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Cytosolic phospholipase A(2) alpha (cPLA(2)alpha) plays a key role in signaling in mammalian cells by releasing arachidonic acid (AA) from glycerophospholipids (GPLs) but the factors determining the specificity of cPLA(2)alpha for AA- containing GPLs are not well understood. Accordingly, we investigated those factors by determining the activity of human cPLA(2)alpha towards a multitude of GPL species present in micelles or bilayers. Studies on isomeric PC sets containing a saturated acyl chain of 6 to 24 carbons in the sn1 or sn2 position in micelles showed an abrupt decrease in hydrolysis when the length of the snl or sn2 chain exceeded 17 carbons suggesting that the acyl binding cavity on the enzyme is of the corresponding length. Notably, the saturated isomer pairs were hydrolyzed identically in micelles as well as in bilayers suggesting promiscuous binding of acyl chains to the active site of cPLA(2)alpha. Such promiscuous binding would explain the previous finding that cPLA(2)alpha has both PLA(1) and PLA(2) activities. Interestingly, increasing the length of either the sn1 or sn2 acyl chain inhibited the hydrolysis in bilayers far more than that in micelles suggesting that with micelles (loosely packed) substrate accommodation at the active site of cPLA(2)alpha is rate-limiting, while with bilayers (tightly packed) upward movement of the substrate from the bilayer (efflux) is the rate-limiting step. With the AA-containing PCs, the length of the saturated acyl chain also had a much stronger effect on hydrolysis in bilayers vs. micelles in agreement with this model. In contrast to saturated PCs, a marked isomer preference was observed for AA-containing PCs both in micelles and bilayers. In conclusion, these data significantly help to understand the mode of action and specificity of cPLA(2)alpha. (C) 2016 Elsevier B.V. All rights reserved.

Regular consumption of low-dose aspirin reduces the occurrence of colorectal, esophageal, stomach, and gastrointestinal cancers. The underlying mechanism is unknown but may be linked to inhibition of angiogenesis. Because the effective doses of aspirin are consistent with the inhibition of cyclooxygenase-1 in platelets, we used liquid chromatography with tandem mass spectrometry analyses and immunoassays of human platelet releasates coupled with angiogenesis assays to search for the mediator(s) of these effects. Blood or platelet-rich plasma from healthy volunteers stimulated with platelet activators produced a broad range of eicosanoids. Notably, preincubation of platelets with aspirin, but not with a P2Y12 receptor antagonist, caused a marked reduction in the production of 11-hydroxyeicosatetraenoic acid (HETE) and 15(S)-HETE, in addition to prostanoids such as thromboxane A2 Releasates from activated platelets caused cell migration and tube formation in cultured human endothelial cells and stimulated the sprouting of rat aortic rings in culture. These proangiogenic effects were absent when platelets were treated with aspirin but returned by coincubation with exogenous 15(S)-HETE. These results reveal 15(S)-HETE as a major platelet cyclooxygenase-1 product with strong proangiogenic effects. Thus, 15(S)-HETE represents a potential target for the development of novel antiangiogenic therapeutics, and blockade of its production may provide a mechanism for the anticancer effects of aspirin.-Rauzi, F., Kirkby, N. S., Edin, M. L., Whiteford, J. Zeldin, D. C., Mitchell, J. A., Warner, T. D. Aspirin inhibits the production of proangiogenic 15(S)-HETE by platelet cyclooxygenase-1.

Background— Secretory phospholipase A 2 (sPLA 2 ) enzymes are considered to play a role in atherosclerosis. sPLA 2 activity encompasses several sPLA 2 isoenzymes, including sPLA 2 -V. Although observational studies show a strong association between elevated sPLA 2 activity and CHD, no assay to measure sPLA 2 -V levels exists, and the only evidence linking the sPLA 2 -V isoform to atherosclerosis progression comes from animal studies. In the absence of an assay that directly quantifies sPLA 2 -V levels, we used PLA2G5 mRNA levels in a novel, modified Mendelian randomization approach to investigate the hypothesized causal role of sPLA 2 -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 sPLA 2 -V levels. We tested the association of this SNP with sPLA 2 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 sPLA 2 activity (difference in geometric mean of sPLA 2 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 sPLA 2 -V levels) and CHD events. The evidence does not support a causal role for sPLA 2 -V in CHD.

Objective— High circulating levels of group IIA secretory phospholipase A 2 (sPLA 2 -IIA) activity and mass are independent cardiovascular risk factors. Therefore, inhibition of sPLA 2 -IIA may be a target for the treatment of atherosclerotic cardiovascular disease. The present study evaluated the effects of sPLA 2 -IIA inhibition with varespladib acid in a novel mouse model, human apolipoprotein B (apoB)/human cholesteryl ester transfer protein (CETP)/human sPLA 2 -IIA triple transgenic mice (TTT) fed a Western-type diet. Approach and Results— sPLA 2 -IIA expression increased atherosclerotic lesion formation in TTT compared with human apoB/human CETP double transgenic mice ( P 2 -IIA activity. Surprisingly, however, administration of varespladib acid to TTT had no impact on atherosclerosis, which could be attributed to a proatherogenic plasma lipoprotein profile that appears in response to sPLA 2 -IIA inhibition because of increased plasma CETP activity. In the TTT model, sPLA 2 -IIA decreased CETP activity by reducing the acceptor properties of sPLA 2 -IIA–modified very low-density lipoproteins specifically because of a significantly lower apoE content. Increasing very low-density lipoprotein-apoE content by means of adenovirus-mediated gene transfer in sPLA 2 -IIA transgenic mice restored the acceptor properties for CETP. Conclusions— These data show that in a humanized triple transgenic mouse model with hypercholesterolemia, sPLA 2 -IIA inhibition increases CETP activity via increasing the very low-density lipoprotein-apoE content, resulting in a proatherogenic lipoprotein profile.

We recently suggested a novel mechanism for the activation of phospholipase A2 (PLA2), with a (catalytically) highly active oligomeric state, which subsequently becomes inactivated by conversion into amyloid. This process can be activated by lysophosphatidylcholine which promotes both oligomerization and amyloid activation/inactivation. The heat shock protein 70 (Hsp70), has been demonstrated to be able to revert the conversion of α-synuclein and Alzheimer β-peptide to amyloid fibrils in vitro. Accordingly, we would expect Hsp70 to sustain the lifetime of the active state of the enzyme oligomer by attenuating the conversion of the enzyme oligomers into inactive amyloid. Here we show that Hsp70 activates PLA2 in vitro, in a manner requiring ATP and Mg2+.

Secreted phospholipase A2 group X (sPLA(2)-X) is one of the most potent enzymes of the phospholipase A(2) lipolytic enzyme superfamily. Its high catalytic activity toward phosphatidylcholine (PC), the major phospholipid of cell membranes and low-density lipoproteins (LDL), has implicated sPLA(2)-X in chronic inflammatory conditions such as atherogenesis. We studied the role of sPLA(2)-X enzyme activity in vitro and in vivo, by generating sPLA(2)-X-overexpressing macrophages and transgenic macrophage-specific sPLA(2)-X mice. Our results show that sPLA(2)-X expression inhibits macrophage activation and inflammatory responses upon stimulation, characterized by reduced cell adhesion and nitric oxide production, a decrease in tumor necrosis factor (TNF), and an increase in interleukin (IL)-10. These effects were mediated by an increase in IL-6, and enhanced production of prostaglandin E-2 (PGE(2)) and 15-deoxy-Delta 12,14-prostaglandin J(2) (PGJ(2)). Moreover, we found that overexpression of active sPLA(2)-X in macrophages strongly increases foam cell formation upon incubation with native LDL but also oxidized LDL (oxLDL), which is mediated by enhanced expression of scavenger receptor CD36. Transgenic sPLA(2)-X mice died neonatally because of severe lung pathology characterized by interstitial pneumonia with massive granulocyte and surfactant-laden macrophage infiltration. We conclude that overexpression of the active sPLA(2)-X enzyme results in enhanced foam cell formation but reduced activation and inflammatory responses in macrophages in vitro. Interestingly, enhanced sPLA(2)-X activity in macrophages in vivo leads to fatal pulmonary defects, suggesting a crucial role for sPLA(2)-X in inflammatory lung disease.

Objective. Phospholipases A(2) (PLA(2)) comprise a family of enzymes probably implicated in the development of acute respiratory distress syndrome (ARDS). The aim was to investigate PLA2 activities and characteristics in bronchoalveolar lavage (BAL) fluid, BAL cells, and plasma from patients with ARDS by a fluorometric method. Design: Prospective, controlled study. Setting: Fourteen-bed polyvalent intensive care unit in a university hospital. Patients: A total of 31 mechanically ventilated patients, 20 with and 11 without ARDS, were studied. Intervention. BAL was performed by fiberoptic bronchoscopy in mechanically ventilated patients with a controlled mechanical ventilation mode. Measurements: PLA(2) and platelet-activating-factor acetylhydrolase were determined in BAL fluid, cells, and plasma. For the classification of PLA(2)-specific inhibitors, Western blot analysis and their biochemical characteristics were used. Results: In ARDS patients, increased PLA(2) levels were detected in BAL fluid, BAL cells, and plasma compared with the control patients. PLA(2) in BAL fluid was mainly type IIA secretory and cytosolic types. In plasma, type IIA secretory and cytosolic and a Ca2+-independent PLA(2) were found. In BAL cells, a cytosolic form, probably a Ca2+-independent intracellular form, and a low activity of type IIA secretory PLA(2) was also observed. Total PLA(2) activity correlated inversely with Pao(2)/Fio(2) ratio and positively with the mortality rate. Patients with direct ARDS exhibited higher PLA(2) activity compared with patients with indirect ARDS. Platelet-activating-factor acetylhydrolase activity was higher in BAL fluid and plasma, but it was lower in BAL cells. Conclusion. Ca2+-dependent secretory, cytosolic, and Ca2+-independent forms of PLA(2) and platelet-activating-factor acetylhydrolase could play important roles in the development or down-regulation of inflammation in ARDS, respectively. Crit Care Med

Modulation of cytosolic phospholipase A2 (PLA2) expression levels and production of its metabolites have been reported in several tumor types, indicating involvement of arachidonic acid and its derivatives in tumorigenesis. Following our demonstration that the PLA2 group IV isoform α (PLA2IVα) controls TSH-independent growth of normal thyroid (PCCl3) cells, we have investigated the mitogenic role of PLA2IVα in rat thyroid cells transformed by the RET/PTC oncogenes (PC-PTC cells). We now report that PLA2IVα acts downstream of the RET/PTC oncogenes in a novel pathway controlling RET-dependent cell proliferation. In addition, we show that PLA2IVα is in its phosphorylated/active form not only in RET/PTC-transformed cells and in cells derived from human papillary carcinomas but also in lysates from tumor tissues, thus relating constitutive activation of PLA2IVα to RET/PTC-dependent tumorigenesis. Moreover, p38 stress-activated protein kinase is the downstream effector of RET/PTC that is responsible for PLA2IVα phosphorylation and activity. In summary, our data elucidate a novel mechanism in the control of thyroid tumor cell growth that is induced by the RET/PTC oncogenes and which is distinguishable from that of other oncogenes, such as BRAF. This mechanism is mediated by PLA2IVα and should be amenable to targeted pharmacologic intervention. [Cancer Res 2007;67(24):11769–78]

Snake presynaptic phospholipase A2 neurotoxins (SPANs) paralyze the neuromuscular junction (NMJ). Upon intoxication, the NMJ enlarges and has a reduced content of synaptic vesicles, and primary neuronal cultures show synaptic swelling with surface exposure of the lumenal domain of the synaptic vesicle protein synaptotagmin I. Concomitantly, these neurotoxins induce exocytosis of neurotransmitters. We found that an equimolar mixture of lysophospholipids and fatty acids closely mimics all of the biological effects of SPANs. These results draw attention to the possible role of local lipid changes in synaptic vesicle release and provide new tools for the study of exocytosis.

Recent studies suggest that antisense phosphorothioate oligonucleotides (APO) are useful tools not only to impair gene expression, but also to modify the splicing of pre-mRNA, as the classical view that they act by suppressing the translation of mature mRNA has been challenged by several examples showing their nuclear site of action. In this work we show that an APO directed against cytosolic phospholipase A2 (cPLA2) mRNA localises in the nucleus and interacts with a specific nuclear protein.