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M. Opreanu, T. A. Lydic, W. J. Esselman, J. V. Busik; Docosahexaenoic Acid (dha 22:6n3) Increases Sphingomyelin Content of Caveolae/ Lipid Rafts Isolated From Human Retinal Vascular Endothelial (hrve) Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3982. doi: https://doi.org/.
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Cholesterol and sphingolipids are two major components of the caveolae/lipid rafts. We have previously demonstrated that docosahexaenoic acid reduces cholesterol content in the caveolae/lipid rafts and thus has an anti-inflammatory effect on human retinal vascular endothelial (hRVE) cells. The purpose of this study was to determine the effect of DHA on sphingolipid content of the caveolae/lipid rafts isolated from hRVE cells.
Acid and neutral sphingomyelinase (ASMase and NSMase) activity was determined by sphingomyelinase assay. Methyl β- Cyclodextrin (MCD) was used for cholesterol depletion. The expression levels of ICAM-1 and VCAM-1 were analyzed by Western Blotting. Gene silencing of ASMase and NSMase was achieved by siRNA treatment. Lipids extracted from fatty acids treated hRVE cells were assayed by electrospray ionization mass spectrometry (MS) and MS/MS.
We have previously shown that DHA decreases ASMase and NSMase activity in hRVE cells. Gene silencing of both ASMase and NSMase reduces adhesion molecules expression in TNF α stimulated hRVE cells. MCD treatement of hRVE cells led to a decrease in cholesterol content of the caveolae/lipid rafts by 50% compared to control, but not to the dissociation of these microdomains. Cholesterol depletion of hRVE cells decreased neutral sphingomyelinase activity by 49% and cholesterol replenishment in DHA treated hRVE cells reversed DHA- induced reduction of NSMase activity. hRVE cells treated with DHA exhibited a 1.3 fold increase in sphingomyelin content in caveolae/lipid rafts compared to control.
DHA might exert its anti-inflammatory effect by displacing cholesterol and promoting formation of sphingomyelin rich caveolae/lipid rafts.
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