Retinoic Acid Improves Keratocyte Phenotype in a 3D Model of the Corneal Stroma

Program Number: 1300 Presentation Time: 8:30 AM–8:45 AM Retinoic Acid Improves Keratocyte Phenotype in a 3D Model of the Corneal Stroma Ricardo M. Gouveia, Fadhilah Z. Abidin, Che J. Connon. Institute of Genetic Medicine, Newcastle University, Newcastle-upon-Tyne, United Kingdom. Purpose: Retinoic Acid (RA) is a metabolite of vitamin A fundamental for the correct function and development of the human eye. However, the role of this signaling molecule in the corneal stroma has been long overlooked. The purpose of this study was to investigate the effects of RA on the phenotype of human corneal keratocytes within a 3D stromal model. Methods: Keratocytes isolated from human corneas were encapsulated within compressed collagen gels to constitute a 3D model of the cornea’s stroma. Cell-gel constructs were then cultured for up to 30 days in serum-free media with and without 1x10-5 M RA. The effects of RA on cell proliferation, survival, and contractile activity within the gel were evaluated. In addition, the expression of keratocyte markers was quantified at the transcriptional and protein levels by quantitative PCR and immunodetection, respectively. Results: As a supplement of serum-free media, RA promoted the proliferation and survival of keratocytes at day 30 while enhancing the expression of keratocyte-characteristic molecular markers (Fig. 1). Specifically, RA increased the expression of keratocan, lumican, and decorin proteoglycans, ALDH1 and ALDH3 crystallins, collagen type-I and V, and CHST6 by more than two-fold, a significant (p<0.05) improvement compared with untreated control conditions. In addition, RA supplementation abrogated the expression of MMP1, MMP3, and MMP9 proteases in αSMAand fibronectin-negative cells. Furthermore, RA significantly inhibited collagen gel contraction compared with untreated gels (p<0.01). Conclusions: Retinoic acid was shown to control the phenotype of human corneal keratocytes cultured in a 3D environment by regulating cell behavior and extracellular matrix composition. These findings contribute to our understanding of corneal stromal biology in health and disease, and will prove useful in optimizing keratocyte cultures for tissue engineering, cell biology, and clinical applications.