Histone deacetylase inhibitors blocked activation and caused senescence of corneal stromal cells.
OBJECTIVE
Corneal myofibroblasts differentiated from activated corneal stromal cells are the major cellular sources of extracellular matrix synthesis for the repair of corneal injury. In this study, the effects of histone deacetylase (HDAC) inhibitors on the activation, proliferation, migration and senescence of corneal stromal cells were evaluated.


METHODS
Primary human and mouse corneal stromal cells were harvested by sequential digestion with dispase and collagenase, and cultured in DMEM/F-12 media under serum-free (keratocytes), serum- (corneal fibroblasts) and TGFbeta1-supplemented (corneal myofibroblasts) conditions. The responses of corneal stromal cells to HDAC inhibitors were characterized by cDNA microarray, real time PCR, immunocytochemistry and western blot analysis. The effects of HDAC inhibitors on corneal fibroblast proliferation, cell cycle distribution, migration and senescence were also assessed in vitro.


RESULTS
Fetal bovine serum and TGFbeta1 activated the transdifferentiation of corneal stromal cells into fibroblasts and myofibroblasts, indicated by cell spreading, renewed assembly of actin filaments and enhanced expression of extracellular matrix components, all of which were suppressed by the addition of HDAC inhibitors. HDAC inhibitors inhibited the proliferation of corneal fibroblasts by decreasing the proportion in the S-phase and increasing the proportion in the G0/G1 and G2/M cell cycle checkpoints. HDAC inhibitors showed a dose-dependent inhibitory effects on the migration of corneal fibroblasts. In addition, HDAC inhibitors induced the senescence of corneal myofibroblasts as shown by enhanced staining of beta-galactosidase and upregulated expression of p16(ink4a).


CONCLUSIONS
HDAC inhibitors may affect corneal stromal cells by inhibiting myofibroblastic differentiation, cell proliferation, migration and by inducing cell senescence. Thus, this has implications for future studies in the development of promising drugs in the prevention or treatment of corneal haze and scar formation.
Zhou
Qingjun
Q
Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Qingdao, China.
Wang
Yao
Y
Yang
Lingling
L
Wang
Ye
Y
Chen
Peng
P
Wang
Yiqiang
Y
Dong
Xiaoguang
X
Xie
Lixin
L
D000818:Animals; D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects*; D003319:Corneal Stroma / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D020869:Gene Expression Profiling; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
D002087:Butyrates; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D053773:Transforming Growth Factor beta1; C012589:trichostatin A