Group V secreted phospholipase A2 plays a protective role against aortic dissection.

Aortic dissection is a life-threatening aortopathy involving separation of the aortic wall, whose underlying mechanisms are still incompletely understood. Epidemiological evidence suggests that unsaturated fatty acids improve cardiovascular health. Here, using quantitative RT-PCR, histological analyses, magnetic cell sorting and flow cytometry assays, and MS-based lipidomics, we show that the activity of a lipid-metabolizing enzyme, secreted phospholipase A2 group V (sPLA2-V), protects against aortic dissection by endogenously mobilizing vasoprotective lipids. Global and endothelial cell-specific sPLA2-V-deficient mice frequently developed aortic dissection shortly after infusion of angiotensin II (AT-II). We observed that in the AT-II-treated aorta, endothelial sPLA2-V mobilized oleic and linoleic acids, which attenuated endoplasmic reticulum stress, increased the expression of lysyl oxidase, and thereby stabilized the extracellular matrix in the aorta. Of note, dietary supplementation with oleic or linoleic acid reversed the increased susceptibility of sPLA2-V-deficient mice to aortic dissection. These findings reveal an unexplored functional link between sPLA2-driven phospholipid metabolism and aortic stability, possibly contributing to the development of improved diagnostic and/or therapeutic strategies for preventing aortic dissection.

Degradation of group V secretory phospholipase A2 in lung endothelium is mediated by autophagy.

Group V secretory phospholipase A2 (gVPLA2) is a potent inflammatory mediator in mammalian tissues that hydrolyzes phospholipids and initiates eicosanoid biosynthesis. Previous work has demonstrated that multiple inflammatory stimuli induce its expression and secretion in several cell types, including the lung endothelium. However, little is known about the mechanism(s) by which gVPLA2 inflammatory signaling is subsequently downregulated. Therefore, in this study we characterized potential clearance mechanisms for gVPLA2 in lung endothelial cells (EC). We observed that exogenous gVPLA2 is taken up rapidly by nutrient-starved human pulmonary artery EC (HPAEC) in vitro, and its cellular expression subsequently is reduced over several hours. In parallel experiments performed in pulmonary vascular EC isolated from mice genetically deficient in gVPLA2, the degradation of exogenously applied gVPLA2 occurs in a qualitatively similar fashion. This degradation is significantly attenuated in EC treated with ammonium chloride or chloroquine, which are lysosomal inhibitors that block autophagic flux. In contrast, the proteasomal inhibitor MG132 fails to prevent the clearance of gVPLA2. Both immunofluorescence microscopy and proximity ligation assay demonstrate the co-localization of LC3 and gVPLA2 during this process, indicating the association of gVPLA2 with autophagosomes. Nutrient starvation, a known inducer of autophagy, is sufficient to stimulate gVPLA2 degradation. These results suggest that a lysosome-mediated autophagy pathway contributes to gVPLA2 clearance from lung EC. These novel observations advance our understanding of the mechanism by which this key inflammatory enzyme is downregulated in the lung vasculature.

Macrophages regulate lung ILC2 activation via Pla2g5-dependent mechanisms.

Group V phospholipase A2 (Pla2g5) is a lipid-generating enzyme necessary for macrophage effector functions in pulmonary inflammation. However, the lipid mediators involved and their cellular targets have not been identified. Mice lacking Pla2g5 showed markedly reduced lung ILC2 activation and eosinophilia following repetitive Alternaria Alternata inhalation. While Pla2g5-null mice had Wt levels of immediate IL-33 release after one Alternaria dose, they failed to upregulate IL-33 in macrophages following repeated Alternaria administration. Unexpectedly, while adoptive transfer of bone marrow-derived (BM)-macrophages restored ILC2 activation and eosinophilia in Alternaria-exposed Pla2g5-null mice, exogenous IL-33 did not. Conversely, transfers of Pla2g5-null BM-macrophages reduced inflammation in Alternaria-exposed Wt mice. Mass spectrometry analysis of free fatty acids (FFAs) demonstrated significantly reduced FFAs (including linoleic acid (LA) and oleic acid (OA)) in lung and BM-macrophages lacking Pla2g5. Exogenous administration of LA or LA+OA to Wt mice sharply potentiated IL-33-induced lung eosinophilia and ILC2 expansion in vitro and in vivo. In contrast, OA potentiated IL-33-induced inflammation and ILC2 expansion in Pla2g5-null mice, but LA was inactive both in vivo and in vitro. Notably, Pla2g5-null ILC2s showed significantly reduced expression of the FFA-receptor-1 compared to Wt ILC2s. Thus, macrophage-associated Pla2g5 contributes significantly to type-2 immunity through regulation of IL-33 induction and FFA-driven ILC2 activation.

The dual role of group V secretory phospholipase A2 in pancreatic ß-cells.

PURPOSE: Group X (GX) and group V (GV) secretory phospholipase A2 (sPLA2) potently release arachidonic acid (AA) from the plasma membrane of intact cells. We previously demonstrated that GX sPLA2 negatively regulates glucose-stimulated insulin secretion (GSIS) by a prostaglandin E2 (PGE2)-dependent mechanism. In this study we investigated whether GV sPLA2 similarly regulates GSIS. METHODS: GSIS and pancreatic islet-size were assessed in wild-type (WT) and GV sPLA2-knock out (GV KO) mice. GSIS was also assessed ex vivo in isolated islets and in vitro using MIN6 pancreatic beta cell lines with or without GV sPLA2 overexpression or silencing. RESULTS: GSIS was significantly decreased in islets isolated from GV KO mice compared to WT mice and in MIN6 cells with siRNA-mediated GV sPLA2 suppression. MIN6 cells overexpressing GV sPLA2 (MIN6-GV) showed a significant increase in GSIS compared to control cells. Though the amount of AA released into the media by MIN6-GV cells was significantly higher, PGE2 production was not enhanced or cAMP content decreased compared to control MIN6 cells. Surprisingly, GV KO mice exhibited a significant increase in plasma insulin levels following i.p. injection of glucose compared to WT mice. This increase in GSIS in GV KO mice was associated with a significant increase in pancreatic islet size and number of proliferating cells in ß-islets compared to WT mice. CONCLUSIONS: Deficiency of GV sPLA2 results in diminished GSIS in isolated pancreatic beta-cells. However, the reduced GSIS in islets lacking GV sPLA2 appears to be compensated by increased islet mass in GV KO mice.

Differential Expression of Inflammation-Related Genes in Children with Down Syndrome.

OBJECTIVE: The aim of the study was to investigate the expression patterns of a specific set of genes involved in the inflammation process in children with Down Syndrome (DS) and children without the syndrome (control group) to identify differences that may be related to the immune abnormalities observed in DS individuals. METHOD: RNA samples were obtained from peripheral blood, and gene expression was quantified using the TaqMan® Array Plate Human Inflammation Kit, which facilitated the investigation into 92 inflammation-related genes and four reference genes using real-time polymerase chain reaction (qPCR). RESULTS: Twenty genes showed differential expression in children with DS; 12 were overexpressed (PLA2G2D, CACNA1D, ALOX12, VCAM1, ICAM1, PLCD1, ADRB1, HTR3A, PDE4C, CASP1, PLA2G5, and PLCB4), and eight were underexpressed (LTA4H, BDKRB1, ADRB2, CD40LG, ITGAM, TNFRSF1B, ITGB1, and TBXAS1). After statistically correcting for the false discovery rate, only the genes BDKRB1 and LTA4H showed differential expression, and both were underexpressed within the DS group. CONCLUSION: DS children showed differential expression of inflammation-related genes that were not located on chromosome 21 compared with children without DS. The BDKRB1 and LTA4H genes may differentiate the case and control groups based on the inflammatory response, which plays an important role in DS pathogenesis.

Epigenetic control of group V phospholipase A2 expression in human malignant cells.

Secreted phospholipases A2 (sPLA2) are suggested to play an important role in inflammation and tumorigenesis. Different mechanisms of epigenetic regulation are involved in the control of group IIA, III and X sPLA2s expression in cancer cells, but group V sPLA2 (GV-PLA2) in this respect has not been studied. Here, we demonstrate the role of epigenetic mechanisms in regulation of GV-PLA2 expression in different cell lines originating from leukaemia and solid cancers. In blood leukocytes from leukaemic patients, levels of GV-PLA2 transcripts were significantly lower in comparison to those from healthy individuals. Similarly, in DU-145 and PC-3 prostate and CAL-51 and MCF-7 mammary cancer cell lines, levels of GV-PLA2 transcripts were significantly lower in relation to those found in normal epithelial cells of prostate or mammary. By sequencing and methylation-specific high-resolution melting (MS-HRM) analyses of bisulphite-modified DNA, distinct CpG sites in the GV-PLA2 promoter region were identified that were differentially methylated in cancer cells in comparison to normal epithelial and endothelial cells. Spearman rank order analysis revealed a significant negative correlation between the methylation degree and the cellular expression of GV-PLA2 (r = -0.697; p = 0.01). The effects of demethylating agent (5-aza-2'-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on GV-PLA2 transcription in the analysed cells confirmed the importance of DNA methylation and histone modification in the regulation of the GV-PLA2 gene expression in leukaemic, prostate and mammary cancer cell lines. The exposure of tumour cells to human recombinant GV-PLA2 resulted in a reduced colony forming activity of MCF-7, HepG2 and PC-3 cells, but not of DU-145 cells suggesting a cell-type-dependent effect of GV-PLA2 on cell growth. In conclusion, our results suggest that epigenetic mechanisms such as DNA methylation and histone modification play an important role in downregulation of GV-PLA2 expression in cancer cells.

Group V secreted phospholipase A2 is upregulated by IL-4 in human macrophages and mediates phagocytosis via hydrolysis of ethanolamine phospholipids.

Studies on the heterogeneity and plasticity of macrophage populations led to the identification of two major polarization states: classically activated macrophages or M1, induced by IFN-γ plus LPS, and alternatively activated macrophages, induced by IL-4. We studied the expression of multiple phospholipase A2 enzymes in human macrophages and the effect that polarization of the cells has on their levels. At least 11 phospholipase A2 genes were found at significant levels in human macrophages, as detected by quantitative PCR. None of these exhibited marked changes after treating the cells with IFN-γ plus LPS. However, macrophage treatment with IL-4 led to strong upregulation of the secreted group V phospholipase A2 (sPLA2-V), both at the mRNA and protein levels. In parallel with increasing sPLA2-V expression levels, IL-4-treated macrophages exhibited increased phagocytosis of yeast-derived zymosan and bacteria, and we show that both events are causally related, because cells deficient in sPLA2-V exhibited decreased phagocytosis, and cells overexpressing the enzyme manifested higher rates of phagocytosis. Mass spectrometry analyses of lipid changes in the IL-4-treated macrophages suggest that ethanolamine lysophospholipid (LPE) is an sPLA2-V-derived product that may be involved in regulating phagocytosis. Cellular levels of LPE are selectively maintained by sPLA2-V. By supplementing sPLA2-V-deficient cells with LPE, phagocytosis of zymosan or bacteria was fully restored in IL-4-treated cells. Collectively, our results show that sPLA2-V is required for efficient phagocytosis by IL-4-treated human macrophages and provide evidence that sPLA2-V-derived LPE is involved in the process.

Phospholipase A2 group v in benign familial fleck retina in a set of triplets.

PURPOSE: To evaluate the association of phospholipase A2, Group V (PLA2G5), with benign familial fleck retina in a consanguineous family with triplets. METHODS: Clinical eye examination, including fundus examination and spectral domain optical coherence tomography, was performed for all the family members. After blood sample collection and DNA extraction, polymerase chain reaction was performed to amplify regions spanning Exons 2, 3, 4, and 5 of PLA2G5. The amplified products were sequenced to observe the presence of any mutations. RESULTS: Fundus examination in two of the triplets revealed discrete yellow-white flecks and both had good vision and absence of night blindness, consistent with benign familial fleck retina. The flecks were hyperautofluorescent. Furthermore, spectral domain optical coherence tomography showed focal thickening of the retinal pigment epithelium because of the presence of these flecks. Molecular investigations showed that PLA2G5 Exons 2, 4, and 5 harbored no misalignments among all family members. However, PLA2G5 Exon 3 showed a p.Gly45Cys mutation for the father and the third triplet who was affected. CONCLUSION: The clinical findings in this family suggest a diagnosis of benign familial fleck retina with excellent prognosis, in which the PLA2G5 gene may play a role.

Group V secretory phospholipase A2 impairs endothelial protein C receptor-dependent protein C activation and accelerates thrombosis in vivo.

BACKGROUND: Endothelial protein C receptor (EPCR) must be bound to a molecule of phosphatidylcholine (PC) to be fully functional, i.e. to interact with protein C/activated protein C (APC) properly. PC can be replaced with other lipids, such as lysophosphatidylcholine or platelet-activating factor, by the action of group V secretory phospholipase A2 (sPLA2-V), an enzyme that is upregulated in a variety of inflammatory conditions. Studies in purified systems have demonstrated that the substitution of PC notably impairs EPCR function in a process called EPCR encryption. OBJECTIVES: To analyze whether sPLA2-V was able to regulate EPCR-dependent protein C activation in vivo, and its impact on thrombosis and the hemostatic system. METHODS: Mice were transfected with sPLA2-V by hydrodynamic gene delivery. The effects on thrombosis were studied with the laser carotid artery occlusion model, and APC generation capacity was measured with ELISA. Global hemostasis was analyzed with thromboelastometry. RESULTS: We found that sPLA2-V overexpression in mice significantly decreased their ability to generate APC. Furthermore, a murine carotid artery laser thrombosis model revealed that higher sPLA2-V levels were directly associated with faster artery thrombosis. CONCLUSIONS: sPLA2-V plays a thrombogenic role by impairing the ability of EPCR to promote protein C activation.

The (G>A) rs11573191 polymorphism of PLA2G5 gene is associated with premature coronary artery disease in the Mexican Mestizo population: the genetics of atherosclerotic disease Mexican study.

Coronary artery disease (CAD) is a multifactorial disorder that results from an excessive inflammatory response. Secretory phospholipase A2-V (sPLA2-V) encoded by PLA2G5 gene promotes diverse proinflammatory processes. The aim of the present study was to analyze if PLA2G5 gene polymorphisms are associated with premature CAD. Three PLA2G5 polymorphisms (rs11573187, rs2148911, and rs11573191) were analyzed in 707 patients with premature CAD and 749 healthy controls. Haplotypes were constructed after linkage disequilibrium analysis. Under dominant, recessive, and additive models, the rs11573191 polymorphism was associated with increased risk of premature CAD (OR = 1.51, P(dom) = 3.5 × 10(-3); OR = 2.95, P(rec) = 0.023; OR = 1.51, P(add) = 1.2 × 10(-3)). According to the informatics software, this polymorphism had a functional effect modifying the affinity of the sequence by the MZF1 transcription factor. PLA2G5 polymorphisms were in linkage disequilibrium and the CGA haplotype was associated with increased risk of premature CAD (OR = 1.49, P = 0.0023) and with hypertension in these patients (OR = 1.75, P = 0.0072). Our results demonstrate the association of the PLA2G5 rs11573191 polymorphism with premature CAD. In our study, it was possible to distinguish one haplotype associated with increased risk of premature CAD and hypertension.

The adipocyte-inducible secreted phospholipases PLA2G5 and PLA2G2E play distinct roles in obesity.

Metabolic disorders, including obesity and insulin resistance, have their basis in dysregulated lipid metabolism and low-grade inflammation. In a microarray search of unique lipase-related genes whose expressions are associated with obesity, we found that two secreted phospholipase A2s (sPLA2s), PLA2G5 and PLA2G2E, were robustly induced in adipocytes of obese mice. Analyses of Pla2g5(-/-) and Pla2g2e(-/-) mice revealed distinct roles of these sPLA2s in diet-induced obesity. PLA2G5 hydrolyzed phosphatidylcholine in fat-overladen low-density lipoprotein to release unsaturated fatty acids, which prevented palmitate-induced M1 macrophage polarization. As such, PLA2G5 tipped the immune balance toward an M2 state, thereby counteracting adipose tissue inflammation, insulin resistance, hyperlipidemia, and obesity. PLA2G2E altered minor lipoprotein phospholipids, phosphatidylserine and phosphatidylethanolamine, and moderately facilitated lipid accumulation in adipose tissue and liver. Collectively, the identification of "metabolic sPLA2s" adds this gene family to a growing list of lipolytic enzymes that act as metabolic coordinators.

Novel genetic approach to investigate the role of plasma secretory phospholipase A2 (sPLA2)-V isoenzyme in coronary heart disease: modified Mendelian randomization analysis using PLA2G5 expression levels.

BACKGROUND: Secretory phospholipase A2 (sPLA2) enzymes are considered to play a role in atherosclerosis. sPLA2 activity encompasses several sPLA2 isoenzymes, including sPLA2-V. Although observational studies show a strong association between elevated sPLA2 activity and CHD, no assay to measure sPLA2-V levels exists, and the only evidence linking the sPLA2-V isoform to atherosclerosis progression comes from animal studies. In the absence of an assay that directly quantifies sPLA2-V levels, we used PLA2G5 mRNA levels in a novel, modified Mendelian randomization approach to investigate the hypothesized causal role of sPLA2-V in coronary heart disease (CHD) pathogenesis. METHODS AND RESULTS: Using data from the Advanced Study of Aortic Pathology, we identified the single-nucleotide polymorphism in PLA2G5 showing the strongest association with PLA2G5 mRNA expression levels as a proxy for sPLA2-V levels. We tested the association of this SNP with sPLA2 activity and CHD events in 4 prospective and 14 case-control studies with 27 230 events and 70 500 controls. rs525380C>A showed the strongest association with PLA2G5 mRNA expression (P=5.1×10(-6)). There was no association of rs525380C>A with plasma sPLA2 activity (difference in geometric mean of sPLA2 activity per rs525380 A-allele 0.4% (95% confidence intervals [-0.9%, 1.6%]; P=0.56). In meta-analyses, the odds ratio for CHD per A-allele was 1.02 (95% confidence intervals [0.99, 1.04]; P=0.20). CONCLUSIONS: This novel approach for single-nucleotide polymorphism selection for this modified Mendelian randomization analysis showed no association between rs525380 (the lead single-nucleotide polymorphism for PLA2G5 expression, a surrogate for sPLA2-V levels) and CHD events. The evidence does not support a causal role for sPLA2-V in CHD.

Human group V secretory phospholipase A2 is associated with lipid rafts and internalized in a flotillin­dependent pathway.

The mechanisms of secretory phospholipase A2 (sPLA2) action are not understood clearly. Previously, it was suggested that sPLA2s are internalized into cells for the targeting of sPLA2 to intracellular action sites. However, the mechanisms for sPLA2 internalization remain to be identified. The present study demonstrated for the first time that human group V sPLA2 (hVPLA2) is associated with lipid rafts and is internalized in a flotillin­dependent pathway. The lipid raft association was probed by cholesterol­sensitive enrichment of hVPLA2 in low­density fractions and co­patching of ganglioside GM1 rafts through cross­linking of hVPLA2 in HEK293 and CHO cells. The hVPLA2 associated with lipid rafts was shown to be internalized into HEK293 cells at a relatively rapid rate (t1/2 =16 min) and this internalization was inhibited by the knockdown of flotillin­1, but not by chlorpromazine, an inhibitor of clathrin­mediated endocytosis. Moreover, internalized hVPLA2 was shown to be colocalized extensively with flotillin­1 in a punctate structure, but not caveolin­1. These data revealed that the internalization of hVPLA2 is mediated by flotillin­1. Attenuation of arachidonic acid release from plasma membrane through the association of hVPLA2 with lipid rafts suggested that this association with lipid rafts may be important in protecting mammalian cells from excessive degradation of plasma membrane and trafficking hVPLA2 into intracellular targets.

Key role of group v secreted phospholipase A2 in Th2 cytokine and dendritic cell-driven airway hyperresponsiveness and remodeling.

BACKGROUND: Previous work has shown that disruption of the gene for group X secreted phospholipase A2 (sPLA2-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA2s in the mammalian genome, the involvement of other sPLA2s in the asthma model is possible - in particular, the group V sPLA2 (sPLA2-V) that like sPLA2-X is highly active at hydrolyzing membranes of mammalian cells. METHODOLOGY AND PRINCIPAL FINDINGS: The allergen-driven asthma phenotype was significantly reduced in sPLA2-V-deficient mice but to a lesser extent than observed previously in sPLA2-X-deficient mice. The most striking difference observed between the sPLA2-V and sPLA2-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA2-V(-/-) mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V(-/-) mice diminishes Th2 cytokine responses in the airways. CONCLUSIONS: This paper illustrates the diverse roles of sPLA2s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA2-V as a potential new therapy to treat asthma and other allergic disorders.

Telmisartan and N-acetylcysteine suppress group V secretory phospholipase A2 expression in TNFα-stimulated human endothelial cells and reduce associated atherogenicity.

Group V secretory phospholipase A2 (sPLA2-V) hydrolyzes phosphatidylcholine in low-density lipoprotein (LDL) to increase lysophosphatidylcholine (LPC) content. Because in human umbilical vein endothelial cells (HUVEC), tumor necrosis factor alpha (TNFα)-induced sPLA2-V expression, and LPC content in LDL and monocyte chemoattractant protein-1 mRNA were enhanced by incubation of LDL with TNFα-stimulated HUVEC, we investigated whether an angiotensin II receptor type 1 blocker, telmisartan, or an antioxidant drug, N-acetylcysteine (NAC), suppressed TNFα-induced sPLA2-V expression. Telmisartan or NAC administered before and during TNFα stimulation diminished the increase of sPLA2-V mRNA in HUVEC and reduced TNFα-induced sPLA2-V protein at 3 days after TNFα stimulation. Angiotensin II did not induce sPLA2-V mRNA, and a peroxisome proliferator-activated receptor-γ antagonist, GW3335, did not influence the inhibitory effect of telmisartan on TNFα-induced sPLA2-V mRNA. At 3 days after TNFα stimulation, 30 µM telmisartan or 20 mM NAC administered before and during TNFα stimulation prevented the enhancement of LPC content in LDL and monocyte chemoattractant protein-1 mRNA by LDL incubation with TNFα-stimulated HUVEC. A 2-month treatment with telmisartan in 29 hypertensive type 2 diabetic patients significantly reduced LPC content in circulating LDL. Telmisartan's suppressive effect on TNFα-induced sPLA2-V expression may have beneficial effects in preventing proatherogenic changes of LDL.

Biallelic mutations in PLA2G5, encoding group V phospholipase A2, cause benign fleck retina.

Flecked-retina syndromes, including fundus flavimaculatus, fundus albipunctatus, and benign fleck retina, comprise a group of disorders with widespread or limited distribution of yellow-white retinal lesions of various sizes and configurations. Three siblings who have benign fleck retina and were born to consanguineous parents are the basis of this report. A combination of homozygosity mapping and exome sequencing helped to identify a homozygous missense mutation, c.133G>T (p.Gly45Cys), in PLA2G5, a gene encoding a secreted phospholipase (group V phospholipase A(2)). A screen of a further four unrelated individuals with benign fleck retina detected biallelic variants in the same gene in three patients. In contrast, no loss of function or common (minor-allele frequency>0.05%) nonsynonymous PLA2G5 variants have been previously reported (EVS, dbSNP, 1000 Genomes Project) or were detected in an internal database of 224 exomes (from subjects with adult onset neurodegenerative disease and without a diagnosis of ophthalmic disease). All seven affected individuals had fundoscopic features compatible with those previously described in benign fleck retina and no visual or electrophysiological deficits. No medical history of major illness was reported. Levels of low-density lipoprotein were mildly elevated in two patients. Optical coherence tomography and fundus autofluorescence findings suggest that group V phospholipase A(2) plays a role in the phagocytosis of photoreceptor outer-segment discs by the retinal pigment epithelium. Surprisingly, immunohistochemical staining of human retinal tissue revealed localization of the protein predominantly in the inner and outer plexiform layers.

Group V phospholipase A2 in bone marrow-derived myeloid cells and bronchial epithelial cells promotes bacterial clearance after Escherichia coli pneumonia.

Group V-secreted phospholipase A(2) (GV sPLA(2)) hydrolyzes bacterial phospholipids and initiates eicosanoid biosynthesis. Here, we elucidate the role of GV sPLA(2) in the pathophysiology of Escherichia coli pneumonia. Inflammatory cells and bronchial epithelial cells both express GV sPLA(2) after pulmonary E. coli infection. GV(-/-) mice accumulate fewer polymorphonuclear leukocytes in alveoli, have higher levels of E. coli in bronchoalveolar lavage fluid and lung, and develop respiratory acidosis, more severe hypothermia, and higher IL-6, IL-10, and TNF-α levels than GV(+/+) mice after pulmonary E. coli infection. Eicosanoid levels in bronchoalveolar lavage are similar in GV(+/+) and GV(-/-) mice after lung E. coli infection. In contrast, GV(+/+) mice have higher levels of prostaglandin D(2) (PGD(2)), PGF(2α), and 15-keto-PGE(2) in lung and express higher levels of ICAM-1 and PECAM-1 on pulmonary endothelial cells than GV(-/-) mice after lung infection with E. coli. Selective deletion of GV sPLA(2) in non-myeloid cells impairs leukocyte accumulation after pulmonary E. coli infection, and lack of GV sPLA(2) in either bone marrow-derived myeloid cells or non-myeloid cells attenuates E. coli clearance from the alveolar space and the lung parenchyma. These observations show that GV sPLA(2) in bone marrow-derived myeloid cells as well as non-myeloid cells, which are likely bronchial epithelial cells, participate in the regulation of the innate immune response to pulmonary infection with E. coli.

Time-dependent changes in the brain arachidonic acid cascade during cuprizone-induced demyelination and remyelination.

Phospholipases A(2) (PLA(2)) are the enzymatic keys for the activation of the arachidonic acid (AA) cascade and the subsequent synthesis of pro-inflammatory prostanoids (prostaglandins and tromboxanes). Prostanoids play critical roles in the initiation and modulation of inflammation and their levels have been reported increased in several neurological and neurodegenerative disorders, including multiple sclerosis (MS). Here, we aimed to determine whether brain expression PLA(2) enzymes and the terminal prostagland in levels are changed during cuprizone-induced demyelination and in the subsequent remyelination phase. Mice were given the neurotoxicant cuprizone through the diet for six weeks to induce brain demyelination. Then, cuprizone was withdrawn and mice were returned to a normal diet for 6 weeks to allow spontaneous remyelination. We found that after 4-6 weeks of cuprizone, sPLA(2)(V) and cPLA(2), but not iPLA(2)(VI), gene expression was upregulated in the cortex, concomitant with an increase in the expression of astrocyte and microglia markers. Cyclooxygenase (COX)-2 gene expression was consistently upregulated during all the demyelination period, whereas COX-1 sporadically increased only at week 5 of cuprizone exposure. However, we found that at the protein level only sPLA(2)(V) and COX-1 were elevated during demyelination, with COX-1 selectively expressed by activated and infiltrated microglia/macrophages and astrocytes. Levels of PGE(2), PGD(2), PGI(2) and TXB(2) were also increased during demyelination. During remyelination, none of the PLA(2) isoforms was significantly changed, whereas COX-1 and -2 were sporadically upregulated only at the gene expression level. PGE(2), PGI(2) and PGD(2) levels returned to normal, whereas TXB(2) was still upregulated after 3 weeks of cuprizone withdrawal. Our study characterizes for the first time time-dependent changes in the AA metabolic pathway during cuprizone-induced demyelination and the subsequent remyelination and suggests that sPLA(2)(V) is the major isoform contributing to AA release.

Group V secretory phospholipase A2 plays a pathogenic role in myocardial ischaemia-reperfusion injury.

AIMS: Group V secretory phospholipase A(2) (sPLA(2)-V) is highly expressed in the heart. This study examined (i) the role of sPLA(2)-V in myocardial ischaemia-reperfusion (I/R) injury and (ii) the cooperative action of sPLA(2)-V and cytosolic PLA(2) (cPLA(2)) in myocardial I/R injury, using sPLA(2)-V knockout (sPLA(2)V(-/-)) mice. METHODS AND RESULTS: Myocardial I/R injury was created by 1 h ligation of the left anterior descending coronary artery, followed by 24 h of reperfusion. The sPLA(2)V(-/-) mice had a 44% decrease in myocardial infarct size, a preservation of echocardiographic LV function (%fractional shortening: 40 ± 3.5 vs. 21 ± 4.6, respectively), and lower content of leucotriene B(4) (LTB(4)) and thromboxane B(2) (TXB(2)) (40 and 37% lower, respectively) in the ischaemic myocardium after I/R compared with wild-type (WT) mice. Intraperitoneal administration of AACOCF3 or MAFP, inhibitors of cPLA(2) activity, decreased myocardial infarct size and myocardial content of LTB(4) and TXB(2) in both genotyped mice. The decrease in myocardial infarct size and content of LTB(4) and TXB(2) after cPLA(2) inhibitor administration was greater in WT mice than in sPLA(2)V(-/-) mice. I/R increased phosphorylation of extracellular signal-related kinase 1/2, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in the ischaemic myocardium in association with cPLA(2) phosphorylation. The I/R-induced increase in the phosphorylation of p38 and cPLA(2) was less in sPLA(2)-V(-/-) mice than in WT mice. Pretreatment with the p38 inhibitor SB202190 suppressed an increase in cPLA(2) phosphorylation after I/R in WT mice. CONCLUSION: sPLA(2)-V plays an important role in the pathogenesis of myocardial I/R injury partly in concert with the activation of cPLA(2).

Group V secreted phospholipase A2 contributes to LPS-induced leukocyte recruitment.

Secreted phospholipases A(2) (sPLA(2)s) are well known for their contribution in the biosynthesis of inflammatory eicosanoids. These enzymes also participate in the inflammatory process by regulating chemokine production and protein expression of adhesion molecules. The majority of sPLA(2) isoforms are up-regulated by proinflammatory stimuli such as bacterial lipopolysaccharide (LPS), which predominantly increases the expression of group V sPLA(2) (sPLA(2)-V). Furthermore, it has recently been shown that sPLA(2)-V is a critical messenger in the regulation of cell migration during allergic airway responsiveness. Herein, we investigated the effect of sPLA(2)-V on LPS-mediated leukocyte recruitment and its capacity to modulate adhesion molecule expression. We conducted our study in the murine air pouch model, using sPLA(2)-V null mice (sPLA(2)-V(-/-)) and control wild-type (WT) littermates. We observed that LPS (1 microg/ml)-mediated leukocyte emigration in sPLA(2)-V(-/-) was attenuated by 52% and 86% upon 6 and 12 h of treatment respectively, as compared to WT mice. In WT mice, treatment with the cell-permeable sPLA(2) inhibitor (12-epi-scalaradial; SLD) reduced LPS-mediated leukocyte recruitment by 67%, but had no additional inhibitory effect in sPLA(2)-V(-/-) mice. Protein analyses from the air pouch skin were carried out upon LPS-challenge, and the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were both significantly reduced in sPLA(2)-V(-/-) mice as compared to control WT mice. Together, our data demonstrate the role of sPLA(2)-V in LPS-induced ICAM-1 and VCAM-1 protein overexpression and leukocyte recruitment, supporting the contribution of sPLA(2)-V in the development of inflammatory innate immune responses.