Trichostatin a inhibits corneal haze in vitro and in vivo.
OBJECTIVE
Trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to suppress TGF-beta-induced fibrogenesis in many nonocular tissues. The authors evaluated TSA cytotoxicity and its antifibrogenic activity on TGF-beta-driven fibrosis in the cornea with the use of in vitro and in vivo models.


METHODS
Human corneal fibroblasts (HSFs) were used for in vitro studies, and New Zealand White rabbits were used for in vivo studies. Haze in the rabbit cornea was produced with photorefractive keratectomy (PRK) using excimer laser. Trypan blue exclusion and MTT assays evaluated TSA cytotoxicity to the cornea. Density of haze in the rabbit eye was graded with slit lamp biomicroscopy. Real-time PCR, immunoblotting, or immunocytochemistry was used to measure alpha-smooth muscle actin (SMA), fibronectin, and collagen type IV mRNA or protein levels. TUNEL assay was used to detect cell death.


RESULTS
TSA concentrations of 250 nM or less were noncytotoxic and did not alter normal HSF morphology or proliferation. TGF-beta1 treatment of HSF significantly increased mRNA and protein levels of SMA (9-fold), fibronectin (2.5-fold), and collagen type IV (2-fold). TSA treatment showed 60% to 75% decreases in TGF-beta1-induced SMA and fibronectin mRNA levels and 1.5- to 3.0-fold decreases in protein levels but had no effect on collagen type IV mRNA or protein levels in vitro. Two-minute topical treatment of TSA on rabbit corneas subjected to -9 D PRK significantly decreased corneal haze in vivo.


CONCLUSIONS
TSA inhibits TGF-beta1-induced accumulation of extracellular matrix and myofibroblast formation in the human cornea in vitro and markedly decreases haze in rabbit cornea in vivo.
Sharma
Ajay
A
Mason Eye Institute, University of Missouri-Columbia, Columbia, Missouri 65212, USA.
Mehan
Maneesh M
MM
Sinha
Sunilima
S
Cowden
John W
JW
Mohan
Rajiv R
RR
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000235:genetics / Q000378:metabolism; D003318:Corneal Opacity / Q000188:drug therapy* / Q000209:etiology / Q000378:metabolism; D003319:Corneal Stroma / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D005355:Fibrosis / Q000139:chemically induced / Q000517:prevention & control; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D018903:Photorefractive Keratectomy / Q000009:adverse effects; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000633:toxicity
D000199:Actins; D024141:Collagen Type IV; D004791:Enzyme Inhibitors; D005353:Fibronectins; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; C012589:trichostatin A