Lyso-PAF, a biologically inactive phospholipid, contributes to RAF1 activation.

Phospholipase A2, group VII (PLA2G7) is widely recognized as a secreted, lipoprotein-associated PLA2 in plasma that converts phospholipid platelet-activating factor (PAF) to a biologically inactive product Lyso-PAF during inflammatory response. We report that intracellular PLA2G7 is selectively important for cell proliferation and tumor growth potential of melanoma cells expressing mutant NRAS, but not cells expressing BRAF V600E. Mechanistically, PLA2G7 signals through its product Lyso-PAF to contribute to RAF1 activation by mutant NRAS, which is bypassed by BRAF V600E. Intracellular Lyso-PAF promotes p21-activated kinase 2 (PAK2) activation by binding to its catalytic domain and altering ATP kinetics, while PAK2 significantly contributes to S338-phosphorylation of RAF1 in addition to PAK1. Furthermore, the PLA2G7-PAK2 axis is also required for full activation of RAF1 in cells stimulated by epidermal growth factor (EGF) or cancer cells expressing mutant KRAS. Thus, PLA2G7 and Lyso-PAF exhibit intracellular signaling functions as key elements of RAS-RAF1 signaling.

Consumption of plant extract supplement reduces platelet activating factor-induced platelet aggregation and increases platelet activating factor catabolism: a randomised, double-blind and placebo-controlled trial.

Platelet-activating factor (PAF) is a potent mediator of inflammation that plays a crucial role in atherosclerosis. The purpose of this study was to evaluate the effect of a dietary supplement containing mainly plant extracts on PAF actions and metabolism in healthy volunteers. A double-blind, placebo-controlled, 8 weeks' duration study was performed. Healthy volunteers were randomly allocated into the supplement or the placebo group and fifty-eight of them completed the study. The supplement contained plant extracts (Aloe gel, grape juice, Polygonum cuspidatum) and vitamins. The activities of PAF metabolic enzymes: the two isoforms of acetyl-CoA:lyso-PAF acetyltransferase, cytidine 5'-diphospho-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-cholinephosphotransferase) and platelet-activating factor-acetylhydrolase (PAF-AH) in leucocytes and lipoprotein associated phospholipase-A2 in plasma were measured along with several markers of endothelial function. Platelet aggregation against PAF, ADP and thrombin receptor activating peptide was measured in human platelet-rich plasma by light transmission aggregometry. No difference was observed on soluble vascular cell adhesion molecule-1, sP-selectin and IL-6 levels at the beginning or during the study period between the two groups. Concerning PAF metabolism enzymes' activity, no difference was observed at baseline between the groups. PAF-AH activity was only increased in the supplement group at 4 and 8 weeks compared with baseline levels. In addition, supplement consumption led to lower platelet sensitivity against PAF and ADP compared with baseline levels. However, a trial effect was only observed when platelets were stimulated by PAF. In conclusion, supplementation with plant extracts and vitamins ameliorates platelet aggregation primarily against PAF and secondarily against ADP and affects PAF catabolism by enhancing PAF-acetylhydrolase activity in healthy subjects.

Platelet Activating Factor (PAF) biosynthesis is inhibited by phenolic compounds in U-937 cells under inflammatory conditions.

Interleukin 1 beta (IL-1ß) induced platelet activating factor (PAF) synthesis in U-937 cells through stimulation of acetyl-CoA:lysoPAF-acetyltransferase (lyso PAF-AT) at 3 h and DTT-independentCDP-choline-1-alkyl-2-acetyl-sn-glycerol cholinophosphotransferase (PAF-CPT) at 0.5 h. The aim of this study was to investigate the effect of tyrosol (T), resveratrol (R) and their acetylated derivatives(AcDs) which exhibit enhanced bioavailability, on PAF synthesis in U-937 after IL-1ß stimulation. The specific activity of PAF enzymes and intracellular levels were measured in cell homogenates. T and R concentration capable of inducing 50% inhibition in IL-1ß effect on lyso PAF-AT was 48 µΜ ± 11 and 157 µΜ ± 77, for PAF-CPT 246 µΜ ± 61 and 294 µΜ ± 102, respectively. The same order of concentration was also observed on inhibiting PAF levels produced by IL-1ß. T was more potent inhibitor than R (p<0.05). AcDs of T retain parent compound inhibitory activity, while in the case of R only two AcDs retain the activity. The observed inhibitory effect by T,R and their AcDs, may partly explain their already reported beneficial role.

Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2.

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.

Upregulation of Platelet-Activating Factor Receptor Expression and Lyso-Platelet-Activating Factor Isoforms in Human Nasal Polyp Tissues.

BACKGROUND: The Platelet-Activating Factor (PAF)/receptor (PAFR) system is involved in asthma and allergic rhinitis; however, its role in chronic rhinosinusitis (CRS) is still unclear. This study aimed to assess the expression of PAFR and the concentration of Lyso-PAF isoforms in the nasal polyps (NP) of patients suffering from CRS with/without comorbidities such as asthma and NSAID-exacerbated respiratory disease (N-ERD) compared to healthy nasal mucosa (NM) from control subjects. METHODS: NM (n = 8) and NP tissues were obtained from patients undergoing surgery for septal deviation/turbinate hypertrophy or endoscopic sinus surgery, respectively. Three phenotypes were studied: CRSwNP with no asthma (n = 6), CRSwNP with non-steroidal anti-inflammatory drug (NSAID)-tolerant asthma (n = 6), and CRSwNP with NSAID-exacerbated respiratory disease (n = 6). PAFR protein and mRNA were assessed via immunochemistry, immunofluorescence, Western blot, and real-time quantitative PCR. Lyso-PAF isoforms (C16, C18, and C18:1) were quantified via mass spectrometry. RESULTS: PAFR protein was expressed in the NM and NP, concretely in epithelial cells and submucosal glands. Compared to NM, PAFR mRNA expression was higher in all NP phenotypes (p < 0.05) while all Lyso-PAF isoform concentrations were higher in the NP from asthmatic patients (p < 0.05). Lyso-PAF C16 and C18 concentrations were higher in the NP from asthmatic patients than in the NP from patients without asthma. CONCLUSIONS: The PAF/PAFR system could play a pathophysiological role in CRSwNP pathogenesis.

Association of PAF and its Metabolic Enzymes with GGT and the Fatty Liver Index in Healthy Volunteers.

BACKGROUND: Platelet-activating-factor (PAF) is a lipid inflammatory mediator implicated in liver disease. Its main biosynthetic enzymes are cytidine diphosphate (CDP)-choline: 1-alkyl-2-acetyl-sn-glycerol-cholinephosphotransferase (PAF-CPT) and acetyl-coenzyme A: lyso-PAF-acetyltransferases (Lyso-PAF-AT). At the same time, PAF acetylhydrolase (PAF-AH) and lipoprotein-associated phospholipase A2 (Lp-PLA2 ) degrade PAF. OBJECTIVE: To explore the relation of PAF metabolism with liver diseases and non-alcoholic fatty liver disease, as reflected by the fatty liver index (FLI). METHODS: In 106 healthy volunteers, PAF concentration, the activity of its metabolic enzymes and gamma-glutamyl transferase (GGT) were measured in whole blood, leukocytes and serum, respectively and the FLI was calculated. Partial correlations and linear regression models were used. RESULTS: In males, serum GGT activity was positively correlated with abdominal fat (as assessed by analysis of a manually defined region of interest in dual-energy X-ray absorptiometry), triacylglycerols, bound-PAF and Lp-PLA2 , while the FLI was positively correlated with Lp-PLA2 activity. In females, serum GGT activity was negatively associated with high-density lipoprotein cholesterol (HDL-C) (age adjusted correlations, all p<0.05). Lp-PLA2 was a significant determinant of serum GGT activity in males after controlling for age, low- density lipoprotein cholesterol (LDL-C) and abdominal fat. The addition of bound-PAF in the model significantly increased the explained variance of serum GGT activity (total variance explanation 30%). CONCLUSION: Bound-PAF and Lp-PLA2 activity predicted serum GGT activity while Lp-PLA2 was also related to FLI. Our findings shed light on the metabolic pathways linking Lp-PLA2 to other atherosclerosis and/or oxidative markers, such as HDL-C, LDL-C, GGT and FLI and underline the important role of PAF.

Alterations in the Plasma Levels of Specific Choline Phospholipids in Alzheimer's Disease Mimic Accelerated Aging.

Alzheimer's disease (AD) is the most common neurodegenerative disease and of continuously rising prevalence. The identification of easy-to-measure biomarkers capable to assist in the prediction and early diagnosis of AD is currently a main research goal. Lipid metabolites in peripheral blood of human patients have recently gained major attention in this respect. Here, we analyzed plasma of 174 participants (not demented at baseline; mean age: 75.70±0.44 years) of the Vienna Transdanube Aging (VITA) study, a longitudinal, population-based birth cohort study, at baseline and after 90 months or at diagnosis of probable AD. We determined the levels of specific choline phospholipids, some of which have been suggested as potential biomarkers for the prediction of AD. Our results show that during normal aging the levels of lysophosphatidylcholine, choline plasmalogen, and lyso-platelet activating factor increase significantly. Notably, we observed similar but more pronounced changes in the group that developed probable AD. Thus, our results imply that, in terms of choline-containing plasma phospholipids, the conversion to AD mimics an accelerated aging process. We conclude that age, even in the comparatively short time frame between 75 and 82.5 years, is a crucial factor in the quest for plasma lipid biomarkers for AD that must be carefully considered in future studies and trials.

Pharmacological inhibition of eicosanoids and platelet-activating factor signaling impairs zymosan-induced release of IL-23 by dendritic cells.

The engagement of the receptors for fungal patterns induces the expression of cytokines, the release of arachidonic acid, and the production of PGE2 in human dendritic cells (DC), but few data are available about other lipid mediators that may modulate DC function. The combined antagonism of leukotriene (LT) B4, cysteinyl-LT, and platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) inhibited IL23A mRNA expression in response to the fungal surrogate zymosan and to a lower extent TNFA (tumor necrosis factor-α) and CSF2 (granulocyte macrophage colony-stimulating factor) mRNA. The combination of lipid mediators and the lipid extract of zymosan-conditioned medium increased the induction of IL23A by LPS (bacterial lipopolysaccharide), thus suggesting that unlike LPS, zymosan elicits the production of mediators at a concentration enough for optimal response. Zymosan induced the release of LTB4, LTE4, 12-hydroxyeicosatetraenoic acid (12-HETE), and PAF C16:0. DC showed a high expression and detectable Ser663 phosphorylation of 5-lipoxygenase in response to zymosan, and a high expression and activity of LPCAT1/2 (lysophosphatidylcholine acyltransferase 1 and 2), the enzymes that incorporate acetate from acetyl-CoA into choline-containing lysophospholipids to produce PAF. Pharmacological modulation of the arachidonic acid cascade and the PAF receptor inhibited the binding of P-71Thr-ATF2 (activating transcription factor 2) to the IL23A promoter, thus mirroring their effects on the expression of IL23A mRNA and IL-23 protein. These results indicate that LTB4, cysteinyl-LT, and PAF, acting through their cognate G protein-coupled receptors, contribute to the phosphorylation of ATF2 and play a central role in IL23A promoter trans-activation and the cytokine signature induced by fungal patterns.

Baseline and 6-Week follow-up levels of PAF and activity of its metabolic enzymes in patients with heart failure and healthy volunteers--a pilot study.

This study aimed at evaluating the changes in platelet-activating factor (PAF) and its metabolic enzymes over a 6-week follow-up period in patients with newly diagnosed heart failure ([HF] n = 12) compared with age-, sex-, and BMI-matched apparently healthy volunteers (n = 10). The PAF, its key biosynthetic enzymes (lyso-PAF acetyltransferase [lyso-PAF-AT] and dithiothreitol [DTT]-insensitive CDP choline: 1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase [PAF-CPT]), and its catabolic isoenzymes (PAF-acetylhydrolase [PAF-AH] and lipoprotein-associated phospholipase A2 [Lp-PLA2]) were measured in serum and leukocytes of participants. At baseline, patients with HF had lower median activities of lyso-PAF-AT (P < .001) and PAF-CPT (P = .07) in parallel with PAF levels (P = .05) and higher activities of PAF-AH (P = .02) and Lp-PLA2 (P < .001) than controls. At follow-up, PAF-CPT and PAF levels marginally increased (P = .1), lyso-PAF-AT (P < .001) remained downregulated, while PAF-AH (P = .004) and Lp-PLA2 (P < .001) remained elevated compared with the controls. Newly diagnosed patients with HF under drug treatment have an affected profile of PAF biosynthetic enzymes and especially lyso-PAF-AT.

A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-induced paw edema in mice.

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase is a key enzyme in the biosynthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) in inflammatory cells. Substances which inhibit this enzyme are of therapeutic interest. In this study, we screened for new inhibitors of lyso-PAF acetyltransferase with anti-inflammatory effects. In a metabolite from Penicillium sp. F33, we isolated an acetyltransferase inhibitor identified as dihydrofumigatin (2-methoxy-1,3,4-trihydroxy-5-methylbenzene) from high resolution mass spectrometer and NMR data. Dihydrofumigatin had strong acetyltransferase inhibitory activity, but was not stable in aqueous solution. Thus, we chemically synthesized its oxidized form fumigatin (3-hydroxy-2-methoxy-5-methyl-1,4-benzoquinone) and derivatives thereof, and evaluated their inhibitory effects. Strong inhibitory activity was observed for saturated fatty acid esters of fumigatin; the order of inhibition was 3-decanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone (termed FUD-7, IC50 = 3 µM)>2-methoxy-5-methyl-3-tetradecanoyloxy-1,4-benzoquinone (termed FUD-8, IC50 = 20 µM)>3-hexanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone (IC50 = 139 µM). Interestingly, these compounds also significantly suppressed the gene expression of lyso-PAF acetyltransferase/LPCAT2 in mouse bone marrow-derived macrophages stimulated by lipopolysaccharide (LPS). We further evaluated the effect of these substances on anti-inflammatory activity in vivo using the carrageenan-induced mouse paw edema test. FUD-7 and FUD-8 at 2.5 mg/kg showed significant, 47.9-51.7%, inhibition stronger than that of prednisolone at 10 mg/kg (41.9%). These results suggest that FUD-7 and FUD-8 are potent inhibitors with anti-inflammatory activity.