Endogenous Platelet-activating Factor Research Articles

Re-evaluation of the canonical PAF pathway in cutaneous anaphylaxis

Platelet-activating factor (PAF) is a potent classical lipid mediator that plays a critical role in various diseases such as allergy and nervous system disorders. In the realm of allergy, previous studies suggested that PAF is generated in response to extracellular stimuli and contributes to allergic reactions via PAF receptor (PAFR). However, the sources of endogenous PAF and its pathophysiological dynamics remain largely elusive in vivo. Here, we report that rapid and local PAF generation completely depends on lysophospholipid acyltransferase 9 (LPLAT9, also known as LPCAT2) expressed in mast cells in IgE-mediated passive cutaneous anaphylaxis. However, we found that LPLAT9 knockout (KO) mice did not display attenuated vascular leakage. Additionally, decreased vascular leakage was observed in PAFR KO mice, but not in endothelial cell-specific mice in this model. These divergences highlight a yet unsolved complexity of the biological functions of PAF and PAFR in a pathophysiological process.

Inhibition of platelet-activating factor (PAF)-induced platelet aggregation by fatty acids from human saliva

Experiments were undertaken to identify the nature of a previously identified inhibitor of PAF-induced platelet aggregation (PA) in human saliva. Human saliva fractionated by preparative thin layer chromatography (TLC) yielded a fraction that co-migrated with fatty acids (FAs) and inhibited PAF-induced aggregation of platelets. Synthetic FAs tested for their capacities to inhibit 0.1 nM PAF-induced PA showed that only the cis-unsaturated compounds were inhibitory with activities of some of the polyunsaturated FAs (PUFA) reaching almost 100% at 20 μM. Eicosapentanoic acid EPA and 8,11,14-eicosatrienoic acid also deaggregated the PAF-induced aggregates. With the exception of oleic acid (OLA), cis-monounsaturated FAs, and elaidic acid, the trans isomer of OLA, were poor inhibitors. In a direct comparison with other platelet agonists, ADP, thrombin, and ionophore A23187, the active saliva fraction and selected individual FA inhibited, to greater or lesser extent, PA induced by each of the agonists. EPA, OLA, linoleic acid (LNA), and the active saliva fraction were potent inhibitors of ADP-induced PA, EPA completely inhibited thrombin-induced PA and the saliva fraction showed only weak – moderate inhibitory activity to both thrombin- and ionophore A23187-induced PA. Other reports of endogenous PAF inhibitors in mammalian tissues are compared to the present results. PAF can trigger and amplify inflammatory cascades suggesting a possible modulation role for cis-unsaturated FAs in some diseases.

Platelet-activating factor increases reactive oxygen species-mediated microbicidal activity of human macrophages infected with Leishmania (Viannia) braziliensis

Platelet-activating factor (PAF) is produced by macrophages during inflammation and infections. We evaluated whether PAF is able to modulate the infection of human macrophages by Leishmania braziliensis, the main Leishmania sp. in Brazil. Monocyte-derived macrophages were incubated with promastigote forms in absence or presence of exogenous PAF. We observed that the treatment of macrophages with low concentrations of PAF prior to infection increased the phagocytosis of L. braziliensis. More importantly, exogenous PAF reduced the parasitism when it was added before, during or after infection. In addition, treatment with a PAF antagonist (PCA 4248) resulted in a significant increase of macrophage infection in a concentration-dependent manner, suggesting that endogenous PAF is important to control L. braziliensis infection. Mechanistically, while exogenous PAF increased production of reactive oxygen species (ROS) treatment with PCA 4248 reduced oxidative burst during L. braziliensis infection. The microbicidal effects of exogenous PAF were abolished when macrophages were treated with apocynin, an NADPH oxidase inhibitor. The data show that PAF promotes the production of ROS induced by L. braziliensis, suggesting that this lipid mediator may be relevant to control L. braziliensis infection in human macrophages.

Glucosamine suppresses platelet-activating factor-induced activation of microglia through inhibition of store-operated calcium influx.

Microglia activation and subsequent release of inflammatory mediators are implicated in the pathophysiology of neurodegenerative diseases. Platelet-activating factor (PAF), a potent lipid mediator synthesized by microglia, is known to stimulate microglia functional responses. In this study, we determined that endogenous PAF exert autocrine effects on microglia activation, as well as the underlying mechanism involved. We also investigated the effect of D-glucosamine (GlcN) on PAF-induced cellular activation in human HMO6 microglial cells. PAF induced sustained intracellular Ca(2+) ([Ca(2+)]i) increase through store-operated Ca(2+) channels (SOC) and reactive oxygen species (ROS) generation. PAF also induced pro-inflammatory markers through NFκB/COX-2 signaling. GlcN significantly inhibited PAF-induced Ca(2+) influx and ROS generation without significant cytotoxicity. GlcN downregulated excessive expression of pro-inflammatory markers and promoted filopodia formation through NFκB/COX-2 inhibition in PAF-stimulated HMO6 cells. Taken together, these data suggest that GlcN may offer substantial therapeutic potential for treating inflammatory and neurodegenerative diseases accompanied by microglial activation.

Effects of endotoxin on neurally-mediated gastric acid secretion in the rat.

The effects of a peripheral administration of E. coli endotoxin on neurally-mediated gastric acid secretion and the role of endogenous opioids or PAF receptors in endotoxin effects have been evaluated in the continuously perfused stomach of the anaesthetized rat. Gastric acid secretion stimulated by distension (20 cm H2O) was reduced dose-dependently by single intravenous bolus injection of endotoxin (0.1-10 microg kg(-1)). Doses of 5 microg kg(-1) induced a peak reduction of distension-stimulated acid output and significantly reduced the secretory response induced by an intravenous bolus of 2-deoxy-D-glucose (150 mg kg(-1)). This dose of endotoxin did not significantly modify mean systemic arterial blood pressure throughout the experimental period. Pretreatment with the opioid receptor antagonist naloxone (1 mg kg(-1) , i.v.) or the platelet-activating factor (PAF) receptor antagonist WEB 2086 (2 mg kg(-1), i.v.) did not reverse the inhibitory effects of endotoxin (5 microg kg(-1) , i.v.) on acid secretion stimulated by both distension and 2-deoxy-D-glucose. These findings suggest that endotoxin-induced acute inhibition of neurally-mediated acid responses, stimulated by gastric distension or administration of 2-deoxy-D-glucose, do not involve the activation of endogenous opioids or PAF receptors.

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.

Platelet Activating Factor Production and Proinflammatory Gene Expression in Endotoxin-Challenged Bovine Mammary Endothelial Cells

The bovine mammary gland responds to gram-negative pathogens by stimulating the production of cytokines and other proinflammatory mediators that orchestrate the migration of leukocytes into tissues. Platelet activating factor (PAF), interleukin 1β (IL-1β), IL-8, and intercellular adhesion molecule 1 (ICAM1) are among the several inflammatory factors involved in the early activation and migration of leukocytes into the mammary gland during the initial stages of coliform mastitis. Several different cell types within the mammary gland are capable of expressing these potent pro-inflammatory mediators. The objective of this study was to characterize the expression profile of vascular-derived inflammatory molecules that may play a role in the pathogenesis of bovine coliform mastitis. Isolated bovine mammary gland endothelial cells were stimulated in culture for up to 12h with endotoxin obtained from Escherichia coli, and the temporal expression of proinflammatory cytokines and adhesion molecules relative to endogenous PAF biosynthesis was evaluated. Results from the in vitro time course experiment showed that vascular-derived PAF biosynthesis began as early as 30min and peaked at 1h following endotoxin challenge. The biosynthesis of PAF preceded the endotoxin-induced IL-1β, IL-8, and ICAM1 mRNA expression that increased after 1h and reached peak expression between 4 and 12h following stimulation. Inhibiting the effects of endogenous PAF with a receptor antagonist suggests that vascular-derived PAF is an early proinflammatory mediator that plays at least a partial role in the subsequent expression of IL-1β, IL-8, and ICAM1 during endotoxin challenge. Furthermore, endotoxin-induced PAF biosynthesis by bovine mammary gland endothelial cells is regulated to some extent by phospholipase D activity and phosphatidic acid production. The results from this study support the contention that mammary gland endothelial cells can contribute to the production of important proinflammatory mediators that are typically associated with coliform mastitis.

Change of PAF and TNF-α in neonatal rat model of necrotizing enterocolitis

Objective To study the change of platelet-activating factor(PAF)and tumor necrosis fac-tor-α(TNF-α)density in neonatal rat model of necrotizing enterocolitis,and analyze the correlation between the PAF、TNF-α levd and the scoring of intestinal injury.The aim is to explore the function of PAF and TNF-α in NEC.Methods According to 2 x 2 factor analysis,32 neonate Sprague-Dewley rats(48 hours olds)were divided into 4 groups(A,B,C and D,8 rats each group).Group A was made into NEC models as follows:separate from mother rats and feeding with rat milk substitute,hypoxia(100%N2)for 90 second and 4℃cold exposure for 10 minutes,twice a day during 3 consecutive days;Group B was given formula feeding alone,not hypoxia and cold exposure.Group C was given rat milk feeding,the same hypoxia and cold exposure as group A,Group D was control group,given rat milk feeding,not hypoxia and cold exposure.On the 4th day all the subjects were sacrificed and the intestine around ileocecal junction were obtained to evaluated by stained with H&E for histological analysis and score.The mean scores more than 2 were considered NEC.The contents of PAF,TNF-α in homogenate of intestinal tissue were measured by ELISA(Kits bought from Shanghai Langka Company).Kruskal-Wallis H test,Spearman correlation analysis were used to analyze difference among various groups.α=0.05 was considered significant.Results The score of histopatholigical and the contents of PAF,TNF-α had significant difference among groups(P＜0.01).By spearman correlation analysis,the relations of the degree of intestinal injury and the contents of PAF,TNF-α in intestinal tissue were positive(rpAF=0.71,rTNF-α=0.81,P＜0.05).Conclusion Endogenous PAF and TNF-α in intestine tissue are probably the critical factors for the development of NEC.As risk factors,they are involed in the development of NEC.It indicated that the concentrations of PAF and TNF-α in intestine tissue had a positive correlation with the severity of NEC and can inflect the degree of intestine mucosa lesions. Key words: Necrotizing enterocolitis; Neonatal rat; Animal model; Platelet-activating factor; Tumor necrosis factor-α

Platelet activating factor (PAF) receptor expression is increased in lungs of neonatal rats with hypoxia‐induced pulmonary hypertension

Endogenous PAF contributes to high vasomotor tone in the fetal pulmonary circulation and has been implicated in the pathogenesis of chronic‐hypoxia (CH)‐induced pulmonary hypertension (PH). We studied the effect of CH on neonatal rat (pup) body weight, right ventricle/left ventricle + septum ratio (RV/LV+S), and lung PAF receptor (PAF‐r) protein expression by Western blotting. Pups were placed in an air‐tight chamber ventilated with 13% oxygen (hypoxia) or room air (normoxia) from 1d to 22d of age. Three groups were studied (12 pups in each group): Group1, pups in Hypoxia; Group2, pups in hypoxia given 5mg/kg WEB 2170 daily, a PAF‐r antagonist, (Hypoxia +WEB); Group3, pups in Normoxia. We found that: a) Pups in Hypoxia gained 50% less weight than pups in Normoxia; WEB treatment in Hypoxia did not improve weight gain; b) RV/LV+S in Hypoxia group was highest at 0.79; 2‐fold higher than in WEB + Hypoxia and Normoxia groups; c) PAF‐r protein expression in lungs of pups in Hypoxia was >3‐fold higher than in WEB+Hypoxia and Normoxia groups. Our findings show that chronic hypoxia induces PAF‐r expression in lungs and suggest that increased PAF‐r expression may be responsible for the right ventricular hypertrophy and PH. Inhibition of PAF effects with a PAF‐r antagonist, WEB, though it did not improve weight gain of the pups in hypoxia, prevented RV hypertrophy in the pups. HL‐077819.

Involvement of endogenous leukotriene B4 and platelet‐activating factor in polymorphonuclear leucocyte recruitment to dermal inflammatory sites in rats

A critical role for leukotriene B(4) (LTB(4)) and/or platelet-activating factor (PAF) in regulating polymorphonuclear cell (PMN) trafficking to inflammatory sites has been reported in a number of experimental inflammatory models. In vitro, newly synthesized LTB(4) and PAF were shown to act in an autocrine/paracrine or intracrine fashion to enhance intracellular arachidonic acid availability and leukotriene biosynthesis. This suggested potentially cooperative effects of these lipid mediators in regulating PMN extravasation. The present study aimed to elucidate whether endogenous LTB(4) and PAF may both act to regulate plasma extravasation and PMN trafficking to inflammatory sites in experimental inflammation. With this aim, we have used selective and potent PAF and LTB(4) receptor antagonist pretreatments in dermal and pulmonary inflammation models in rats. Our results show additive inhibitory effects of dual LTB(4) and PAF receptor blockade in either PAF- or LTB(4)-elicited cutaneous PMN accumulation compared to single-drug administration. Furthermore, the combined administration of the drugs inhibited the PMN accumulation induced by the chemically unrelated soluble agonists tumour necrosis factor-alpha and C5a. Finally, in a model of pulmonary inflammation induced by the intravenous injection of Sephadex beads, lung neutrophilia was reduced by 63% following the administration of LTB(4) and PAF antagonists, in contrast with the lack of effect of single drug administration. Our results strongly support a role of both endogenous LTB(4) and PAF in regulating PMN trafficking to inflammatory sites in various experimental conditions.

Acute Pancreatitis: Bench to the Bedside

Acute Pancreatitis: Bench to the Bedside

Down-Regulation of Platelet-Activating Factor Receptor Gene Expression During Focal Reversible Cerebral Ischemia in Rats

Platelet-activating factor (PAF) is an endogenous potent phospholipid mediator in stroke and related to the post-ischemic brain damage. The aim of this study was to investigate the regulation and mechanisms of PAF receptor gene expression in the perifocal regions of cerebral infarction after middle cerebral artery occlusion/reperfusion. Sixty mature Wistar rats were randomly divided into 12 groups: sham-operated control group, simple ischemia 90 min group, 6, 12, 18 h, 1 day (1 d), 2, 3, 4, 5, 6, 7 d reperfusion groups. After the right middle cerebral artery occluded, the rats were suffered from ischemia for 90 min, and then reperfusion was allowed for different time courses. Reverse transcription-polymerase chain reaction (RT-PCR) and radioimmunoassay were applied to evaluate the PAF receptor messenger RNA (mRNA) expression and PAF levels in the perifocal regions of cerebral infarction respectively. RT-PCR analysis revealed that PAF receptor mRNA was 0.95 +/- 0.15 in control group. However, following ischemia-reperfusion, the levels of PAF receptor mRNA progressively decreased until 2 d of reperfusion (0.54 +/- 0.10), then returned to control group's levels gradually. Compared with the control group's (582 +/- 72 pg/g wet weight), the PAF concentrations of simple ischemic and 6, 12, 18 h, 1, 2 d reperfusion group were significantly higher than that of any other groups. These results indicate that PAF receptor gene expression may be subject to down-regulation in the perifocal regions of cerebral infarction after cerebral ischemia-reperfusion and relative to the increase of endogenous PAF concentrations.

Vascular permeability induced by VEGF family members in vivo: Role of endogenous PAF and NO synthesis

We previously reported that vascular endothelial growth factor (VEGF) increases vascular permeability through the synthesis of endothelial platelet-activating factor (PAF), while others reported the contribution of nitric oxide (NO). Herein, we addressed the contribution of VEGF receptors and the role played by PAF and NO in VEGF-induced plasma protein extravasation. Using a modified Miles assay, intradermal injection in mice ears of VEGF-A(165), VEGF-A(121), and VEGF-C (1 microM) which activate VEGFR-2 (Flk-1) receptor increased vascular permeability, whereas a treatment with VEGFR-1 (Flt-1) analogs; PlGF and VEGF-B (1 microM) had no such effect. Pretreatment of mice with PAF receptor antagonist (LAU8080) or endothelial nitric oxide synthase (eNOS) inhibitor (L-NAME) abrogated protein extravasation mediated by VEGF-A(165). As opposed to PAF (0.01-1 microM), treatment with acetylcholine (ACh; up to 100 microM; inducer of NO synthesis) or sodium nitroprusside (SNP; up to 1 microM; NO donor) did not induce protein leakage. Simultaneous pretreatment of mice with eNOS and protein kinase A (PKA) inhibitors restored VEGF-A(165) vascular hyperpermeability suggesting that endogenous NO synthesis leads to PKA inhibition, which support maintenance of vascular integrity. Our data demonstrate that VEGF analogs increase vascular permeability through VEGFR-2 activation, and that both endogenous PAF and NO synthesis contribute to VEGF-A(165)-mediated vascular permeability. However, PAF but not NO directly increases vascular permeability per se, thereby, suggesting that PAF is a direct inflammatory mediator, whereas NO serves as a cofactor in VEGF-A(165) proinflammatory activities.

Lipopolysaccharide induces CXCL2/macrophage inflammatory protein‐2 gene expression in enterocytes via NF‐κB activation: independence from endogenous TNF‐α and platelet‐activating factor

CXCL2 (macrophage inflammatory protein-2 (MIP-2)), a critical chemokine for neutrophils, has been shown to be produced in the rat intestine in response to platelet-activating factor (PAF) and to mediate intestinal inflammation and injury. The intestinal epithelium, constantly exposed to bacterial products, is the first line of defence against micro-organisms. It has been reported that enterocytes produce proinflammatory mediators, including tumour necrosis factor (TNF) and PAF, and we showed that lipopolysaccharide (LPS) and TNF activate nuclear factor (NF)-kappaB in enterocytes. However, it remains elusive whether enterocytes release CXCL2 in response to LPS and TNF via a NF-kappaB-dependent pathway and whether this involves the endogenous production of TNF and PAF. In this study, we found that TNF and LPS markedly induced CXCL2 gene expression in IEC-6 cells, TNF within 30 min, peaking at 45 min, while LPS more slowly, peaking after 2 hr. TNF- and LPS- induced CXCL2 gene expression and protein release were completely blocked by pyrrolidine dithiocarbamate (PDTC) and helenalin, two potent NF-kappaB inhibitors. NEMO-binding domain peptide, a specific inhibitor of inhibitor protein kappaB kinase (IKK) activation, a major upstream kinase mediating NF-kappaB activation, significantly blocked CXCL2 gene expression and protein release induced by LPS. WEB2170 (PAF antagonist) and anti-TNF antibodies had no effect on LPS-induced CXCL2 expression. In conclusion, CXCL2 gene is expressed in enterocytes in response to both TNF and LPS. LPS-induced CXCL2 expression is dependent on NF-kappaB activation via the IKK pathway. The effect of LPS is independent of endogenous TNF and PAF.

Influence of platelet activating factor and its antagonist on portal hypertension associated with liver cirrhosis: an experiment with rats

To investigate the influence of platelet activating factor (PAF) and its antagonist BN52021 on portal hypertension associated with liver cirrhosis. Ten SD rats were injected intraperitoneally with carbon tetrachloride to establish a liver cirrhosis model and 10 rats were injected with olive oil as controls. The concentrations of PAF in the blood and liver was examined by rapid (3)H-PAF scintillation proximity assay and the hepatic PAF binding capacity was examined by receptor saturation binding technique. The pressure of portal vein and pressure of femoral artery were measured by intubation method. BN52021 was infused into the portal vein to observe its influence on the blood pressure. The hepatic PAF levels of the cirrhotic rats was 4.0 ng/g +/- 0.4 ng/g, significantly higher than that of the control rats (2.7 ng/g +/- 0.5 ng/g, P < 0.01). The hepatic efflux PAF level of the cirrhotic rats was 6.3 ng/g +/- 0.6 ng/g, significantly higher than that of the control rats (3.4 ng/g +/- 0.6 ng/g, P < 0.01). The hepatic output PAF levels of the cirrhotic rats was 1.0 ng/g +/- 0.6 ng/g, significantly lower than that of the control rats (0.3 ng/g +/- 0.5 ng/g, P < 0.01). The maximum PAF binding capacity of the cirrhotic rats was 2.8 +/- 0.21 fmol/microg protein, significantly higher than that of the control rats (0.9 +/- 0.06 fmol/microg protein, P < 0.01). However, the receptor affinity was not significantly different between these 2 groups. The portal pressure of the cirrhotic rats was 12.2 mm Hg +/- 0.7 mm Hg, significantly higher than that of the control rats (5.3 mm Hg +/- 0.6 mm Hg, P < 0.01). The femoral arterial pressure of the cirrhotic rats was 82 mm Hg +/- 10 mm Hg, significantly lower than that of the control rats (114 mm Hg +/- 9 mm Hg, P < 0.01). Infusion of PAF via the portal vein increased the portal pressure from 12.1 mm Hg +/- 0.6 mm Hg with an increase of 32% (P < 0.01) in the cirrhotic rats, and from 7.7 mm Hg +/- 0.8 mm Hg with an increase of 23%. The PAF response of the cirrhotic rats was 4.1 mm Hg +/- 1.0 mm Hg (227%), significantly higher than that of the control rats (1.8 mm Hg +/- 0.3 mm Hg, P < 0.01). The femoral artery pressure decreased from 82 mm Hg +/- 10 mm Hg to 48 mm Hg +/- 4 mm Hg (P < 0.01) in the cirrhotic rats, and from 114 mm Hg +/- 9 mm Hg to 52 mm Hg +/- 4 mm Hg (P < 0.01). After portal infusion of BN52021 the portal pressure decreased from 14.6 mm Hg +/- 1.6 mm Hg to 12.3 mm Hg +/- 0.8 mm Hg (P < 0.05) with a decrease of 16%, however, did not significantly influenced the femoral arterial pressure in the cirrhotic rats, and did not significantly influenced the portal pressure and femoral arterial pressure in the control rats. The increased hepatic PAF synthesis in cirrhotic is the major source of elevated circulating PAF It upregulates the hepatic hemodynamics that contributes to portal hypertension. The increased portal pressure by endogenous PAF can be decreased to a certain extent by BN52021 which may be used in treatment of portal hypertension.

Synergistic interaction of endogenous platelet-activating factor and vasopressin in generating angina in rats

We examined the involvement of endogenous vasopressin and platelet-activating factor (PAF) in the pathogenesis of two types of experimental angina in urethane-anaesthetised male Wistar rats. In the first model, epinephrine (10 μg kg −1) was injected into the tail vein, followed at the development of the maximum blood pressure response, i.e., 30 s later, by phentolamine (15 mg kg −1). In the second model, the vasopressin V 1 receptor agonist ornithine-vasopressin (ornipressin; 0.5 IU kg −1, i.v.) was administered. The heart rate, mean arterial blood pressure and surface electrocardiogram (ECG, standard lead II) were registered simultaneously. As a measure of myocardial ischaemia, at 1 min after phentolamine or ornipressin administration, we found significant ST-segment depression, lasting for more than 10 or 5 min, respectively. Pretreatment (15 min, s.c.) with the vasopressin V 1 receptor antagonist Mca 1,Tyr(Me) 2AVP (the Manning peptide; 0.02–0.2 μg kg −1) or the platelet-activating factor receptor antagonist ginkgolide B (BN 52021; 0.25–2.5 mg kg −1) alone caused a dose-dependent reduction of the ST-segment depression. Concurrent administration of the two antagonists in their threshold doses (0.02 μg kg −1 and 0.25 mg kg −1) also attenuated the ST-segment depression in both models. Neither antagonist affected the blood pressure or heart rate changes throughout the studies. Our results suggest that endogenous vasopressin and platelet-activating factor interact synergistically in provoking myocardial ischaemia in vivo in experimental angina in the rat.

VEGF-mediated endothelial P-selectin translocation: role of VEGF receptors and endogenous PAF synthesis

AbstractThe acute increase in vascular permeability produced by vascular endothelial growth factor (VEGF-A165) requires activation of endothelial Flk-1 receptors (VEGFR-2) and stimulation of platelet-activating factor (PAF) synthesis. Like PAF, VEGF-A165 promotes translocation of P-selectin to the endothelial cell (EC) surface. However, the mechanisms involved remain unknown. By treating human umbilical vein endothelial cells (HUVECs) with VEGF analogs, we show that activation of VEGFR-1 or VEGFR-2 or both induced a rapid and transient translocation of endothelial P-selectin and neutrophil adhesion to activated ECs. The effects mediated by VEGF-A165 and VEGF-A121 (VEGFR-1/VEGFR-2 agonists) were blocked by a selective VEGFR-2 inhibitor, SU1498. VEGF-A165 was twice as potent as VEGF-A121, which can be explained by the binding capacity of VEGF-A165 to its coreceptor neuropilin-1 (NRP-1). Indeed, treatment with NRP-1 antagonist (GST-Ex7) reduced the effect of VEGF-A165 to the levels observed upon stimulation with VEGF-A121. Finally, the use of selective PAF receptor antagonists reduced VEGF-A165–mediated P-selectin translocation. Together, these data show that maximal P-selectin translocation and subsequent neutrophil adhesion was mediated by VEGF-A165 on the activation of VEGFR-2/NRP-1 complex and required PAF synthesis.

Fertilization potential of human sperm is correlated with endogenous platelet-activating factor content.

Platelet-activating factor (PAF) is a potent signaling phospholipid that is found in mammalian sperm and has a positive correlation with fertility. Whereas PAF is present in human sperm, there are no relational reports on its content and the cells fertilization potential. Therefore, the study objective was to determine if PAF content in capacitated-induced sperm is related to fertilization potential as determined by the sperm penetration assay (SPA). Endogenous sperm lipids were measured for PAF content by a specific radioimmunoassay following insemination of zona pellucida-free hamster ova. Data were analyzed by regression analysis and Student's t test. Regression analysis revealed a positive and significant relation (R2 = 0.806; P < 0.05) between PAF content in human sperm and SPA outcome (pass: > or = 5.0; fail: < 5.0, penetrations/ova). Patients that passed (22.61 +/- 5.21 picomoles/10(6)) the SPA had significantly (P < 0.01) higher PAF levels in their sperm than patients that failed (12.91 +/- 1.76 picomoles/10(6) cells) the test. PAF content in capacitated-induced sperm has a significant and positive relationship with fertilization potential. Fertilization potential may be predicted by measuring PAF levels in capacitation-induced human sperm. Determining PAF content in capacitated human sperm may be a beneficial diagnostic tool for the infertility specialist.

The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the generation of platelet-activating factor and leukotriene B 4 in hypoxic–ischemic brain in young mice

Platelet-activating factor (PAF), leukotriene B 4 (LTB 4) and other cytokines have been indicated to be responsible for the neuronal damage in hypoxic–ischemic brain. Diets in omega-3 (n-3) fatty acids appear to have an antiinflammatory effect, which is thought to be due to decrease in active prostaglandins and leukotrienes production after incorporation of these fatty acids into cell membrane phospholipids. We investigated the effect of dietary supplementation with n-3 fatty acids on endogenous PAF and LTB 4 biosynthesis in hypoxic–ischemic brain of young mice. Young mice were randomly divided into four groups: Group 1 mice were fed standard chow (n-3 polyunsaturated fatty acids free); Group 2 and Group 3 mice were given standard diet supplemented with 10% by weight of fish oil, as source of n-3 polyunsaturated fatty acids, for 3 and 6 weeks, respectively. Group 4 mice served as control. We injured the right cerebral hemisphere of young mice by ligating the right common carotid artery and exposing the mice to 8% oxygen for 60 min. Approximately 10-fold increase in PAF concentration was determined in hypoxic–ischemic brain tissue of Group 1 mice. Tissue concentration of PAF showed a profound decline in Group 3 mice compared to Groups 1 and 2 ( P<0.01, P<0.05, respectively). LTB 4 was also significantly elevated in the brain of Group 1 mice when compared to the brain of control mice ( P<0.001). A striking decline was observed in the concentration of LTB 4 in both Group 2 and Group 3 mice compared to Group 1 mice ( P<0.05, P<0.01, respectively). The present study shows that n-3 fatty-acid-enriched diet inhibits endogenous PAF and LTB 4 generation in hypoxic–ischemic brain tissue; however it demonstrates that 6 weeks of dietary supplementation with n-3 fatty acids results in a significant decrease in tissue level of PAF in the brain.

Lipopolysaccharide-induced leukocyte lipid body formation in vivo: innate immunity elicited intracellular Loci involved in eicosanoid metabolism.

Lipid bodies are rapidly inducible, specialized cytoplasmic domains for eicosanoid-forming enzyme localization, which we hypothesize to have specific roles in enhanced inflammatory mediator production during pathological conditions, including sepsis. However, little is known about the origins, composition, or functions of lipid bodies in vivo. We show that lipid body numbers were increased in leukocytes from septic patients in comparison with healthy subjects. Analogously, the intrathoracic administration of LPS into mice induced a dose- and time-dependent increase in lipid body numbers. Pretreatment with anti-CD14 or anti-CD11b/CD18 mAb drastically inhibited LPS-induced lipid body formation. Moreover, LPS failed to form lipid bodies in C3H/HeJ (TLR4 mutated) mice, demonstrating a requisite role for LPS receptors in lipid body formation. LPS-induced lipid body formation was also inhibited by the platelet-activating factor-receptor antagonists, suggesting a role for endogenous platelet-activating factor. The eicosanoid-forming enzymes, 5-lipoxygenase and cyclooxygenase-2, were immunolocalized within experimentally induced (LPS in mice) or naturally occurring (septic patients) lipid bodies. The proinflammatory cytokine involved in the pathogenesis of sepsis, TNF-alpha, was also shown to colocalize within lipid bodies. Prior stimulation of leukocytes to form lipid bodies enhanced the capacity of leukocytes to produce leukotriene B(4) and PGE(2). In conclusion, our studies indicate that lipid bodies formed after LPS stimulation and sepsis are sites for eicosanoid-forming enzymes and cytokine localization and may develop and function as structurally distinct, intracellular sites for paracrine eicosanoid synthesis during inflammatory conditions.