Effect of HDAC Inhibitors on Corneal Keratocyte Mechanical Phenotypes in 3-D Collagen Matrices

PURPOSE Histone deacetylase inhibitors (HDAC) have been shown to inhibit the TGFβ-induced myofibroblast transformation of corneal fibroblasts in 2-D culture. However, the effect of HDAC inhibitors on keratocyte spreading, contraction, and matrix remodeling in 3-D culture has not been directly assessed. The goal of this study was to investigate the effects of the HDAC inhibitors Trichostatin A (TSA) and Vorinostat (SAHA) on corneal keratocyte mechanical phenotypes in 3-D culture using defined serum-free culture conditions. METHODS Rabbit corneal keratocytes were plated within standard rat tail type I collagen matrices (2.5 mg/ml) or compressed collagen matrices (~100 mg/ml) and cultured for up to 4 days in serum-free media, PDGF BB, TGFβ1, and either 50 nM TSA, 10 μM SAHA, or vehicle (DMSO). F-actin, α-SM-actin, and collagen fibrils were imaged using confocal microscopy. Cell morphology and global matrix contraction were quantified digitally. The expression of α-SM-actin was assessed using western blotting. RESULTS Corneal keratocytes in 3-D matrices had a quiescent mechanical phenotype, as indicated by a dendritic morphology, a lack of stress fibers, and minimal cell-induced matrix remodeling. This phenotype was generally maintained following the addition of TSA or SAHA. TGFβ1 induced a contractile phenotype, as indicated by a loss of dendritic cell processes, the development of stress fibers, and significant matrix compaction. In contrast, cells cultured in TGFβ1 plus TSA or SAHA remained dendritic and did not form stress fibers or induce ECM compaction. Western blotting showed that the expression of α-SM actin after treatment with TGFβ1 was inhibited by TSA and SAHA. PDGF BB stimulated the elongation of keratocytes and the extension of dendritic processes within 3-D matrices without inducing stress fiber formation or collagen reorganization. This spreading response was maintained in the presence of TSA or SAHA. CONCLUSIONS Overall, HDAC inhibitors appear to mitigate the effects of TGFβ1 on the transformation of corneal keratocytes to a contractile, myofibroblast phenotype in both compliant and rigid 3-D matrices while preserving normal cell spreading and their ability to respond to the pro-migratory growth factor PDGF.