title: Suberoylanilide hydroxamic acid (vorinostat): its role on equine  corneal fibrosis and matrix metalloproteinase activity.
abstract: OBJECTIVE
  To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis.
  
  
  METHODS
  Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFβ1 to induce myofibroblast formation. The cultures were treated with either 5 μm or 10 μm SAHA for 72 h in the presence of TGFβ1. Real-time PCR and immunocytochemistry were used to determine the antifibrotic efficacy of SAHA by quantifying α-smooth muscle actin (αSMA), a marker of myofibroblast formation and fibrosis. Real-time PCR was used to determine the effects of SAHA on MMP2 and MMP9 expression. Cytotoxicity of SAHA was evaluated with phase contrast microscopy and trypan blue exclusion assays.
  
  
  RESULTS
  Suberoylanilide hydroxamic acid (SAHA) significantly attenuated TGFβ1-induced differentiation of equine fibroblasts to myofibroblasts as indicated by 3- to 3.5-fold (P < 0.001) decrease in αSMA mRNA and 86-88% (P < 0.001) decrease in αSMA+ immunocytochemical staining. SAHA treatment also resulted in 4.5- to 5.5-fold (P < 0.01) decrease in MMP9 expression. A dose-dependent bimodal effect of SAHA on MMP2 expression was noted (3.5-fold increase with 5 μm dose; 0.5-fold decrease with 10 μm dose). No change in fibroblast viability was observed with a 5 μm SAHA dose, whereas a 10 μm dose resulted in a moderate 17% decrease in cell viability.
  
  
  CONCLUSIONS
  Suberoylanilide hydroxamic acid (SAHA) can effectively inhibit TGFβ-induced differentiation of equine corneal fibroblasts to myofibroblasts and modulates MMP production in vitro.
authors.forename: Kevin S
authors.initials: KS
authors.lastname: Donnelly
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Giuliano
authors.forename: Ajay
authors.initials: A
authors.lastname: Sharm
authors.forename: Rajiv R
authors.initials: RR
authors.lastname: Mohan
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002921:Cicatrix / Q000188:drug therapy / Q000201:enzymology / Q000662:veterinary*; D065306:Corneal Injuries / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology / Q000662:veterinary*; D060527:Corneal Keratocytes / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D056572:Histone Deacetylase Inhibitors / Q000627:therapeutic use*; D006734:Horse Diseases / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D006736:Horses; D006877:Hydroxamic Acids / Q000627:therapeutic use*; D066298:In Vitro Techniques / Q000662:veterinary; D020782:Matrix Metalloproteinases / Q000187:drug effects / Q000378:metabolism*; D000077337:Vorinostat
chemical_list: D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D000077337:Vorinostat; D020782:Matrix Metalloproteinases