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.