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Synergistic effect of platelet-activating factor and tumor necrosis factor-alpha on corneal myofibroblast apoptosis.
Invest Ophthalmol Vis Sci. 2006 Mar; 47(3):883-91.IO

Abstract

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)-alpha 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-alpha-smooth muscle actin antibody was used to identify the cell phenotype. Immunofluorescence was performed to localize PAF and TNF-alpha 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-alpha (20 ng/mL), and TNF-alpha+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-alpha 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-alpha 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-alpha. 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-alpha suggests that during corneal stromal wound healing, PAF acting in conjunction with other cytokines could play an important role in eliminating these cells.

Authors+Show Affiliations

He J
Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center School of Medicine, New Orleans, Louisiana, USA.
Bazan HE
No affiliation info available

MeSH

AnimalsApoptosisBlotting, WesternCell MembraneCells, CulturedCorneaCytoplasmDihydropyridinesDrug CombinationsDrug SynergismFibroblastsFluorescent Antibody Technique, IndirectIn Situ Nick-End LabelingMicroscopy, FluorescenceNuclear EnvelopePlatelet Activating FactorPlatelet Membrane GlycoproteinsReceptors, G-Protein-CoupledReceptors, Tumor Necrosis Factor, Type IReceptors, Tumor Necrosis Factor, Type IISwineTumor Necrosis Factor-alpha

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

16505020

Citation

He, Jiucheng, and Haydee E P. Bazan. "Synergistic Effect of Platelet-activating Factor and Tumor Necrosis Factor-alpha On Corneal Myofibroblast Apoptosis." Investigative Ophthalmology & Visual Science, vol. 47, no. 3, 2006, pp. 883-91.
He J, Bazan HE. Synergistic effect of platelet-activating factor and tumor necrosis factor-alpha on corneal myofibroblast apoptosis. Invest Ophthalmol Vis Sci. 2006;47(3):883-91.
He, J., & Bazan, H. E. (2006). Synergistic effect of platelet-activating factor and tumor necrosis factor-alpha on corneal myofibroblast apoptosis. Investigative Ophthalmology & Visual Science, 47(3), 883-91.
He J, Bazan HE. Synergistic Effect of Platelet-activating Factor and Tumor Necrosis Factor-alpha On Corneal Myofibroblast Apoptosis. Invest Ophthalmol Vis Sci. 2006;47(3):883-91. PubMed PMID: 16505020.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Synergistic effect of platelet-activating factor and tumor necrosis factor-alpha on corneal myofibroblast apoptosis. AU - He,Jiucheng, AU - Bazan,Haydee E P, PY - 2006/3/1/pubmed PY - 2006/4/4/medline PY - 2006/3/1/entrez SP - 883 EP - 91 JF - Investigative ophthalmology & visual science JO - Invest Ophthalmol Vis Sci VL - 47 IS - 3 N2 - 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)-alpha 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-alpha-smooth muscle actin antibody was used to identify the cell phenotype. Immunofluorescence was performed to localize PAF and TNF-alpha 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-alpha (20 ng/mL), and TNF-alpha+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-alpha 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-alpha 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-alpha. 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-alpha suggests that during corneal stromal wound healing, PAF acting in conjunction with other cytokines could play an important role in eliminating these cells. SN - 0146-0404 UR - https://www.unboundmedicine.com/medline/citation/16505020/Synergistic_effect_of_platelet_activating_factor_and_tumor_necrosis_factor_alpha_on_corneal_myofibroblast_apoptosis_ DB - PRIME DP - Unbound Medicine ER -