Role of 5\u27TG3\u27-Interacting Factors (TGIFs) in Vorinostat (HDAC Inhibitor)-Mediated Corneal Fibrosis Inhibition

Purpose: We have previously reported that vorinostat, an FDA-approved, clinically used histone deacetylase (HDAC) inhibitor, attenuates corneal fibrosis in vivo in rabbits by blocking transforming growth factor β (TGFβ). The 5′TG3′-interacting factors (TGIFs) are transcriptional repressors of TGFβ1 signaling via the Smad pathway. The present study was designed to explore the expression of TGIFs in human corneal fibroblasts and to investigate their role in mediating the antifibrotic effect of vorinostat. Methods: Human corneal fibroblast cultures were generated from donor corneas. RNA isolation, cDNA preparation, and PCR were performed to detect the presence of TGIF1 and TGIF2 transcripts. The cultures were exposed to vorinostat (2.5 μM) to test its effect on TGIF mRNA and protein levels using qPCR and immunoblotting. Myofibroblast formation was induced with TGFβ1 (5 ng/ml) treatment under serum-free conditions. The changes in fibrosis parameters were quantified by measuring fibrosis marker α-smooth muscle actin (αSMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Smad2/3/4 and TGIF knockdowns were performed using pre-validated RNAi/siRNAs and a commercially available transfection reagent. Results: Human corneal fibroblasts showed the expression of TGIF1 and TGIF2. Vorinostat (2.5 μM) caused a 2.8–3.3-fold increase in TGIF1 and TGIF2 mRNA levels and a 1.4–1.8-fold increase in TGIF1 and TGIF2 protein levels. Vorinostat treatment also caused a significant increase in acetylhistone H3 and acetylhistone H4. Vorinostat-induced increases in TGIF1 and TGIF2 were accompanied by a concurrent decrease in corneal fibrosis, as indicated by a decrease in αSMA mRNA by 83±7.7% and protein levels by 97±5%. The RNAi-mediated knockdown of Smad2, Smad3, and Smad4 markedly attenuated TGFβ1-evoked transdifferentiation of fibroblasts to myofibroblasts. The siRNA-mediated knockdown of TGIF1 and TGIF2 neutralized vorinostat-evoked decreases in αSMA mRNA by 31%–45% and protein levels by 12%–23%. Conclusions: Human corneal fibroblasts demonstrate the expression of TGIF1 and TGIF2 transcription factors. These transcriptional repressors are critical, at least partially, in mediating the antifibrotic effect of vorinostat in the cornea

Vector Delivery Technique Affects Gene Transfer in the Cornea \u3cem\u3ein vivo\u3c/em\u3e

Purpose: This study tested whether controlled drying of the cornea increases vector absorption in mouse and rabbit corneas in vivo and human cornea ex vivo, and studied the effects of corneal drying on gene transfer, structure and inflammatory reaction in the mouse cornea in vivo. Methods: Female C57 black mice and New Zealand White rabbits were used for in vivo studies. Donor human corneas were used for ex vivo experiments. A hair dryer was used for drying the corneas after removing corneal epithelium by gentle scraping. The corneas received no, once, twice, thrice, or five times warm air for 10 s with a 5 s interval after each 10 s hair dryer application. Thereafter, balanced salt solution (BSS) was topically applied immediately on the cornea for 2 min using a custom-cloning cylinder. The absorbed BSS was quantified using Hamilton microsyringes. The adenoassociated virus 8 (AAV8) vector (1.1×108 genomic copies/μl) expressing marker gene was used to study the effect of corneal drying on gene transfer. Animals were sacrificed on day 14 and gene expression was analyzed using commercial staining kit. Morphological changes and infiltration of inflammatory cells were examined with H & E staining and immunocytochemistry. Results: Mice, rabbit or human corneas subjected to no or 10 s drying showed 6%–8% BSS absorption whereas 20, 30, or 50 s corneal drying showed significantly high 14%–19% (pin vivowith mild-to-moderate changes in corneal morphology. The 30 s of drying also showed significantly (pin vivowithout jeopardizing corneal morphology whereas 10 or 20 s drying showed moderate degree of gene transfer with no altered corneal morphology. Corneas that underwent 50 s drying showed high CD11b-positive cells (p Conclusions: Controlled corneal drying with hair dryer increases vector absorption significantly. The dispensing of efficacious AAV serotype into cornea with optimized minimally invasive topical application technique could provide high and targeted expression of therapeutic genes in the stroma in vivo without causing significant side effects

Molecular Mechanisms of Suberoylanilide Hydroxamic Acid in the Inhibition of TGF-β1-Mediated Canine Corneal Fibrosis

Objective—To investigate molecular mechanisms mediating anti-fibrotic effect of SAHA in the canine cornea using an in vitro model. We hypothesized that SAHA attenuates corneal fibrosis by modulating Smad-dependent and, to a lesser extent, Smad-independent signaling pathways activated by TGF-β1, as well as matrix metalloproteinase (MMP) activity. Methods—Cultured canine corneal fibroblasts (CCF) were incubated in the presence/absence of TGF-β1 (5ng/ml) and SAHA (2.5μM) for 24hrs. Western blot analysis was used to quantify non-phosphorylated and phosphorylated isoforms of Smad2/3, p38 MAP kinase (MAPK), ERK1/2 and JNK1. Real-time PCR and zymography were utilized to quantify MMP1, MMP2, MMP8 and MMP9 mRNA expression and MMP2 and MMP9 protein activity, respectively. Results—TGF-β1 treatment caused a significant increase in phospho-Smad2/3 and phospho-p38 MAPK. SAHA treatment reduced TGF-β1-induced phosphorylation of Smad2/3 but not of p38 MAPK. TGF-β1 did not modulate the phosphorylation of ERK1/2 or JNK1. SAHA caused a significant reduction in phospho-ERK1/2 expression regardless of concurrent TGF-β1 treatment. Neither SAHA alone nor in combination with TGF-β1 altered phospho-JNK1 expression. TGF-β1 significantly increased MMP1 and MMP9 mRNA expression but did not alter MMP2 mRNA. SAHA treatment attenuated TGF-β1-induced MMP9 mRNA expression while significantly enhancing TGF-β1-induced MMP1 mRNA expression. Zymography detected reduced expression of MMP2 and MMP9 proteins in untreated control CCF. TGF-β1 treatment did not alter their expression but SAHA treatment +/−TGF-β1 significantly increased MMP2 and MMP9 protein expression. Conclusions—The corneal anti-fibrotic effects of SAHA involve multiple mechanisms including modulation of canonical and non-canonical components of TGF-β1 intracellular signaling and MMP activity

AAV Serotype Influences Gene Transfer in Corneal Stroma \u3cem\u3ein vivo\u3c/em\u3e

This study evaluated the cellular tropism and relative transduction efficiency of three AAV serotypes, AAV6, AAV8 and AAV9, for corneal gene delivery using mouse cornea in vivo and donor human cornea ex vivo. The AAV6, AAV8 and AAV9 serotypes having AAV2 plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Viral vectors (109 vg/μl) were topically applied onto mouse cornea in vivo and human cornea ex vivo after removing the epithelium. Human corneas were processed for transgene delivery at day 5 after viral vector application. Mouse corneas were harvested at 4, 14 and 30 days after vector application for AP staining. Transduction efficiency was calculated by quantifying pixels of AP-stained area using Image J software and also confirmed by functional AP enzyme activity in the corneal lysates. Cellular toxicity of the three AAV serotypes was tested with TUNEL assay. Inflammatory response was detected by immunostaining for CD11b and F4/80. All three AAV serotypes successfully transduced mouse and human corneas. The order of transduction efficiency was AAV9\u3eAAV8\u3eAAV6. The transduction efficiency of AAV9 was 1.1–1.4 fold higher (p\u3e0.05) as compared to AAV8 and 3.5–5.5 fold higher (p\u3c0.01) as compared to AAV6. The level of transgene expression for all the three serotypes was greater at 14 days compared to 4 days and this high level of transgene expression was maintained up to the tested time point of 30 days. Corneas exposed to any of the three AAV serotypes did not show significant TUNEL positive cells or any inflammatory response as tested by CD11b or F4/80 staining suggesting that tested AAV serotypes do not induce cell death or inflammation and are safe for corneal gene therapy

PDGF-driven proliferation, migration, and IL8 chemokine secretion in human corneal fibroblasts involve JAK2-STAT3 signaling pathway

Purpose: Platelet-derived growth factor (PDGF) is associated with corneal fibroblast migration and proliferation and plays an important role in corneal wound healing. However, the intracellular mechanisms of PDGF-mediated functions in corneal fibroblasts are poorly understood. We tested the hypothesis that PDGF functional activities in the cornea involve the Janus kinase-2/signal transducers and activators of transcription-3 (JAK2-STAT3) signaling pathway and whether PDGF induces the expression of suppressors of cytokine signaling 3 (SOCS3), belonging to the novel family of feedback regulators of cytokine and growth factor activities. Methods: Human corneal fibroblast (HSF) cultures were used as an in vitro model for functional analysis. Real-time polymerase chain reactions were performed to quantify gene expression. Immunoprecipitation and immunoblotting techniques were used to measure protein expression. Cell growth, migration, and ELISA assays were used for functional validation. Results: Low endogenous levels of STAT3 and SOCS3 mRNA and protein expression were noted in HSFs. PDGF treatment of HSF significantly induced SOCS3 mRNA (3.0–4.5 fold) and protein (1.5–2.5 fold) expression in a timedependent manner. Similarly, PDGF treatment of HSF significantly increased STAT3 protein expression at two tested time points (2.5–2.96 fold). Cultures exposed to vehicle (control) did not show any change in SOCS3 and STAT3 mRNA or protein expression. An addition of AG-490, a selective inhibitor of the JAK2-STAT3 pathway, significantly inhibited PDGF-mediated STAT3 induction and cell growth and migration in HSF. We also observed that PDGF induced interleukin-8 (IL8) chemokine secretion (2 fold) and AG-490 inhibited IL8 secretion. Conclusions: Our data showed that PDGF induced STAT3, SOCS3, and IL8 chemokine secretion in human corneal fibroblasts. Further, PDGF-induced cell growth, migration, and IL8 secretion in corneal fibroblast involve the JAK2- STAT3 signaling pathway

Development of a Novel \u3cem\u3eEx Vivo\u3c/em\u3e Equine Corneal Model

Objective To develop an ex vivo equine corneal organ culture model. Specifically, to assess the equine cornea\u27s extracellular matrix and cellularity after 7 days using two different culture techniques: either (i) immersion system or (ii) air/liquid interface system, to determine the best ex vivo equine corneal model. Animals Studied Fourteen healthy equine corneas of various breeds. Procedures Equine corneas with 2 mm of perilimbal sclera were freshly harvested from 7 horses undergoing humane euthanasia. One corneal–scleral ring (CSR) from each horse was randomly placed in the (i) immersion condition organ culture system (IC), with the contralateral CSR being placed in the (ii) air/liquid interface organ culture system (ALC) for 7 days. All corneas were evaluated using serial daily gross photography, histology, qPCR, and TUNEL assay. Results corneal–scleral rings placed in the IC (i) had complete loss of corneal transparency on gross photography by 7 days, showed a significant level of corneal stromal disorganization, significantly increased α‐SMA levels on qPCR, and apoptosis on TUNEL assay compared to controls. The ALC (ii) had weak stromal disorganization on histopathologic examination and was not significantly different from normal equine corneal controls on all other evaluated parameters. Conclusions The air–liquid interface organ culture system maintains the equine cornea\u27s extracellular matrix and preserves corneal transparency, while the immersion condition results in near complete degradation of normal equine corneal architecture after 7 days in culture. The air–liquid organ culture is a viable option to maintain a healthy equine cornea in an ex vivo setting for wound healing studies

Efficacy and Safety of Mitomycin C as an Agent to Treat Corneal Scarring in Horses Using an \u3cem\u3ein vitro\u3c/em\u3e Model

Objective—Mitomycin C (MMC) is used clinically to treat corneal scarring in human patients. We investigated the safety and efficacy of MMC to treat corneal scarring in horses by examining its effects at the early and late stages of disease using an in-vitro model. Procedure—An in-vitro model of equine corneal fibroblast (ECF) developed was used. The equine corneal fibroblast or myofibroblast cultures were produced by growing primary ECF in the presence or absence of transforming growth factor beta-1 (TGFβ1) under serum-free conditions. The MMC dose for the equine cornea was defined with dose-dependent trypan blue exclusion and MTT [(3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays after applying MMC to the cultures once for 2 minutes. The efficacy of MMC to control corneal scarring in horses was determined by measuring mRNA and protein expression of corneal scarring markers (α-smooth muscle actin and F-actin) with western blotting, immunocytochemistry and/or quantitative realtime polymerase chain reactions. Results—A single 2 minutes treatment of 0.02% or less MMC did not alter ECF phenotype, viability, or cellular proliferation whereas 0.05% or higher MMC doses showed mild-to-moderate cellular toxicity. The TGFβ1 at 1ng/ml showed significant myofibroblast formation in ECF under serum-free conditions. A single 2 minute, 0.02% MMC treatment 24 hours (early) after TGFβ1 stimulation significantly reduced conversion of ECF to myofibroblasts, however, a single 0.02% MMC treatment 11 days after TGFβ1 stimulation showed moderate myofibroblast inhibition. Conclusions—That MMC safely and effectively reduced scarring in ECF by reducing the degree of transdifferentiation of corneal fibroblasts to myofibroblasts in vitro. Further clinical invivo investigations are warranted using MMC in horses

Isolation and Cultivation of Equine Corneal Keratocytes, Fibroblasts and Myofibroblasts

Objective—To establish an in vitro model for the investigation of equine corneal wound healing. To accomplish this goal, a protocol to isolate and culture equine corneal keratocytes, fibroblasts and myofibroblasts was developed. Animal material—Equine corneal buttons were aseptically harvested from healthy research horses undergoing humane euthanasia for reasons unrelated to this study. Slit-lamp biomicroscopy was performed prior to euthanasia by a board-certified veterinary ophthalmologist to ensure that all samples were harvested from horses free of anterior segment disease. Procedure—Equine corneal stroma was isolated using mechanical techniques and stromal subsections were then cultured. Customized media at different culture conditions was used to promote growth and differentiation of corneal stromal cells into keratocytes, fibroblasts and myofibroblasts. Results—Cell culture techniques were successfully used to establish a method for the isolation and culture of equine corneal keratocytes, fibroblasts and myofibroblasts. Immunohistochemical staining for alpha-smooth muscle and F-actin was used to definitively differentiate the three cell types. Conclusion—Equine corneal stromal keratocytes, fibroblasts and myofibroblasts can be predictably isolated and cultured in vitro using this protocol

Gene Delivery in the Equine Cornea: A Novel Therapeutic Strategy

Objective—To determine if hybrid adeno-associated virus serotype 2/5 (AAV5) vector can effectively deliver foreign genes into the equine cornea without causing adverse side effects. The aims of this study were to: (i) evaluate efficacy of AAV5 to deliver therapeutic genes into equine corneal fibroblasts (ECFs) using enhanced green fluorescent protein (EGFP) marker gene and (ii) establish the safety of AAV5 vector for equine corneal gene therapy. Animal Material—Primary ECF cultures were harvested from healthy donor equine corneas. Cultures were maintained at 370C in humidified atmosphere with 5% CO2. Procedure—AAV5 vector expressing EGFP under control of hybrid cytomegalovirus (CMV) + chicken β-actin (CBA) promoter was applied topically to ECF. Expression of delivered EGFP gene in ECF was quantified using fluorescent microscopy. Using DAPI staining, the total number of cells and transduction efficiency of tested AAV vector was determined. Phase contrast microscopy, trypan blue and TUNEL assays were used to determine toxicity and safety of AAV5 for ECFs. Results—Topical AAV5 application successfully transduced significant numbers of ECFs. Transduction efficiency was 13.1%. Tested AAV5 vector did not cause phenotype change or significant cell death and cell viability was maintained. Conclusions—Tested AAV5 vector is effective and safe for gene therapy in ECFs in vitro. Clinical Relevance—Tested AAV5 vector has potential to extend a novel gene therapy approach to treat equine corneal disease in vivo

Localization of Angiotensin Converting Enzyme in Rabbit Cornea and Its Role in Controlling Corneal Angiogenesis \u3cem\u3ein vivo\u3c/em\u3e

Purpose: The renin angiotensin system (RAS) has been shown to modulate vascular endothelial growth factor and angiogenesis. In this study we investigated (i) the existence of the RAS components angiotensin converting enzyme (ACE) and angiotensin II receptors (AT1 and AT2) in the rabbit cornea using in vitro and ex vivo models and (ii) the effect of enalapril, an ACE inhibitor, to inhibit angiogenesis in rabbit cornea in vivo. Methods: New Zealand White rabbits were used. Cultured corneal fibroblasts and corneal epithelial cells were used for RNA isolation and cDNA preparation using standard molecular biology techniques. PCR was performed to detect the presence of ACE, AT1, and AT2 gene expression. A corneal micropocket assay to implant a vascular endothelial growth factor (VEGF) pellet in the rabbit cornea was used to induce corneal angiogenesis. Rabbits of the control group received sterile water, and the treated group received 3 mg/kg enalapril intramuscularly once daily for 14 days starting from day 1 of pellet implantation. The clinical eye examination was performed by slit-lamp biomicroscopy. We monitored the level of corneal angiogenesis in live animals by stereomicroscopy at days 4, 9, and 14 after VEGF pellet implantation. Collagen type IV and lectin immunohistochemistry and fluorescent microscopy were used to measure corneal angiogenesis in tissue sections of control and enalapril-treated corneas of the rabbits. Image J software was used to quantify corneal angiogenesis in the rabbit eye in situ. Results: Our data demonstrated the presence of ACE, AT1, and AT2 expression in corneal fibroblasts. Cells of corneal epithelium expressed AT1 and AT2 but did not show ACE expression. Slit-lamp examination did not show any significant difference between the degree of edema or cellular infiltration between the corneas of control and enalapril-treated rabbits. VEGF pellet implantation caused corneal angiogenesis in the eyes of vehicle-treated control rabbits, and the mean area of corneal neovascularization was 1.8, 2.8, and 3.2 mm2 on days 4, 9, and 14, respectively. Enalapril treatment caused a notable decrease in corneal neovascularization of 44% (1 mm2), 28% (2.1 mm2), and 31% (2.2 mm2) on the three tested time points, respectively. The immunostaining of corneal tissue sections with collagen type IV and lectin confirmed the presence of blood vessels, with enalapril-treated rabbit corneas showing a lesser degree of blood vessel staining. Conclusions: Corneal cells show expression of tissue RAS components, such as ACE, AT1, and AT2. Treatment with ACE inhibitor enalapril markedly decreased corneal angiogenesis in a rabbit model of VEGF-induced corneal neovascularization, suggesting that ACE inhibitors may represent a novel therapeutic strategy to treat corneal angiogenesis