Platelet-activating Factor Receptor mRNA Research Articles

Upregulation of Platelet-Activating Factor Receptor Expression and Lyso-Platelet-Activating Factor Isoforms in Human Nasal Polyp Tissues

Background: The Platelet-Activating Factor (PAF)/receptor (PAFR) system is involved in asthma and allergic rhinitis; however, its role in chronic rhinosinusitis (CRS) is still unclear. This study aimed to assess the expression of PAFR and the concentration of Lyso-PAF isoforms in the nasal polyps (NP) of patients suffering from CRS with/without comorbidities such as asthma and NSAID-exacerbated respiratory disease (N-ERD) compared to healthy nasal mucosa (NM) from control subjects. Methods: NM (n = 8) and NP tissues were obtained from patients undergoing surgery for septal deviation/turbinate hypertrophy or endoscopic sinus surgery, respectively. Three phenotypes were studied: CRSwNP with no asthma (n = 6), CRSwNP with non-steroidal anti-inflammatory drug (NSAID)-tolerant asthma (n = 6), and CRSwNP with NSAID-exacerbated respiratory disease (n = 6). PAFR protein and mRNA were assessed via immunochemistry, immunofluorescence, Western blot, and real-time quantitative PCR. Lyso-PAF isoforms (C16, C18, and C18:1) were quantified via mass spectrometry. Results: PAFR protein was expressed in the NM and NP, concretely in epithelial cells and submucosal glands. Compared to NM, PAFR mRNA expression was higher in all NP phenotypes (p < 0.05) while all Lyso-PAF isoform concentrations were higher in the NP from asthmatic patients (p < 0.05). Lyso-PAF C16 and C18 concentrations were higher in the NP from asthmatic patients than in the NP from patients without asthma. Conclusions: The PAF/PAFR system could play a pathophysiological role in CRSwNP pathogenesis.

New transcriptome and clinical findings of platelet-activating factor in chronic spontaneous urticaria: Pathogenic and treatment relevance.

The objective of the study was to assess the pathogenic and treatment relevance of Platelet Activating factor (PAF) in chronic spontaneous urticaria (CSU). The expression and cellular location of PAF receptor (PAFR) and serum levels of PAF and PAF acetylhydrolase (PAF-AH) in patients with moderate/severe CSU (n=45) and healthy controls (HCs, n=17) were studied. Skin samples from the active wheal (LS-CSU, 13 samples for qPCR and 33 for immunohistochemistry) and non-lesional skin (NLS-CSU, 13 samples) of CSU patients and HCs (13 samples and 5 for immunohistochemistry) were analyzed. Serum PAF and PAF-AH levels were measured by ELISA and compared between HC (10 samples) and CSU patients (25 samples) and, among them, between those refractory and non-refractory to second-generation H1 -antihistamines (sgAH). PAFR mRNA expression was significantly higher in LS-CSU versus HCs (p=0.014). PAFR positive staining in immunohistochemistry was mainly found in the epidermal basal layer in HCs, whereas it was broadly present along the epidermis in LS-CSU samples. Endothelial cells showed PAFR expression exclusively in LS-CSU and NLS-CSU samples. PAFR expression was observed in the nerves of HC, LS-CSU, and NLS-CSU samples. Double PAFR/CD43 expression showed that T-lymphocytes were the main cell type from the wheal inflammatory infiltrate expressing PAFR. A significantly lower PAF-AH/PAF ratio was observed in sgAH non-responders versus responders (6.1 vs. 12.6; p=0.049). Our study confirms that PAF is a mediator of wheal pathogenesis in CSU. The significantly lower PAF-AH/PAF ratio in sgAH non-responders vs responders suggests that PAF could be a potential biomarker of sgAH refractoriness.

A Possible Role for Platelet-Activating Factor Receptor in Amyotrophic Lateral Sclerosis Treatment.

Amyotrophic lateral sclerosis (ALS) is the third most prevalent neurodegenerative disease affecting upper and lower motor neurons. An important pathway that may lead to motor neuron degeneration is neuroinflammation. Cerebrospinal Fluids of ALS patients have increased levels of the inflammatory cytokine IL-18. Because IL-18 is produced by dendritic cells stimulated by the platelet-activating factor (PAF), a major neuroinflammatory mediator, it is expected that PAF is involved in ALS. Here we show pilot experimental data on amplification of PAF receptor (PAFR) mRNA by RT-PCR. PAFR is overexpressed, as compared to age matched controls, in the spinal cords of transgenic ALS SOD1-G93A mice, suggesting PAF mediation. Although anti-inflammatory drugs have been tested for ALS before, no clinical trial has been conducted using PAFR specific inhibitors. Therefore, we hypothesize that administration of PAFR inhibitors, such as Ginkgolide B, PCA 4248 and WEB 2086, have potential to function as a novel therapy for ALS, particularly in SOD1 familial ALS forms. Because currently there are only two approved drugs with modest effectiveness for ALS therapy, a search for novel drugs and targets is essential.

Platelet-Activating Factor Receptor Ligands Protect Tumor Cells from Radiation-Induced Cell Death.

Irradiation generates oxidized phospholipids that activate platelet-activating factor receptor (PAFR) associated with pro-tumorigenic effects. Here, we investigated the involvement of PAFR in tumor cell survival after irradiation. Cervical cancer samples presented higher levels of PAF-receptor gene (PTAFR) when compared with normal cervical tissue. In cervical cancer patients submitted to radiotherapy (RT), the expression of PTAFR was significantly increased. Cervical cancer-derived cell lines (C33, SiHa, and HeLa) and squamous carcinoma cell lines (SCC90 and SCC78) express higher levels of PAFR mRNA and protein than immortalized keratinocytes. Gamma radiation increased PAFR expression and induced PAFR ligands and prostaglandin E2 (PGE2) in these tumor cells. The blocking of PAFR with the antagonist CV3938 before irradiation inhibited PGE2 and increased tumor cells death. Similarly, human carcinoma cells transfected with PAFR (KBP) were more resistant to radiation compared to those lacking the receptor (KBM). PGE2 production by irradiated KBP cells was also inhibited by CV3988. These results show that irradiation of carcinoma cells generates PAFR ligands that protect tumor cells from death and suggests that the combination of RT with a PAFR antagonist could be a promising strategy for cancer treatment.

MRNA expression of platelet activating factor receptor (PAFR) in peripheral blood mononuclear cells is associated with albuminuria and vascular dysfunction in patients with type 2 diabetes

AimsRenal dysfunction in addition to diabetes is a serious risk factor for cardiovascular events. We hypothesized that some of the changes in gene expression in blood cells cause renal dysfunction and macrovascular disease through impaired endothelial function. This study aimed to define which changes in gene expression in peripheral blood mononuclear cells (PBMCs) are related to renal function parameters and endothelial function of large arteries in patients with type 2 diabetes mellitus (T2DM). MethodsWe recruited 95 patients with T2DM. After matching for gender, age, BMI and HbA1c levels, the patient cohort included 42 with normoalbuminuria, 28 with microalbuminuria, and 25 with macroalbuminuria. All patients in the three groups were assessed for urinary albumin to creatinine ratio (ACR), estimated glomerular filtration rate (eGFR), flow-mediated dilatation (FMD), and mRNA expression in PBMCs. ResultsThe mRNA expression of platelet activating factor receptor (PAFR) differed most markedly between the three groups and was significantly higher in the macroalbuminuric group (p < 0.001 vs. normoalbuminuric group; p < 0.05 vs. microalbuminuric group). PAFR mRNA expression significantly correlated with log transformed ACR (ρ = 0.424, p < 0.001) but not eGFR. PAFR mRNA expression also had a significant negative correlation with FMD (ρ = −0.379, p < 0.001). Furthermore, the prevalence of macrovascular complications, particularly stroke, was significantly higher in patients with elevated PAFR mRNA expression in PBMCs. ConclusionsPAFR overexpression in PBMCs may link diabetic nephropathy to macroangiopathy through impairment of endothelial function in patients with T2DM.

Exposure to welding fumes and lower airway infection with Streptococcus pneumoniae

Welders are at increased risk of pneumococcal pneumonia. The mechanism for this association is not known. The capacity of pneumococci to adhere to and infect lower airway cells is mediated by host-expressed platelet-activating factor receptor (PAFR). We sought to assess the effect of mild steel welding fumes (MS-WF) on PAFR-dependent pneumococcal adhesion and infection to human airway cells invitro and on pneumococcal airway infection in a mouse model. The oxidative potential of MS-WF was assessed by their capacity to reduce antioxidants invitro. Pneumococcal adhesion and infection of A549, BEAS-2B, and primary human bronchial airway cells were assessed by means of quantitative bacterial culture and expressed as colony-forming units (CFU). After intranasal instillation of MS-WF, mice were infected with Streptococcus pneumoniae, and bronchoalveolar lavage fluid (BALF) and lung CFU values were determined. PAFR protein levels were assessed by using immunofluorescence and immunohistochemistry, and PAFR mRNA expression was assessed by using quantitative PCR. PAFR was blocked by CV-3988, and oxidative stress was attenuated by N-acetylcysteine. MS-WF exhibited high oxidative potential. In A549 and BEAS-2B cells MS-WF increased pneumococcal adhesion and infection and PAFR protein expression. Both CV-3988 and N-acetylcysteine reduced MS-WF-stimulated pneumococcal adhesion and infection of airway cells. MS-WF increased mouse lung PAFR mRNA expression and increased BALF and lung pneumococcal CFU values. In MS-WF-exposed mice CV-3988 reduced BALF CFU values. Hypersusceptibility of welders to pneumococcal pneumonia is in part mediated by the capacity of welding fumes to increase PAFR-dependent pneumococcal adhesion and infection of lower airway cells.

Inhibitory effects of the platelet-activating factor receptor antagonists, CV-3988 and Ginkgolide B, on alkali burn-induced corneal neovascularization

Purpose: Platelet-activating factor (PAF) has been found in various ocular tissues; the activity of PAF depends on the binding to its specific receptor, PAF-receptor. We investigated the therapeutic effects of PAF-receptor antagonists (CV-3988 and Ginkgolide B) on alkali burn-induced corneal neovascularization (CNV).Methods: CNV was induced by applying a 0.2 N sodium hydroxide (3 µl, NaOH) solution directly on mice corneas. CV-3988 (1 mM/10 µl) and Ginkgolide B (1 mM/10 µl) were administered topically on the corneas three times daily for three consecutive days. CNV was evaluated under a slit-lamp microscope. Corneas were processed for histological, immunohistochemical and reverse transcription polymerase chain reaction analysis. Human umbilical vein endothelial cells were used for the migration and tube formation assay.Results: Application of CV-3988 and Ginkgolide B inhibited CNV caused by alkali burn. CV-3988 and Ginkgolide B attenuated the expression of PAF-receptor mRNA. Alkali injury induced a massively increased intraocular mRNA expression of an angiogenic factor in cornea tissues, whereas these increments were attenuated by the application of CV-3988 and Ginkgolide B.Conclusions: CV-3988 and Ginkgolide B reversed opacity and neovascularization in alkali burn-induced corneas. Our findings suggest that CV-3988 and Ginkgolide B may be therapeutically useful in the treatment of CNV and inflammation.

Bronchial platelet-activating factor receptor in chronic obstructive pulmonary disease

Bacteria expressing phosphorylcholine (ChoP) co-opt host-expressed platelet-activating factor receptor (PAFR) to adhere to lower airway cells. Cigarette smoke and rhinovirus (RV) infection upregulate PAFR-dependent bacterial adhesion to airway cells in vitro, and in healthy adults smoking increases the proportion of PAFR positive bronchial epithelial cells. To date the effect of chronic obstructive pulmonary disease (COPD) on smoke-induced PAFR is unknown. We therefore sought to test the hypothesis that bronchial PAFR mRNA expression is increased in smokers with chronic obstructive pulmonary disease (COPD), and further increases after RV infection. Endobronchial biopsies were obtained by fibreoptic bronchoscopy from healthy non-smokers, smokers without airway obstruction, and smokers with COPD, before and after infection with rhinovirus (RV) serotype 16. Endobronchial PAFR mRNA expression was assessed by quantitative PCR and expressed as a ratio of glyceraldehyde-3-phosphate dehydrogenase. The distribution of PAFR was assessed by immunohistochemistry. Baseline PAFR mRNA expression was increased (p < 0.05) in smokers (n = 16), and smokers with COPD (n = 14) compared with non-smokers (n = 18). In RV16 infected subjects there was no increase in PAFR mRNA expression in either non-smokers (n = 9), smokers (n = 8), or smokers with COPD (n = 7). PAFR immunoreactivity in all 3 groups was predominately restricted to the bronchial epithelium and submucosal glands. Endobronchial PAFR mRNA is increased in both smokers without airway obstruction and smokers with COPD. We found preliminary evidence that RV16 infection does not increase PAFR mRNA expression in either smokers or smokers with COPD.

Platelet-activating factor synthesis and receptor-mediated signaling are downregulated in ovine newborn lungs: relevance in postnatal pulmonary adaptation and persistent pulmonary hypertension of the newborn.

Platelet-activating factor (PAF) is a phospholipid with a wide range of biological activities. We studied PAF metabolism and PAF receptor (PAFR) signaling in perinatal ovine lungs to understand PAF's role in transition of the perinatal pulmonary hemodynamics and pathophysiology of persistent pulmonary hypertension of the newborn. We hypothesized that downregulation of PAF synthesis with upregulation of PAF catabolism by acetylhydrolase (PAF-Ah) in the newborn lung is needed for fetus-to-newborn pulmonary adaptation. Studies were conducted on fetal and newborn lamb pulmonary arteries (PA), veins (PV) and smooth muscle cells (SMC). PAF metabolism, PAFR binding and cell proliferation were studied by cell culture; gene expression was studied by qPCR. Fetal lungs synthesized 60% more PAF than newborn lungs. Compared with the fetal PVs and SMCs, PAF-Ah activity in newborn was 40-60% greater. PAF-Ah mRNA expression in newborn vessels was different from the expression by fetal PA. PAF-Ah gene clone activity confirmed deletion of hypoxia-sensitive site. PAFR mRNA expression by the PVs and SMC-PV of the fetus and newborn was greater than by corresponding PAs and SMC-PA. Q-PCR study of PAFR expression by the SMC-PV of both groups was greater than SMC-PA. Fetal SMCs bound more PAF than the newborn SMCs. PAFR antagonist, CV-3988, inhibited PAFR binding and DNA synthesis by the fetal SMCs, but augmented binding and DNA synthesis by newborn cells. We show different PAF-PAFR mediated effects in perinatal lungs, suggesting both transcriptional and translational regulation of PAF-Ah and PAFR expression in the perinatal lamb lungs. These indicate that the downregulation of PAF-mediated effects postnatally protects against persistent pulmonary hypertension of the newborn.

Genomics of corneal wound healing: a review of the literature

Corneal wound healing is a complex process: its mechanisms and the underlying genetic control are not fully understood. It involves the integrated actions of multiple growth factors, cytokines and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells and lacrimal gland cells. Following an epithelial insult, multiple cytokines are released triggering a cascade of events that leads to repair the epithelial defect and remodelling of the stroma to minimize the loss of transparency and function. In this review, we examine the literature surrounding the genomics of corneal wound healing with respect to the following topics: epithelial and stromal wound healing (including inhibition); corneal neovascularisation; the role of corneal nerves in wound healing; the endothelium; the role of aquaporins and aptamers. We also examine the effect of ectasia on corneal wound healing with regard to keratoconus and following corneal surgery. A better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design treatments that selectively modulate key phases of the healing process resulting in scars that more closely resemble normal corneal architecture.

Up-Regulation of Platelet-Activating Factor Synthases and its Receptor in Spinal Cord Contribute to Development of Neuropathic Pain following Peripheral Nerve Injury

BackgroundPlatelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a lipid mediator derived from cell membrane. It has been reported that PAF is involved in various pathological conditions, such as spinal cord injury, multiple sclerosis, neuropathic pain and intrathecal administration of PAF leads to tactile allodynia. However, the expression of PAF synthases and its receptor in the spinal cord following peripheral nerve injury is unknown.MethodsUsing the rat spared nerve injury (SNI) model, we investigated the expression of PAF synthases (LPCAT1 and 2) and PAF receptor (PAFr) mRNAs in the spinal cord. Reverse transcription polymerase chain reaction (RT-PCR) and double-labeling analysis of in situ hybridization histochemistry (ISHH) with immunohistochemistry (IHC) were employed for the analyses. Pain behaviors were also examined with PAFr antagonist (WEB2086).ResultsRT-PCR showed that LPCAT2 mRNA was increased in the ipsilateral spinal cord after injury, but not LPCAT1 mRNA. Double-labeling of ISHH with IHC revealed that LPCAT1 and 2 mRNAs were constitutively expressed by a subset of neurons, and LPCAT2 mRNA was increased in spinal microglia after nerve injury. RT-PCR showed that PAFr mRNA was dramatically increased in the ipsilateral spinal cord after nerve injury. Double-labeling analysis of ISHH with IHC revealed that after injury PAFr mRNA was predominantly colocalized with microglia in the spinal cord. Continuous intrathecal administration of the PAFr antagonist suppressed mechanical allodynia following peripheral nerve injury. Delayed administration of a PAFr antagonist did not reverse the mechanical allodynia.ConclusionsOur data show the histological localization of PAF synthases and its receptor in the spinal cord following peripheral nerve injury, and suggest that PAF/PAFr signaling in the spinal cord acts in an autocrine or paracrine manner among the activated microglia and neurons, thus contributing to development of neuropathic pain.

PAF effects on MCP-1 and IL-6 secretion in U-937 monocytes in comparison with OxLDL and IL-1β effects

ObjectiveTo study the effects of PAF, in comparison with OxLDL and IL-1β on MCP-1 and IL-6 secretion from U-937 monocytes and to investigate the mechanism of its action. MethodsU-937 cell line was cultured in the presence or absence of PAF or OxLDL or IL-1β. Secretion of IL-6 and MCP-1 was measured by ELISA method, mRNA levels of MCP-1 and PAFR was measured using real-time PCR. In order to investigate the mechanism of mediator's action signal transduction appropriate inhibitors was used and oxidant status of cells by measurement the total cellular thiols content and glutathione was determined. Results and conclusionNone of the tested mediators induced the secretion of IL-6. On the other hand PAF and OxLDL caused a short-term while IL-1β caused a long-term secretion and expression of MCP-1. Reduced total thiol levels and GSH/GSSG ratio indicate that the above mediators induce oxidative stress. The signal transduction of all mediators is mediated through G-proteins, protein kinases (PKC, serine–threonine kinase and tyrosine kinase) and NF-κB activation. In addition, PAF, OxLDL, IL-1β activates monocytes leading to increased PAF receptor mRNA levels. These results indicate that PAF and OxLDL, in a different pattern from that of IL-1β, regulate MCP-1 expression via pathways that involve changes in cell redox status.

Mechanism and dose-effect of Ginkgolide B on severe acute pancreatitis of rats

To determine the optimal dosage and mechanism of Ginkgolide B (BN52021) on severe acute pancreatitis (SAP) of rats. Seventy male Wistar rats were randomly divided into seven groups (10 for each group). Sham-operation group (SO), SAP model group (SAP), dimethyl sulfoxide (DMSO) contrast group (DMSO), and groups treated with 2.5 mg/kg BN52021 (BN1), 5 mg/kg BN52021 (BN2), 10 mg/kg BN52021 (BN3), and 20 μg/kg Sandostatin (SS). The SAP model was established in Wistar rats by injecting 5% sodium taurocholate retrogradely into the common bilio-pancreatic duct. The rats of SO, DMSO and BN52021 were injected with 0.9% NaCl, 0.5% DMSO and BN52021 through femoral vein 15 min after the operation. The SS group was injected with Sandostatin subcutaneously. All rats were anaesthetized at 6 h after operation, and venous blood was collected to determine the levels of serum amylase and phospholipase A2 (PLA₂), and pancreas tissue was harvested and stained. There was no significant difference between the SAP and DMSO groups in serum amylase level, PLA₂, ascites and pathologic score, but significant difference was found in SAP/DMSO groups compared with those in SO group (P < 0.05) and the levels of serum amylase, PLA₂, ascites, and pathologic score were lower in the BN1, BN2, BN3 and SS groups than in the SAP and DMSO groups (P < 0.05). However, among BN1, BN2, BN3 and SS groups, BN2 had the best effect in decreasing the levels of serum amylase and PLA₂ (P < 0.05). Expression of platelet activating factor (PAF) receptor (PAFR) mRNA and protein showed no significant difference between the SAP and DMSO groups, or among BN1, BN2, BN3 and SS groups, but there was remarkable difference between SAP/DMSO group and SO group (P < 0.05), and expression of PAFR mRNA and protein was higher in the BN1, BN2, BN3 and SS groups than in the SAP and DMSO groups (P < 0.05). PAFR expression was observed in the nucleus and cytoplasm of pancreatic islet cells in Wistar rats by immunohistochemistry. By iv injection, 5 mg/kg of BN52021 is the optimal dosage for SAP rats. BN52021 may inhibit the interaction/binding of PAF with PAFR.

Prolonged hypoxia modulates platelet activating factor receptor-mediated responses by fetal ovine pulmonary vascular smooth muscle cells

Hypoxia augments PAF receptor (PAFr) binding and PAFr protein expression in venous SMC (SMC-PV). We compared effect of acute and prolonged hypoxia (pO 2 < 40 torr) on PAFr-mediated responses in arterial SMC (SMC-PA) and SMC-PV. Cells were studied for 30 min (acute) or for 48 h (prolonged) hypoxia and compared to normoxic (pO 2 ~ 100 torr) conditions. PAF binding was quantified in fmol/10 6 cells (mean ± SEM). PAF binding in normoxia were SMC-PA, 5.2 ± 0.2 and in SMC-PV, 19.3 ± 1.1; values in acute hypoxia were SMC-PA, 7.7 ± 0.4 and in SMC-PV, 27.8 ± 1.7. Prolonged hypoxia produced 6-fold increase in binding in SMC-PA, but only 2-fold increase in SMC-PV, but binding in SMC-PV was still higher. Acute hypoxia augmented inositol phosphate release by 50% and 40% in SMC-PA and SMC-PV, respectively. During normoxia, PAFr mRNA expression by both cell types was similar, but expression in hypoxia by SMC-PA was greater. In SMC-PA, hypoxia and PAF augmented intracellular calcium flux. Re-exposure of cells to 30 min normoxia after 48 h hypoxia decreased binding by 45–60%, suggesting immediate down-regulation of hypoxia-induced PAFr-mediated effects. We speculate that re-oxygenation immediately reverses hypoxia effect probably due to oxygen tension-dependent reversibility of PAFr activation and suggest that exposure of the neonate to prolonged state of hypoxia will vilify oxygen exchange capacity of the neonatal lungs.

Expression and significance of platelet-acfivating factor in human hepatocellular carcinoma

Objective To investigate the expression of platelet-activating factor(PAF)gene in human hepatocellular carcinoma(hHCC).Methods The samples were obtained from 10 cases of hHCC specimens.Immunohistochemistry and semi-quantitative RT-PCR techniques were used for detecting the PAF positive-staining and PAF receptor mRNA expression in 10 samples and adjacent noncanerous tissues.Results PAF pesitive-staining and PAF receptor mRNA were detected in 90% of hHCC samples and 20% adjacent noncanerous tissues respectively(P ＜ 0.05).Conclusion The PAF expression is increased in middle-advance hHCC,which may play a role in the pathogenesis of hHCC. Key words: Carcinoma; hepatocellular; PAF

Platelet‐activating factor‐stimulated production of reactive oxygen species in ovarian granulosa cells from periovulatory follicles

The platelet-activating factor (PAF) is a proinflammatory lipid present in the fluid of ovarian Graafian follicle. Ovarian blockage of the PAF receptor (PAFr) reduces ovulations in the rat whereas underlying mechanism is poorly understood. Mural granulosa cells (MGC) were mechanically isolated from the theca interna of bovine periovulatory follicle. The mRNA abundance for PAFr, progesterone receptor and cyclooxygenase-2 were measured by real-time PCR. Cytosolic calcium (Ca2+) concentration was assayed by microscopy using Fura-2 AM as indicator, 8-isoprostaglandin F(2alpha) (8-isoPGF(2alpha)) by an ELISA kit. Fluorescent products arising from intracellular oxidation of hydroethidine (HE) and dihydrorhodamine (DHR) were quantified by flow cytometry. The cells expressed PAFr mRNA and PAFr protein and responded to cPAF (nonhydrolyzable form of PAF) with a pulsating increase in Ca2+, demonstrating functional PAFr. Elevation of Ca2+ was reversed by WEB-2086, an inverse PAFr agonist. cPAF elevated the level of 8-isoPGF(2alpha) in the medium of MGC cultured with luteinizing hormone (LH). cPAF alone had no significant influence on the oxidation of HE and DHR, or 8-isoPGF(2alpha) level. In MGC from vital periovulatory follicle, PAF and LH signaling plays an important role in regulating the production of excessive oxidants. Blockage of PAFr seems to interfere with these regulatory processes essential for ovulation.

Polyunsaturated Fatty Acid Supplementation Alters Proinflammatory Gene Expression and Reduces the Incidence of Necrotizing Enterocolitis in a Neonatal Rat Model

Although supplementation of preterm formula with polyunsaturated fatty acids (PUFA) has been shown to reduce the incidence of necrotizing enterocolitis (NEC) in animal models and clinical trials, the mechanisms remain elusive. We hypothesized that the protective effect of PUFA on NEC may be due to the ability of PUFA to suppress Toll-like receptor (TLR) 4 and platelet-activating factor receptor (PAFR) gene expression (molecules that are important in the pathogenesis of NEC) in epithelial cells. To investigate the efficacy of different PUFA preparations on NEC in a neonatal rat model, we compared the incidence of NEC among the four PUFA supplemented groups--A: arachidonic acid and docosahexaenoic acid (AA+DHA), B: egg phospholipids (EP), C: DHA, and D: control without PUFA. PUFA supplementation reduced the incidence of NEC and inhibited intestinal PAFR and TLR4 gene expression compared with the controls. To validate the in vivo observations, IEC-6 cells were exposed to PAF after pretreatment with AA or DHA. Both AA and DHA supplementation blocked PAF-induced TLR4 and PAFR mRNA expression in these enterocytes. These results suggest that PUFA modulates gene expression of key factors involved in experimental NEC pathogenesis. These effects might in part explain the protective effect of PUFA on neonatal NEC.

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.

Significance of platelet activating factor receptor expression in pancreatic tissues of rats with severe acute pancreatitis and effects of BN52021

To investigate the dynamic changes and significance of platelet activating factor receptor (PAF-R) mRNA and protein in pancreatic tissues of rats with severe acute pancreatitis (SAP) and effects of BN52021 (Ginkgolide B). Wistar male rats were randomly assigned to the negative control group (NC group), SAP model group (SAP group), and BN52051-remedy group (BN group), and each of the groups was divided into 6 subgroups at different time points after operation (1 h, 2 h, 3 h, 6 h, 12 h, and 24 h) (n = 10 in each). PT-PCR and Western blot methods were used to detect PAF-RmRNA and protein expression in pancreatic tissues of rats respectively. Pathological examination of pancreatic tissues was performed and the serum amylase change was detected. Serum amylase and pathological results showed the that SAP model was successfully prepared, BN52021 was able to decrease serum amylase, and the pathological ratings in BN group at 3 h, 6 h, and 12 h significantly decreased compared with those in the SAP group (8.85 +/- 0.39 vs 5.95 +/- 0.19, 9.15 +/- 0.55 vs 5.55 +/- 0.36, 10.10 +/- 0.65 vs 6.72 +/- 0.30, P < 0.05). The result of PAF-mRNA showed dynamic changes in SAP and BN groups, which increased gradually in early stage, reached a peak at 3 h (0.71 +/- 0.14 vs 0.54 +/- 0.14, 0.69 +/- 0.13 vs 0.59 +/- 0.04, P < 0.05), and decreased gradually later. There were significant differences at each time point except 1 h and 2 h, when compared with those in the NC group (0.71 +/- 0.14 or 0.69 +/- 0.13 vs 0.47 +/- 0.10, 0.38 +/- 0.08 or 0.59 +/- 0.04 vs 0.47 +/- 0.09, 0.25 +/- 0.07 or 0.29 +/- 0.05 vs 0.46 +/- 0.10, 0.20 +/- 0.06 or 0.20 +/- 0.04 vs 0.43 +/- 0.09, P < 0.05), whereas there was no significant difference between BN and SAP groups at each time point. The result of PAF-R protein showed that the change of PAF-R protein in the SAP group and the BN group was consistent with that of PAF-R mRNA. There were significant differences at each time point except 1 h, when compared with those in the NC group (0.90 +/- 0.02 or 0.80 +/- 0.05 vs 0.48 +/- 0.02, 1.69 +/- 0.06 or 1.58 +/- 0.02 vs 0.48 +/- 0.03, 1.12 +/-0.10 or 0.98 +/- 0.03 vs 0.49 +/- 0.09, 1.04 +/- 0.14 or 0.87 +/- 0.02 vs 0.52 +/- 0.08, 0.97 +/- 0.16 or 0.90 +/- 0.05 vs 0.49 +/- 0.10, P < 0.05), whereas there was no significant difference between the BN group and the SAP group. PAF-R plays an important role in occurrence and development of SAP. BN52021 exerts biological effects through competitively inhibiting the binding of increased both PAF and PAF-R expression rather than through decreasing PAF-R expression in pancreatic tissues.

HCl-induced inflammatory mediators in cat esophageal mucosa and inflammatory mediators in esophageal circular muscle in an in vitro model of esophagitis

Platelet-activating factor (PAF) and interleukin-6 (IL-6) are produced in the esophagus in response to HCl and affect ACh release, causing changes in esophageal motor function similar to esophagitis (Cheng L, Cao W, Fiocchi C, Behar J, Biancani P, and Harnett KM. Am J Physiol Gastrointest Liver Physiol 289: G418-G428, 2005). We therefore examined HCl-activated mechanisms for production of PAF and IL-6 in cat esophageal mucosa and circular muscle. A segment of normal mucosa was tied at both ends, forming a mucosal sac (Cheng L, Cao W, Fiocchi C, Behar J, Biancani P, and Harnett KM. Am J Physiol Gastrointest Liver Physiol 289: G860-G869, 2005) that was filled with acidic Krebs buffer (pH 5.8) or normal Krebs buffer (pH 7.0) as control and kept in oxygenated Krebs buffer for 3 h. The supernatant of the acidic sac (MS-HCl) abolished contraction of normal muscle strips in response to electric field stimulation. The inhibition was reversed by the PAF antagonist CV3988 and by IL-6 antibodies. PAF and IL-6 levels in MS-HCl and mucosa were significantly elevated over control. IL-6 levels in mucosa and supernatant were reduced by CV3988, suggesting that formation of IL-6 depends on PAF. PAF-receptor mRNA levels were not detected by RT-PCR in normal mucosa, but were significantly elevated after exposure to HCl, indicating that HCl causes production of PAF and expression of PAF receptors in esophageal mucosa and that PAF causes production of IL-6. PAF and IL-6, produced in the mucosa, are released to affect the circular muscle layer. In the circular muscle, PAF causes production of additional IL-6 that activates NADPH oxidase to induce production of H(2)O(2). H(2)O(2) causes formation of IL-1beta that may induce production of PAF in the muscle, possibly closing a self-sustaining cycle of production of inflammatory mediators.