Increased platelet-activating factor receptor gene expression by corneal epithelial wound healing

PURPOSE. Platelet activating factor (PAF) is a potent inflammatory mediator the synthesis of which increases in the cornea after injury. The effects o f PAF are mediated by receptors (PAF-R), which are present in target cells. This study was undertaken to investigate the effects of wound healing, PAF , and growth factors on modulating PAF-R mRNA levels in corneal epithelial cells. METHODS. Cultures Of rabbit corneal epithelial (RCE), rabbit limbal epithel ial (RLE), rabbit corneal fibroblast (RCF), and rabbit corneal endothelial (RCEn) cells, as well as rabbit corneal keratocytes (RCKs) were used. For t he in vivo wound-healing experiments, a 7-mm central corneal deepithelializ ation was performed in anesthetized rabbits. For the in vitro experiments, wounded rabbit corneas were maintained in organ culture. Corneas were stimu lated with 120 nM PAI: or preincubated with PAF antagonists, cyclohexamide (CHX) or actinomycin D (AcD) before adding PAF. RCE cells were stimulated w ith transforming growth factor (TGF)-beta 1, -beta 2, and, -beta 3, basic f ibroblast growth factor (bFGF), keratinocyte growth factor ((KGF); and hepa tocyte growth factor (HGF). Total RNA was isolated and PAF-R expression eva luated by reverse transcription-polymerase chain reaction CRT-PCR), Norther n blot analysis, and quantitative RT-PCR. RESULTS. PAF-R mRNA was expressed in RCE, RLE, and RCEn cells and RCKs, but not in RCFs. Alter epithelial injury, PAF-R expression increased from 2.5 to 4 times, both in vitro and in vivo. Addition of cPAF further stimulated PAF-R gene expression in epithelium, which was abolished by PAF; antagonist s. Quantitative RT-PCR revealed that PAF stimulated PAF-R mRNA threefold af ter injury. The induction of PAF-R by its agonist required previous injury and was inhibited by AcD but not by CHX. Treatment of RCE cells with TGF-be ta 1, -beta 2, or -beta 3, HGF, and KGF increased mRNA in PAF-R; however, b FGF had no effect. CONCLUSIONS. Corneal injury produces changes in PAF-R mRNA expression. Wher eas stroma fibroblastic cells lost the PAF-R gene expression found in kerat ocytes, corneal epithelial injury upregulated PAF-R mRNA. These results sug gest that activation of selective growth factors and increases in PAF synth esis after injury stimulate PAF-R gene transcription and constitute importa nt feedback mechanisms needed to maintain the inflammatory process and regu late epithelial wound healing.