Ample experimental evidence suggests that early diabetic retinopathy is a low-grade chronic inflammatory disease.49-56
Under diabetic conditions, retinal endothelial cells were shown to be activated to produce adhesion molecules such as ICAM-1 and VCAM-1. Adhesion molecules interact with their counterpart receptors on activated leukocytes to govern the adherence and transmigration of leukocytes to the endothelium and to mediate inflammation. Although n-3 PUFAs suppress cytokine induced adhesion molecule expression in endothelial cells,48,57-61
the functional mechanism for this suppression is not well understood. Our results demonstrate that the anti-inflammatory effect of the most abundant n-3 PUFA in the retina, DHA22:6,n3
, on retinal endothelial cells may be mediated through modification of the important membrane signaling microdomains, caveolae/lipid rafts.
Caveolae/lipids rafts are important in mediating cytokine-induced inflammatory signaling.31,62-66
Several signaling molecules implicated in the development of diabetic retinopathy, such as VEGFR2,27,28,67
have been shown to localize to the caveolae/lipid raft fractions in various cell types, including endothelial cells. Diabetes and hypertension lead to increased caveolin-1 expression in retinal pericytes and endothelial cells.69
Moreover, hyperglycemia has been shown to induce caveolin-1 expression in a bovine aortic endothelial cell model.70
We have previously demonstrated that DHA22:6,n3
inhibits IL-1β–, TNF-α–, and VEGF-induced inflammatory signaling through inhibition of the NF-κB pathway upstream of IκBα phosphorylation and degradation in hRVE cells.48
The data provided in this study show that the lipid integrity of caveolae/lipid rafts is related to the intactness of cytokine-induced NF-κB activation (). Thus, DHA22:6,n3
could oppose hyperglycemia-induced proinflammatory changes by modifying caveolae/lipid raft structure and function in the diabetic retina.
The Src family kinase inhibitor PP2 inhibited the cytokine-induced expression of adhesion molecules, implicating the involvement of Src kinases in cytokine-induced inflammatory signaling. In fact, a growing body of literature has demonstrated the requirement of SFKs in TNF receptor–mediated34,35
and IL-1β receptor–mediated36,37
inflammatory signaling. The recruitment of specific SFKs to VEGFR2 on VEGF binding was also documented in several cell types.38-41
Although the detailed evaluation of the role of Src family kinases in cytokine signaling is beyond the scope of this study, we used Src family kinases as a model to study the effect of DHA22:6,n3
on signaling components of the caveolae/lipid rafts in hRVE cells.
In agreement with previous studies, dual-acylated SFKs such as Fyn and c-Yes were localized to the caveolae/lipid raft fraction. c-Srk itself, which is only myristoylated because it is missing the cysteine3
palmitoylation site, was excluded from caveolae/lipid rafts.32,33
Thus, the highly ordered lipid structure of the caveolae/raft and the posttranslational modification of proteins that direct protein into or out of rafts are likely important regulatory features controlling signal transduction. In this study, we demonstrate that DHA22:6,n3
enrichment in hRVE cells is associated with displacement of SFKs Fyn and c-Yes from caveolae/lipid rafts.
Altered lipid environment and altered protein acylation are two possible routes by which PUFA can affect protein targeting to lipid rafts. In hRVE cells, DHA22:6,n3
treatment resulted in its incorporation into phospholipids in the caveolae/lipid rafts. These data are in agreement with reports in T lymphocytes, suggesting that the substitution of fatty acyl chains by PUFA in phospholipids of lipid rafts could change the lipid environment and thus affect the association of proteins dually acylated by saturated fatty acids.9,10,13
Moreover, in vivo feeding studies demonstrated increased n-3 PUFAs in the lipid rafts from T lymphocytes and colons of mice fed a diet enriched in n-3 PUFAs collectively with displacement of important signaling molecules, such as Ras, caveolin-1, and eNOS.5,71,72
incorporated primarily into the phosphatidylcholine fraction of phospholipids in hRVE cells. This is different from the reports in T cells showing EPA20:5n3
incorporating into phosphatidylcholine and phosphatidylethanolamine,9,73
underscoring the difference in lipid metabolism between retinal vascular endothelial cells and other cell types. Substitution of phospholipid fatty acyl chains by DHA22:6,n3
in total plasma membranes was much higher than in caveolae/lipid rafts, suggesting that cells tend to maintain the higher lipid-ordered structure of caveolae/lipid rafts by maintaining a saturated fatty acyl environment. Nevertheless, incorporation of DHA22:6,n3
resulted in a considerable (35%) increase in unsaturation in acyl chains of neutral phospholipids in caveolae/lipid rafts of DHA22:6,n3
-treated hRVE cells ().
The enrichment of DHA22:6,n3
in raft phospholipids is associated with a decline in arachidonic acid20:4,n6
(). When released from membranes, arachidonic acid20:4,n6
serves as a precursor of inflammatory mediators, including leukotrienes, thromboxanes, and prostaglandins, and other bioactive lipid mediators, such as hydroxy and epoxy fatty acids.74-76
Our previous studies indicated that treatment of hRVE cells with arachidonic acid20:4,n6
leads to a lipoxygenase-dependent increase in ICAM/VCAM expression.43
This effect of arachidonic acid20:4,n6
was observed after 12-hour treatment of hRVE cells. The fact that the effects of cholesterol depletion/replenishment on NF-κB signaling and adhesion molecule expression are obvious in 30 minutes argues against a decline in arachidonic acid20:4,n6
as a primary mechanism for the inhibitory effect of arachidonic acid20:4,n6
on caveolae/lipid raft signaling. However, a reduction of arachidonic acid20:4,n6
phospholipid content by DHA22:6,n3
could represent an additional mechanism of anti-inflammatory effect of DHA22:6,n3
in hRVE cells.
Enrichment of phospholipids with DHA22:6,n3 dramatically altered the lipid environment in caveolae/lipid rafts. This is particularly evident in the 70% decrease in caveolae/lipid raft–associated cholesterol. The effect of DHA22:6,n3 on raft cholesterol is specific because significant changes in the overall plasma membrane cholesterol content were not detected (). Cholesterol depletion using MCD mimicked the inhibitory effects on DHA22:6,n3 on cytokine-induced IκBα phosphorylation and ICAM-1 expression. Moreover, cholesterol replenishment reversed the inhibitory effect of DHA22:6,n3 on cytokine signaling in hRVE cells.
Cholesterol is the major structural lipid in caveolae/lipid rafts that is required to maintain the ordered state of the raft membrane. Cholesterol sterol rings tightly bind the ceramide moiety of sphingomyelin in the exoplasmic leaflet of caveolae/lipid rafts, promoting caveolae/lipid raft lateral separation.77-79
In the cytoplasmic leaflet that lacks sphingolipids, the interaction of phospholipid-saturated acyl chains with cholesterol is required to maintain organization of caveolae/lipid rafts in a liquid-ordered phase.77,80
Cholesterol interacts differentially with different membrane lipids and has a particularly strong association with saturated phospholipids and sphingolipids and a weak association with highly unsaturated lipid species (for a review, see Silvius21
). Thus, substitutions of acyl chains of the phospholipids by DHA22:6,n3
could dramatically affect cholesterol interaction and cause a decreased lipid order in the caveolae/lipid rafts. The mechanism of specific cholesterol depletion in caveolae/lipid rafts induced by DHA22:6,n3
enrichment is unclear. It may involve a spontaneous redistribution between membranes because of changes of lipid environment in caveolae/lipid rafts.21
Decreases in the sphingomyelin content have also been reported in lipid rafts isolated from T lymphocytes and the colons of mice fed a diet enriched in n3-PUFA.5,71,72
Treating cells with sphingomyelinase, which cleaves the phosphorylcholine head group of sphingomyelin, leading to ceramide production, causes a significant displacement of cholesterol from lipid raft membranes and a modification of lipid raft signaling.81-83
was shown to activate neutral sphingomyelinase in cultured MDA-MB-231 breast cancer cells,84
the activation of sphingomyelinase followed by ceramide production could represent a potential mechanism for the DHA22:6,n3
-induced cholesterol depletion from caveolae/lipid raft reported here. Sphingomyelinase activity and sphingomyelin and ceramide content were not analyzed in this article and will be the focus of future study.
As reported in Cos-1 cells, Fyn can be acylated by fatty acids other than myristate and palmitate.7
Acylation of Fyn by unsaturated fatty acids caused the displacement of Fyn from membrane rafts in Cos-1 cells.7
Determining whether SFKs can be acylated by DHA22:6,n3
, which would provide an additional mechanism for selective displacement of Fyn and c-Yes from caveolae/lipid rafts in endothelial cells, is beyond the scope of this study.
In conclusion, we have characterized the involvement of caveolae/lipid rafts in mediating cytokine-induced proinflammatory signaling in primary cultures of retinal vascular endothelial cells. The modification of phospholipids residing in the caveolae/lipid rafts and the depletion of caveolae/lipid raft-specific cholesterol provide a compelling model to explain the molecular mechanism of DHA22:6,n3-induced displacement of Src family kinases and thus the immunomodulatory effect of DHA22:6,n3 on endothelial cells.