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

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

Targeted AAV5-Smad7 Gene Therapy Inhibits Corneal Scarring \u3cem\u3ein vivo\u3c/em\u3e

Corneal scarring is due to aberrant activity of the transforming growth factor β (TGFβ) signaling pathway following traumatic, mechanical, infectious, or surgical injury. Altered TGFβ signaling cascade leads to downstream Smad (Suppressor of mothers against decapentaplegic) protein-mediated signaling events that regulate expression of extracellular matrix and myogenic proteins. These events lead to transdifferentiation of keratocytes into myofibroblasts through fibroblasts and often results in permanent corneal scarring. Hence, therapeutic targets that reduce transdifferentiation of fibroblasts into myofibroblasts may provide a clinically relevant approach to treat corneal fibrosis and improve long-term visual outcomes. Smad7 protein regulates the functional effects of TGFβ signaling during corneal wound healing. We tested that targeted delivery of Smad7 using recombinant adeno-associated virus serotype 5 (AAV5-Smad7) delivered to the corneal stroma can inhibit corneal haze post photorefractive keratectomy (PRK) in vivo in a rabbit corneal injury model. We demonstrate that a single topical application of AAV5-Smad7 in rabbit cornea post-PRK led to a significant decrease in corneal haze and corneal fibrosis. Further, histopathology revealed lack of immune cell infiltration following AAV5-Smad7 gene transfer into the corneal stroma. Our data demonstrates that AAV5-Smad7 gene therapy is relatively safe with significant potential for the treatment of corneal disease currently resulting in fibrosis and impaired vision

Efficacious and Safe Tissue-Selective Controlled Gene Therapy Approaches for the Cornea

Untargeted and uncontrolled gene delivery is a major cause of gene therapy failure. This study aimed to define efficient and safe tissue-selective targeted gene therapy approaches for delivering genes into keratocytes of the cornea in vivo using a normal or diseased rabbit model. New Zealand White rabbits, adeno-associated virus serotype 5 (AAV5), and a minimally invasive hair-dryer based vector-delivery technique were used. Fifty microliters of AAV5 titer (6.5×1012 vg/ml) expressing green fluorescent protein gene (GFP) was topically applied onto normal or diseased (fibrotic or neovascularized) rabbit corneas for 2-minutes with a custom vector-delivery technique. Corneal fibrosis and neovascularization in rabbit eyes were induced with photorefractive keratectomy using excimer laser and VEGF (630 ng) using micropocket assay, respectively. Slit-lamp biomicroscopy and immunocytochemistry were used to confirm fibrosis and neovascularization in rabbit corneas. The levels, location and duration of delivered-GFP gene expression in the rabbit stroma were measured with immunocytochemistry and/or western blotting. Slot-blot measured delivered-GFP gene copy number. Confocal microscopy performed in whole-mounts of cornea and thick corneal sections determined geometric and spatial localization of delivered-GFP in three-dimensional arrangement. AAV5 toxicity and safety were evaluated with clinical eye exam, stereomicroscopy, slit-lamp biomicroscopy, and H&E staining. A single 2-minute AAV5 topical application via custom delivery-technique efficiently and selectively transduced keratocytes in the anterior stroma of normal and diseased rabbit corneas as evident from immunocytochemistry and confocal microscopy. Transgene expression was first detected at day 3, peaked at day 7, and was maintained up to 16 weeks (longest tested time point). Clinical and slit-lamp eye examination in live rabbits and H&E staining did not reveal any significant changes between AAV5-treated and untreated control corneas. These findings suggest that defined gene therapy approaches are safe for delivering genes into keratocytes in vivo and has potential for treating corneal disorders in human patients

Targeted AAV5-Smad7 gene therapy inhibits corneal scarring in vivo.

Corneal scarring is due to aberrant activity of the transforming growth factor β (TGFβ) signaling pathway following traumatic, mechanical, infectious, or surgical injury. Altered TGFβ signaling cascade leads to downstream Smad (Suppressor of mothers against decapentaplegic) protein-mediated signaling events that regulate expression of extracellular matrix and myogenic proteins. These events lead to transdifferentiation of keratocytes into myofibroblasts through fibroblasts and often results in permanent corneal scarring. Hence, therapeutic targets that reduce transdifferentiation of fibroblasts into myofibroblasts may provide a clinically relevant approach to treat corneal fibrosis and improve long-term visual outcomes. Smad7 protein regulates the functional effects of TGFβ signaling during corneal wound healing. We tested that targeted delivery of Smad7 using recombinant adeno-associated virus serotype 5 (AAV5-Smad7) delivered to the corneal stroma can inhibit corneal haze post photorefractive keratectomy (PRK) in vivo in a rabbit corneal injury model. We demonstrate that a single topical application of AAV5-Smad7 in rabbit cornea post-PRK led to a significant decrease in corneal haze and corneal fibrosis. Further, histopathology revealed lack of immune cell infiltration following AAV5-Smad7 gene transfer into the corneal stroma. Our data demonstrates that AAV5-Smad7 gene therapy is relatively safe with significant potential for the treatment of corneal disease currently resulting in fibrosis and impaired vision

Enteral Multiple Micronutrient Supplementation in Preterm and Low Birth Weight Infants: A Systematic Review and Meta-analysis

OBJECTIVES To assess effects of supplementation with 3 or more micronutrients (multiple micronutrients; MMN) compared to no MMN in human milk-fed preterm and low birth weight (LBW) infants. RESULTS Data on a subgroup of 414 preterm or LBW infants from 2 randomized controlled trials (4 reports) were included. The certainty of evidence ranged from low to very low. For growth outcomes in the MMN compared to the non-MMN group, there was a small increase in weight-for-age (2 trials, 383 participants) and height-for-age z-scores (2 trials, 372 participants); a small decrease in wasting (2 trials, 398 participants); small increases in stunting (2 trials, 399 participants); and an increase in underweight (2 trials, 396 participants). For neurodevelopment outcomes at 78 weeks, we found small increases in Bayley Scales of Infant Development, Version III (BISD-III), scores (cognition, receptive language, expressive language, fine motor, gross motor) in the MMN compared to the non-MMN group (1 trial, 27 participants). There were no studies examining dose or timing of supplementation. CONCLUSIONS Evidence is insufficient to determine whether enteral MMN supplementation to preterm or LBW infants who are fed mother's own milk is associated with benefit or harm. More trials are needed to generate evidence on mortality, morbidity, growth, and neurodevelopment.publishedVersio

Novel Combination BMP7 and HGF Gene Therapy Instigates Selective Myofibroblast Apoptosis and Reduces Corneal Haze In Vivo.

PurposeWe tested the potential of bone morphogenic protein 7 (BMP7) and hepatocyte growth factor (HGF) combination gene therapy to treat preformed corneal fibrosis using established rabbit in vivo and human in vitro models.MethodsEighteen New Zealand White rabbits were used. Corneal fibrosis was produced by alkali injury. Twenty-four hours after scar formation, cornea received topically either balanced salt solution (BSS; n = 6), polyethylenimine-conjugated gold nanoparticle (PEI2-GNP)-naked plasmid (n = 6) or PEI2-GNP plasmids expressing BMP7 and HGF genes (n = 6). Donor human corneas were used to obtain primary human corneal fibroblasts and myofibroblasts for mechanistic studies. Gene therapy effects on corneal fibrosis and ocular safety were evaluated by slit-lamp microscope, stereo microscopes, quantitative real-time PCR, immunofluorescence, TUNEL, modified MacDonald-Shadduck scoring system, and Draize tests.ResultsPEI2-GNP-mediated BMP7+HGF gene therapy significantly decreased corneal fibrosis in live rabbits in vivo (Fantes scale was 0.6 in BMP7+HGF-treated eyes compared to 3.3 in -therapy group; P 104 gene copies per microgram DNA of BMP7 and HGF genes. The recombinant HGF rendered apoptosis in corneal myofibroblasts but not in fibroblasts. Localized topical BMP7+HGF therapy showed no ocular toxicity.ConclusionsLocalized topical BMP7+HGF gene therapy treats corneal fibrosis and restores transparency in vivo mitigating excessive healing and rendering selective apoptosis in myofibroblasts