lyso-PAF AT Research Articles

Consumption of Enriched Yogurt with PAF Inhibitors from Olive Pomace Affects the Major Enzymes of PAF Metabolism: A Randomized, Double Blind, Three Arm Trial

Platelet-activating factor (PAF), a proinflammatory lipid mediator, plays a crucial role in the formation of the atherosclerotic plaque. Therefore, the inhibition of endothelium inflammation by nutraceuticals, such as PAF inhibitors, is a promising alternative for preventing cardiovascular diseases. The aim of the present study was to evaluate the impact of a new functional yogurt enriched with PAF inhibitors of natural origin from olive oil by-products on PAF metabolism. Ninety-two apparently healthy, but mainly overweight volunteers (35–65 years) were randomly allocated into three groups by block-randomization. The activities of PAF’s biosynthetic and catabolic enzymes were measured, specifically two isoforms of acetyl-CoA:lyso-PAF acetyltransferase (LPCATs), cytidine 5′-diphospho-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT) and two isoforms of platelet activating factor acetylhydrolase in leucocytes (PAF-AH) and plasma (lipoprotein associated phospholipase-A2, LpPLA2). The intake of the enriched yogurt resulted in reduced PAF-CPT and LpPLA2 activities. No difference was observed in the activities of the two isoforms of lyso PAF-AT. In conclusion, intake of yogurt enriched in PAF inhibitors could favorably modulate PAF biosynthetic and catabolic pathways.

Postprandial effects of wine consumption on Platelet Activating Factor metabolic enzymes

Platelet Activating factor (PAF) is a potent inflammatory mediator that is involved in the initiation and the prolongation of atherosclerosis. The purpose of the study was to investigate the effect of wine consumption on the activity of PAF metabolic enzymes and on IL-6 levels as a cytokine inflammatory marker. Healthy men participated in 4 daily trials and consumed a standardized meal along with Robola wine (trial R), or Cabernet Sauvignon (trial CS), or ethanol solution (trial E), or water (trial W).A significant trial effect was found in the activity of lyso-PAF acetyltransferase (Lyso-PAF AT) (ptrial=0.01). In specific, R trial decreased enzyme activity compared to E trial (p=0.03) while a trend for differentiation was observed between CS trial and E one (p=0.06) as well as between R trial and W one (p=0.07). Concerning PAF-cholinephosphotransferase (PAF-CPT) activity, a significant trial effect was found (ptrial<0.00). Specifically, both R (p=0.002) and CS (p=0.001) trials decreased enzyme activity compared to E trial. Concerning lipoprotein-associated phospholipase A2 (LpPLA2) no time either trial effect was observed. Concerning IL-6 levels a significant time effect was found (ptime<0.00) while no trial effect was revealed.In conclusion, the protective effect of wine consumption could partly be explained through the modulation of PAF metabolism by wine micro-constituents that lead to lower PAF levels.

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

Interleukin 1 beta (IL-1β) induced platelet activating factor (PAF) synthesis in U-937 cells through stimulation of acetyl-CoA:lysoPAF-acetyltransferase (lyso PAF-AT) at 3h and DTT-independentCDP-choline-1-alkyl-2-acetyl-sn-glycerol cholinophosphotransferase (PAF-CPT) at 0.5h.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-1β 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-1β effect on lyso PAF-AT was 48μΜ±11 and 157μΜ±77, for PAF-CPT 246μΜ±61 and 294μΜ±102, respectively. The same order of concentration was also observed on inhibiting PAF levels produced by IL-1β. 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.

In Vitro Effects of Anti-Glaucomatous Eye Drops on Platelet-Activating Factor and its Metabolism

Purpose: The purpose of this study is to determine the effect of various commonly used antiglaucoma eye drops on inflammatory mediators such as the platelet activating factor (PAF). Methods: Various intraocular pressure (IOP) lowering drops were tested to examine their inhibitory effect on PAF. Multiple eye drops were tested in washed rabbit platelets (WRPs) in order to determine the interaction between these eye drops and the inhibition of PAF in the PAF-induced platelet aggregation model. In addition, we examined the eyedrops’ effect on PAF-metabolism, through in vitro analysis on PAF basic metabolic enzymes (PAF-CPT, lyso PAF-AT, and PAF-AH). Results: Latanoprost (Xalatan) was found to be the most potent in inhibiting PAF, suggesting that it is the most effective in decreasing IOP amongst the eye drops tested. Conversely, dorzolamide hydrochloride-timolol (Cosopt) exhibited the least anti-PAF action. Conclusions: This is the first study examine the relationship between PAF activity and glaucoma medication. Potency in PAF inhibition may be related to drop efficacy.

In vitro effects of vitamin supplements on platelet-activating factor and its metabolism in age-related macular degeneration

Objective: The purpose of our study was to investigate for the first time a series of vitamin supplements used for age-related macular degeneration (AMD) as potential inhibitors of platelet-activating factor (PAF).Materials and methods: Various vitamin supplements were tested in washed rabbit platelets (WRPs), in order to investigate the interaction between vitamin supplements (InShape, Nutrof, Ocuvite, Vitalux) and inhibition of PAF-induced platelet aggregation. Additionally, we examined their ability to affect PAF-metabolism, through their in vitro effect on PAF basic metabolic enzymes (PAF-CPT, lyso PAF-AT, and PAF-AH).Results: Nutrof exhibited the strongest anti-PAF activity, while Vitalux was the most potent anti-inflammatory factor.Conclusion: This is the first study to bring in surface potent anti-inflammatory and anti-angiogenic activities of some vitamin supplements used against AMD, through their in vitro anti-PAF effects in WRPs and the rabbit plasma and leukocyte PAF metabolism, suggesting a promising role of vitamin supplements and especially resveratrol, concerning its potent anti-angiogenic activity in AMD.

HCl-induced and ATP-dependent upregulation of TRPV1 receptor expression and cytokine production by human esophageal epithelial cells

The pathogenesis of gastroesophageal reflux disease (GERD) remains elusive, but recent evidence suggests that early secretion of inflammatory cytokines and chemokines by the mucosa leads to influx of immune cells followed by tissue damage. We previously showed that exposure of esophageal mucosa to HCl causes ATP release, resulting in activation of acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT), the enzyme responsible for the production of platelet-activating factor (PAF). In addition, HCl causes release of IL-8 from the esophageal mucosa. We demonstrate that esophageal epithelial cells secrete proinflammatory mediators in response to HCl and that this response is mediated by ATP. Monolayers of the human esophageal epithelial cell line HET-1A were exposed to acidified cell culture medium (pH 5) for 12 min, a total of seven times over 48 h, to simulate the recurrent acid exposure clinically occurring in GERD. HCl upregulated mRNA and protein expression for the acid-sensing transient receptor potential cation channel, subfamily vanilloid member 1 (TRPV1), lyso-PAF AT, IL-8, eotaxin-1, -2, and -3, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1. The chemokine profile secreted by HET-1A cells in response to repeated HCl exposure parallels similar findings in erosive esophagitis patients. In HET-1A cells, the TRPV1 agonist capsaicin reproduced these findings for mRNA of the inflammatory mediators lyso-PAF AT, IL-8, and eotaxin-1. These effects were blocked by the TRPV1 antagonists iodoresiniferatoxin and JNJ-17203212. These effects were imitated by direct application of ATP and blocked by the nonselective ATP antagonist suramin. We conclude that HCl/TRPV-induced ATP release upregulated secretion of various chemoattractants by esophageal epithelial cells. These chemoattractants are selective for leukocyte subsets involved in acute inflammatory responses and allergic inflammation. The data support the validity of HET-1A cells as a model of the response of the human esophageal mucosa in GERD.

Platelet‐activating factor and distinct chemokines are elevated in mucosal biopsies of erosive compared with non‐erosive reflux disease patients and controls

A distinction between symptomatic non-erosive reflux disease (NERD) and erosive esophagitis (EE) patients is supported by the presence of inflammatory response in the mucosa of EE patients, leading to a damage of mucosal integrity. To explore the underlying mechanism of this difference, we assessed inflammatory mediators in mucosal biopsies from EE and NERD patients and compared them with controls. Nineteen NERD patients, 15 EE patients, and 16 healthy subjects underwent endoscopy after a 3-week washout from PPI or H(2) antagonists. Biopsies obtained from the distal esophagus were examined by quantitative real-time polymerase chain reaction (qPCR) and multiplex enzyme-linked immunosorbent assay for selected chemokines and lyso-PAF acetyltransferase (LysoPAF-AT), the enzyme responsible for production of platelet-activating factor (PAF). Expression of LysoPAF-AT and multiple chemokines was significantly increased in mucosal biopsies derived from EE patients, when compared with NERD patients and healthy controls. Upregulated chemokines included interleukin 8, eotaxin-1, -2, and -3, macrophage inflammatory protein-1α (MIP-1α), and monocyte chemoattractant protein-1 (MCP-1). LysoPAF-AT and the chemokine profile in NERD patients were comparable with healthy controls. Levels of selected cytokines and Lyso-PAF AT were significantly higher in the esophageal mucosa of EE patients compared with NERD and control patients. This difference may explain the distinct inflammatory response occurring in EE patients' mucosa. In contrast, as no significant differences existed between the levels of all mediators in NERD and control subjects, an inflammatory response does not appear to play a major role in the pathogenesis of the abnormalities found in NERD patients.

Sa1373 Selective Activation of Transcription Factors in Models of Erosive and of Non-Erosive Gastro-Esophageal Reflux Disease

Gastro-esophageal reflux disease (GERD) is a common disorder in the US. Up to 60% of GERD patients have non erosive reflux disease (NERD) i.e. reflux-associated pain with normal-appearing esophageal mucosa. In contrast erosive esophagitis (EE) is characterized by inflammation, i.e. infiltration of leukocytes in the mucosa. We have shown that increased expression of the acid-sensitive receptor TRPV1 occurs in both groups of patients, but only biopsies from EE patients exhibit upregulation of cytokines and mucosal infiltration by leukocytes. The mechanisms responsible for these changes were examined in human esophageal epithelial (HET-1A) cells, and compared to data obtained in patient biopsies. We show that repeated (7 times in 48 h) acid exposure (pH 5.0, 12 min) (EE protocol) results in mRNA upregulation for TRPV1, Lyso-PAF AT, IL-8, eotaxin-1, -2, and -3, macrophage inflammatory protein-1alpha and monocyte chemoattractant protein-1, as it occurs in EE biopsies. In contrast exposure (7 times in 48 h) of esophageal epithelial cells to a less acidic pH (5.8) for shorter time periods (8 min) (NERD protocol) induces mRNA upregulation for TRPV1 only, mimicking changes found in NERD biopsies. We next examined the transcription factors activated by the two HCl exposure protocols. CREB, a cAMP response element-binding protein, is a Ca2+-dependent transcription factor. TRPV1 activation induces Ca2+ influx into neurons and esophageal epithelial cells, leading to activation of Ca2+mediated signal transduction, including activation of Ca2+/calmodulin-dependent protein kinase (CaMK). CaMK activates CREB via phosphorylation of Ser-133, that is required for CREB-activated transcription. We find that CREB phosphorylation and TRPV1 upregulation occurs both in the NERD and EE model of HCl exposure. NF-kB is a family of Rel domaincontaining proteins that is thought to participate in production of cytokines. In the cytoplasm NF-kB is in an inactive state, containing an IkBalpha (inhibitor of kappa Β) protein. IkBalpha inactivates the NF-kB transcription factor by masking the nuclear localization signals of NFkB proteins and keeping them sequestered in an inactive state in the cytoplasm. NF-kB is activated by proteolitic cleavage allowing removal of the phosphorylated IkB and subsequent translocation of Rel dimers to the nucleus. Phosphorylation of IkB occurs through phosphorylation/activation of an IkB kinase IkkB. We find that NF-kB activation, demonstrated by phosphorylation/activation of IkkB occurs in the EE but not in the NERD model of HCl exposure. These data are consistent with CREB-dependent transcription affecting TRPV1 upregulation in the NERD and EE models of HCL exposure, and NF-kB-dependent transcription affecting EE and related cytokine/LysoPAF AT upregulation. β

ATP: a mediator for HCl-induced TRPV1 activation in esophageal mucosa

In esophageal mucosa, HCl causes TRPV1-mediated release of calcitonin gene-related peptide (CGRP) and substance P (SP) from submucosal neurons and of platelet-activating factor (PAF) from epithelial cells. CGRP and SP release was unaffected by PAF antagonists but reduced by the purinergic antagonist suramin. ATP caused CGRP and SP release from esophageal mucosa, confirming a role of ATP in the release. The human esophageal epithelial cell line HET-1A was used to identify epithelial cells as the site of ATP release. HCl caused ATP release from HET-1A, which was reduced by the TRPV1 antagonist 5-iodoresiniferatoxin. Real-time PCR demonstrated the presence of mRNA for several P2X and P2Y purinergic receptors in epithelial cells. HCl also increased activity of lyso-PAF acetyl-CoA transferase (lyso-PAF AT), the enzyme responsible for production of PAF. The increase was blocked by suramin. ATP caused a similar increase, confirming ATP as a mediator for the TRPV1-induced increase in enzyme activity. Repeated exposure of HET-1A cells to HCl over 2 days caused upregulation of mRNA and protein expression for lyso-PAF AT. Suramin blocked this response. Repeated exposure to ATP caused a similar mRNA increase, confirming ATP as a mediator for upregulation of the enzyme. Thus, HCl-induced activation of TRPV1 causes ATP release from esophageal epithelial cells that causes release of CGRP and SP from esophageal submucosal neurons and activation of lyso-PAF AT, the enzyme responsible for the production of PAF in epithelial cells. Repeated application of HCl or of ATP causes upregulation of lyso-PAF AT in epithelial cells.

Identification of a Novel Noninflammatory Biosynthetic Pathway of Platelet-activating Factor*

Platelet-activating factor (PAF) is a potent lipid mediator playing various inflammatory and physiological roles. PAF is biosynthesized through two independent pathways called the de novo and remodeling pathways. Lyso-PAF acetyltransferase (lyso-PAF AT) was believed to biosynthesize PAF under inflammatory conditions, through the remodeling pathway. The first isolated lyso-PAF AT (LysoPAFAT/LPCAT2) had consistent properties. However, we show in this study the finding of a second lyso-PAF AT working under noninflammatory conditions. We partially purified a Ca(2+)-independent lyso-PAF AT from mouse lung. Immunoreactivity for lysophosphatidylcholine acyltransferase 1 (LPCAT1) was detected in the active fraction. Lpcat1-transfected Chinese hamster ovary cells exhibited both LPCAT and lyso-PAF AT activities. We confirmed that LPCAT1 transfers acetate from acetyl-CoA to lyso-PAF by the identification of an acetyl-CoA (and other acyl-CoAs) interacting site in LPCAT1. We further showed that LPCAT1 activity and expression are independent of inflammatory signals. Therefore, these results suggest the molecular diversity of lyso-PAF ATs is as follows: one (LysoPAFAT/LPCAT2) is inducible and activated by inflammatory stimulation, and the other (LPCAT1) is constitutively expressed. Each lyso-PAF AT biosynthesizes inflammatory and physiological amounts of PAF, depending on the cell type. These findings provide important knowledge for the understanding of the diverse pathological and physiological roles of PAF.

LPT1 Encodes a Membrane-bound O-Acyltransferase Involved in the Acylation of Lysophospholipids in the Yeast Saccharomyces cerevisiae

Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156-22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Deltalpt1 mutant strain. Although the Deltalpt1 mutant strain did not show other detectable defects, the Deltalpt1 Deltaslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.

Mo-W14:1 Novel approaches and new biomarkers for CHD risk assessment

Platelet Activating factor (PAF) is a potent inflammatory mediator that is involved in the initiation and the prolongation of atherosclerosis. The purpose of the study was to investigate the effect of wine consumption on the activity of PAF metabolic enzymes and on IL-6 levels as a cytokine inflammatory marker. Healthy men participated in 4 daily trials and consumed a standardized meal along with Robola wine (trial R), or Cabernet Sauvignon (trial CS), or ethanol solution (trial E), or water (trial W).A significant trial effect was found in the activity of lyso-PAF acetyltransferase (Lyso-PAF AT) (ptrial = 0.01). In specific, R trial decreased enzyme activity compared to E trial (p = 0.03) while a trend for differentiation was observed between CS trial and E one (p = 0.06) as well as between R trial and W one (p = 0.07). Concerning PAF-cholinephosphotransferase (PAF-CPT) activity, a significant trial effect was found (ptrial < 0.00). Specifically, both R (p = 0.002) and CS (p = 0.001) trials decreased enzyme activity compared to E trial. Concerning lipoprotein-associated phospholipase A2 (LpPLA2) no time either trial effect was observed. Concerning IL-6 levels a significant time effect was found (ptime < 0.00) while no trial effect was revealed.In conclusion, the protective effect of wine consumption could partly be explained through the modulation of PAF metabolism by wine micro-constituents that lead to lower PAF levels.

Effect of the inhibition of PLA2 and PAF on the neutrophilic respiratory burst and apoptosis

Background Since the exact pathogenesis of sepsis-induced ARDS has not been elucidated, the mechanisms of enhanced neutrophilic respiratory burst were probed in endotoxin primed neutrophils associated with the roles of phospholipase A2 (PLA2), platelet activating factor (PAF) and apoptosis. Methods In isolated fresh human neutrophils, effects of the inhibition of PLA2 and PAF on the apoptosis were examined by the method of Annexin-FITC/dual PI flow cytometry. The roles of PLA2 and PAF on the neutrophilic respiratory burst were also examined by measuring oxidant generation in cytochrome-c reduction assay. Activities of the PLA2 and lysoPAF acetyltransferase (lysoPAF AT) of the neutrophils were determined to understand the effect of endotoxin on these enzymatic activities which may be related to the neutrophilic respiratory burst and apoptosis. In addition, the role roles of PLA2 and PAF in neutrophilic adhesion to bovine endothelial cells were examined in vitro by neutrophil adhesion assay. To investigate the effect of oxidants on pulmonary surfactant, cytochemical ultrastructural microscopy was performed. To inhibit PLA2 and PAF, non-specific PLA2 inhibitor mepacrine (100 nM) and WEB 2086 (100 nM) or ketotifen fumarate (10 kg/ml) were used respectively in all in vitro experimental sets. WEB 2086 is PAF receptor antagonist, and ketotifen fumarate is a lyso PAF AT inhibitor. Results The mapacrine treatment, provided after the endotoxin (ETX) treatment, resulted in increased apoptosis of neutrophils (p<0.001) while treatments of WEB 2086 and ketotifen did not. The inhibition of PLA2 and PAF decreased (p<0.001) production of oxidants from PMA-stimulated neutrophils. While endotoxin increased the PLA2 activity of neutrophils (p<0.01), mepacrine supressed (p<0.001) the activity, provided after treatment of ETX. The lyso PAF actyltransferase activity (lyso PAF AT) increased(p<0.01) after treatment of ETX. In contrast, mepacrine, WEB 2086 and ketotifen showed a tendency of decreasing the activity after treatment of ETX. The Treatment of ETX increased (p<0.001) neutrophil adhesion to endothelial cells, which was reversed by inhibition of PLA2 and PAF (p<0.001). The binding of oxidants to pulmonary surfactant was identified histologically. Conclusions The enhanced neutrophilic respiratory burst by ETX plays a pivotal role in the pathogenesis of ARDS in term of oxidayive oxidative stress. Increased production of oxidants form neutrophils is mediated by the activations of PLA2 and lyso PAF AT.