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Y.–G. He, B. Zhao, J.S. Owen, J.M. Johnston; Detection of Platelet–Activating Factor in Cultured Human Retinal Pigment Epithelial Cell . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1377.
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A considerable amount of evidence has accrued which would link age–related macular degeneration (AMD) to the inflammatory process. Platelet–activating factor (PAF) is the most potent proinflammatory lipid mediator described and is produced by platelets, macrophages, and endothelial cells. It has been well established that PAF is involved in a number of disease processes, including cardiac infarction, cerebral ischemia, etc. We have therefore hypothesized that PAF may play a pathogenic role in the development of AMD. We have previously reported the expression of PAF receptor in human RPE and choroidal cells. We now examined the presence of PAF in cultured human retinal pigment epithelial (RPE) cells in an attempt to further support the pathogenic role of PAF in AMD.
Human RPE cells were grown in 100 mm dishes to ∼80% confluence, then washed and scraped from the dish. The lipids were extracted using the method of Bligh and Dyer following the addition of 400 pg of deuterated PAF as an internal standard. Aliquots of the lipid extracts were assayed for total phosphorus after perchloric acid digestion. PAF was determined in the lipid extracts by normal–phase HPLC and measured by C18 reversed–phase HPLC with detection by electrospray tandem mass spectrometry (LC–MS/MS) in a multiple–reaction monitoring mode. Three commonly occurring PAF molecular species were determined: 1–O–hexadecyl–, 1–O–octadecyl–, and 1–O–octadecenyl–2–acetyl–sn–glycero–phosphocholine.
Cultured RPE cells contained 15.3 +/– 1.8 pg of PAF per 100 mm dish (mean+/– SEM; n = 5). Normalized to lipid phosphorus, the PAF content was 71.3 +/– 8.5 pg of PAF per micromole of total phospholipid. Consistent with observations in other cell types, the 1–O–hexadecyl molecular species was the most abundant species measured. 1–O–Hexadecyl PAF comprised 77–85% of the total PAF measured; the remainder present was 1–O–octadecyl. The 1–O–octadecenyl species was not present in the lipid extracts in detectable quantities (i.e., <1 pg per sample).
This work demonstrates that RPE cells grown in culture contain PAF. We have previously shown that these cells express the PAF receptor and PAF involves in VEGF regulation in these cells. Based on these findings we suggest that PAF may play an important mediator role in retinal cell physiology and pathology. PAF assay of cultured RPE may provide a useful in vitro experimental model to dissect the molecular basis for the pathogenesis of retinal disorders, such as AMD.
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