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

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

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

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

Synthesis and vectorial functionalisation of pyrazolo[3,4- c ]pyridines

Heterocycles are a cornerstone of fragment-based drug discovery (FBDD) due to their prevalence in biologically active compounds. However, novel heterocyclic fragments are only valuable if they can be suitably elaborated to compliment a chosen target protein. Here we describe the synthesis of 5-halo-1H-pyrazolo[3,4-c]pyridine scaffolds and demonstrate how these compounds can be selectively elaborated along multiple growth-vectors. Specifically, N-1 and N-2 are accessed through protection-group and N-alkylation reactions; C-3 through tandem borylation and Suzuki–Miyaura cross-coupling reactions; C-5 through Pd-catalysed Buchwald–Hartwig amination; and C-7 through selective metalation with TMPMgCl.LiCl followed by reaction with electrophiles or transmetalation to ZnCl2 and Negishi cross-coupling. Linking multiple functionalisation strategies emulates a hit-to-lead pathway and demonstrates the utility of pyrazolo[3,4-c]pyridines to FBDD

Retinal detachment post-phacoemulsification in Bichons Frises: A retrospective study of 54 dogs

Objective—To compare rates of retinal detachment (RD) post phacoemulsification in American Bichons Frises with and without prophylactic retinopexy. Procedures—Medical records of 54 Bichons Frises undergoing phacoemulsification with or without prophylactic retinopexy between 2003–2013 in one or both eyes were reviewed from five Midwestern university veterinary teaching hospitals. Inclusion criteria were pre-operative ERG, at least 6 months of follow up post phacoemulsification, and absence of pre-existing RD as determined by ophthalmic examination and/or ultrasound. Statistical analyses used chi-squared and Wilcoxon rank-sum tests and Wilson confidence intervals with the p value Results—Phacoemulsification was performed without retinopexy in 79 eyes (42 dogs, non-PR group) and with prophylactic retinopexy in 23 eyes (12 dogs, PR group). Incidence of diabetes mellitus was 10/42 and 3/12 in the non-PR and the PR groups respectively (p=0.93). Intraocular lens implantation was performed in 40/42 non-PR dogs and 11/12 PR dogs, (p=0.63, 73/79 versus 21/23 eyes). At final re-examination, RD occurred in 4/79 eyes without retinopexy, compared to 0/23 RD in the retinopexy group. There was no statistically significant difference in RD rates between the two groups (p=0.27) Conclusions—These data provide no statistical evidence to support prophylactic retinopexy in Bichon Frises. Due to the low rate of retinal detachment following phacoemulsification without prophylactic retinopexy, the procedure appears to offer limited benefit to offset cost, procedural risk and risk of extended or repeated anesthesia in Bichons Frises

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

Social Networks, High-Risk anal Hpv and Coinfection With Hiv in Young Sexual Minority Men

OBJECTIVES: Young sexual minority men (SMM) exhibit a high prevalence and incidence of high-risk genotypes of human papillomavirus (hrHPV) anal infections and a confluence of a high prevalence of HIV and rectal STIs. Social determinants of health (SDOHs) are linked to social network contexts that generate and maintain racial disparities in HIV and STIs. A network perspective was provided to advance our knowledge of drivers of genotype-specific hrHPV infection and coinfection with HIV. The study also examined whether socially connected men are infected with the same high-risk HPV genotypes and, if so, whether this tendency is conditioned on coinfection with HIV. METHODS: Our sample included 136 young SMM of predominantly black race and their network members of other races and ethnicities, aged 18-29 years, who resided in Houston, Texas, USA. These participants were recruited during 2014-2016 at the baseline recruitment period by network-based peer referral, where anal exfoliated cells and named social and sexual partners were collected. Exponential random graph models were estimated to assess similarity in genotype-specific hrHPV anal infection in social connections and coinfection with HIV in consideration of the effects of similarity in sociodemographic, sexual behavioural characteristics, SDOHs and syphilis infection. RESULTS: Pairs of men socially connected to each other tend to be infected with the same hrHPV genotypes of HPV-16, HPV-45 and HPV-51 or HPV-16 and/or HPV-18. The tendency of social connections between pairs of men who were infected with either HPV-16 or HPV-18 were conditioned on HIV infection. CONCLUSIONS: Networked patterns of hrHPV infection could be amenable to network-based HPV prevention interventions that engage young SMM of predominantly racial minority groups who are out of HIV care and vulnerable to high-risk HPV acquisition