accepted that the major cause of POAG is the dysregulation of the trabecular meshwork (TM), which regulates the resistance to aqueous humour outflow. Intraocular pressure is elevated with increasing outflow resistance in the conventional pathway, which consists of the TM and Schlemm's canal. The TM is a filter made up of extracellular matrix (e.g., collagens), most of which is organized into a network of beams covered by endothelial-like trabecular cells. Currently, lack of effective anti-glaucoma drugs acting on TM to normalize trabecular outflow represents a bottleneck for POAG therapy. Atorvastatin, a lipid-lowering drug, has been proven to be of benefit for POAG. The present study aimed to investigate the possible mechanisms of action of atorvastatin on the TM by using a porcine aqueous humour outflow model in vivo and TM cells in vitro. Perfusion of enucleated porcine eyes with atorvastatin (50-200 µM) for 2 h increased aqueous humour outflow (P

Purpose To examine the effects of losartan on scar formation after trabeculectomy and on fibrotic changes of human Tenon's fibroblasts (HTFs). Methods Trabeculectomy was performed on New Zealand rabbits. They were randomized to receive one of the following treatments: 0.9% normal saline, mitomycin-C, or one of the three doses of losartan. Bleb morphology, IOP, and histopathology examination were performed. Primary cultured HTFs were treated with losartan or vehicle, with or without angiotensin II (Ang II). Cell proliferation was assessed by Cell Counting Kit-8 assay, and cell migration was detected by scratch wound and transwell assay. Transdifferentiation was evaluated through the expression of α-smooth muscle actin (α-SMA) by immunofluorescence, real-time PCR, and Western blot. The expression of fibronectin (FN) was evaluated by real-time PCR and Western blot. Results An amount of 5 mg/mL of losartan subconjunctival injection significantly decreased IOP postoperatively and attenuated wound healing of the filtering bleb in the rabbit model. Immunostaining results showed less myofibroblast and collagen deposition around the bleb area in the losartan-treated eyes. Losartan (10-5 M) in vitro significantly attenuated Ang II's stimulatory effects on proliferation and migration of HTFs. Expressions of α-SMA and FN in these cells were also decreased by losartan pretreatment. Conclusions Losartan attenuates scar formation of filtering bleb after trabeculectomy likely via decreasing proliferation, migration, transdifferentiation, and extracellular matrix deposition of Tenon's fibroblasts. These results indicate that losartan may be an effective therapeutic agent in preventing bleb scar formation and in improving surgical outcome after trabeculectomy.