Inhibition of cytokine signaling in human retinal endothelial cells through modification of caveolae/lipid rafts by docosahexaenoic acid

PURPOSE. Docosahexaenoic acid (DHA(22:6,n3)) is the principal n3 polyunsaturated fatty acid (PUFA) in the retina. The authors previously demonstrated that DHA(22: 6,n3) inhibited cytokine-induced adhesion molecule expression in primary human retinal vascular endothelial (hRVE) cells, the target tissue affected by diabetic retinopathy. Despite the importance of vascular inflammation in diabetic retinopathy, the mechanisms underlying anti-inflammatory effects of DHA(22:6,n3) in vascular endothelial cells are not understood. In this study the authors address the hypothesis that DHA(22:6,n3) acts through modifying lipid composition of caveolae/lipid rafts, thereby changing the outcome of important signaling events in these specialized plasma membrane microdomains. METHODS. hRVE cells were cultured in the presence or absence of DHA(22:6,n3). Isolated caveolae/lipid raft-enriched detergent-resistant membrane domains were prepared using sucrose gradient ultracentrifugation. Fatty acid composition and cholesterol content of caveolae/lipid rafts before and after treatment were measured by HPLC. The expression of Src family kinases was assayed by Western blotting and immunohistochemistry. RESULTS. Disruption of the caveolae/lipid raft structure with a cholesterol-depleting agent, methyl-cyclodextrin (MCD), diminished cytokine-induced signaling in hRVE cells. Growth of hRVE cells in media enriched in DHA(22:6,n3) resulted in significant incorporation of DHA(22:6,n3) into the major phospholipids of caveolae/lipid rafts, causing an increase in the unsaturation index in the membrane microdomain. DHA(22:6,n3) enrichment in the caveolae/raft was accompanied by a 70% depletion of cholesterol from caveolae/lipid rafts and displacement of the SFK, Fyn, and c-Yes from caveolae/lipid rafts. Adding water-soluble cholesterol to DHA(22:6,n3)-treated cells replenished cholesterol in caveolae/lipid rafts and reversed the effect of DHA(22:6,n3) on cytokine-induced signaling. CONCLUSIONS. Incorporation of DHA(22:6,n3) into fatty acyl chains of phospholipids in caveolae/lipid rafts, followed by cholesterol depletion and displacement of important signaling molecules, provides a potential mechanism for anti-inflammatory effect of DHA(22:6,n3) in hRVE cells.