Lipids in ultraviolet radiation-induced immune modulation.

Ultraviolet (UV) radiation-mediated immune suppression is a key mechanism conferring both detrimental and beneficial impacts of sun exposure on human health. Suppression of anti-tumour responses promotes the development and progression of UV-induced skin cancers. In contrast, suppression of dysregulated immune responses facilitate the therapeutic success of phototherapy treatment for skin disorders and is postulated to be responsible for UV protection from autoimmune diseases. While some of the molecular and cellular mechanisms underlying UV-suppression of the immune system are known, a relatively unexplored area is immunomodulatory lipids. Cutaneous UV exposure changes lipids both locally in the skin, increasing platelet-activating factor (PAF) production and decreasing free triglyceride levels, and systemically reducing adipose tissue mass. There is growing recognition that bioactive lipids and lipid metabolism directly affect immune cell phenotype and function. Manipulation of immunomodulatory lipid pathways are effective strategies in modifying systemic immune responses. Indeed, the PAF pathway is a key initiator of UV-induced immune suppression and antagonism of PAF-receptors restores immune function and reduces skin cancer development in mice. This review focuses on the known effects of UV on lipids and proposes how this may in turn be involved in the modulation of the immune system.

Before platelets: the production of platelet-activating factor during growth and stress in a basal marine organism.

Corals and humans represent two extremely disparate metazoan lineages and are therefore useful for comparative evolutionary studies. Two lipid-based molecules that are central to human immunity, platelet-activating factor (PAF) and Lyso-PAF were recently identified in scleractinian corals. To identify processes in corals that involve these molecules, PAF and Lyso-PAF biosynthesis was quantified in conditions known to stimulate PAF production in mammals (tissue growth and exposure to elevated levels of ultraviolet light) and in conditions unique to corals (competing with neighbouring colonies over benthic space). Similar to observations in mammals, PAF production was higher in regions of active tissue growth and increased when corals were exposed to elevated levels of ultraviolet light. PAF production also increased when corals were attacked by the stinging cells of a neighbouring colony, though only the attacked coral exhibited an increase in PAF. This reaction was observed in adjacent areas of the colony, indicating that this response is coordinated across multiple polyps including those not directly subject to the stress. PAF and Lyso-PAF are involved in coral stress responses that are both shared with mammals and unique to the ecology of cnidarians.

Stereochemistry- and concentration-dependent effects of phosphatidylserine enrichment on platelet function.

Platelets are small (1-2µm in diameter), circulating anuclear cell fragments with important roles in hemostasis and thrombosis that provide an excellent platform for studying the role of membrane components in cellular communication. Platelets use several forms of communication including exocytosis of three distinct granule populations, formation of bioactive lipid mediators, and shape change (allowing for adhesion). This work explores the role of stereochemistry and concentration of exogenous phosphatidylserine (PS) on platelet exocytosis and adhesion. PS, most commonly found in the phosphatidyl-l-serine (l-PS) form, is exposed on the outer leaflet of the cell membrane after the platelet is activated. Knowledge about the impact of exogenous phosphatidylserine on cell-to-cell communication is limited (particularly concentration and stereochemistry effects). This study found that platelets incubated in l-PS or phosphatidyl-d-serine (d-PS) are enriched to the same extent with their respective incubated PS. All levels of l-PS enrichment also showed an increase in platelet cholesterol, but only the 50µM d-PS incubation showed an increase in cholesterol. The uptake of d-PS induced the secretion of granules and manufactured platelet activating factor (PAF) in otherwise unstimulated platelets. The uptake of l-PS had a greater impact on platelet stimulation by decreasing both the amount of δ-granule secretion and the amount of PAF that was manufactured.

Lipopolysaccharide acts synergistically with the dengue virus to induce monocyte production of platelet activating factor and other inflammatory mediators.

BACKGROUND: Platelet Activating Factor (PAF) has been shown to be an important mediator of vascular leak in acute dengue. Antibody dependent enhancement (ADE) and microbial translocation has also shown to contribute to severe dengue. Since monocytes are one of the primary targets of the dengue virus (DENV) we sought to investigate if monocytes were a source of PAF, and the effect of ADE and microbial endotoxin (LPS) on DENV infected monocytes. METHODS: PAF and cytokine levels were evaluated in serial blood samples, in patients with acute dengue infection. The effect of ADE and LPS in production of PAF and cytokines from DENV infected primary human monocytes derived macrophages (MDMθ) was assessed. Gene expression analysis was undertaken to investigate mechanisms by which LPS potentiates PAF and cytokine production by DENV infected MDMθ. RESULTS: Serum PAF levels significantly correlated with both TNF-α (p < 0.0001) and IL-1ß (p < 0.0001) in patients with acute DENV infection. Although primary human MDMθ produced inflammatory cytokines following infection with the DENV, they did not produce PAF following in vitro DENV infection alone, or in the presence of dengue immune serum. Levels of PAF produced by DENV infected MDMθ co-cultured with LPS was significantly higher than uninfected MDMθs co-cultured with LPS. Although TLR-4 was upregulated in uninfected MDMθs co-cultured with LPS, this upregulation was not significant in DENV infected MDMθ. Only expression of RIG-I was significantly up regulated (p < 0.05) when DENV infected MDMθ were co-cultured with LPS. CONCLUSION: LPS acts synergistically with the DENV to induce production of PAF and other inflammatory cytokines, which suggests that microbial translocation that has shown to occur in acute dengue, could contribute to dengue disease severity.

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.

Epidermal growth factor induces platelet-activating factor production through receptors transactivation and cytosolic phospholipase A2 in ovarian cancer cells.

BACKGROUND: Among the pro-inflammatory lipid mediators, platelet-activating factor (PAF) is a major primary and secondary messenger that binds to the PAF-receptor (PAFR). Epidermal growth factor (EGF) is a polypeptide growth factor that binds to the EGF-receptor (EGFR). Evidence suggests that both PAF and EGF play a significant role in oncogenic transformation, tumor growth, neoangiogenesis and metastasis, including ovarian cancer. PAF has the potential to transactivate EGFR in ovarian cancer cells. This study explores the mechanisms involved in EGF-induced PAF production. METHODS: The effect of EGF on PAF production in ovarian cancer cells was observed using enzyme-linked immunosorbent assay. The receptors transactivation and the role of cytosolic phospholipase A2 (cPLA2) in modulating PAF production induced by EGF was assessed using pharmacological inhibitors, si-RNA knockdown, targeted gene overexpression and immunocytochemistry. The signaling pathways invovled in PAF production induced by EGF in ovarian cancer cells were assessed. RESULTS: We demonstrate that EGF increases the production of PAF in CAOV3 and SKOV3 ovarian cancer cell lines. EGF induces the transactivation of PAFR, which can be blocked by an EGFR inhibitor. Inhibition of EGFR and/or PAFR blocks PAF production in response to EGF. EGF-induced PAF production involves the phosphorylation of extracellular-regulated protein kinase (ERK) and cytosolic phospholipase A2 (cPLA2). A cPLA2 inhibitor blocks EGF-induced PAF production as well as si-cPLA2, while overexpression of cPLA2 increases PAF production. CONCLUSIONS: These results indicate that EGF stimulates PAF production in ovarian cancer cells in a manner that requires cPLA2. We have also determined that crosstalk can occur bidirectionally between EGFR and PAFR, suggesting that EGF-induced PAF production could result in positive feedback that acts on the PAF-receptor to promote ovarian cancer progression.

Platelet-activating factor induces proliferation in differentiated keratinocytes.

Increased levels of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) are found in several inflammatory dermatoses, but PAF's exact role in epidermis is uncertain. In order to better understand the physiological consequences of excess PAF production in epidermis, we examined the gene regulatory effects of PAF short-term stimulation in differentiated HaCaT keratinocytes by transcriptional profiling. Even though PAF induces COX2 expression, we found that PAF regulates only few genes associated with inflammation in differentiated keratinocytes. Rather, we show that natural PAF rapidly regulates genes involved in proliferation, (anti)-apoptosis and migration, all sub-processes of re-epithelialization and wound healing. Moreover, profiling of phosphorylated kinases, cellular wound-scratch experiments, resazurin assay and flow cytometry cell cycle phase analysis all support a role for PAF in keratinocyte proliferation and epidermal re-epithelialization. In conclusion, these results suggest that PAF acts as an activator of proliferation and may, therefore, function as a connector between inflammation and proliferation in differentiated keratinocytes.

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.

Alkylglycerol monooxygenase as a potential modulator for PAF synthesis in macrophages.

Alkylglycerol monooxygenase (AGMO, glyceryl ether monooxygenase) is an enzyme known to catalyze the cleavage of the O-alkyl bond of glyceryl ether lipids. Identification of the gene encoding AGMO was reported recently, however, the involvement of AGMO in modulating cellular lipids has not been reported until now. In this report, we investigate a possible role for AGMO in macrophage platelet-activating factor (PAF) production. AGMO mRNA expression levels decreased with lipopolysaccharide (LPS) treatments in mouse peritoneal macrophages and RAW264.7 cells. Tetrahydrobiopterin-dependent conversion of lyso-PAF to glycerophosphocholine in the microsomal fraction was also reduced in LPS-treated RAW264.7 cells. In the LPS-treated cells, both lyso-PAF and PAF levels increased. Moreover, exogenously expressed AGMO caused a reduction in cellular lyso-PAF and PAF levels in HEK293 cells. Collectively, our results suggest a possible mechanism for AGMO in modulating macrophage PAF production by regulating cellular lyso-PAF levels.

Interleukin-1beta stimulates platelet-activating factor production in U-937 cells modulating both its biosynthetic and catabolic enzymes.

Interleukin-1beta (IL-1ß) is a potent agonist of platelet-activating factor (PAF) synthesis. The monocyte-derived PAF may amplify the inflammatory and thrombotic processes. The IL-1ß-induced enzymatic alterations leading to increased PAF synthesis are ill-defined. In the present study the last enzymatic activities of the remodeling (acetyl-CoA:lyso-PAF acetyltransferase) and de novo (DTT-insensitive CDP-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase) biosynthetic routes of PAF and its main catabolic enzyme, PAF acetylhydrolase, along with the intracellular and extracellular PAF levels were determined in homogenates and medium of U-937 after their stimulation with recombinant IL-1ß. IL-1ß at 2.5ng/mL induced an early (0.5-3h) and a late (12h) elevation of intracellular PAF levels (2-fold). Only a small portion of intracellular PAF (∼10%) was released to the extracellular medium. IL-1ß increased lyso-PAF acetyltrasnferase activity which was peaked at 3h and kept elevated till 12h. A rapid 1.5-fold increase of cholinephosphotransferase activity was observed in IL-1ß stimulated cells. Finally, a transient stimulation of intracellular PAF-AH was induced by IL-1ß at 3h while incubation of U-937 with the PAF acetylhydrolase inhibitor pefabloc in the presence or absence of IL-1ß led to a strong sustained increase of intracellular PAF levels. In conclusion, both biosynthetic routes of PAF, along with its degradation can be modulated by IL-1ß in a time-specific manner. The inhibition of PAF acetylhydrolase strongly augments PAF's intracellular levels implying its crucial role for the regulation of cellular PAF. The regulation of PAF's enzymatic machinery under inflammatory conditions is more complicated than we thought to be.

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

BACKGROUND: 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. METHODS: 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). KEY RESULTS: 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. CONCLUSIONS & INFERENCES: 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.

Albuterol isomers modulate platelet-activating factor synthesis and receptor signaling in human bronchial smooth muscle cells.

BACKGROUND: Racemic albuterol is a 50:50 mixture of the (R)- and (S)-enantiomers of albuterol. Its clinical efficacy resides in the (R)-enantiomer (levalbuterol). Studies have shown that (S)-albuterol induces human bronchial smooth muscle cell (HBSMC) proliferation via a pathway linked to platelet-activating factor (PAF), but the underlying mechanism by which (S)-albuterol augments PAF effects is not clear. In this study, we compared effect of levalbuterol and (S)-albuterol on PAF receptor (PAFr)-mediated signaling and PAF metabolism by HBSMCs after incubation with the albuterol isomers. METHODS: PAF binding and inositol phosphate (IP(3)) release were studied on adherent cultured cells. PAFr protein expression was measured by Western blotting, PAF synthesis and catabolism were measured in membrane and cytosolic proteins of cells incubated with albuterol isomers. RESULTS: Compared to control conditions, (S)-albuterol increased PAF binding by 70% after 30 min of preincubation and by 150% after 24 h of preincubation. Levalbuterol had no effect on PAF binding under both conditions. (S)-albuterol also augmented PAF stimulation of IP(3) release, while levalbuterol and the racemic mixture had no effect. WEB 2170, a PAFr antagonist, inhibited the ability of (S)-albuterol to increase PAF binding or stimulate IP(3) release. (S)-albuterol stimulated PAFr protein expression. With PAF metabolism, (S)-albuterol treatment augmented PAF synthesis, but significantly inhibited PAF catabolism. CONCLUSIONS: Our data suggest that one mechanism by which (S)-albuterol stimulates HBSMC proliferation involves upregulation of PAFr-mediated effects including increased PAF synthesis and decreased PAF catabolism.

Fish polar lipids retard atherosclerosis in rabbits by down-regulating PAF biosynthesis and up-regulating PAF catabolism.

BACKGROUND: Platelet activating factor (PAF) has been proposed as a key factor and initial trigger in atherosclerosis. Recently, a modulation of PAF metabolism by bioactive food constituents has been suggested. In this study we investigated the effect of fish polar lipid consumption on PAF metabolism. RESULTS: The specific activities of four PAF metabolic enzymes; in leukocytes, platelets and plasma, and PAF concentration; either in blood cells or plasma were determined. Samples were acquired at the beginning and at the end of a previously conducted study in male New Zealand white rabbits that were fed for 45 days with atherogenic diet supplemented (group-B, n = 6) or not (group-A, n = 6) with gilthead sea bream (Sparus aurata) polar lipids.The specific activity of PAF-Acetylhydrolase (PAF-AH); a catabolic enzyme of PAF, was decreased in rabbits' platelets of both A and B groups and in rabbits' leukocytes of group A (p < 0.05). On the other hand the specific activity of Lipoprotein-associated Phospholipase A2 (Lp-PLA2); the catabolic enzyme of PAF in plasma was increased in both A and B groups in both leukocytes and platelets (p < 0.05). PAF-cholinephosphotransferase (PAF-CPT); a biosynthetic enzyme of PAF showed increased specific activity only in rabbits' leukocytes of group A (p < 0.05). Neither of the two groups showed any change in Lyso-PAF-acetyltransferase (Lyso-PAF-AT) specific activity (p > 0.05). Free and bound PAF levels increased in group A while decreased in group B (p < 0.05). CONCLUSIONS: Gilthead sea bream (Sparus aurata) polar lipids modulate PAF metabolism upon atherosclerotic conditions in rabbits leading to lower PAF levels and activity in blood of rabbits with reduced early atherosclerotic lesions compared to control group.

Platelet activating factor: the good and the bad in the ischemic/reperfused heart.

The present review is focused on the dual role played by platelet-activating factor (PAF) in ischemia and reperfusion (I/R) injury of the heart. Although the involvement of PAF in the pathogenesis of myocardial reperfusion injury is well established, in the last few years it has emerged that very low concentrations of PAF exert cardioprotective effects, comparable to that afforded by ischemic preconditioning (IP). PAF is a potent phosphoglyceride involved in different pathophysiological conditions affecting the cardiovascular system, including the development of myocardial I/R injury. PAF is released from the I/R myocardium in concentrations (1-10 nmol/L) high enough to negatively modulate coronary circulation as well as electrical and contractile activities. PAF may act either directly, via generation of secondary mediators, or through the activation of inflammatory cells like platelets and polymorphonuclear neutrophils, which exacerbate postischemic myocardial injury. The effects of PAF are mediated through specific receptors (PAFRs) that belong to the superfamily of G protein-coupled receptors. Since cardiomyocytes not only produce PAF but also possess PAFRs, it is likely that PAF acts as an autocrine/paracrine mediator. Although the negative effects exerted by high concentrations of PAF are well established, several recent findings from our and other laboratories have demonstrated that very low concentrations (pmol/L) of PAF infused before ischemia induce cardioprotective effects similar to those afforded by IP, and that endogenous PAF production participates in the induction of IP itself. The IP-like action exerted by low concentrations of PAF is due to the activation/phosphorylation of kinases included in the reperfusion injury salvage kinase (RISK) pathway, such as protein kinase C, Akt/PkB and nitric oxide synthase. Together with the activation of mitochondrial K(ATP) channels, these events may allow prevention of mitochondrial permeability transition pores opening at reperfusion. Moreover, the nitric oxide-dependent S-nitrosylation of L-type Ca(2+) channels induced by PAF reduces intracellular Ca(2+) overload.

Urothelial cell platelet-activating factor production mediated by calcium-independent phospholipase A2γ.

OBJECTIVES: To determine the effect of phospholipase A(2) (PLA(2)) inhibitors on urothelial cell platelet-activating factor (PAF) production in response to tryptase stimulation. METHODS: Urothelial cells isolated from normal human ureters were immortalized with the human papillomavirus type 16E6E7 cell line (TEU-2 cells). PLA(2) activity in TEU-2 cells was measured using (16:0, [(3)H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence and absence of calcium. [(3)H]PAF production was measured in TEU-2 cells prelabeled with [(3)H] acetic acid. PAF-acetylhydrolase activity was measured by determining the amount of [(3)H] acetate hydrolyzed from [(3)H]PAF incubated with TEU-2 cellular protein. Adherence of human polymorphonuclear leukocyte (PMN) to TEU-2 cells was assessed by measuring myeloperoxidase activity in adherent PMNs after incubation with TEU-2 cells. RESULTS: Most PLA(2) activity measured in TEU-2 cells was determined to be membrane-associated, calcium-independent PLA(2) and selective for plasmenylcholine substrate. Stimulation of TEU-2 cells with tryptase results in increased production of PAF and increased PMN adherence that were inhibited completely by pretreatment with the membrane-associated, calcium-independent PLA(2)γ-selective inhibitor (R)-bromoenol lactone. Pretreatment with the cytosolic PLA(2) inhibitor methyl arachidonyl fluorophosphonate resulted in potentiation of tryptase-stimulated PAF production and PMN adherence to TEU-2 cells that is a result of PAF-acetylhydrolase inhibition. CONCLUSIONS: Tryptase stimulation of TEU-2 cells results in activation of membrane-associated, calcium-independent PLA(2)γ, leading to an increase in PAF production and increased PMN adherence. Inhibition of TEU-2 cell PAF-acetylhydrolase activity with methyl arachidonyl fluorophosphonate potentiated tryptase-stimulated PAF production and PMN adherence.

Identification of VEGF receptor-2 tyrosine phosphorylation sites involved in VEGF-mediated endothelial platelet-activating factor synthesis.

Vascular endothelial growth factor (VEGF)-mediated inflammation requires the synthesis of acute platelet-activating factor (PAF) by endothelial cells (ECs). We previously reported that VEGF-mediated PAF synthesis involves the activation of the homodimeric tyrosine kinase receptor VEGFR-2/R-2, leading to the recruitment of p38 and p42/p44 mitogen-activated protein kinases (MAPKs) and activation of secreted group V phospholipase A2 (sPLA2-V). We have also reported that VEGF-A165-mediated prostacyclin (PGI2) synthesis requires VEGFR-1/R-2 heterodimeric receptor activation. Selective activation of VEGF receptors can coordinate the synthesis of pro-PAF and anti-PGI2 inflammatory factors. It is unknown which VEGFR-2 tyrosine phosphorylation site(s) contribute(s) to PAF synthesis. Bovine aortic endothelial cells (BAECs) were transfected with pcDNA vectors encoding for native VEGF receptor-2 (VEGFR-2) cDNA or VEGFR-2 cDNA containing tyrosine phosphorylation sites mutated into phenylalanine residues (Y801F, Y1059F, Y1175F, Y1214F); an empty pcDNA vector was used as a negative control. Treatment of pcDNA-transfected BAECs with VEGF (10⁻9 mol/L) for 15 min increased PAF synthesis by 180%. In BAECs transfected with pcDNA vectors encoding mutated Y801F, Y1059F, Y1175F, or Y1214F VEGFR-2 cDNA, we observed a marked reduction of VEGF-mediated PAF synthesis by 38%, 46%, 69%, and 31%, respectively, compared with BAECs transfected with pcDNA vector encoding VEGFR-2 cDNA. Our data provide a novel insight as to the mechanisms by which VEGF promotes PAF synthesis.

Phosphorylation of lysophosphatidylcholine acyltransferase 2 at Ser34 enhances platelet-activating factor production in endotoxin-stimulated macrophages.

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions under physiological and pathological conditions. We have recently identified two enzymes involved in PAF production: lysophosphatidylcholine acyltransferase-1 (LPCAT1) and LPCAT2. We found that LPCAT2 is highly expressed in inflammatory cells and is activated by lipopolysaccharide (LPS) treatment through Toll-like receptor 4. However, the molecular mechanism for the activation remains elusive. In this study, Phos-tag SDS-PAGE revealed the LPS-induced phosphorylation of LPCAT2. Furthermore, mass spectrometry and mutagenesis analyses identified Ser(34) of LPCAT2 as the phosphorylation site to enhance the catalytic activities. The experiments using inhibitors and siRNA against MAPK cascades demonstrated that LPCAT2 phosphorylation through LPS-TLR4 signaling may directly depend on MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2). These findings develop a further understanding of both PAF production and phospholipid remodeling triggered by inflammatory stimuli. Specific inhibition of the PAF biosynthetic activity by phosphorylated LPCAT2 will provide a novel target for the regulation of inflammatory disorders.

Endothelial cell prostaglandin I(2) and platelet-activating factor production are markedly attenuated in the calcium-independent phospholipase A(2)beta knockout mouse.

Damage and activation of lung endothelium can lead to interstitial edema, infiltration of inflammatory cells into the interstitium and airways, and production of inflammatory metabolites, all of which propagate airway inflammation in a variety of diseases. We have previously determined that stimulation of human microvascular endothelial cells from lung (HMVEC-L) results in activation of a calcium-independent phospholipase A(2) (iPLA(2)), and this leads to arachidonic acid release and production of prostaglandin I(2) (PGI(2)) and platelet-activating factor (PAF). We stimulated lung endothelial cells isolated from iPLA(2)beta-knockout (KO) and wild type (WT) mice with thrombin and tryptase to determine the role of iPLA(2)beta in endothelial cell membrane phospholipid hydrolysis. Thrombin or tryptase stimulation of WT lung endothelial cells resulted in increased arachidonic acid release and production of PGI(2) and PAF. Arachidonic acid release and PGI(2) production by stimulated iPLA(2)beta-KO endothelial cells were significantly reduced compared to WT. Measured PLA(2) activity and PGI(2) production by iPLA(2)beta-KO cells were suppressed by pretreatment with (R)-bromoenol lactone (R-BEL), which is a selective inhibitor of iPLA2gamma. In contrast to the increase in PAF production induced by stimulation of WT endothelial cells, none was observed for KO cells, and this suggests that endothelial PAF production is entirely dependent on iPLA(2)beta activity. Because inflammatory cell recruitment involves the interaction of endothelial cell PAF with PAF receptors on circulating cells, these data suggest that iPLA(2)beta may be a suitable therapeutic target for the treatment of inflammatory lung diseases.

Lipoxygenase metabolites modulate vascular-derived platelet activating factor production following endotoxin challenge.

Endotoxin promotes the production of potent pro-inflammatory lipid mediators, such as platelet activating factor (PAF) and eicosanoids, which contribute to the pathophysiology of endotoxic shock. Endothelial cells are both a target for and producers of these lipid mediators so it is vital to understand the pathways leading to their production in these cells. Previous research suggested a positive feedback loop between eicosanoids and PAF during endotoxemia. This study sought to determine if eicosanoids derived from the 15-lipoxygenase (15-LOX1) pathway can modulate the biosynthesis of PAF in cultured bovine aortic endothelial cells (BAEC) following endotoxin stimulation. Endotoxin stimulation increased the production of 15-LOX1-derived eicosanoids prior to PAF in primary BAEC. Exogenous addition of specific 15-LOX1 eicosanoids, as well as overexpression of 15-LOX1 in endotoxin-stimulated BAEC, further increased the endotoxin-induced production of PAF. Whereas increased expression of 15-LOX1 activity can further exacerbate endotoxin-induced PAF biosynthesis, inhibition of 15-LOX1 activity is not capable of abrogating the initial onset of endotoxin-induced PAF production. The results indicate that 15-LOX1 activity is not necessary for the initial induction of PAF following endotoxin stimulation. There may exist, however, a role for elevated 15-LOX1 activity in further escalating the extent of PAF biosynthesis in BAEC during endotoxic shock. Determining factors that can potentiate endotoxin-induced vascular dysfunction may lead to the development of novel therapeutic targets to diminish the pathophysiological effects of endotoxic shock.