Omega-3 fatty acid (FA) emulsions reduce LPS-stimulated murine macrophage TNF-alpha production, but the exact mechanism has yet to be defined. The purpose of this study was to determine the mechanism for omega-3 FA inhibition of macrophage TNF-alpha production following LPS stimulation. RAW 264.7 cells were pretreated with isocaloric emulsions of omega-3 FA (Omegaven), omega-6 FA (Lipovenos), or DMEM and subsequently exposed to LPS. IkappaB-alpha and phospho-IkappaB-alpha were determined by Western blotting. NF-kappaB binding was assessed using the electromobility shift assay, and activity was measured using a luciferase reporter vector. RT-PCR and ELISA quantified TNF-alpha mRNA and protein levels, respectively. Pretreatment with omega-3 FA inhibited IkappaB phosphorylation and significantly decreased NF-kappaB activity. Moreover, omega-3-treated cells demonstrated significant decreases in both TNF-alpha mRNA and protein expression by 47 and 46%, respectively. These experiments demonstrate that a mechanism for proinflammatory cytokine inhibition in murine macrophages by omega-3 FA is mediated, in part, through inactivation of the NF-kappaB signal transduction pathway secondary to inhibition of IkappaB phosphorylation.

Abstract-Angiopoietin-1 (Ang1) exerts a vascular endothelial barrier protective effect by blocking the action of permeability-increasing mediators such as vascular endothelial growth factor (VEGF) through unclear mechanisms. Because VEGF may signal endothelial hyperpermeability through the phospholipase C (PLC)-IP 3 pathway that activates extracellular Ca 2ϩ entry via the plasmalemmal store-operated channel transient receptor potential canonical-1 (TRPC1), we addressed the possibility that Ang1 acts by inhibiting this Ca 2ϩ entry mechanism in endothelial cells. Studies in endothelial cell monolayers demonstrated that Ang1 inhibited the VEGF-induced Ca 2ϩ influx and increase in endothelial permeability in a concentration-dependent manner. Inhibitors of the PLC-IP 3 Ca 2ϩ signaling pathway prevented the VEGF-induced Ca 2ϩ influx and hyperpermeability similar to the inhibitory effects seen with Ang1. Ang1 had no effect on PLC phosphorylation and IP 3 production, thus its permeability-decreasing effect could not be ascribed to inhibition of PLC activation. However, Ang1 interfered with downstream IP 3 -dependent plasmalemmal Ca 2ϩ entry without affecting the release of intracellular Ca 2ϩ stores. Anti-TRPC1 antibody inhibited the VEGF-induced Ca 2ϩ entry and the increased endothelial permeability. TRPC1 overexpression in endothelial cells augmented the VEGF-induced Ca

No abstract

Omega-3 fatty acids (FAs), which include eicosapentaenoic acid (EPA) and docosahexaenoic acid, are found in fish oils and have long been investigated as components of therapy for various disease states. Population studies initially revealed the cardioprotective and anti-inflammatory effects of omega-3 FAs and EPA, with subsequent clinical studies supporting the therapeutic role of omega-3 FAs in cardiovascular and chronic inflammatory conditions. Prospective randomized placebo-controlled trials have also demonstrated the utility of omega-3 FA supplementation in malignancy and cancer cachexia. In recent years, in vitro and animal studies have elucidated some of the mechanistic explanations underlying the wide range of biological effects produced by omega-3 FAs and EPA, including their antiproliferative and anticachectic actions in malignancy. In this review, the authors discuss the recent progress made with omega-3 FAs, focusing on the advances in mechanistic understanding and the results of clinical trials.

NO is a crucial mediator of the inflammatory response, but its in vivo role as a determinant of lung inflammation remains unclear. We addressed the in vivo role of NO in regulating the activation of NF-κB and expression of inflammatory proteins using an in vivo mouse model of sepsis induced by i.p. injection of Escherichia coli. We observed time-dependent degradation of IκB and activation of NF-κB accompanied by increases in inducible NOS, macrophage inflammatory protein-2 (MIP-2), and ICAM-1 expression after E. coli challenge, which paralleled the ability of lung tissue to produce high-output NO. To determine the role of NO in this process, mice were pretreated with the NO synthase (NOS) inhibitor NG-methyl-l-arginine. Despite having relatively modest effects on NF-κB activation and ICAM-1 or inducible NOS expression, the NOS inhibitor almost completely inhibited expression of MIP-2 in response to E. coli challenge. These responses were associated with the inhibition of migration of neutrophils in lung tissue and increased permeability induced by E. coli. In mice pretreated with NG-methyl-l-arginine, coadministration of E. coli with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate substantially restored MIP-2 expression but decreased ICAM-1 expression. The results suggest that NO generated after administration of E. coli serves as an important proinflammatory signal to up-regulate MIP-2 expression in vivo. Thus, NO production in high quantities may be important in the mechanism of amplification of the lung inflammatory response associated with sepsis.