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Xiang Ma, Haydee E. P. Bazan; Increased Platelet-Activating Factor Receptor Gene Expression by Corneal Epithelial Wound Healing. Invest. Ophthalmol. Vis. Sci. 2000;41(7):1696-1702.
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purpose. Platelet activating factor (PAF) is a potent inflammatory mediator the
synthesis of which increases in the cornea after injury. The effects of
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 epithelial
(RLE), rabbit corneal fibroblast (RCF), and rabbit corneal
endothelial (RCEn) cells, as well as rabbit corneal keratocytes (RCKs)
were used. For the in vivo wound-healing experiments, a 7-mm central
corneal de-epithelialization was performed in anesthetized rabbits. For
the in vitro experiments, wounded rabbit corneas were maintained in
organ culture. Corneas were stimulated with 120 nM PAF or preincubated
with PAF antagonists, cyclohexamide (CHX) or actinomycin D (AcD) before
adding PAF. RCE cells were stimulated with transforming growth factor
(TGF)-β1, -β2, and, -β3, basic fibroblast growth factor (bFGF),
keratinocyte growth factor (KGF); and hepatocyte growth factor (HGF).
Total RNA was isolated and PAF-R expression evaluated by reverse
transcription–polymerase chain reaction (RT-PCR), Northern blot
analysis, and quantitative RT-PCR.
results. PAF-R mRNA was expressed in RCE, RLE, and RCEn cells and RCKs, but not
in RCFs. After 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 antagonists. Quantitative RT-PCR revealed that PAF stimulated PAF-R
mRNA threefold after 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-β1, -β2, or -β3, HGF, and KGF
increased mRNA in PAF-R; however, bFGF had no effect.
conclusions. Corneal injury produces changes in PAF-R mRNA expression. Whereas
stroma fibroblastic cells lost the PAF-R gene expression found in
keratocytes, corneal epithelial injury upregulated PAF-R mRNA. These
results suggest that activation of selective growth factors and
increases in PAF synthesis after injury stimulate PAF-R gene
transcription and constitute important feedback mechanisms needed to
maintain the inflammatory process and regulate epithelial wound
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