Vorinostat: a potent agent to prevent and treat laser-induced corneal haze

Abstract

Purpose: This study investigated the efficacy and safety of vorinostat, a deacetylase (HDAC) inhibitor, in the treatment of laser-induced corneal haze following photorefractive keratectomy (PRK) in rabbits in vivo and transforming growth factor beta 1 (TGFβ1) -induced corneal fibrosis in vitro.

Methods: Corneal haze in rabbits was produced with -9.00 diopters (D) PRK. Fibrosis in cultured human and rabbit corneal fibroblasts was activated with TGFβ1. Vorinostat (25 μm) was topically applied once for 5 minutes on rabbit cornea immediately after PRK for in vivo studies. Vorinostat (0 to 25 μm) was given to human/rabbit corneal fibroblasts for 5 minutes or 48 hours for in vitro studies. Slit-lamp microscopy, TUNEL assay, and trypan blue were used to determined vorinostat toxicity, whereas real-time polymerase chain reaction, immunocytochemistry, and immunoblotting were used to measure its efficacy.

Results: Single 5-minute vorinostat (25 μm) topical application on the cornea following PRK significantly reduced corneal haze (P<.008) and fibrotic marker proteins (α-smooth muscle actin and f-actin; P<.001) without showing redness, swelling, or inflammation in rabbit eyes in vivo screened 4 weeks after PRK. Vorinostat reduced TGFβ1-induced fibrosis in human and rabbit corneas in vitro in a dose-dependent manner without altering cellular viability, phenotype, or proliferation.

Conclusions: Vorinostat is non-cytotoxic and safe for the eye and has potential to prevent laser-induced corneal haze in patients undergoing PRK for high myopia.

Figure 1

Dose-dependent effects of vorinostat 5-minute treatment on human corneal fibroblasts viability determined with trypan blue assay. None of the tested doses reduced cellular viability significantly.

Figure 2

Effect of vorinostat treatment on smooth muscle actin (SMA) and fibronectin mRNA quantified with real-time polymerase chain reaction. Corneal fibroblast cultures grown in transforming growth factor beta 1 (TGFβ1) under serum-free conditions were exposed to vorinostat (10 or 25 μm) for 5 minutes. Vorinostat significantly reduced mRNA levels of SMA (80% to 86%, respectively) and fibronectin (54% to 64%, respectively). ^#P<.001 compared to control; *P<.001 compared to TGFβ1.

Figure 3

Effect of vorinostat on smooth muscle actin (SMA) inhibition determined with A) western blot and B) quantification. Human corneal fibroblasts grown in transforming growth factor beta 1 (TGFβ1) under serum-free conditions and exposed to vorinostat (10 or 25 μm) for 5 minutes showed marked decrease in SMA (45% to 50%, respectively). Beta tubulin was used as a housekeeping gene. ^ζP<.001 compared to control; *P<.001 compared to TGFβ1.

Figure 4

Dose-response effect of long-term vorinostat exposure on smooth muscle actin (SMA) reduction determined with A) immunocytochemistry and B) quantification. Rabbit corneal fibroblasts grown to 80% confluence were incubated with transforming growth factor beta 1 (TGFβ1) with/without vorinostat (0 to 10 μm) under serum-free conditions for 48 hours. Vorinostat showed a significant dose-dependent decrease in SMA at all tested doses with highest inhibition at 10 μm. Original magnification ×100. *P<.05 no treatment vs TGFβ1, and **P<.05 TGFβ1 vs vorinostat.

Figure 5

A) Representative slit-lamp microscopy images demonstrating haze density in rabbit corneas treated with photorefractive keratectomy (PRK) with/without vorinostat (25 μm) 4 weeks postoperatively. B) Haze quantification showed that a single 5-minute topical application of vorinostat significantly decreased haze in postoperative PRK corneas in vivo. *P<.008.

Figure 6

Representative immunohistochemistry images 4 weeks after photorefractive keratectomy showing A, B) smooth muscle actin (SMA) and C, D) f-actin levels in corneas treated with/without vorinostat (25 μm). Blue = DAPI-stained nuclei, Green = SMA+, red = f-actin+ cells. Vorinostat-treated corneas showed a significant decrease in SMA (B) and f-actin (D) compared to untreated corneas (A, C). *P<.001. Scale bar = 100 μm

Figure 7

Quantification of smooth muscle actin (SMA)+ cells 4 weeks after photorefractive keratectomy (PRK) in corneas treated with/without vorinostat (25 μm). Vorinostat treatment significantly decreased SMA+ cells (*P<.001).

Figure 8

Representative images showing TUNEL+ cells in rabbit corneas treated with/without vorinostat (25 μm) collected A, B) 6 hours or C, D) 4 weeks after photorefractive keratectomy (PRK). No significant differences in TUNEL+ cells in rabbit cornea with/without vorinostat collected at early (6 hours) or end-point (4 weeks) suggests that a single 5-minute, 25-μm topical dose of vorinostat is safe for the rabbit eye. The amounts of TUNEL+ cells detected 6 hours after PRK in the anterior stroma or 4 weeks after PRK in the epithelium are consistent with earlier reported findings.^, Red = TUNEL+ cells, Blue = DAPI-stained nuclei. Scale bar = 100 μm