title: The effects of retinoic acid on human corneal stromal keratocytes cultured in vitro under serum-free conditions.
abstract: OBJECTIVE
  Retinoic acid (RA) is a metabolite of vitamin A that plays a fundamental role in the development and function of the human eye. The purpose of this study was to investigate the effects of RA on the phenotype of corneal stromal keratocytes maintained in vitro for extended periods under serum-free conditions.
  
  
  METHODS
  Keratocytes isolated from human corneas were cultured up to 21 days in serum-free media supplemented with RA or DMSO vehicle. The effects of RA and of its removal after treatment on cell proliferation and morphology were evaluated. In addition, the expression of keratocyte markers was quantified at the transcriptional and protein levels by quantitative PCR and immunoblotting or ELISA, respectively. Furthermore, the effects of RA on keratocyte migration were tested using scratch assays.
  
  
  RESULTS
  Keratocytes cultured with RA up to 10 × 10(-6) M showed enhanced proliferation and stratification, and reduced mobility. RA also promoted the expression of keratocyte-characteristic proteoglycans, such as keratocan, lumican, and decorin, and increased the amounts of collagen type-I in culture while significantly reducing the expression of matrix metalloproteases 1, 3, and 9. RA effects were reversible, and cell phenotype reverted to that of control after removal of RA from media.
  
  
  CONCLUSIONS
  Retinoic acid was shown to control the phenotype of human corneal keratocytes cultured in vitro by regulating cell behavior and extracellular matrix composition. These findings contribute to our understanding of corneal stromal biology in health and disease, and may prove useful in optimizing keratocyte cultures for applications in tissue engineering, cell biology, and medicine.
authors.affiliation: School of Chemistry, Food and Pharmacy, University of Reading, Whiteknights, Reading, United Kingdom.
authors.forename: Ricardo Martins
authors.initials: RM
authors.lastname: Gouveia
authors.forename: Che John
authors.initials: CJ
authors.lastname: Connon
mesh_terms: D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D060527:Corneal Keratocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D003319:Corneal Stroma / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D016895:Culture Media, Serum-Free; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000096:biosynthesis* / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects / Q000473:pathology; D005786:Gene Expression Regulation; D006801:Humans; D007641:Keratolytic Agents / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D016895:Culture Media, Serum-Free; D005136:Eye Proteins; D007641:Keratolytic Agents; D014212:Tretinoin; D012313:RNA
keywords: cell-matrix interactions; corneal disease; corneal stroma; keratocytes; retinoic acid