title: Therapeutic potential of Pirfenidone for treating equine corneal scarring.
abstract: OBJECTIVE
  To evaluate the safety and efficacy of Pirfenidone (PFD) in the treatment of equine corneal fibrosis using an in vitro model.
  
  
  METHODS
  Healthy donor equine corneas were collected and used to generate primary equine corneal fibroblasts (ECFs) by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Equine corneal myofibroblasts (ECMs), used as a model of equine corneal fibrosis, were produced by growing ECF cultures in serum-free medium containing transforming growth factor β1 (1 ng/mL). Trypan blue viability assays and changes in ECF morphology were utilized to determine the optimal PFD dose for this in vitro model. Trypan blue viability, phase-contrast microscopy, and TUNEL assays were used to evaluate the cytotoxicity of PFD. Scratch and MTT assays were used to evaluate the effect of PFD on cellular migration and proliferation. Real-time PCR, immunoblot analysis, and immunocytochemistry were employed to determine the efficacy of PFD to inhibit ECM formation in vitro.
  
  
  RESULTS
  Topical PFD application at 200 μg/mL successfully decreased αSMA expression when compared to the TGFβ1 only treatment group (P < 0.01). PFD application ≤ 200 μg/mL did not affect ECF phenotype or cellular viability and did not result in significant cytotoxicity.
  
  
  CONCLUSIONS
  Pirfenidone safely and effectively inhibits TGFβ1-induced equine corneal fibrosis in vitro. In vivo studies are warranted.
authors.affiliation: Harry S. Truman Memorial Veterans Hospital, Columbia, MO, USA; College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Fink
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Giuliano
authors.forename: Ashish
authors.initials: A
authors.lastname: Tandon
authors.forename: Rajiv R
authors.initials: RR
authors.lastname: Mohan
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000627:therapeutic use*; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology; D065306:Corneal Injuries / Q000188:drug therapy / Q000662:veterinary*; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects; D006734:Horse Diseases / Q000188:drug therapy*; D006736:Horses; D011728:Pyridones / Q000627:therapeutic use*
chemical_list: D000199:Actins; D000894:Anti-Inflammatory Agents, Non-Steroidal; D011728:Pyridones; C093844:pirfenidone
keywords: cornea; equine; fibroblasts; fibrosis; myofibroblasts; pirfenidone