Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by platelet-activating factor.
OBJECTIVE
To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing.


METHODS
A 7-mm central de-epithelializing wound was produced in rabbit corneas, and the tissue was incubated with 125 nM carbamyl PAF (cPAF), an analogue of PAF. Rabbit corneal epithelial and stromal cells were also cultured in the presence of cPAF. Cell adhesion, proliferation, and migration assays were conducted. Apoptosis was assayed by TUNEL staining on preparations of corneal tissue sections and in cells in culture.


RESULTS
Twenty-four hours after injury, 50% of the wounded area was covered by new epithelium, whereas only 30% was covered in the presence of cPAF. At 48 hours, the epithelium completely closed the wound, but only 45% of the original wound was covered in corneas treated with cPAF. Similar inhibition of epithelial wound closure was found with human corneas incubated with PAF in organ culture. Moreover, addition of several growth factors involved in corneal wound healing, such as epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, could not overcome the inhibitory action of PAF in wound closure. Three PAF antagonists, BN50727, BN50730, and BN50739, abolished the effect of PAF. A significant increase in TUNEL-positive staining occurred in corneal stromal cells (keratocytes), which was inhibited by preincubating the corneas with PAF antagonists. However, no TUNEL-positive staining was found in epithelial cells. TUNEL-staining results in cultured stromal cells (keratocytes) and epithelial cells in first-passage cell culture were similar to those in organ-cultured corneas. In addition, PAF caused 35% to 56% inhibition of adhesion of epithelial cells to proteins of the extracellular matrix: collagen I and IV, fibronectin, and laminin. There were no significant changes in proliferation or migration of epithelial cells induced by the lipid mediator.


CONCLUSIONS
The results suggest PAF plays an important role in preventing corneal wound healing by affecting adhesion of epithelial cells and increasing apoptosis in stromal cells. PAF antagonists could be of therapeutic importance during prolonged ocular inflammation, helping to avoid loss of corneal transparency and visual acuity.
Chandrasekher
Gudiseva
G
Department of Ophthalmology, Louisiana State University Health Sciences Center, 2020 Gravier Street, New Orleans, LA 70112, USA.
Ma
Xiang
X
Lallier
Thomas E
TE
Bazan
Haydee E P
HE
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000187:drug effects* / Q000473:pathology; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000293:injuries; D005347:Fibroblasts / Q000187:drug effects / Q000473:pathology; D006801:Humans; D020287:In Situ Nick-End Labeling; D009924:Organ Culture Techniques; D010742:Phospholipid Ethers / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D011817:Rabbits; D014945:Wound Healing / Q000187:drug effects*
D010742:Phospholipid Ethers; C053479:1-O-hexadecyl-2-N-methylcarbamylphosphatidylcholine