Platelet-activating Factor Synthesis Research Articles

Kupffer cells are a major source of increased platelet activating factor in the CCl 4-induced cirrhotic rat liver

Background/Aims: Endothelin-1 (ET-1) stimulates the synthesis of platelet-activating factor (PAF) by Kupffer cells in vitro. Hepatic concentrations of both ET-1 (a potent vasoconstrictor) and PAF (a mediator of hepatic vasoconstriction and the cirrhotic hyperdynamic state) increase in cirrhosis. The aim of this study was to determine if the responsiveness of Kupffer cells to produce PAF upon ET-1 challenge is modified by cirrhosis. Methods: Kupffer cells, isolated from the livers of control and CCl 4-induced cirrhotic rats, were placed in serum-free medium after overnight culture. PAF and ET-1 receptors, ET-1-induced PAF synthesis, and PAF- and ET-1-induced prostaglandin E 2 (PGE 2) synthesis were determined 24 h later. Results: Both basal and ET-1-stimulated PAF synthesis was increased in cirrhotic Kupffer cells as indicated by increased cell-associated and released PAF. Cirrhotic Kupffer cells also had elevated densities of functional receptors for both PAF and ET-1 (exclusively ET B), as measured by ligand binding, mRNA expression of the respective receptors, and ligand-stimulated PGE 2 synthesis. Conclusions: Cirrhosis sensitizes Kupffer cells to both ET-1 and PAF by elevating their respective receptor levels. Since both mediators individually cause portal hypertension, an increase in ET-1-stimulated PAF synthesis in Kupffer cells will exacerbate the hepatic and extrahepatic complications of cirrhosis.

Experimental Trypanosoma cruzi infection in platelet-activating factor receptor-deficient mice

The generation of an inflammatory response driven by Trypanosoma cruzi or its subproducts appears to be essential for tissue injury and disease pathogenesis. However, this inflammatory response is also relevant in the control of T. cruzi replication. The lipid mediator platelet-activating factor (PAF) has been implicated in a number of pathological conditions characterized by tissue inflammation. In the present study, we aimed at evaluating the role of PAF during T. cruzi infection by using mice that were genetically deficient in the PAF receptor. We observed that infected hearts of PAFR –/– mice had an increased number of parasite nests, associated with a more intense inflammatory infiltrate. This was associated with greater parasitemia and lethality. When wild-type and PAFR –/– mice were compared, there were no marked changes in the kinetics of the expression of MCP-1, RANTES, IFN-γ and TNF-α in heart tissue of infected animals. Moreover, serum concentrations of TNF-α, nitrate and parasite-specific IgM were similar in both groups of mice. In vitro, macrophages from PAFR –/– animals did not phagocytose trypomastigote forms when activated with PAF, leukotriene B 4 or MCP-1 and produced less nitric oxide when infected and activated with IFN-γ. These results are consistent with the hypothesis that endogenous synthesis of PAF and activation of PAF receptors control T. cruzi replication in mice in great part via facilitation of the uptake of the parasite and consequent activation of macrophages.

IL-4 primes human endothelial cells for secondary responses to histamine.

Interleukin-4 (IL-4) is a multifunctional cytokine, which is involved in numerous disease states, including atopic asthma. IL-4 not only induces direct responses in cells but can also prime for secondary responses to stimuli. Little is known about the priming effects of IL-4 on endothelial cells; therefore, we chose to examine the ability of IL-4 to prime endothelial cells for platelet-activating factor (PAF) synthesis and prostaglandin E(2) (PGE(2)) release. IL-4 alone did not enhance PAF synthesis or PGE(2) release; however, pretreatment with IL-4 primed for PAF synthesis and PGE(2) release in response to subsequent stimulation with histamine. In contrast, tumor necrosis factor alpha (TNF-alpha), oncostatin M (OSM), and IL-1beta did not prime endothelial cells for PAF synthesis in response to histamine. The priming effects of IL-4 occurred without any detectable changes in the requirement for signaling pathways upstream of PGE(2) release. IL-4 treatment increased the expression of mRNA for histamine receptor 1 (HR1) and shifted the inhibition curve for pyrilamine, a specific HR1 antagonist. In addition, the dose-response curve for histamine-induced elevations in intracellular calcium was shifted following IL-4 stimulation. Together, these data indicate that HR1 is up-regulated in IL-4-stimulated human umbilical vein endothelial cells (HUVEC) and suggest that this up-regulation may contribute to the enhanced responsiveness of IL-4-stimulated HUVEC to histamine.

Regulation of platelet-activating factor synthesis in human monocytes by dipalmitoyl phosphatidylcholine.

Platelet-activating factor (PAF) has a major role in inflammatory responses within the lung. This study investigates the effect of pulmonary surfactant on the synthesis of PAF in human monocytic cells. The pulmonary surfactant preparation Curosurf significantly inhibited lipopolysaccharide (LPS)-stimulated PAF biosynthesis (P<0.01) in a human monocytic cell line, Mono mac-6 (MM6), as determined by (3)H PAF scintillation-proximity assay. The inhibitory properties of surfactant were determined to be associated, at least in part, with the 1,2-dipalmitoyl phosphatidylcholine (DPPC) component of surfactant. DPPC alone also inhibited LPS-stimulated PAF biosynthesis in human peripheral blood monocytes. DPPC treatment did not affect LPS-stimulated phospholipase A(2) activity in MM6 cell lysates. However, DPPC significantly inhibited LPS-stimulated coenzyme A (CoA)-independent transacylase and acetyl CoA:lyso-PAF acetyltransferase activity. DPPC treatment of MM6 cells decreased plasma membrane fluidity as demonstrated by electron paramagnetic resonance spectroscopy coupled with spin labeling. Taken together, these findings indicate that pulmonary surfactant, particularly the DPPC component, can inhibit LPS-stimulated PAF production via perturbation of the cell membrane, which inhibits the activity of specific membrane-associated enzymes involved in PAF biosynthesis.

Platelet-activating factor synthesis by neutrophils, monocytes, and endothelial cells is modulated by nitric oxide production.

Nitric oxide (NO) and platelet-activating factor (PAF) can modulate the interaction between endothelial lining and circulating leukocytes. Several studies implicated the production of PAF and NO in the pathogenesis of microcirculatory alterations occurring in septic shock. However, the reciprocal interaction between PAF and NO has not been fully elucidated. In the present study, we evaluated whether the basal synthesis of NO could modulate the production of PAF by neutrophils (PMN), monocytes (MO), and endothelial cells (EC) unstimulated or stimulated with lipopolysaccharides (LPS) or tumor necrosis factor (TNF). PMN, MO, and EC, when incubated with N(omega)-nitro-L-arginine methyl ester (L-NAME) spontaneously synthesized PAF, with an early peak at 30 min. The effective inhibition of NO production was visualized on MO cells as generation of fluorescence reactivity by cell-permeable NO reactive dye DAF-2 DA. Also, monomethyl-L-arginine (L-NMMA) induced PAF synthesis by PMN, whereas the biologically inactive D-enantiomers of NAME (D-NAME) and of NMMA (D-NMMA) did not. Stimulation of PMN with L-NAME in presence of the exogenous NO donor nitroprusside, of the NO secondary mediator cGMP, or of the NO synthase substrate L-arginine reduced PAF synthesis, suggesting the involvement of an NO-dependent pathway on the modulation of PAF synthesis. The synthesis of PAF was enhanced by combined treatment with L-NAME and TNF or LPS. These results indicate an inhibitor effect of NO on the spontaneous and TNF or LPS-induced synthesis of PAF by human PMN, MO, and EC.

Secretory phospholipases A2 activate selective functions in human eosinophils.

Secretory phospholipases A(2) (sPLA(2)s) are released in large amounts in the blood of patients with systemic inflammatory diseases and accumulate at sites of chronic inflammation, such as the airways of patients with bronchial asthma. Blood eosinophils or eosinophils recruited in inflammatory areas therefore can be exposed in vivo to high concentrations of sPLA(2). We have examined the effects of two structurally different sPLA(2)s (group IA and group IIA) on several functions of eosinophils isolated from normal donors and patients with hypereosinophilia. Both group IA and IIA sPLA(2) induced a concentration-dependent release of beta-glucuronidase, IL-6, and IL-8. Release of the two cytokines was associated with the accumulation of their specific mRNA. In addition, sPLA(2)s induced the surface expression of CD44 and CD69, two major activation markers of eosinophils. In contrast, none of the sPLA(2)s examined induced the production of IL-5, the de novo synthesis of leukotriene C(4) and platelet-activating factor, or the generation of superoxide anion from human eosinophils. Incubation of eosinophils with the major enzymatic products of the sPLA(2)s (arachidonic acid, lysophosphatidylcholine, or lysophosphatidic acid) did not reproduce any of the enzymes' effects. In addition, inactivation of sPLA(2) enzymatic activity by bromophenacyl bromide did not influence the release of beta-glucuronidase or of cytokines. Stimulation of eosinophils by sPLA(2)s was associated with activation of extracellular signal-regulated kinases 1/2. These results indicate that sPLA(2)s selectively activate certain proinflammatory and immunoregulatory functions of human eosinophils through mechanism(s) independent from enzymatic activity and from the generation of arachidonic acid.

The Long Pentraxin Ptx3 Is Synthesized in IgA Glomerulonephritis and Activates Mesangial Cells

The long pentraxin PTX3 has been recently involved in amplification of the inflammatory reactions and regulation of innate immunity. In the present study we evaluated the expression and role of PTX3 in glomerular inflammation. PTX3 expression was investigated in the IgA, type I membranoproliferative, and diffuse proliferative lupus glomerulonephritis, which are characterized by inflammatory and proliferative lesions mainly driven by resident mesangial cells, and in the membranous glomerulonephritis and the focal segmental glomerular sclerosis, where signs of glomerular inflammation are usually absent. We found an intense staining for PTX3 in the expanded mesangial areas of renal biopsies obtained from patients with IgA glomerulonephritis. The pattern of staining was on glomerular mesangial and endothelial cells. Scattered PTX3-positive cells were also detected in glomeruli of type I membranoproliferative glomerulonephritis. The concomitant expression of CD14 suggests an inflammatory origin of these cells. Normal renal tissue and biopsies from patients with the other glomerular nephropathies studied were mainly negative for PTX3 expression in glomeruli. However, PTX3-positive cells were detected in the interstitium of nephropathies showing inflammatory interstitial injury. In vitro, cultured human mesangial cells synthesized PTX3 when stimulated with TNF-alpha and IgA and exhibited specific binding for recombinant PTX3. Moreover, stimulation with exogenous PTX3 promoted mesangial cell contraction and synthesis of the proinflammatory lipid mediator platelet-activating factor. In conclusion, we provide the first evidence that mesangial cells may both produce and be a target for PTX3. The detection of this long pentraxin in the renal tissue of patients with glomerulonephritis suggests its potential role in the modulation of glomerular and tubular injury.

Regulation of Activities of Cytidine 5'-Diphospho-Choline: 1-O-Alkyl-2-Acetyl-sn-Glycerol Cholinephosphotransferase, an Enzyme Responsible for de novo Synthesis of Platelet-Activating Factor, by Membrane Phospholipids.

Cytidine 5′-diphospho (CDP)-choline: 1-O-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (AAG-CPT), an enzyme responsible for de novo synthesis of platelet-activating factor (PAF), was solubilized from porcine spleen microsomes using digitonin. Although the activity of the solubilized enzyme was relatively stable, further purification by sequential chromatography on Toyopearl HW-65 gel filtration and diethylaminoethyl (DEAE)-Toyopearl 650 caused a remarkable decrease in enzyme activity, which was partially recovered by the exogenously addition of phospholipids such as egg phosphatidylcholine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, and dioleoylphosphatidylserine. In contrast, dioleoylphosphatidic acid (DOPA) showed an inhibitory effect on enzyme activity. In addition, lysophospholipids such as monooleoylphosphatidylcholine, monooleoylphosphatidylethanolamine, monooleoylphosphatidylglycerol, and monooleoylphosphatidic acid showed an inhibitory effect on the enzyme activity. When DOPA was concomitantly added with DOPC, the enzyme activity reactivated by DOPC decreased with the ratio of DOPA to DOPC. Furthermore, we examined whether phosphatidic acid (PA) or lysophospholipids had an inhibitory effect on AAG-CPT activity under more physiological conditions. Treatment of microsomes with exogenously added phospholipase D or phospholipase A2 resulted in a decrease in AAG-CPT. In addition, when endogenous phospholipase D was activated by fatty acids such as oleic acid and arachidonic acid to generate PA in the porcine microsomes, the enzyme activity was significantly inhibited. The molecular weight of the enzyme solubilized from porcine spleen microsomes was estimated to be 440 kDa based on gel-filtration column chromatography on Toyopearl HW-65, suggesting that this enzyme formed a complex with other protein molecules and membrane phospholipids, and that these phospholipids were necessary to maintain the enzyme activity. Our results indicate that environmental membrane phospholipids containing PA and/or lysophospholipids are important factors in the regulation of the enzyme for de novo synthesis of PAF.

Overexpression of Phospholipid Hydroperoxide Glutathione Peroxidase Modulates Acetyl-CoA, 1-O-Alkyl-2-lyso-sn-glycero-3-phosphocholine Acetyltransferase Activity

The synthesis of platelet-activating factor (PAF) by -stimulated RBL-2H3 cells was significantly suppressed by overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx). When the cells overexpressing PHGPx (L9 cells) were pretreated with diethyl maleate, which reduces PHGPx activity, PAF synthesis upon stimulation rose to levels seen in mock-transfected cells (S1 cells). Hydroperoxide levels, which are reduced in L9 cells, are involved in regulating PAF synthesis, because the addition of hydroperoxyeicosatetraenoic acid increased PAF production in -stimulated L9 cells to control cell levels. The activity of acetyl-CoA:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase, which is involved in the last step of PAF synthesis, is also reduced in L9 cells. p38 kinase inhibitors block acetyltransferase activity in normal -stimulated cells, suggesting that p38 kinase is involved in regulating acetyltransferase activity. Recombinant active p38 kinase activates acetyltransferase, whereas alkaline phosphatase reverses this, suggesting p38 kinase directly phosphorylates acetyltransferase. p38 kinase phosphorylation is blocked in L9 cells, indicating that high hydroperoxide levels are needed for the activation of p38 kinase. Thus, intracellular hydroperoxide levels participate in regulating p38 kinase phosphorylation, which in turn controls the activation of acetyltransferase and thus the synthesis of PAF. These observations suggest that PHGPx is an important component of the mechanisms regulating inflammation.

Immediate and delayed VEGF-mediated NO synthesis in endothelial cells: role of PI3K, PKC and PLC pathways.

1. The mechanism(s) by which vascular endothelial growth factor (VEGF) induces endothelial nitric oxide synthase (eNOS) activation remain(s) unclear up to a certain extent. Therefore, we sought to evaluate the contribution of numerous pathways in VEGF-induced nitric oxide (NO) synthesis by measuring cGMP production. In addition, as VEGF induces the synthesis of NO and platelet-activating factor (PAF), we wanted to assess if the induction of PAF and NO is contributing to the synthesis of each other. 2. Herein, we show that a treatment of endothelial cells with a phospholipase C (PLC) inhibitor (U73122), a calmodulin antagonist (W-7) or with intracellular calcium chelators (EGTA/AM, BAPTA/AM) prevented VEGF-mediated eNOS Ser(1177)-phosphorylation and NO synthesis measured by cGMP production. 3. Pretreatment with phosphatidylinositol 3-kinase (PI3K) (Wortmannin, LY294002) or protein kinase C (PKC) (GF109203X, Ro318220) inhibitors attenuated eNOS Ser(1177)-phosphorylation mediated by VEGF, but did not alter immediate (0-10 min) cGMP synthesis induced by VEGF, but abrogated by up to 84% the delayed (10-30 min) cGMP synthesis. 4. Pretreatment with PAF synthesis inhibitors or with PAF receptor antagonists did not abrogate neither eNOS Ser(1177)-phosphorylation nor cGMP synthesis mediated by VEGF. 5. In conclusion, VEGF induces an immediate cGMP synthesis through the PLC-Ca2+/CaM pathway, and that the induction of delayed cGMP synthesis implies Akt and PKC activity.

Molecular Mechanisms for Lipopolysaccharide-induced Biphasic Activation of Nuclear Factor-κB (NF-κB)

The nuclear factor-kappaB (NF-kappaB) is an important transcription factor necessary for initiating and sustaining inflammatory and immune reactions. The inducers of NF-kappaB are well characterized, but the molecular mechanisms underlying multiple in vivo NF-kappaB activation processes are poorly understood. The injection of lipopolysaccharide resulted in a biphasic activation of NF-kappaB during the 18-h observation period in various organs of mice. The early and late phases of NF-kappaB activation occurred at 0.5-2 h and 8-12 h, respectively. Platelet-activating factor, which is released in response to lipopolysaccharide injection, was responsible for the activation of the early phase of NF-kappaB. The early NF-kappaB activity led to the induction of proinflammatory cytokines, tumor necrosis factor (TNF), and interleukin (IL)-1beta, which are known to be efficient inducers of NF-kappaB. Using the TNF knockout and IL-1 receptor knockout mice, we found that TNF and IL-1beta had a role in the second phase activation of NF-kappaB. These cytokines did promote the synthesis of platelet-activating factor, which in turn induced the secondary activation of NF-kappaB. These observations describe a novel autoregulatory molecular mechanism for the biphasic activation of NF-kappaB.

Nitric oxide/platelet activating factor cross-talk in mesangial cells modulates the interaction with leukocytes

Nitric oxide/platelet activating factor cross-talk in mesangial cells modulates the interaction with leukocytes.BackgroundPlatelet activating factor (PAF) and nitric oxide (NO) exert opposite effects on adherence and activation of circulating leukocytes to endothelium. Several studies have implicated the production of PAF and NO by mesangial cells in the regulation of glomerular filtration, permeability and inflammation. However, the reciprocal interaction between PAF and NO in mesangial cells and their role in leukocyte adhesion has not been investigated.MethodsWe evaluated whether blockade of constitutive production of NO by two different NO synthase (NOS) inhibitors (L-NAME and L-NMMA) could modulate PAF synthesis, and conversely whether exogenous PAF could influence the production of NO by mesangial cells. We evaluated whether modulation of PAF synthesis by NOS inhibitors could affect leukocyte adhesion to mesangial cells. The effect of PAF-receptor antagonist WEB2170, of anti-β2 integrins and intracellular adhesion molecule-1 (ICAM-1) blocking antibodies and of soluble Sialyl-Lewis-a also was evaluated.ResultsBlockade of NO synthesis by NOS inhibitors induced a spontaneous synthesis of PAF that was conversely inhibited by NO generation. On the other hand, PAF inhibited both the basal and L-arginine induced synthesis of NO by mesangial cells. Moreover, NOS inhibition promoted the adhesion of polymorphonuclear cells and monocytes to mesangial cells by a mechanism dependent on the synthesis of PAF and on the interaction of β2 integrins and ICAM-1.ConclusionsThese data indicate that PAF and NO exhibit a bi-directional effect on their respective synthesis in human mesangial cells, and suggest that their reciprocal regulation may be relevant for leukocyte adhesion to glomerular mesangial cells. Nitric oxide/platelet activating factor cross-talk in mesangial cells modulates the interaction with leukocytes. Platelet activating factor (PAF) and nitric oxide (NO) exert opposite effects on adherence and activation of circulating leukocytes to endothelium. Several studies have implicated the production of PAF and NO by mesangial cells in the regulation of glomerular filtration, permeability and inflammation. However, the reciprocal interaction between PAF and NO in mesangial cells and their role in leukocyte adhesion has not been investigated. We evaluated whether blockade of constitutive production of NO by two different NO synthase (NOS) inhibitors (L-NAME and L-NMMA) could modulate PAF synthesis, and conversely whether exogenous PAF could influence the production of NO by mesangial cells. We evaluated whether modulation of PAF synthesis by NOS inhibitors could affect leukocyte adhesion to mesangial cells. The effect of PAF-receptor antagonist WEB2170, of anti-β2 integrins and intracellular adhesion molecule-1 (ICAM-1) blocking antibodies and of soluble Sialyl-Lewis-a also was evaluated. Blockade of NO synthesis by NOS inhibitors induced a spontaneous synthesis of PAF that was conversely inhibited by NO generation. On the other hand, PAF inhibited both the basal and L-arginine induced synthesis of NO by mesangial cells. Moreover, NOS inhibition promoted the adhesion of polymorphonuclear cells and monocytes to mesangial cells by a mechanism dependent on the synthesis of PAF and on the interaction of β2 integrins and ICAM-1. These data indicate that PAF and NO exhibit a bi-directional effect on their respective synthesis in human mesangial cells, and suggest that their reciprocal regulation may be relevant for leukocyte adhesion to glomerular mesangial cells.

Nitric oxide/platelet activating factor cross-talk in mesangial cells modulates the interaction with leukocytes

Platelet activating factor (PAF) and nitric oxide (NO) exert opposite effects on adherence and activation of circulating leukocytes to endothelium. Several studies have implicated the production of PAF and NO by mesangial cells in the regulation of glomerular filtration, permeability and inflammation. However, the reciprocal interaction between PAF and NO in mesangial cells and their role in leukocyte adhesion has not been investigated. We evaluated whether blockade of constitutive production of NO by two different NO synthase (NOS) inhibitors (L-NAME and L-NMMA) could modulate PAF synthesis, and conversely whether exogenous PAF could influence the production of NO by mesangial cells. We evaluated whether modulation of PAF synthesis by NOS inhibitors could affect leukocyte adhesion to mesangial cells. The effect of PAF-receptor antagonist WEB2170, of anti-beta(2) integrins and intracellular adhesion molecule-1 (ICAM-1) blocking antibodies and of soluble Sialyl-Lewis-a also was evaluated. Blockade of NO synthesis by NOS inhibitors induced a spontaneous synthesis of PAF that was conversely inhibited by NO generation. On the other hand, PAF inhibited both the basal and l-arginine induced synthesis of NO by mesangial cells. Moreover, NOS inhibition promoted the adhesion of polymorphonuclear cells and monocytes to mesangial cells by a mechanism dependent on the synthesis of PAF and on the interaction of beta(2) integrins and ICAM-1. These data indicate that PAF and NO exhibit a bi-directional effect on their respective synthesis in human mesangial cells, and suggest that their reciprocal regulation may be relevant for leukocyte adhesion to glomerular mesangial cells.

Effects of atorvastatin 10 mg on triglycerides in diabetics type 2 patients, a double blind randomized placebo controlled study

We investigated the effect of sodium nitroprusside (SNP), a donor of nitric oxide, on the formation of platelet-activating factor (PAF) and uterine contractility in mouse uterine horns from mice treated with estrogen. Because the major pathway of PAF synthesis is the remodeling pathway in uterine tissue, we evaluated the incorporation of 14C-acetate into PAF-like molecules. Our results showed that SNP (100–300 μmol/L) caused a transient increase in the synthesis of PAF, which remained cell-associated.The addition of SNP (100–300 μmol/L) to a mouse uterine horn in an isolated organ bath preparation evoked a transient increase in contractility, which was inhibited by hemoglobin (2 μg/mL), a nitric oxide scavenger, but not by methylene blue (10 μmol/L), a guanylate cyclase inhibitor. The pharmacological characteristics of the contractions evoked by SNP resembled those evoked after mast cell activation, in that they were blocked by ritodrine (a β2adrenergic agonist, 0.1 μmol/L); indomethacin (a cyclooxygenase inhibitor, 10 μmol/L); ketotifen (a mast cell stabilizer, 1.0 μmol/L); cromolyn sodium (a mast cell stabilizer, 100 μmol/L); pyrilamine (an H1 antagonist, 10 μmol/L); and ketanserine (5HT2 antagonist, 0.1 μmol/L).These data demonstrate that nitric oxide generated from SNP stimulated the synthesis of PAF and evoked contractility in uterine horns from mice treated with estrogen. This result suggests the possibility that these tissue conditions might be favorable for the generation of peroxynitrites, possible mediators of both effects. It is also shown that the contractility evoked by the addition of SNP was not due to production of PAF, because its antagonist, WEB 2086 (10–30 μmol/L, a concentration that blocked contractions evoked by PAF 1 nmol/L), had no effect on the SNP-evoked contractions.

Antisense inhibition of Flk-1 by oligonucleotides composed of 2'-deoxy-2'-fluoro-beta-D-arabino- and 2'-deoxy-nucleosides.

The design of new antisense oligomers with improved binding affinity for targeted RNA, while still activating RNase H, is a major research area in medicinal chemistry. RNase H recognizes the RNA-DNA duplex and cleaves the complementary mRNA strand, providing the main mechanism by which antisense oligomers elicit their activities. It has been shown that configuration inversion at the C2' position of the DNA sugar moiety (arabinonucleic acid, ANA), combined with the substitution of the 2'OH group by a fluorine atom (2'F-ANA) increases the oligomer's binding affinity for targeted RNA. In the present study, we evaluated the antisense activity of mixed-backbone phosphorothioate oligomers composed of 2'-deoxy-2'-fluoro-beta-D-arabinose and 2'-deoxyribose sugars (S-2'F-ANA-DNA chimeras). We determined their abilities to inhibit the protein expression and phosphorylation of Flk-1, a vascular endothelial growth factor receptor (VEGF), and VEGF biological effects on endothelial cell proliferation, migration, and platelet-activating factor synthesis. Treatment of endothelial cells with chimeric oligonucleotides reduced Flk-1 protein expression and phosphorylation more efficiently than with phosphorothioate antisenses (S-DNA). Nonetheless, these two classes of antisenses inhibited VEGF activities equally. Herein, we also demonstrated the capacity of the chimeric oligomers to elicit RNase H activity and their improved binding affinity for complementary RNA as compared with S-DNA.

Distinct pathways of lipopolysaccharide priming of human neutrophil respiratory burst: role of lipid mediator synthesis and sensitivity to interleukin-10.

Exposure of neutrophils to low doses of bacterial lipopolysaccharides enhances their readiness to respond with inflammatory mediator generation including oxygen radical formation to a subsequently applied inflammatory stimulus ("priming"). In the present study, we investigated the role of lipid mediator synthesis and the impact of the anti-inflammatory cytokine interleukin-10 on the lipopolysaccharide-dependent priming of human neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine. Prospective, experimental study. Research laboratory at a university hospital. Isolated neutrophils from healthy volunteers. Incubation of isolated neutrophils with endotoxin. Evidence for two distinct priming mechanisms was obtained. The first was strictly serum component dependent, proceeded via CD14, and was not inhibited by even high concentrations of interleukin-10. The second priming mechanism was serum component independent but nevertheless proceeded via CD14. It was linked with neutrophil synthesis of the platelet activating factor and resulted in the appearance of leukotrienes, in particular leukotriene B4, as far as exogenous arachidonic acid was provided. The employment of a platelet-activating factor receptor antagonist (WEB 2086) blocked leukotriene synthesis, and both WEB 2086 and a 5-lipoxygenase inhibitor (MK-886) suppressed the respiratory burst linked with this second priming pathway. This sequence of priming events was inhibited by interleukin-10, when this cytokine was coadministered with the priming agent lipopolysaccharide, whereas late interleukin-10 admixture was ineffective. We conclude that two mechanisms of lipopolysaccharide priming of human neutrophil respiratory burst can be differentiated. One displays serum component dependence, is independent of neutrophil lipid mediator generation, and is not affected by interleukin-10. The other is serum independent although being operated via CD14, employs autocrine loops of platelet-activating factor and leukotriene B4 synthesis, and is sensitive to the inhibitory capacity of interleukin-10. These features may be relevant when the goal is to pharmacologically modify neutrophil functions in septic events.

Regulation of platelet-activating factor synthesis in human neutrophils by MAP kinases

Human neutrophils (PMN) are potentially a major source of platelet-activating factor (PAF) produced during inflammatory responses. The stimulated synthesis of PAF in PMN is carried out by a phospholipid remodeling pathway involving three enzymes: acetyl-CoA:lyso-PAF acetyltransferase (acetyltransferase), type IV phospholipase A 2 (cPLA 2) and CoA-independent transacylase (CoA-IT). However, the coordinated actions and the regulatory mechanisms of these enzymes in PAF synthesis are poorly defined. A23187 has been widely used to activate the remodeling pathway, but it has not been shown how closely its actions mimic those of physiological stimuli. Here we address this important problem and compare responses of the three remodeling enzymes and PAF synthesis by intact cells. In both A23187- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN, acetyltransferase activation is blocked by SB 203580, a p38 MAP kinase inhibitor, but not by PD 98059, which blocks activation of the ERKs. In contrast, either agent attenuated cPLA 2 activation. Correlating with these results, SB 203580 decreased stimulated PAF formation by 60%, whereas PD 98059 had little effect. However, the combination of both inhibitors decreased PAF formation to control levels. Although a role for CoA-IT in PAF synthesis is recognized, we did not detect activation of the enzyme in stimulated PMN. CoA-IT thus appears to exhibit full activity in resting as well as stimulated cells. We conclude that the calcium ionophore A23187 and the receptor agonist fMLP both act through common pathways to stimulate PAF synthesis, with p38 MAP kinase regulating acetyltransferase and supplementing ERK activation of cPLA 2.

Metabolism of platelet activating factor by intrapulmonary vascular smooth muscle cells. Effect of oxygen on phospholipase A 2 protein expression and activities of acetyl-CoA acetyltransferase and cholinephosphotransferase

We have demonstrated that platelet activating factor (PAF) plays an important physiological role in the maintenance of high pulmonary vascular tone in fetal lambs, a role attributable to increased PAF receptor binding (J. Appl. Physiol. 85 (1998) 1079; Am J. Physiol. 278 (2000) H1168). In this study, we examined the possibility that increased PAF synthesis via de novo and remodeling pathways as well as decreased PAF catabolism in hypoxic state of fetal lungs may account for the PAF action in vivo. We investigated effect of oxygen tension on PAF synthesis by ovine fetal intrapulmonary venous (PV) and arterial (PA) smooth muscle cells pulsed with [ 3 H ]choline (de novo), or [ 3 H ]acetate (remodeling), while PAF catabolism was studied by assay of acetylhydrolase (PAF-Ah) activity. Hypoxia stimulated PAF synthesis by choline incorporation (pmol/10 6 cells) in both PVSMC (1.14±0.13 vs 0.53±0.05) and PASMC (0.39±0.12 vs 0.22±0.04). Hypoxia stimulated PAF synthesis via remodeling pathway only in PVSMC (408±32 vs 225±17) which was 5-fold greater than in PASMC (77±15 vs 105±24), however, with A23187 in remodeling pathway, PAF synthesis increased 5-fold compared to baseline conditions and then synthesis in hypoxia was greater than in normoxia in both cell types. Phospholipase A 2 protein expression was significantly higher in hypoxia in both cells and was ∼2-fold higher in PVSMC. PAF-Ah activity (nmol lyso-PAF/min/mg protein) was greater in hypoxia vs normoxia in PVSMC (0.81±0.24 vs 0.44±0.088), but in PASMC activity was less in hypoxia vs normoxia (1.68±0.24 vs 3.93±0.44). Compared to PVSMC PAF-Ah activity in PASMC was 4-fold higher in hypoxia. Our data demonstrate that (1) PAF synthesis in intrapulmonary SMC of fetal lambs occurs by both de novo studied by choline incorporation and remodeling pathways, the latter being predominant. (2) There is heterogeneity in PAF synthetic and catabolic activities in lung vasculature of fetal lambs. We conclude that increased PAF synthesis in veins by the two synthetic pathways coupled with decreased catabolism will result in a higher venous PAF levels in the hypoxic environment of fetal lungs. We speculate that in vivo, a high PAF level in veins will make more PAF available for binding to its receptors so as to sustain the desired high venous tone in the fetal pulmonary circulation.

Synthesis of platelet-activating factor (PAF) in transformed cell lines of a different origin

Interest in the possible involvement of the platelet-activating factor (PAF) in tumor growth and invasiveness has been stimulated by the recognition that PAF influences various biological responses relevant to metastatic diffusion, such as angiogenesis, adhesiveness to endothelia and cellular motility. In the present study, we investigated the extent to which PAF is synthesized by a series of human and murine transformed cell lines of a different histotype. Synthesis of PAF was studied by combining the 14 C -acetate incorporation into PAF with the quantitative analysis of PAF performed by a procedure based on gas chromatography–mass spectrometry with a negative ion chemical ionization. In the presence of the Ca 2+ ionophore A23187, cultures of human melanoma (Hs294T), fibrosarcoma (HT1080) and colon carcinoma (LS180) cell lines synthesized conspicuous amounts of PAF, comparable to those produced by resident peritoneal macrophages. Substantial quantities of PAF were also synthesized by the murine melanoma (F10-M3 cells). PAF synthesis was rather limited in RSV-transformed Balb/c3T3 (B77-3T3) cells and in one of their high metastatic variants (B77-AA6 cells), although it was more abundant in the latter. We also investigated whether certain cytokines, such as TNFα and IFNγ might induce PAF synthesis in our systems of cell lines which we found to express mRNAs encoding receptors for these cytokines. We observed that PAF synthesis was enhanced in human melanoma and colon carcinoma cell lines and in the murine B77-AA6 cells to levels comparable to those obtained with the Ca 2+ ionophore. Synthesis of PAF was not inducible by TNFα in murine F10-M3 melanoma cells. IFNγ also stimulated PAF synthesis in human and murine melanoma lines, and in human LS180 colon carcinoma line, but not in the B77-AA6 cells. PAF synthesis was also inducible by exogenous PAF in the human and murine melanoma lines, and in the human LS180 colon carcinoma line, all of which expressed cell surface PAF receptors. PAF synthesis was not inducible by exogenous PAF in the B77-AA6 cells, which do not express PAF receptors.

Omega-3 fatty acids suppress monocyte adhesion to human endothelial cells: role of endothelial PAF generation.

Monocyte-endothelium interaction is a fundamental process in many acute and chronic inflammatory diseases. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are fish oil-derived alternative (omega-3) precursor fatty acids implicated in the suppression of inflammatory events. We investigated their influence on rolling and adhesion of monocytes to human umbilical vein endothelial cells (HUVEC) under laminar flow conditions in vitro. Exposure of HUVEC to tumor necrosis factor (TNF-alpha) strongly increased 1) surface expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin, 2) platelet-activating factor (PAF) synthesis as assessed by thrombin challenge, and 3) rate of rolling and adhesion of monocytes. Preincubation of HUVEC with EPA or DHA markedly suppressed PAF synthesis, monocyte rolling, and adherence, whereas expression of endothelial adhesion molecules was unchanged. Also, PAF receptor antagonists markedly suppressed the adhesion rate of monocytes, and EPA or DHA revealed no additional inhibitory capacity. In contrast, arachidonic acid partially reversed the effect of the antagonist. We conclude that omega-3 fatty acids suppress rolling and adherence of monocytes on activated endothelial cells in vitro by affecting endothelial PAF generation.