HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by He, J. Articles by Bazan, H. E. P. Search for Related Content PubMed PubMed Citation Articles by He, J. Articles by Bazan, H. E. P.

Synergistic Effect of Platelet-Activating Factor and Tumor Necrosis Factor- on Corneal Myofibroblast Apoptosis

From the Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center School of Medicine, New Orleans, Louisiana.

PURPOSE. Elimination of myofibroblasts after repair of corneal injury is essential for the maintenance of corneal transparency. In the current study, the role of platelet-activating factor (PAF) in combination with tumor necrosis factor (TNF)- in corneal myofibroblast apoptosis was explored.

METHODS. Porcine corneal myofibroblasts (PCMs) were obtained from subcultured fibroblasts plated at a low density (5 cells/mm²). Mouse anti--smooth muscle actin antibody was used to identify the cell phenotype. Immunofluorescence was performed to localize PAF and TNF- receptors in those cells. The reactivity of the antibodies was characterized by Western blot analysis. To induce myofibroblast apoptosis, PCMs were treated for 24 to 72 hours with methylcarbamyl-PAF (cPAF, 300 nM), a nonhydrolyzable PAF analogue, TNF- (20 ng/mL), and TNF-+cPAF, with or without LAU-0901 (150 nM), a novel PAF antagonist. Apoptosis was assayed by Hoechst 33258 and TUNEL staining and DNA laddering. 6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Images were recorded by fluorescence microscope.

RESULTS. Immunofluorescence with a PAF-receptor (N terminus) polyclonal antibody showed that the receptor was expressed in both plasma and nuclear membranes of myofibroblasts. TNF- receptor II (TNF-RII) was localized in the cytoplasm, whereas TNF-receptor I (TNF-RI) was found in both cytoplasm and plasma membrane. Treatment with TNF- for 24, 48, and 72 hours induced apoptosis in 18%, 24%, and 32%, respectively, of the myofibroblasts. Western blot analysis showed expression of single bands corresponding to the molecular weights of the receptors. Treatment with cPAF induced apoptosis in 10%, 18%, and 26% of the cells, respectively. However, treatment with both cytokines induced apoptosis in 42%, 78%, and 86%, respectively, of the cells, demonstrating a synergistic action between PAF and TNF-. Blocking the PAF receptor with LAU-0901 inhibited the synergistic effect induced by PAF.

CONCLUSIONS. Corneal myofibroblasts express a PAF receptor in the nuclear membrane, and they also express TNF-RI and RII. The synergistic effect on myofibroblast apoptosis by PAF and TNF- suggests that during corneal stromal wound healing, PAF acting in conjunction with other cytokines could play an important role in eliminating these cells.

This article has been cited by other articles:

				J. He and H. E. P. Bazan Epidermal Growth Factor Synergism with TGF-{beta}1 via PI-3 Kinase Activity in Corneal Keratocyte Differentiation Invest. Ophthalmol. Vis. Sci., July 1, 2008; 49(7): 2936 - 2945. [Abstract] [Full Text] [PDF]

				C. Ramos, M. Montano, C. Becerril, J. Cisneros-Lira, L. Barrera, V. Ruiz, A. Pardo, and M. Selman Acidic fibroblast growth factor decreases {alpha}-smooth muscle actin expression and induces apoptosis in human normal lung fibroblasts Am J Physiol Lung Cell Mol Physiol, November 1, 2006; 291(5): L871 - L879. [Abstract] [Full Text] [PDF]

				D. Reigada, W. Lu, and C. H. Mitchell Glutamate acts at NMDA receptors on fresh bovine and on cultured human retinal pigment epithelial cells to trigger release of ATP J. Physiol., September 15, 2006; 575(3): 707 - 720. [Abstract] [Full Text] [PDF]