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 ≥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-κB-binding site in the COX-2 promoter. EMSAs confirmed that DHA attenuated NF-κB 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-κB subunit translocation by decreasing cytokine-stimulated reactive oxygen species and ERK1/2 activation by effects on both NAD(P)H oxidase and PKCε 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

Essential thrombocythemia (ET) is characterized by enhanced platelet generation and thrombotic complications. Once-daily low-dose aspirin incompletely inhibits platelet thromboxane A₂ (TXA₂) in the majority of ET patients. In the present study, we investigated the determinants of aspirin-insensitive platelet TXA₂ biosynthesis and whether it could be further suppressed by changing the aspirin dose, formulation, or dosing interval. In 41 aspirin-treated ET patients, the immature platelet count predicted serum TXB₂ independently of platelet count, age, JAK-2 V617F mutation, or cytoreduction (β = 3.53, P = .001). Twenty-one aspirin-treated patients with serum TXB₂ ≥ 4 ng/mL at 24 hours after dosing were randomized to the following 7-day regimens in a crossover design: enteric-coated aspirin 100 mg twice daily, enteric-coated aspirin 200 mg once daily, or plain aspirin 100 mg once daily. A twice-daily regimen caused a further 88% median (IQR, 78%-92%, P < .001) TXB₂ reduction and normalized the functional platelet response to aspirin, as assessed by urinary 11-dehydro-TXB₂ excretion and the VerifyNow Aspirin assay. Doubling the aspirin dose reduced serum TXB₂ only partially by 39% median (IQR, 29%-54%, P < .05). We conclude that the abnormal megakaryopoiesis characterizing ET accounts for a shorter-lasting antiplatelet effect of low-dose aspirin through faster renewal of platelet cyclooxygenase-1, and impaired platelet inhibition can be rescued by modulating the aspirin dosing interval rather than the dose.

To examine the role of cyclooxygenase (COX) isozymes in prostaglandin formation and oxidant stress in inflammation, we administered to volunteer subjects placebo or bolus injections of lipopolysaccharide (LPS), which caused a dose-dependent increase in temperature, heart rate, and plasma cortisol. LPS caused also dose-dependent elevations in urinary excretion of 2,3-dinor 6-keto PGF_1α (PGI-M) and 11-dehydro thromboxane B₂ (Tx-M). Platelet COX-1 inhibition by chronic administration of low-dose aspirin before LPS did not alter the symptomatic and febrile responses to LPS, but the increment in urinary PGI-M and Tx-M were both partially depressed. Pretreatment with ibuprofen, a nonspecific COX inhibitor, attenuated the febrile and systemic response to LPS and inhibited prostanoid biosynthesis. Both celecoxib, a selective COX-2 inhibitor, and ibuprofen attenuated the pyrexial, but not the chronotropic, response to LPS. Experimental endotoxemia caused differential expression of the COX isozymes in monocytes and polymorphonuclear leucocytes ex vivo. LPS also increased urinary iPF_2α-III, iPF_2α-VI, and 8,12-iso-iPF_2α-VI, isoprostane (iP) indices of lipid peroxidation, and none of the drugs blunted this response. These studies indicate that (a) although COX-2 predominates, both COX isozymes are induced and contribute to the prostaglandin response to LPS in humans; (b) COX activation contributes undetectably to lipid peroxidation induced by LPS; and (c) COX-2, but not COX-1, contributes to the constitutional response to LPS in humans.

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype and proliferate and synthesize fibrosis components. Endothelin-1 (ET-1), which inhibited the growth of human myofibroblastic HSC, increased the formation of two NF-kappaB DNA binding complexes; this effect was also observed with tumor necrosis factor-alpha (TNF-alpha). The complexes were identified as the p50/p50 and p50/p65 NF-kappaB dimers. Activation of NF-kappaB was associated with the degradation of the inhibitory protein IkappaB-alpha; no IkappaB-beta was detected. Activation of NF-kappaB and degradation of IkappaB-alpha were prevented by the NF-kappaB inhibitors sodium salicylate and MG-132. In addition to cyclooxygenase-1 (COX-1), COX-2 is also constitutively expressed in human HSC, and the use of dexamethasone and of SC-58125, a selective COX-2 inhibitor, revealed that COX-2 accounts for basal COX activity. Moreover, COX-2 mRNA and protein were up-regulated by ET-1 and TNF-alpha, whereas COX-1 was unaffected. Induction of COX-2 and stimulation of COX activity by ET-1 and TNF-alpha were prevented by sodium salicylate and MG-132, suggesting that activation of NF-kappaB by either factor is needed for stimulation of COX-2. Finally, SC-58125 and dexamethasone reduced the growth inhibitory effect of ET-1 and TNF-alpha, indicating that activation of COX-2 is required for inhibition of HSC proliferation. Taken together, our results suggest that NF-kappaB, by inducing COX-2 expression, may play an important role in the negative regulation of human myofibroblastic HSC proliferation.