Effects of prostaglandins and nitric oxide on rat macrophage lipid metabolism in culture: implications for arterial wall-leukocyte interplay in atherosclerosis (original) (raw)
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EXCLI journal, 2015
Oxidized low-density lipoprotein (ox-LDL) is assumed to be a major causal agent in hypercholesteraemia-induced atherosclerosis. Because the proliferation of lipid-loaden macrophages within atherosclerotic lesions has been described, we investigated the dependence of macrophage proliferation on the inhibition of inducible nitric oxide synthase (iNOS) by hypochlorite oxidized LDL. Ox-LDL induces a dose dependent inhibition of inducible nitric oxide synthesis in lipopolysaccharide-interferon stimulated mouse macrophages (J774.A1) with concomitant macrophage proliferation as assayed by cell counting, tritiated-thymidine incorporation and measurement of cell protein. Native LDL did not influence macrophage proliferation and inducible nitric oxide synthesis. iNOS protein and mRNA was reduced by HOCl-oxidized LDL (0-40 µg/ml) as revealed by immunoblotting and competitive semiquantitative PCR. Macrophage proliferation was increased by the addition of the iNOS inhibitor L-NAME. The addition ...
Macrophage activation induces formation of the anti-inflammatory lipid cholesteryl-nitrolinoleate
Biochemical Journal, 2009
Nitroalkene derivatives of fatty acids act as adaptive, antiinflammatory signalling mediators, based on their high-affinity PPARγ (peroxisome-proliferator-activated receptor γ ) ligand activity and electrophilic reactivity with proteins, including transcription factors. Although free or esterified lipid nitroalkene derivatives have been detected in human plasma and urine, their generation by inflammatory stimuli has not been reported. In the present study, we show increased nitration of cholesteryllinoleate by activated murine J774.1 macrophages, yielding the mononitrated nitroalkene CLNO 2 (cholesteryl-nitrolinoleate). CLNO 2 levels were found to increase ∼ 20-fold 24 h after macrophage activation with Escherichia coli lipopolysaccharide plus interferon-γ ; this response was concurrent with an increase in the expression of NOS2 (inducible nitric oxide synthase) and was inhibited by the • NO (nitric oxide) inhibitor L-NAME (N G -nitro-L-arginine methyl ester). Macrophage (J774.1 and bone-marrowderived cells) inflammatory responses were suppressed when activated in the presence of CLNO 2 or LNO 2 (nitrolinoleate). This included: (i) inhibition of NOS2 expression and cytokine secretion through PPARγ and • NO-independent mechanisms; (ii) induction of haem oxygenase-1 expression; and (iii) inhibition of NF-κB (nuclear factor κB) activation. Overall, these results suggest that lipid nitration occurs as part of the response of macrophages to inflammatory stimuli involving NOS2 induction and that these by-products of nitro-oxidative reactions may act as novel adaptive down-regulators of inflammatory responses.
Mediators of Inflammation, 1994
Activated cholesterol-laden macrophages in atherosclerotic lesions are believed to influence the progression of this disease. The induction of nitric oxide synthase (iNOS) activity was investigated in control and cholesterol-laden J774 macrophages, obtained by pre-incubation with oxidized or acetylated low density lipoproteins (oxLDL, acLDL). Loading with oxLDL caused a small induction of NOS activity in unstimulated cells, as indicated by nitrite and citrulline accumulation in the supernatant. However, it suppressed the iNOS activity resulting from stimulation of the cells with lipopolysaccharide with or without interferon-γ. AcLDL had no inhibitory effect, indicating that cholesterol accumulation as such was not responsible. Since the induction of NOS in macrophages is inhibited by glucocorticoids, the possibility that a glucocorticoid-like factor, formed during oxidation of LDL, may cause the inhibition, was investigated. However, addition of the glucocorticoid receptor antagonis...
Journal of Surgical Research, 2008
Background-Omega-3 FA (ω-3 FA) have been demonstrated to have anti-inflammatory properties; postulated to occur through several principal mechanisms, including 1) displacement of arachidonic acid from the cellular membrane, 2) shifting of PGE 2 and LTB 4 production and 3) molecular level alterations including decreased activation of NF-κB and AP-1. An additional regulator that is likely associated is the production of nitric oxide (NO) by nitric oxide synthetase. NO is a short-lived free radical involved in many biological functions. However, excessive NO production can lead to complications, suggesting that decreased NO production is a potential target for some inflammatory diseases. We hypothesized that pretreating with an ω-3 FA lipid emulsion would decrease the production of NO in macrophages and that this effect would occur through alterations in inducible nitric oxide synthetase (iNOS). Methods-Greiss reagent was used to assess NO production in RAW 264.7 macrophages following ω-3 or ω-6 FA treatment alone or in combination with LPS-stimulation for 12 hr/24 hr. iNOS levels were determined by western blot. TNF-alpha levels were determined by ELISA. Results-Following LPS-stimulation, ω-3 FA pretreatment at 12 and 24 hrs produced significantly less NO (p<0.05) compared to ω-6 FA or media-only conditions. ω-3 FA pretreatment at 12 and 24 hrs also had less iNOS protein expression compared to ω-6 FA or media-only conditions. TNF-α production was significantly decreased with ω-3 FA treatment compared to ω-6 FA treatment (p<0.05) after 24 hrs LPS-stimulation.
Fatty acid control of nitric oxide production by macrophages
Febs Letters, 2006
Modulation of macrophage functions by fatty acids 11 (FA) has been studied by several groups, but the effect of FA 12 on nitric oxide production by macrophages has been poorly 13 examined. In the present study the effect of palmitic, stearic, 14 oleic, linoleic, arachidonic, docosahexaenoic and eicosapentae-15 noic acids on NF-jB activity and NO production in J774 cells 16 (a murine macrophage cell line) was investigated. All FA tested 17 stimulated NO production at low doses (1-10 lM) and inhibited 18 it at high doses (50-200 lM). An increase of iNOS expression 19 and activity in J774 cells treated with a low concentration of 20 FA (5 lM) was observed. The activity of NF-jB was time-depen-21 dently enhanced by the FA treatment. The inhibitory effect of FA 22 on NO production may be due to their cytotoxicity, as observed 23 by loss of membrane integrity and/or increase of DNA fragmen-24 tation in cells treated for 48 h with high concentrations. The re-25 sults indicate that, at low concentrations FA increase NO 26 production by J774 cells, whereas at high concentrations they 27 cause cell death. 28
Cardiovascular Research, 2002
Objective: Oxidized low-density lipoproteins (ox-LDL) or their components suppress macrophage inflammatory response by down-regulating cytokine synthesis, nitric oxide synthase and inducible cyclooxygenase (Cox-2). This event is crucial for the pathophysiological process leading to the formation of atherosclerotic plaque. Our present study focused on the mechanisms through which oxidized phospholipids inhibit LPS-induced Cox-2 expression in human macrophages. Methods: Macrophages were incubated with a mixture of oxidized fragmented phospholipids (ox-PAPC), present in modified LDL, and then exposed to LPS. Cox-2 was evaluated in terms of protein levels, mRNA and activity. Results: Ox-PAPC dose-dependently inhibited Cox-2 protein, mRNA and activity by preventing NF-kB binding to DNA. This effect was consequent to alterations of the degradation pattern of IkBa. Moreover, ox-PAPC markedly prevented extracellular signal-regulated kinase (ERK2) activation, leading to Cox-2 expression, whereas activation of the transcription factor peroxisome proliferator-activated receptors (PPARs) was not influenced. Conclusion: ox-PAPC down-regulates LPS-induced Cox-2 expression in human macrophages by targeting both NF-kB/IkB and ERK2 pathways. An altered inflammatory response by macrophages within atheromata may contribute to the progression of atherosclerosis.
Modulation of PGE2 and Tnfα by Nitric Oxide in Resting and LPS-Activated Raw 264.7 Cells
Cytokine, 2002
Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNF play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNF synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNF release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE 2 production, while the LPS-stimulated production was slightly increased only by PAPA/ NO. The modulation of PGE 2 synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules.
The Journal of Immunology, 2001
NO produced by the inducible NO synthase (NOS2) and prostanoids generated by the cyclooxygenase (COX) isoforms and terminal prostanoid synthases are major components of the host innate immune and inflammatory response. Evidence exists that pharmacological manipulation of one pathway could result in cross-modulation of the other, but the sense, amplitude, and relevance of these interactions are controversial, especially in vivo. Administration of 6 mg/kg LPS to rats i.p. resulted 6 h later in induction of NOS2 and the membrane-associated PGE synthase (mPGES) expression, and decreased constitutive COX (COX-1) expression. Low level inducible COX (COX-2) mRNA with absent COX-2 protein expression was observed. The NOS2 inhibitor aminoguanidine (50 and 100 mg/kg i.p.) dose dependently decreased both NO and prostanoid production. The LPS-induced increase in PGE 2 concentration was mediated by NOS2-derived NO-dependent activation of COX-1 pathway and by induction of mPGES. Despite absent COX-2 protein, SC-236, a putative COX-2-specific inhibitor, decreased mPGES RNA expression and PGE 2 concentration. Ketoprofen, a nonspecific COX inhibitor, and SC-236 had no effect on the NOS2 pathway. Our results suggest that in a model of systemic inflammation characterized by the absence of COX-2 protein expression, NOS2-derived NO activates COX-1 pathway, and inhibitors of COX isoforms have no effect on NOS2 or NOS3 (endothelial NOS) pathways. These results could explain, at least in part, the deleterious effects of NOS2 inhibitors in some experimental and clinical settings, and could imply that there is a major conceptual limitation to the use of NOS2 inhibitors during systemic inflammation.