Platelet-activating Factor Synthesis Research Articles

Lysophospholipid Acyltransferase 9 Promotes Emphysema Formation via Platelet-activating Factor.

Cigarette smoking is known to be the leading cause of chronic obstructive pulmonary disease (COPD). However, the detailed mechanisms have not been elucidated. PAF (platelet-activating factor), a potent inflammatory mediator, is involved in the pathogenesis of various respiratory diseases such as bronchial asthma and COPD. We focused on LPLAT9 (lysophospholipid acyltransferase 9), a biosynthetic enzyme of PAF, in the pathogenesis of COPD. LPLAT9 gene expression was observed in excised COPD lungs and single-cell RNA sequencing data of alveolar macrophages (AMs). LPLAT9 was predominant and upregulated in AMs, particularly monocyte-derived AMs, in patients with COPD. To identify the function of LPLAT9/PAF in AMs in the pathogenesis of COPD, we exposed systemic LPLAT9-knockout (LPALT9-/-) mice to cigarette smoke (CS). CS increased the number of AMs, especially the monocyte-derived fraction, which secreted MMP12 (matrix metalloprotease 12). Also, CS augmented LPLAT9 phosphorylation/activation on macrophages and, subsequently, PAF synthesis in the lung. The LPLAT9-/- mouse lung showed reduced PAF production after CS exposure. Intratracheal PAF administration accumulated AMs by increasing MCP1 (monocyte chemoattractant protein-1). After CS exposure, AM accumulation and subsequent pulmonary emphysema, a primary pathologic change of COPD, were reduced in LPALT9-/- mice compared with LPLAT9+/+ mice. Notably, these phenotypes were again worsened by LPLAT9+/+ bone marrow transplantation in LPALT9-/- mice. Thus, CS-induced LPLAT9 activation in monocyte-derived AMs aggravated pulmonary emphysema via PAF-induced further accumulation of AMs. These results suggest that PAF synthesized by LPLAT9 has an important role in the pathogenesis of COPD.

Severe Type 2 Inflammation Leads to High Platelet-Activating-Factor-Associated Pathology in Chronic Rhinosinusitis with Nasal Polyps-A Hierarchical Cluster Analysis Using Bulk RNA Barcoding and Sequencing.

Platelet-activating factor (PAF) is a phospholipid-derived inflammatory mediator that triggers various inflammatory conditions, including eosinophil activation and recruitment. This study aimed to evaluate the expressions of PAF-metabolism-associated genes, namely genes coding the enzymes involved in PAF synthesis (LPCAT1, LPCAT2, LPCAT3, and LPCAT4), PAF degradation (PAFAH1B2, PAFAH1B3, and PAFAH2), and the gene for the PAF receptor (PTAFR) in subtypes of CRSwNP classified by clinical- or hierarchal-analysis-based classifications. Transcriptomic analysis using bulk RNA barcoding and sequencing (BRB-seq) was performed with CRSwNP, including eosinophilic CRS (ECRS) (n = 9), nonECRS (n = 8), ECRS with aspirin-exacerbated respiratory disease (Asp) (n = 3), and controls with a normal uncinate process mucosa (n = 6). PTAFR was only upregulated in ECRS and nonECRS. In the hierarchical cluster analysis with clusters 1 and 2 reflecting patients with low-to-moderate and high levels of type 2 inflammation, respectively, cluster 1 exhibited a significant downregulation of LPCAT2 and an upregulation of PTAFR expression, while cluster 2 showed an upregulation of LPCAT1, PAFAH1B2, and PTAFR and downregulation of PAFAH2 expression. Understanding this strong PAF-associated pathophysiology in the severe type 2 inflammation group could provide valuable insights into the treatment and management of CRSwNP.

Biochemical and Signaling Functions of Plasmalogens in the Norm and in Various Diseases

Plasmalogens (PL) are alkenyl ether glycerophospholipids that perform many physiologically important functions in the body and have a variety of properties. The role of PLs as endogenous antioxidants is well studied, as well as PLs that determine the physicochemical properties of biomembranes, deposit polyunsaturated fatty acids, participate in cholesterol biosynthesis, the process of ferroptosis and adaptation of cells to hypoxia, are sources for the synthesis of platelet activating factor and act as its antagonists. At the same time, LPs are involved in the pathogenesis of neurodegenerative, inflammatory, oncological diseases and a number of other pathologies. These conditions are united by the development of chronic inflammation and oxidative stress, accompanied by a change in the activity of synthesis and the content of PL in cells. Despite the fact that PL replacement therapy inhibits inflammation, the molecular mechanisms of the association of PL with inflammation have not been fully elucidated. The purpose of this review is to summarize the latest literature data on the structure, biosynthesis, and functions of PL, their relationship with signaling cascades involved in the pathogenesis of chronic inflammatory and neuroinflammatory diseases, as well as to show current achievements and future prospects for the use of PL in the treatment of certain diseases.

Development and Validation of UV-Visible spectroscopic method for estimation of Nimesulide in bulk and its pharmaceutical formulation

The aim of the present work was to develop and validate a simple UV Spectroscopic method for the determination of Nimesulide, which is the inhibitor of prostaglandin synthesis from arachidonic acid and platelet aggregation. It has the ability to exert analgesic and anti-inflammatory actions through various mechanisms (ie, COX independent pathways) and also it inhibits the release of tumour necrosis factor (TNFα) and interleukins, and stops the release of histamine from mast cells and reduce the synthesis of platelet activating factor (PAF) from the basophil cells. The UV visible Spectrophotometric analysis was performed using systronics UV-Spectrophotometer 2704 X visible double beam by using solvent KOH. Detection was performed at a wavelength of 395nm. Method validation was carried out according to ICH Q2R1 guidelines by taking the parameters such as accuracy, linearity, precision, ruggedness, and robustness, LOD and LOQ. The UV spectrophotometric was found linear in the range 5- 25µg/ml. The method was rugged and robust with % relative standard deviation less than 2. The extraction recoveries was found to be higher than 99.19% in all experimental conditions. Based upon the performance characteristics, the proposed method was found accurate, precise, rapid and suitable for the determination of nimesulide for routine analysis. The aim of the present work was to develop and validate a simple UV Spectroscopic method for the determination of Nimesulide, which is the inhibitor of prostaglandin synthesis from arachidonic acid and platelet aggregation. It has the ability to exert analgesic and anti-inflammatory actions through various mechanisms (ie, COX independent pathways) and also it inhibits the release of tumour necrosis factor (TNFα) and interleukins, and stops the release of histamine from mast cells and reduce the synthesis of platelet activating factor (PAF) from the basophil cells. The UV visible Spectrophotometric analysis was performed using systronics UV-Spectrophotometer 2704 X visible double beam by using solvent KOH. Detection was performed at a wavelength of 395nm. Method validation was carried out according to ICH Q2R1 guidelines by taking the parameters such as accuracy, linearity, precision, ruggedness, and robustness, LOD and LOQ. The UV spectrophotometric was found linear in the range 5- 25µg/ml. The method was rugged and robust with % relative standard deviation less than 2. The extraction recoveries was found to be higher than 99.19% in all experimental conditions. Based upon the performance characteristics, the proposed method was found accurate, precise, rapid and suitable for the determination of nimesulide for routine analysis.

Neutrophil Extracellular Traps, Angiogenesis and Cancer.

Human neutrophils, the most abundant circulating leukocytes, are fundamental components of the host response against different pathogens. Until a few years ago, neutrophils received limited attention in cancer immunology. Recently, it was discovered that both circulating, and tumor-associated, neutrophils possess functional plasticity when exposed to various inflammatory stimuli and in the tumor microenvironment. Neutrophils and their mediators can exert several pro-tumor activities in cancer and promote metastasis through different mechanisms. Angiogenesis plays a pivotal role in inflammation and tumor growth. Activated human neutrophils release several angiogenic factors [vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (ANGPT1), CXCL8, hepatocyte growth factor (HGF), and metalloproteinase 9 (MMP-9)] and form neutrophil extracellular traps (NETs). NETs promote tumor growth and metastasis formation through several mechanisms: they can awake dormant cancer cells, capture circulating tumor cells, coat and shield cancer cells, thus preventing CD8+- and natural killer (NK) cell-mediated cytotoxicity. ANGPTs released by endothelial and periendothelial mural cells induce platelet-activating factor (PAF) synthesis and neutrophil adhesion to endothelial cells. NETs can directly exert several proangiogenic activities in human endothelial cells and NETs induced by ANGPTs and PAF increase several aspects of angiogenesis in vitro and in vivo. A better understanding of the pathophysiological functions of NETs in cancer and angiogenesis could be of importance in the early diagnosis, prevention and treatment of tumors.

New Insights Into the Pathologic Roles of the Platelet-Activating Factor System.

Described almost 50 years ago, the glycerophosphocholine lipid mediator Platelet-activating factor (PAF) has been implicated in many pathologic processes. Indeed, elevated levels of PAF can be measured in response to almost every type of pathology involving inflammation and cell damage/death. In this review, we provide evidence for PAF involvement in pathologic processes, with focus on cancer, the nervous system, and in photobiology. Importantly, recent insights into how PAF can generate and travel via bioactive extracellular vesicles such as microvesicle particles (MVP) are presented. What appears to be emerging from diverse pathologies in different organ systems is a common theme where pro-oxidative stressors generate oxidized glycerophosphocholines with PAF agonistic effects, which then trigger more enzymatic PAF synthesis via the PAF receptor. A downstream consequence of PAF receptor activation is the generation and release of MVP which provide a mechanism to transmit PAF as well as other bioactive agents. The knowledge gaps which when addressed could result in novel therapeutic strategies are also discussed. Taken together, an enhanced understanding of the PAF family of lipid mediators is essential in our improved comprehension of the relationship amongst the diverse cutaneous, cancerous, neurologic and systemic pathologic processes.

Human Eosinophils Express, Relative to Other Circulating Leukocytes, Large Amounts of Secretory 14-kD Phospholipase A2

Human eosinophils perform several functions dependent on phospholipase A2 (PLA2) activity, most notably the synthesis of platelet-activating factor (PAF) and leukotriene C4 (LTC4). Several forms of PLA2 have been identified in mammalian cells. In the present study, the 14-kD, secretory form of PLA2 was detected in human eosinophils by immunocytochemical staining with the specific monoclonal antibody (MoAb) 4A1. In contrast, preparations of neutrophils, monocytes, lymphocytes, and basophils did not show detectable staining. With two MoAbs in a sandwich enzyme-linked immunosorbent assay (ELISA), large amounts of sPLA2 were detected in lysates of eosinophils, that were 20-fold to 100-fold higher than in the other circulating leukocytes (ie, neutrophils, basophils, monocytes, and lymphocytes). In addition, with a commercially available sPLA2 activity assay kit, we were able to show high activity of sPLA2 in human eosinophils relative to neutrophils. Investigations at the ultrastructural level showed that sPLA2 in eosinophils is mainly located in specific granules. Immunoelectron microscopy also visualized sPLA2 within phagosomes after addition of opsonized particles to the eosinophils. However, sPLA2 was not detected in the cell-free supernatants of activated eosinophils, in contrast to eosinophil-cationic protein (ECP), which colocalizes with sPLA2 in resting eosinophils. These findings warrant further studies into the role of sPLA2 in eosinophil function.

Alterations in Phospholipase A2‐Mediated Pathways in Smokers: A Potential Mediator of Skin Cancer Development

Cigarette smoking is a major preventable cause of disease that maintains a disproportionate prevalence in populations distinguished by socioeconomic status, race, and gender. Much remains unknown about the biochemical effects of smoking on the development of skin cancers including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). However, a recent meta‐analysis study demonstrated that cigarette smoking is associated with a 52% increase in the risk for SCC, but does not appear to modify the risk for BCC. Our previous studies have demonstrated that cigarette smoking is associated with changes in phospholipase A2 (PLA2)‐mediated pathways that are implicated in cancer development and progression. More specifically, PLA2 activation leads to increased platelet‐activating factor (PAF) and prostaglandin E2 (PGE2) synthesis, metabolites that are increased in cancer cells and mediate tumor growth, survival and invasion. To assess the effects of cigarette smoking in non‐melanoma skin cancer, we analyzed PAF and PGE2 expression in skin biopsies from six smokers and six non‐smokers with SCC, and six smokers and six non‐smokers with BCC using immunohistochemistry. PAF was not detectable in BCC patient samples. SCC patient samples revealed positive PAF expression with nuclear localization in all patient samples, but there was no significant difference between expression in samples of smokers when compared to nonsmokers. PGE2 expression was detected in BCC patient samples, with no difference between smokers and nonsmokers. PGE2 expression was greater in SCC when compared to BCC, with a 50% increase in expression in SCC samples from smokers compared to nonsmokers. Increased PGE2 accumulation was accompanied by a 25% increase in COX‐2 expression in SCC samples from smokers compared to nonsmokers. Increased COX‐2 and PGE2 production can stimulate the production of vascular endothelial growth factor (VEGF), accelerating tumor growth, survival and invasion. We detected a 40% increase in VEGF expression in SCC samples from smokers when compared to nonsmokers. COX‐2 and VEGF expression remained unchanged between BCC samples taken from smokers or nonsmokers. Taken together, our data suggest that increased PGE2 accumulation may contribute to the increased risk of SCC in smokers.

A New Class of Acetyl‐TAG Present in Seed Oils of Polygala Species

A New Class of Acetyl‐TAG Present in Seed Oils of <i>Polygala</i> Species

Platelet-Activating Factor-Induced Reduction in Contact Hypersensitivity Responses Is Mediated by Mast Cells via Cyclooxygenase-2-Dependent Mechanisms.

Platelet-activating factor (PAF) stimulates numerous cell types via activation of the G protein-coupled PAF receptor (PAFR). PAFR activation not only induces acute proinflammatory responses, but it also induces delayed systemic immunosuppressive effects by modulating host immunity. Although enzymatic synthesis and degradation of PAF are tightly regulated, oxidative stressors, such as UVB, chemotherapy, and cigarette smoke, can generate PAF and PAF-like molecules in an unregulated fashion via the oxidation of membrane phospholipids. Recent studies have demonstrated the relevance of the mast cell (MC) PAFR in PAFR-induced systemic immunosuppression. The current study was designed to determine the exact mechanisms and mediators involved in MC PAFR-mediated systemic immunosuppression. By using a contact hypersensitivity model, the MC PAFR was not only found to be necessary, but also sufficient to mediate the immunosuppressive effects of systemic PAF. Furthermore, activation of the MC PAFR induces MC-derived histamine and PGE2 release. Importantly, PAFR-mediated systemic immunosuppression was defective in mice that lacked MCs, or in MC-deficient mice transplanted with histidine decarboxylase- or cyclooxygenase-2-deficient MCs. Lastly, it was found that PGs could modulate MC migration to draining lymph nodes. These results support the hypothesis that MC PAFR activation promotes the immunosuppressive effects of PAF in part through histamine- and PGE2-dependent mechanisms.

Synthesis of Human Neutrophil Extracellular Traps Contributes to Angiopoietin-Mediated In Vitro Proinflammatory and Proangiogenic Activities.

Neutrophil extracellular traps (NETs) are composed of nuclear DNA in a web-like structure extruded from neutrophils in response to either bacterial infection or inflammation. We previously reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of both angiopoietins (Ang1 and Ang2) to induce proinflammatory activities, such as synthesis and release of platelet-activating factor, upregulation of β2 integrin complex (CD11/CD18), and neutrophil chemotaxis. In contrast, only Ang1 but not Ang2 is capable of promoting translational and transcriptional activities in neutrophils. In this article, we addressed whether Ang1 and/or Ang2 could modulate the release of NETs and if they contribute to angiopoietin-mediated proinflammatory activities. We observed that Ang1 and Ang2, alone or combined (10 nM, 3 h), increase NET synthesis and release by ≈2.5-fold as compared with PBS-treated neutrophils. The release of NETs is Tie2 dependent and requires downstream intracellular participation of PI3K, p38, and p42/44 MAPK pathways; reactive oxygen species production; intracellular calcium store depletion; and protein arginine deiminase 4 activation. These isolated NETs induced neutrophil and endothelial cell activation, leading to neutrophil adhesion onto human extracellular matrix and HUVEC and in vitro formation of capillary-like tubes by endothelial cells. Our study reports the capacity of Ang1 and Ang2 to promote the release of NETs and that these NETs contribute to angiopoietin-mediated in vitro proinflammatory and proangiogenic activities.

Enhanced Platelet-Activating Factor Synthesis Facilitates Acute and Delayed Effects of Ethanol-Intoxicated Thermal BurnInjury.

Thermal burn injuries in patients who are alcohol-intoxicated result in greater morbidity and mortality. Murine models combining ethanol and localized thermal burn injury reproduce the systemic toxicity seen in human subjects, which consists of both acute systemic cytokine production with multiple organ dysfunction, as well as a delayed systemic immunosuppression. However, the exact mechanisms for these acute and delayed effects are unclear. These studies sought to define the role of the lipid mediator platelet-activating factor in the acute and delayed effects of intoxicated burn injury. Combining ethanol and thermal burn injury resulted in increased enzymatic platelet-activating factor generation in a keratinocyte cell line invitro, human skin explants exvivo, as well as in murine skin invivo. Further, the acute increase in inflammatory cytokines, such as IL-6, and the systemic immunosuppressive effects of intoxicated thermal burn injury were suppressed in mice lacking platelet-activating factor receptors. Together, these studies provide a potential mechanism and treatment strategies for the augmented toxicity and immunosuppressive effects of thermal burn injury in the setting of acute ethanol exposure, which involves the pleotropic lipid mediator platelet-activating factor.

978 Enhanced platelet-activating factor synthesis facilitates acute and delayed effects of intoxicated thermal burn injury

978 Enhanced platelet-activating factor synthesis facilitates acute and delayed effects of intoxicated thermal burn injury

FAR2 is associated with kidney disease in mice and humans.

Mesangial matrix expansion is an important process in the initiation of chronic kidney disease, yet the genetic factors driving its development are unknown. Our previous studies have implicated Far2 as a candidate gene associated with differences in mesangial matrix expansion between mouse inbred strains. Consistent with the hypothesis that increased expression of Far2 leads to mesangial matrix expansion through increased production of platelet-activating factor precursors, we show that FAR2 is capable of mediating de novo platelet-activating factor synthesis in vitro and driven by the transcription factor NKX3.2. We demonstrate that knockdown of Far2 in mice delays the progression of mesangial matrix expansion with at least six months (equivalent to ~15 yr in human). Furthermore, we show that increased FAR2 expression in human patients is associated with diabetic nephropathy, lupus nephritis, and IgA nephropathy. Taken together, these results highlight FAR2's role in the development of mesangial matrix expansion and chronic kidney disease.

PAF Expression in Skin Tumors of Smokers and Never Smokers: a Potential Role in Skin Cancer Development

Significant progress has been made in the management and understanding of cancer, but there remains a large disparity in cancer health within our population. One of the most well‐known cancer risk factors, cigarette smoking, maintains a disproportionate prevalence in populations distinguished by socioeconomic status, race, and gender. Previous studies have demonstrated that cigarette smoking is associated with changes in phospholipase A2 (PLA2)‐mediated pathways that are implicated in cancer development and progression. More specifically, PLA2 activation leads to increased platelet‐activating factor (PAF) synthesis, a metabolite that is increased in cancer cells. PAF accumulation is further enhanced with cigarette smoke via inhibition of PAF‐acetylhydrolase, the enzyme responsible for PAF hydrolysis and inactivation. Much remains unknown about the biochemical effects of smoking on the development of skin cancers including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). However, a recent meta‐analysis study demonstrated that cigarette smoking is associated with a 52% increase in the risk for SCC, but does not appear to modify the risk for BCC. In order to assess the effects of cigarette smoking in these two skin cancers, we analyzed PAF expression in skin biopsies from six smokers and non‐smokers with SCC, and six smokers and non‐smokers with BCC. The biopsy samples were immunohistochemically stained with antibodies specific to PAF and PAF‐receptor. Immunohistochemistry revealed positive PAF and PAF‐R expression with nuclear localization in all patient samples, including SCC and adjacent normal sun exposed skin. There was no significant difference between the PAF or PAF‐R expression in samples of smokers when compared to nonsmokers. However, immunohistochemical analysis demonstrated a significant difference in PAF expression between SCC and BCC cells. SCC cells expressed nuclear PAF, but PAF was not detectable in BCC samples. PAF signal was also not detectable in the basal cell epidermal layer in all patient samples. These findings support a biochemical difference in SCC and BCC cells in association with PAF, which is traditionally upregulated with cigarette smoking.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pneumolysin mediates heterotypic aggregation of neutrophils and platelets in vitro

Platelets orchestrate the inflammatory activities of neutrophils, possibly contributing to pulmonary and myocardial damage during severe pneumococcal infection. This study tested the hypothesis that the pneumococcal toxin, pneumolysin (Ply), activates production of platelet-activating factor (PAF) and thromboxane A2 (TxA2) by neutrophils, these bioactive lipids being potential mediators of neutrophil:platelet (NP) networking. The effects of recombinant Ply (10-80ngmL-1) on the production of PAF and TxA2 by isolated neutrophils were measured using ELISA procedures, and NP aggregation by flow cytometry. Exposure of neutrophils to Ply induced production of PAF and, to a lesser extent, TxA2, achieving statistical significance at ≥20ngmL-1 of the toxin. In the case of NP interactions, Ply promoted heterotypic aggregation which was dependent on upregulation of P-selectin (CD62P) and activation of protease-activated receptor 1 (PAR1), attaining statistical significance at ≥10ngmL-1 of the toxin, but did not involve either PAF or TxA2. Ply induces synthesis of PAF and TxA2, by human neutrophils, neither of which appears to contribute to the formation of NP heterotypic aggregates invitro, a process which is seemingly dependent on CD62P and PAR1. These pro-inflammatory activities of Ply may contribute to the pathogenesis of pulmonary and myocardial injury during severe pneumococcal infection.

Escherichia coli Braun Lipoprotein (BLP) exhibits endotoxemia – like pathology in Swiss albino mice

The endotoxin lipopolysaccharide (LPS) promotes sepsis, but bacterial peptides also promote inflammation leading to sepsis. We found, intraperitoneal administration of live or heat inactivated E. coli JE5505 lacking the abundant outer membrane protein, Braun lipoprotein (BLP), was less toxic than E. coli DH5α possessing BLP in Swiss albino mice. Injection of BLP free of LPS purified from E. coli DH5α induced massive infiltration of leukocytes in lungs and liver. BLP activated human polymorphonuclear cells (PMNs) ex vivo to adhere to denatured collagen in serum and polymyxin B independent fashion, a property distinct from LPS. Both LPS and BLP stimulated the synthesis of platelet activating factor (PAF), a potent lipid mediator, in human PMNs. In mouse macrophage cell line, RAW264.7, while both BLP and LPS similarly upregulated TNF-α and IL-1β mRNA; BLP was more potent in inducing cyclooxygenase-2 (COX-2) mRNA and protein expression. Peritoneal macrophages from TLR2−/− mice significantly reduced the production of TNF-α in response to BLP in contrast to macrophages from wild type mice. We conclude, BLP acting through TLR2, is a potent inducer of inflammation with a response profile both common and distinct from LPS. Hence, BLP mediated pathway may also be considered as an effective target against sepsis.

New Insights into the Pro-Inflammatory Activities of Ang1 on Neutrophils: Induction of MIP-1β Synthesis and Release.

We reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of angiopoietins (Ang1 and Ang2) to induce pro-inflammatory activities, such as platelet-activating factor synthesis, β2-integrin activation and neutrophil migration. Recently, we observed differential effects between both angiopoietins, namely, the capacity of Ang1, but not Ang2, to promote rapid interleukin-8 synthesis and release, as well as neutrophil viability. Herein, we addressed whether Ang1 and/or Ang2 could modulate the synthesis and release of macrophage inflammatory protein-1β (MIP-1β) by neutrophils. Neutrophils were isolated from blood of healthy volunteers; intracellular and extracellular MIP-1β protein concentrations were assessed by ELISA. After 24 hours, the basal intracellular and extracellular MIP-1β protein concentrations were ≈500 and 100 pg/106 neutrophils, respectively. Treatment with Ang1 (10 nM) increased neutrophil intracellular and extracellular MIP-1β concentrations by 310 and 388% respectively. Pretreatment with PI3K (LY294002), p38 MAPK (SB203580) and MEK (U0126) inhibitors completely inhibited Ang1-mediated increase of MIP-1β intracellular and extracellular protein levels. Pretreatment with NF-κB complex inhibitors, namely Bay11-7085 and IKK inhibitor VII or with a transcription inhibitor (actinomycin D) and protein synthesis inhibitor (cycloheximide), did also abrogate Ang1-mediated increase of MIP-1β intracellular and extracellular protein levels. We validated by RT-qPCR analyses the effect of Ang1 on the induction of MIP-1β mRNA levels. Our study is the first one to report Ang1 capacity to induce MIP-1β gene expression, protein synthesis and release from neutrophils, and that these effects are mediated by PI3K, p38 MAPK and MEK activation and downstream NF-κB activation.

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.

The relation of diet with PAF and its metabolic enzymes in healthy volunteers

Platelet-activating factor (PAF), a potent inflammatory mediator, is implicated in atherosclerosis. Its key biosynthetic enzymes are lyso-PAF acetyltransferases (lyso-PAF-AT), responsible for PAF synthesis through the remodeling route and a specific CDP-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT), responsible for its de novo biosynthesis. PAF acetylhydrolase (PAF-AH) and its extracellular isoform lipoprotein-associated phospholipase A₂ catabolize PAF. The impact of diet on PAF metabolism is ill-defined. The aim was to investigate associations between PAF, its enzymes and dietary factors. One-hundred and six (n = 106) healthy volunteers were recruited. Food-frequency questionnaires, dietary recalls, lifestyle and biochemical variables were collected. Food groups, macronutrient intake, a priori (MedDietScore) and a posteriori defined food patterns with PCA analysis, dietary antioxidant capacity (DAC), glycemic index (GI) and glycemic load were assessed. PAF was inversely correlated with antioxidant-rich foods (herbal drinks and coffee), the DAC as well as a dietary pattern characterized by legumes, vegetables, poultry and fish (all Ps < 0.05). PAF was positively correlated to % fat intake. Lyso-PAF-AT was also negatively associated with healthy patterns (fruits, nuts and herbal drinks, and a pattern rich in olive oil and whole-wheat products), as well as the DAC and % monounsaturated fatty acids. PAF-CPT was negatively associated with GI and coffee intake and positively with dietary cholesterol. PAF-AH was negatively associated with coffee and positively associated with alcohol consumption (all Ps < 0.05). In conclusion, the DAC and healthy dietary patterns were inversely associated with PAF or its biosynthetic enzymes, suggesting potential new mechanisms of the diet-disease associations.