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J. He, H.E. Bazan; Differential Expression of Platelet-Activating Factor Receptor (PAF-R) in Rabbit Corneal Stromal Cells during Wound Healing . Invest. Ophthalmol. Vis. Sci. 2003;44(13):877.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Corneal keratocytes residing between the collagen lamellae are normally in a quiescent state. After injury, however, keratocytes activate and migrate to the wound site where they differentiate into fibroblasts and myofibroblasts, both of which have an important role in the process of corneal wound healing. Previous studies in our laboratory showed by RT-PCR that PAF-R mRNA is expressed in freshly isolated rabbit corneal keratocytes, but not in fibroblasts (IOVS 2000;41:1696). The present study was to investigate by immunofluorescence staining the expression of PAF-R in cultures of rabbit corneal keratocytes, fibroblasts, and myofibroblasts. Methods: Rabbit corneal keratocytes (RCK) were prepared according to the procedure of Jester et al.(IOVS.1999;40:1959), with some modifications. Rabbit corneal fibroblasts (RCF) were obtained from stromal explant cultures. Rabbit corneal myofibroblasts (RCM) were obtained from subcultured fibroblasts by plating at a low density (1000cells/ml). Immunofluorescence was performed with a PAF-R antibody that recognizes the C-terminus of the receptor. To identify the cell phenotype, anti-vimentin and the anti-smooth muscle α-actin (α-SM), a myofibroblast marker, were used. DAPI was used for nuclear staining. Images were made using MetaVue imaging software. RT-PCR was performed to examine PAF-R mRNA expression. Results: Newly-isolated RCK could be maintained in serum-free DMEM/F12 for more than 1 week without much change in shape. When RCF were plated at low density in DMEM/F12 supplemented with 5% fetal bovine serum (FBS), about 70% of cells transformed into RCM as identified by immunodetection of α-SM. The PAF-R antibody produced positive staining in RCK and RCM, which probably corresponds to an endosomal compartment that interacts with the cell surface. No staining was detected in subcultured RCF of 3-4 passages. RT-PCR confirmed that PAF-R mRNA was expressed in RCK and RCM, but not in the subcultured RCF. Conclusion: The presence of PAF-R in myofibroblasts suggests that PAF may have an important role in regulating the activities of this phenotype of cells during corneal stromal remodeling.
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