Govindaraj Anumanthan

Epigenetic Modification Prevents Excessive Wound Healing and Scar Formation After Glaucoma Filtration Surgery

Purpose The purpose of this study was to determine the efficacy of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACi), in prevention of excessive wound healing and scar formation in a rabbit model of glaucoma filtration surgery (GFS). Methods A rabbit model of GFS was used. Rabbits that underwent GFS received balanced salt solution, or SAHA (50 μM), or mitomycin C (0.02%). Clinical scores of IOP, bleb vascularity, and slit-lamp examination were performed. On postoperative day 14, rabbits were killed and the bleb tissues were collected for evaluation of tissue fibrosis with hematoxylin and eosin, Masson trichrome, α-smooth muscle actin (αSMA), and F-actin staining. Furthermore, SAHA-mediated acetylation of histones in corneal fibroblasts and conjunctiva were determined by Western blot analysis. Results Suberoylanilide hydroxamic acid treatment after GFS showed no signs of edema, corneal opacity, endophthalmitis, or cataract formation. Morphometric analysis of SAHA-treated eyes showed higher bleb length (P < 0.001), bleb area (P < 0.05), lower IOP (P < 0.01), and decreased vascularity compared to control. Furthermore, SAHA treatment showed significantly reduced levels of αSMA (P < 0.001), F-actin (P < 0.01), and collagen deposition (P < 0.05) at the sclerotomy site. In addition, SAHA treatment increased the acetylation status of H3 and H4 histones in corneal fibroblasts and conjunctiva. Conclusions This study demonstrates that HDAC inhibition is an attractive pharmacologic target to modulate GFS wound healing, and SAHA, an HDACi, can be a useful adjunct to improve the GFS outcome.

Blockade of KCa3.1: A novel target to treat TGF-β1 induced conjunctival fibrosis

Abstract Postoperative conjunctival fibrosis is common in patients after glaucoma filtration surgery. The calcium activated potassium (KCa3.1) channel has been shown to inhibit fibrosis in many non‐ocular tissues. However, its potential in treating ocular fibrosis remains unknown. We tested the anti‐fibrotic potential of TRAM34, a selective blocker of KCa3.1 channel, in treating conjunctival fibrosis. Primary human conjunctival fibroblast (HCF) cultures derived from donor tissues. Myofibroblasts causing conjunctival fibrosis were generated by growing HCFs in the presence of TGF&bgr;1 for 72 h. KCa3.1 mRNA and protein expression in HCF was examined with PCR and western blot. The anti‐fibrotic potential of TRAM34 was examined by measuring fibrotic gene expression with quantitative PCR (qPCR), immunofluorescence, and western blotting in HCFs in ± TGF&bgr;1 (5 ng/ml) and TRAM34 (0–25 &mgr;M). The cytotoxicity of Tram34 was analyzed with trypan blue assay and its role in Smad signaling was studied with immunofluorescence. Expression of KCa3.1 mRNA and protein was detected in HCFs and TGF&bgr;1 treatment to HCFs significantly increased expression of KCa3.1. TRAM34 treatment attenuated transcription of fibrotic markers, &agr;SMA (p < .001), fibronectin (p < .05), collagen I (p < .001) and collagen IV (p < .001) in TGF&bgr;1‐induced HCFs. Further, TRAM34 significantly inhibited TGF&bgr;1‐stimulated &agr;SMA protein expression (p < .01) and nuclear translocation of fibrotic Smad2/3 in HCFs and showed no significant cytotoxicity (p < .05). The KCa3.1 potassium channel plays a significant role in the prevention of conjunctival fibrosis and TRAM34 has potential to control post surgical bleb fibrosis in patients. In vivo studies are warranted. HighlightsGlaucoma filtration surgery (GFS) is a standard of care for glaucoma.Postoperative ocular scarring from excessive wound healing at bleb site is a common complication.Calcium (Ca2+) activated potassium (K+) channel’s (KCa3.1) role in ocular fibrosis remains unknown.Suppression of KCa3.1 by a selective KCa3.1 inhibitor, TRAM34, reduces conjunctival fibrosis in an in vitro model.TRAM34 has potential to control GFS‐induced ocular fibrosis.

KCa3.1 ion channel: A novel therapeutic target for corneal fibrosis

Vision impairment from corneal fibrosis is a common consequence of irregular corneal wound healing after injury. Intermediate-conductance calmodulin/calcium-activated K+ channels 3.1 (KCa3.1) play an important role in cell cycle progression and cellular proliferation. Proliferation and differentiation of corneal fibroblasts to myofibroblasts can lead to corneal fibrosis after injury. KCa3.1 has been shown in many non-ocular tissues to promote fibrosis, but its role in corneal fibrosis is still unknown. In this study, we characterized the expression KCa3.1 in the human cornea and its role in corneal wound healing in vivo using a KCa3.1 knockout (KCa3.1-/-) mouse model. Additionally, we tested the hypothesis that blockade of KCa3.1 by a selective KCa3.1 inhibitor, TRAM-34, could augment a novel interventional approach for controlling corneal fibrosis in our established in vitro model of corneal fibrosis. The expression of KCa3.1 gene and protein was analyzed in human and murine corneas. Primary human corneal fibroblast (HCF) cultures were used to examine the potential of TRAM-34 in treating corneal fibrosis by measuring levels of pro-fibrotic genes, proteins, and cellular migration using real-time quantitative qPCR, Western blotting, and scratch assay, respectively. Cytotoxicity of TRAM-34 was tested with trypan blue assay, and pro-fibrotic marker expression was tested in KCa3.1-/-. Expression of KCa3.1 mRNA and protein was detected in all three layers of the human cornea. The KCa3.1-/- mice demonstrated significantly reduced corneal fibrosis and expression of pro-fibrotic marker genes such as collagen I and α-smooth muscle actin (α-SMA), suggesting that KCa3.1 plays an important role corneal wound healing in vivo. Pharmacological treatment with TRAM-34 significantly attenuated corneal fibrosis in vitro, as demonstrated in HCFs by the inhibition TGFβ-mediated transcription of pro-fibrotic collagen I mRNA and α-SMA mRNA and protein expression (p<0.001). No evidence of cytotoxicity was observed. Our study suggests that KCa3.1 regulates corneal wound healing and that blockade of KCa3.1 by TRAM-34 offers a potential therapeutic strategy for developing therapies to cure corneal fibrosis in vivo.

Efficacy and Safety Comparison Between Suberoylanilide Hydroxamic Acid and Mitomycin C in Reducing the Risk of Corneal Haze After PRK Treatment In Vivo

PURPOSE This study compared the efficacy and safety of suberoylanilide hydroxamic acid (SAHA) and mitomycin C (MMC) up to 4 months in the prevention of corneal haze induced by photorefractive keratectomy (PRK) in rabbits in vivo. METHODS Corneal haze in rabbits was produced with -9.00 diopter PRK. A single application of SAHA (25 μM) or MMC (0.02%) was applied topically immediately after PRK. Effects of the two drugs were analyzed by slit-lamp microscope, specular microscope, TUNEL assay, and immunofluorescence. RESULTS Single topical adjunct use of SAHA (25 μM) or MMC (0.02%) after PRK attenuated more than 95% corneal haze and myofibroblast formation (P < .001). SAHA did not reduce keratocyte density, cause keratocyte apoptosis, or increase immune cell infiltration compared to MMC (P < .01 or .001). Furthermore, SAHA dosing did not compromise corneal endothelial phenotype, density, or function in rabbit eyes, whereas MMC application did (P < .01 or .001). CONCLUSIONS SAHA and MMC significantly decreased corneal haze after PRK in rabbits in vivo. SAHA exhibited significantly reduced short- and long-term damage to the corneal endothelium compared to MMC in rabbits. SAHA is an effective and potentially safer alternative to MMC for the prevention of corneal haze after PRK. Clinical trials are warranted. [J Refract Surg. 2017;33(12):834-839.].