The omega-3 fatty acid docosahexaenoate attenuates endothelial cyclooxygenase-2 induction through both NADP(H) oxidase and PKC epsilon inhibition

Abstract

A high intake of the omega-3 fatty acid docosahexaenoate [docosahexaenoic acid (DHA)] has been associated with systemic antiinflammatory effects and cardiovascular protection. Cyclooxygenase (COX)-2 is responsible for the overproduction of prostaglandins (PG) at inflammatory sites, and its expression is increased in atheroma. We studied the effects of DHA on COX-2 expression and activity in human saphenous vein endothelial cells challenged with proinflammatory stimuli. A>or=24-h exposure to DHA reduced COX-2 expression and activity induced by IL-1, without affecting COX-1 expression. DHA effect depended on the NF-kappaB-binding site in the COX-2 promoter. EMSAs confirmed that DHA attenuated NF-kappaB activation. Because MAPK, PKC, and NAD(P)H oxidase all participate in IL-1-mediated COX-2 expression, we also tested whether these enzymes were involved in DHA effects. Western blots showed that DHA blocked nuclear p65 NF-kappaB subunit translocation by decreasing cytokine-stimulated reactive oxygen species and ERK1/2 activation by effects on both NAD(P)H oxidase and PKCepsilon activities. Finally, to address the question whether DHA itself or DHA-derived products were responsible for these effects, we inhibited the most important enzymes involved in polyunsaturated fatty acid metabolism, showing that 15-lipoxygenase-1 products mediate part of DHA effects. These studies provide a mechanistic basis for antiinflammatory and possibly plaque-stabilizing effects of DHA.

Conflict of interest statement

Conflict of interest statement: R.D.C. has received research support and honoraria for lecturing on omega-3 fatty acids in cardiovascular disease through Pharmacia-Pfizer (Milan, Italy), Società Prodotti Antibiotici (SPA; Milan, Italy), Sigma-Tau (Pomezia, Italy), and Pronova (Lysaker, Norway).

Figures

Fig. 1.

Inhibition of IL-1α- and PMA-mediated induction of COX-2 activity and protein by DHA. (A) HSVEC were preincubated in the absence (vehicle) or presence of 25 μmol/liter DHA for 0–48 h before stimulation with 10 ng/ml IL-1α for 12 h, then medium was collected and 6-ketoPGF1α measured by RIA. 6-ketoPGF1α is shown as percent of maximum response to IL-1α as a function of DHA preincubation time. Each bar represents the mean of eight determinations repeated in three separate experiments. ∗, P < 0.05; ∗∗, P < 0.01 vs. stimulated control. (B) HSVEC were preincubated with DHA, stimulated with 10 PMA for 12 h, then medium was collected and 6-ketoPGF1α measured and expressed as picogram per 1,000 cells for each of two DHA concentrations (25 and 50 μmol/liter). Each bar represents the mean of n = 8 determinations, repeated in three separate experiments. ∗∗, P < 0.01 vs. PMA alone. (C) HSVEC were preincubated with 25 μmol/liter DHA for 48 h, stimulated with 10 ng/ml IL-1α for 12 h, and then whole-cell lysates were analyzed by Western blot using antibodies specific for COX-1 and -2. Values of COX-2 are shown as percent of maximal control response (IL-1α alone). The blot depicted is representative of three similar ones. (D) HSVEC were preincubated with DHA for 48 h, stimulated with 10 nmol/liter PMA for 12 h, and cell lysates prepared and analyzed as in C. The blot depicted is representative of three similar ones. (E) HSVEC were preincubated with 25 μmol/liter for 48 h or 5 μmol/liter DPI, an inhibitor of NAD(P)H oxidase, for 30 min, then stimulated with 10 ng/ml IL-1α for 12 h, after which cells were fixed and immunostained as described in Materials and Methods. For C and D, densitometric values of COX-1 and -2 expression are reported as percent of maximal control response (IL-1α alone).

Fig. 2.

Involvement of ERK1/2 in the stimulated expression of COX-2 and effect of DHA on ERK1/2 activation. (A) HSVEC were treated with MEK1 inhibitor PD 98059 at indicated concentrations for 30 min, then 10 ng/ml IL-1α was added for 12 h. Whole-cell lysates were analyzed by Western blot using an antibody specific for COX-2. Values of COX-2 are reported as percent of maximal control response (IL-1α alone). The blot shown is representative of three similar ones. (B) HSVEC were treated with 10 μmol/liter PD 98059 for 30 min, and then 10 nmol/liter of PMA was added for 12 h. Whole-cell lysates were used for Western blot as in A. The blot depicted is representative of three similar ones. (C) HSVEC were pretreated with 10 μmol/liter PD 98059 for 30 min and then stimulated with 10 ng/ml IL-1α. After 1 h, nuclear proteins were prepared and assayed by Western blot using a specific antibody against the p65 NF-κB subunit. Values of p65 expression are reported as units of OD. The blot shown is representative of three similar ones. (D) HSVEC were pretreated with 25 μmol/liter DHA for 48 h and then stimulated with 10 ng/ml IL-1α for 30 min. Whole-cell lysates were prepared and used for Western blot analysis using a specific antibody against phosphorylated ERK1/2. To ascertain that the total level of the ERK1/2 remained unchanged, the same blots were reprobed with an anti-ERK1/2 antibody that recognizes both phosphorylated and nonphosphorylated forms. Values of ERK1/2 phosphorylation are reported as percent of maximal control response (IL-1α alone). This blot is representative of three similar ones.

Fig. 3.

Effect of DHA on ROS production and p47phox translocation induced by IL-1α. (A) HSVEC were pretreated with DPI, apocynin, or N-acetylcysteine for 30 min before IL-1α stimulation for 12 h. Whole-cell lysates were subjected to Western blot for COX-2. Values of COX-2 are reported as percent of maximal control response (IL-1α alone). The blot shown is representative of three similar ones. (B) HSVEC were pretreated with 25 μmol/ liter DHA for 48 h and then stimulated with 10 ng/ml IL-1α for 20 min. Subcellular fractions (soluble and particulate) were isolated and Western blots performed with an antibody specific for p47phox. Values are in units of OD. The blot depicted is representative of two similar ones. (C) HSVEC were pretreated with 25 μmol/liter DHA for 48 h and then stimulated with 10 ng/ml IL-1α for 1 h. Monolayers were then washed and loaded with reduced dichlorofluorescein for 30 min and imaged as described in Materials and Methods. (Upper) Original microphotographs, where sides of each square are 300 μm long. (Lower) Corresponding pseudocolor transformation of digitalized images, where the yellow color indicates a low generation of ROS, and darker colors indicate increased ROS generation, proportional to color intensity. (D) Quantitative analysis of the effect of DHA on ROS production by IL-1α, as measured by dichlorofluorescein (DCF) fluorescence emission. Subconfluent HSVEC were treated with DHA at 25 and 50 μmol/liter for 48 h in 96-well plates, stimulated with 10 ng/ml IL-1α for 1 h, and finally loaded with reduced DCF. After 30 min, fluorescence was measured with a plate reader as described. More than eight replicates were used for each condition. Results are expressed as arbitrary fluorescence units ± SD. ∗, P < 0.05; ∗∗, P < 0.01 vs. IL-1α-stimulated control. This experiment is representative of a series of four, with similar results.

Fig. 4.

Effects of DHA on PKC isoform translocations. (A) HSVEC were treated with Ro318220 for 30 min before IL-1α stimulation for 12 h. Whole-cell lysates were subjected to Western blot using an antibody specific for COX-2, and the value obtained at densitometric analysis is reported as percent of maximal control response (IL-1α alone). The blot is representative of three similar ones. (B) HSVEC were pretreated with PMA for 24 h before IL-1α stimulation for 12 h to down-regulate PKC activity. Whole-cell lysates were subjected to Western blot using an antibody specific for COX-2, as in A. The blot is representative of three similar ones. (C) HSVEC were pretreated as in B but then stimulated with PMA. The blot is representative of three similar ones. (D) HSVEC were pretreated with 25 μmol/liter DHA for 48 h and then stimulated with 10 nmol/liter PMA for 20 min. Subcellular fractions (soluble and particulate) were isolated, and Western blots were performed using anti-PKCα, -ε, or -ζ antibodies. Values of PKC translocations are reported as units of OD at densitometric analysis. The blot is representative of two similar ones.

Fig. 5.

Proposed molecular model of dietary omega-3 fatty acid interference with IL-1 signaling pathways leading to COX-2 induction in EC. IL-1 binds to the IL-1 receptor type I (IL-1RI), which heterodimerizes with the IL-1 receptor accessory protein (IL-1RAcP). The IL-1R-associated kinases (IRAK) are then recruited and associated by the adapter proteins myeloid differentiation factor(MyD)88 and Toll-interacting protein (Tollip). The signaling pathway also includes the production of ROS (H2O2) through the activation of NAD(P)H oxidase by IRAK activation, as well as the activation of PKC, both contributing to NF-κB activation. DHA, by interfering with the production of ROS (through the inhibition of p47phox translocation and/or the scavenging of ROS by its multiple double bonds), would prevent the formation of H2O2, thus limiting all of the downstream cascade leading to COX-2 gene expression. Furthermore, DHA reduces PKCε activation, thus inhibiting ERK1/2 activation, also leading to NF-κB activation and COX-2 expression. TRAF, TNF receptor-associated factor; TAK-1, TGFβ-activated kinase 1; TAB-2, TAK1-binding protein 2; NIK, NF-κB-inducing kinase; IKK, IκB kinase.