Structural identification of oxidized acyl-phosphatidylcholines that induce platelet activation

Oxidation of low-density lipoprotein (LDL) generates proinflammatory and prothrombotic mediators that may play a crucial role in cardiovascular and inflammatory diseases. In order to study platelet-activating components of oxidized LDL 1-stearoyl-2-arachidonoyl-sn-glycero-3- phosphocholine, a representative of the major phospholipid species in LDL, the 1-acyl-phosphatidylcholines (PC), was oxidized by CuCl2 and H2O2. After separation by high-performance liquid chromatography, three compounds were detected which induced platelet shape change at low micromolar concentrations. Platelet activation by these compounds was distinct from the pathways stimulated by platelet-activating factor, lysophosphatidic acid, lyso-PC and thromboxane A(2), as evidenced by the use of specific receptor antagonists. Further analyses of the oxidized phospholipids by electrospray ionization mass spectrometry structurally identified them as 1-stearoyl-2-azelaoyl-sn-glycero-3-phosphocholine (m/z 694; SAzPC), 1-stearoyl-2-glutaroyl-snglycero-3- phosphocholine (m/z 638; SGPC), and 1-stearoyl-2-( 5-oxovaleroyl)-sn-glycero-3-phosphocholine (m/z 622; SOVPC). These observations demonstrate that novel 1-acyl-PC which had previously been found to stimulate interaction of monocytes with endothelial cells also induce platelet activation, a central step in acute thrombogenic and atherogenic processes. Copyright (C) 2005 S. Karger AG, Basel

Generation and dietary modulation of anti-inflammatory electrophilic omega-3 fatty acid derivatives

Dietary ω-3 polyunsaturated fatty acids (PUFAs) decrease cardiovascular risk via suppression of inflammation. The generation of electrophilic α,β-unsaturated ketone derivatives of the ω-3 PUFAs docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) in activated human macrophages is catalyzed by cyclooxygenase-2 (Cox-2). These derivatives are potent pleiotropic anti-inflammatory signaling mediators that act via mechanisms including the activation of Nrf2- dependent phase 2 gene expression and suppression of pro-inflammatory NF-κB-driven gene expression. Herein, the endogenous generation of ω-3 PUFAs electrophilic ketone derivatives and their hydroxy precursors was evaluated in human neutrophils. In addition, their dietary modulation was assessed through a randomized clinical trial. Methods: Endogenous generation of electrophilic omega-3 PUFAs and their hydroxy precursors was evaluated by mass spectrometry in neutrophils isolated from healthy subjects, both at baseline and upon stimulation with calcium ionophore. For the clinical trial, participants were healthy adults 30-55 years of age with a reported EPA+DHA consumption of ≤ 300 mg/day randomly assigned to parallel groups receiving daily oil capsule supplements for a period of 4 months containing either 1.4 g of EPA+DHA (active condition, n = 24) or identical appearing soybean oil (control condition, n = 21). Participants and laboratory technicians remained blinded to treatment assignments. Results: 5-lypoxygenase-dependent endogenous generation of 7-oxo-DHA, 7-oxo-DPA and 5-oxo-EPA and their hydroxy precursors is reported in human neutrophils stimulated with calcium ionophore and phorbol 12-myristate 13-acetate (PMA). Dietary EPA+DHA supplementation significantly increased the formation of 7-oxo-DHA and 5-oxo-EPA, with no significant modulation of arachidonic acid (AA) metabolite levels. Conclusions: The endogenous detection of these electro.©2014 Cipollina et al

Impact of Anti-Inflammatory Agents on the Gene Expression Profile of Stimulated Human Neutrophils: Unraveling Endogenous Resolution Pathways

Adenosine, prostaglandin E2, or increased intracellular cyclic AMP concentration each elicit potent anti-inflammatory events in human neutrophils by inhibiting functions such as phagocytosis, superoxide production, adhesion and cytokine release. However, the endogenous molecular pathways mediating these actions are poorly understood. In the present study, we examined their impact on the gene expression profile of stimulated neutrophils. Purified blood neutrophils from healthy donors were stimulated with a cocktail of inflammatory agonists in the presence of at least one of the following anti-inflammatory agents: adenosine A2A receptor agonist CGS 21680, prostaglandin E2, cyclic-AMP-elevating compounds forskolin and RO 20-1724. Total RNA was analyzed using gene chips and real-time PCR. Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression. We identified 15 genes for which the anti-inflammatory agents altered mRNA levels. The agents affected the expression profile in remarkably similar fashion, suggesting a central mechanism limiting cell activation. We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation. Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution