Therapeutic potential of the anti-fibrotic drug Suberanilohydroxamic acid (SAHA) in canine corneal fibrosis

Abstract

Purpose: The aims were to (1) investigate the molecular mechanisms mediating the antifibrotic effect of SAHA in the canine cornea using an in vitro model and (2) to develop a novel in vivo model of corneal fibrosis in dogs utilizing alkali burn and assess the ability of SAHA to inhibit fibrosis using this canine model. Methods: In study 1, canine corneal fibroblasts were incubated for 24hrs +/- Transforming Growth Factor- ?1 (TGF-[beta]1) and SAHA. Western blot was used to quantify isoforms of intracellular signaling proteins. Real-time PCR and zymography quantified matrix metalloproteinases (MMP) mRNA and protein expression, respectively. In study 2, a corneal alkali burn was created in Beagles using 1 N NaOH topically. Dogs were randomly and equally assigned into 2 groups: A) vehicle (DMSO); B) anti-fibrotic treatment (SAHA). Degree of corneal opacity and efficacy of SAHA were determined utilizing the Fantes grading scale, optical coherence tomography, corneal histopathology, immunohistochemistry and transmission electron microscopy. Results: In study 1, SAHA treatment reduced phosphorylation of Smad2/3, ERK1/2 and altered MMP protein and gene expression. In study 2, [alpha]-smooth muscle staining and minimum and maximum interfibrillar distances were significantly greater in burned corneas. Conclusion: Corneal anti-fibrotic effects of SAHA involve modulation of canonical and non-canonical components of TGF-[beta]1 intracellular signaling and MMP activity. The alkali burn generates corneal opacity without damaging the limbus, and induces reliable fibrosis. Additional in vivo SAHA dosing studies with larger sample size are warranted.