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Increased platelet-activating factor receptor gene expression by corneal epithelial wound healing
1Department of Ophthalmology and Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, USA.
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 deepithelialization 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)-beta1, -beta2, and, -beta3, 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-beta1, -beta2, or -beta3, 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 healing.
Insights
Corneal injury increases Platelet Activating Factor Receptor (PAF-R) mRNA in epithelial cells, modulated by growth factors. This suggests a feedback mechanism regulating inflammation and epithelial wound healing.
Area of Science:
- Ophthalmology
- Molecular Biology
- Wound Healing Research
Background:
- Platelet-activating factor (PAF) is a key inflammatory mediator.
- PAF's effects are mediated by Platelet-Activating Factor Receptors (PAF-R).
- PAF synthesis and PAF-R expression are altered in corneal injury.
Purpose of the Study:
- To investigate how wound healing, PAF, and growth factors influence PAF-R mRNA levels in corneal epithelial cells.
- To understand the role of PAF-R in corneal wound repair.
Main Methods:
- Utilized rabbit corneal cells (epithelial, fibroblast, endothelial) and keratocytes.
- Induced corneal injury in vivo and in vitro.
- Stimulated cells with PAF, PAF antagonists, and various growth factors (TGF-β, HGF, KGF, bFGF).
- Assessed PAF-R mRNA expression using RT-PCR and Northern blot analysis.
Main Results:
- PAF-R mRNA was detected in corneal epithelial, limbal, endothelial cells, and keratocytes, but not fibroblasts.
- Corneal injury significantly upregulated PAF-R mRNA (2.5-4 fold) in epithelial cells.
- PAF further increased PAF-R mRNA post-injury, an effect inhibited by PAF antagonists.
- Specific growth factors (TGF-β1, -β2, -β3, HGF, KGF) upregulated PAF-R mRNA in corneal epithelial cells.
Conclusions:
- Corneal injury alters PAF-R mRNA expression, upregulating it in epithelial cells.
- Stromal fibroblasts lose PAF-R expression, while keratocytes retain it.
- Growth factor activation and increased PAF synthesis post-injury contribute to PAF-R gene transcription, supporting inflammatory responses and epithelial wound healing.