Corneal epithelial wound healing is delayed by platelet activating factor treatment

Abstract

To investigate the role of PAF in corneal epithelial wound healing and its mechanisms. Rabbit corneal epithelial (RCE) and keratocyte (RCK) cells were cultured and used at passage 2 to 3. For the in vitro wound healing experiments, a 7 mm central corneal de-epithelialization was performed in rabbit corneas. Corneas were incubated in the presence of 100 nmol/L PAF; 4 micro mol/L of the PAF antagonist BN was added 30 min before PAF. The area of the cornea uncovered by the epithelium was measured after 48 h using an imaging system. Cell adhesion, proliferation and migration were analyzed using CyQUANT fluorescence binding assay and Boyden chamber respectively. RNA was extracted and RT-PCR and Northern blot analyses were performed using HGF, KGF, EGF and HGF-R primers and probes. Forty-eight hours after the wound, the uncovered area (in arbitrary units) for control corneas was (6.0 +/- 1.5) U. PAF dramatically inhibited corneal epithelial wound closure, and the uncovered area was (58.0 +/- 7.0) U. The PAF antagonist completely abolished the effect of PAF with values similar to controls (5.0 +/- 1.0) U. PAF significantly increased the cell adhesion among RCE cells, while at the concentration of 100 nmol/L, PAF had no effect on the proliferation and migration of RCE cells. RT-PCR results revealed that PAF at 100 nmol/L decreased the HGF mRNA expression 4.1 times in RCK and HGF-R 3.5 times in RCE at 24 h compared to control group, while PAF had no effect on the expression of KGF and EGF in RCE cells. Northern blot data confirmed these results. Our results suggest that PAF causes significant delay of corneal epithelial wound healing. This effect may partially be due to the increased RCE cell adhesion and suppressed expression of HGF gene in RCK and HGF-R. The PAF antagonist BN can completely abolish the effect of PAF and enhance the corneal wound repair.