Search

Background: In mouse models of atherosclerosis, knockout of the PLA2G2A gene has been shown to reduce the volume of atherosclerotic plaques. Clinical trials have demonstrated the potential of using the sPLA2 inhibitor Varespladib in combination with statins to reduce lipid levels. However, this approach has not yielded the expected results in reducing the risk of cardiovascular events. Therefore, it is necessary to further investigate the mechanisms of PLA2G2A., Methods: Single-cell transcriptome data from two sets of carotid plaques, combined with clinical patient information. were used to describe the expression characteristics of PLA2G2A in carotid plaques at different stages. In order to explore the mechanisms of PLA2G2A, we conducted enrichment analysis, cell-cell communication analysis and single-cell regulatory network inference and clustering analyses. We validated the above findings at the cellular level., Results: Our findings indicate that PLA2G2A is primarily expressed in vascular fibroblasts and shows significant cell interactions with macrophages in the early-stage, especially in complement and inflammation-related pathways. We also found that serum sPLA2 levels have stronger diagnostic value in patients with mild carotid artery stenosis. Subsequent comparisons of single-cell transcriptomic data from early and late-stage carotid artery plaques corroborated these findings and predicted transcription factors that might regulate the progression of early carotid atherosclerosis (CA) and the expression of PLA2G2A., Conclusions: Our study discovered and validated that PLA2G2A is highly expressed by vascular fibroblasts and promotes plaque progression through the activation of macrophage complement and coagulation cascade pathways in the early-stage of CA., (© 2024. The Author(s).)

We previously demonstrated a positive relation of secretory phospholipase A2 group IIA (sPLA2-IIA) with circulating high-density lipoprotein cholesterol (HDL-C) in patients with coronary artery disease, and sPLA2-IIA increased cholesterol efflux in THP-1 cells through peroxisome proliferator-activated receptor-γ (PPAR-γ)/liver X receptor α/ATP-binding cassette transporter A1 (ABCA1) signaling pathway. The aim of the present study was to examine the role of sPLA2-IIA over-expression on lipid profile in a transgenic mouse model. Fifteen apoE -/- and C57BL/7 female mice received bone marrow transplantation from transgenic SPLA2-IIA mice, and treated with specific PPAR-γ inhibitor GW9662. High fat diet was given after one week of bone marrow transplantation, and animals were sacrificed after twelve weeks. Immunohistochemical staining showed over-expression of sPLA2-IIA protein in the lung and spleen. The circulating level of HDL-C, but not that of low-density lipoprotein cholesterol (LDL-C), total cholesterol, or total triglyceride, was increased by sPLA2-IIA over-expression, and was subsequently reversed by GW9662 treatment. Over-expression of sPLA2-IIA resulted in augmented expression of cholesterol transporter ABCA1 at mRNA level in the aortas, and at protein level in macrophages, co-localized with macrophage specific antigen CD68. GW9662 exerted potent inhibitory effects on sPLA2-IIA-induced ABCA1 expression. Conclusively, we demonstrated the effects of sPLA2-IIA on circulating HDL-C level and the expression of ABCA1, possibly through regulation of PPAR-γ signaling in transgenic mouse model, that is in concert with the conditions in patients with coronary artery disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Non-small cell lung cancer (NSCLC) stands prominent among the prevailing and formidable oncological entities. The immune and metabolic-related molecule Phospholipase A2, group IID (PLA2G2D) exerts promotional effects on tumor progression. However, its involvement in cancer angiogenesis remains elusive. Therefore, this investigation delved into the functional significance of PLA2G2D concerning angiogenesis in NSCLC. This study analyzed the expression and enriched pathways of PLA2G2D in NSCLC tissues through bioinformatics analysis, and measured the expression of PLA2G2D in NSCLC cells using qRT-PCR and western blot (WB). Subsequently, the viability and angiogenic potential of NSCLC cells were assessed employing CCK-8 and angiogenesis assays, respectively. The expression profile of angiogenic factors was analyzed through WB. Finally, the expression of glycolysis pathway-related genes, extracellular acidification rate and oxygen consumption rate, and the levels of pyruvate, lactate, citrate, and malate were analyzed in NSCLC cells using qRT-PCR, Seahorse XF 96, and related kits. Bioinformatics analysis revealed the upregulation of PLA2G2D in NSCLC tissues and its association with VEGF and glycolysis signaling pathways. Molecular and cellular experiments demonstrated that upregulated PLA2G2D promoted the viability, angiogenic ability, and glycolysis pathway of NSCLC cells. Rescue assays revealed that the effects of high expression of PLA2G2D on the viability, angiogenic ability, and glycolysis of NSCLC cells were weakened after the addition of the glycolysis inhibitor 2-DG. In summary, PLA2G2D plays a key role in NSCLC angiogenesis through aerobic glycolysis, displaying great potential as a target for anti-angiogenesis therapy.

Delirium is a common psychiatric complication of chronic obstructive pulmonary disease (COPD). The relief of delirium is considered one of the beneficial ways to treat COPD. However, there are currently no specific drugs that alleviate delirium in COPD patients. Our research aimed to elucidate the specific mechanisms underlying delirium in COPD mice, while also seeking more effective therapeutic targets. In our study, bioinformatics analysis and qRT PCR were used to identify key factors in the development of delirium in COPD animal models. Open field and elevated plus maze tests were used to detect delirium in mice. Tunel staining and HE staining were used to analyze the apoptosis of mouse hippocampus cells. EdU and CCK-8 experiments were used to analyze PC-12 cells vitality and proliferation. JASPAR online database, dual luciferase reporting experiments, ChIP experiments, and IF staining were used to analyze the interaction between RXRA and PLA2G2A. RXRA is highly expressed in the brain tissue of COPD mice with delirium symptoms. The downregulation of RXRA inhibits the delirium state in COPD mice. This is mainly due to the reduction of endoplasmic reticulum stress and cell apoptosis by inhibiting the expression of RXRA. In addition, we also confirmed that RXRA is a transcription factor of PLA2G2A. RXRA has an inhibitory effect on the expression of PLA2G2A. In vitro experiments have confirmed that inhibition of the RXRA/PLA2G2A axis reduces cell apoptosis, thereby alleviating the occurrence and development of delirium in COPD mice. Inhibition of the RXRA/PLA2G2A axis reduces endoplasmic reticulum stress and cell apoptosis. This process alleviates the development of delirium in COPD mice.

High levels of human group IIA secreted phospholipase A 2 (hGIIA) have been associated with various inflammatory disease conditions. We have recently shown that hGIIA activity and concentration are increased in the plasma of patients with hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) and negatively correlate with C1-INH plasma activity. In this study, we analyzed whether the presence of both hGIIA and C1-INH impairs their respective function on immune cells. hGIIA, but not recombinant and plasma-derived C1-INH, stimulates the production of IL-6, CXCL8, and TNF-α from peripheral blood mononuclear cells (PBMCs). PBMC activation mediated by hGIIA is blocked by RO032107A, a specific hGIIA inhibitor. Interestingly, C1-INH inhibits the hGIIA-induced production of IL-6, TNF-α, and CXCL8, while it does not affect hGIIA enzymatic activity. On the other hand, hGIIA reduces the capacity of C1-INH at inhibiting C1-esterase activity. Spectroscopic and molecular docking studies suggest a possible interaction between hGIIA and C1-INH but further experiments are needed to confirm this hypothesis. Together, these results provide evidence for a new interplay between hGIIA and C1-INH, which may be important in the pathophysiology of hereditary angioedema., (© 2022. The Author(s).)

Oncogenic K-ras is often activated in pancreatic ductal adenocarcinoma (PDAC) due to frequent mutation (>90%), which drives multiple cellular processes, including alterations in lipid metabolism associated with a malignant phenotype. However, the role and mechanism of the altered lipid metabolism in K-ras-driven cancer remains poorly understood. In this study, using human pancreatic epithelial cells harboring inducible K-rasG12D (HPNE/K-rasG12D) and pancreatic cancer cell lines, we found that the expression of phospholipase A2 group IIA (PLA2G2A) was upregulated by oncogenic K-ras. The elevated expression of PLA2G2A was also observed in pancreatic cancer tissues and was correlated with poor survival of PDAC patients. Abrogation of PLA2G2A by siRNA or by pharmacological inhibition using tanshinone I significantly increased lipid peroxidation, reduced fatty acid synthase (FASN) expression, and impaired mitochondrial function manifested by a decrease in mitochondrial transmembrane potential and a reduction in ATP production, leading to the inhibition of cancer cell proliferation. Our study suggests that high expression of PLA2G2A induced by oncogenic K-ras promotes cancer cell survival, likely by reducing lipid peroxidation through its ability to facilitate the removal of polyunsaturated fatty acids from lipid membranes by enhancing the de novo fatty acid synthesis and energy metabolism to support cancer cell proliferation. As such, PLA2G2A might function as a downstream mediator of K-ras and could be a potential therapeutic target.

Newborns require early generation of effective innate immunity as a primary physiological mechanism for survival. The neonatal Lin28 + Let7 - developmental pathway allows increased generation of Th2-type cells and B1a (B-1 B) cells compared to adult cells and long-term maintenance of these initially generated innate cells. For initial B1a cell growth from the neonatal to adult stage, Th2-type IL-5 production from ILC2s and NKT2 cells is important to increase B1a cells. The Th17 increase is dependent on extracellular bacteria, and increased bacteria leads to lower Th2-type generation. Secreted group IIA-phospholipase A2 (sPLA2-IIA) from the Pla2g2a gene can bind to gram-positive bacteria and degrade bacterial membranes, controlling microbiota in the intestine. BALB/c mice are Pla2g2a + , and express high numbers of Th2-type cells and B1a cells. C57BL/6 mice are Pla2g2a-deficient and distinct from the SLAM family, and exhibit fewer NKT2 cells and fewer B1a cells from the neonatal to adult stage. We found that loss of Pla2g2a in the BALB/c background decreased IL-5 from Th2-type ILC2s and NKT2s but increased bacterial-reactive NKT17 cells and MAIT cells, and decreased the number of early-generated B1a cells and MZ B cells and the CD4/CD8 T cell ratio. Low IL-5 by decreased Th2-type cells in Pla2g2a loss led to low early-generated B1a cell growth from the neonatal to adult stage. In anti-thymocyte/Thy-1 autoreactive μκ transgenic (ATAμκ Tg) Pla2g2a + BALB/c background C.B17 mice generated NKT2 cells that continuously control CD1d + B1 B cells through old aging and lost CD1d in B1 B cells generating strong B1 ATA B cell leukemia/lymphoma. Pla2g2a-deficient ATAμκTg C57BL/6 mice suppressed the initial B1a cell increase, with low/negative spontaneous leukemia/lymphoma generation. These data confirmed that the presence of Pla2g2a to control bacteria is important to allow the neonatal to adult stage. Pla2g2a promotes innate Th2-type immunity lymphocytes to increase early generated B1a cells., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Besides promoting inflammation by mobilizing lipid mediators, group IIA secreted phospholipase A2 (sPLA2-IIA) prevents bacterial infection by degrading bacterial membranes. Here, we show that, despite the restricted intestinal expression of sPLA2-IIA in BALB/c mice, its genetic deletion leads to amelioration of cancer and exacerbation of psoriasis in distal skin. Intestinal expression of sPLA2-IIA is reduced after treatment with antibiotics or under germ-free conditions, suggesting its upregulation by gut microbiota. Metagenome, transcriptome, and metabolome analyses have revealed that sPLA2-IIA deficiency alters the gut microbiota, accompanied by notable changes in the intestinal expression of genes related to immunity and metabolism, as well as in the levels of various blood metabolites and fecal bacterial lipids, suggesting that sPLA2-IIA contributes to shaping of the gut microbiota. The skin phenotypes in Pla2g2a-/- mice are lost (a) when they are cohoused with littermate WT mice, resulting in the mixing of the microbiota between the genotypes, or (b) when they are housed in a more stringent pathogen-free facility, where Pla2g2a expression in WT mice is low and the gut microbial compositions in both genotypes are nearly identical. Thus, our results highlight a potentially new aspect of sPLA2-IIA as a modulator of gut microbiota, perturbation of which affects distal skin responses.

Secreted phospholipase A2-IIA (sPLA2-IIA) hydrolyzes phospholipids to liberate lysophospholipids and fatty acids. Given its poor activity toward eukaryotic cell membranes, its role in the generation of proinflammatory lipid mediators is unclear. Conversely, sPLA2-IIA efficiently hydrolyzes bacterial membranes. Here, we show that sPLA2-IIA affects the immune system by acting on the intestinal microbial flora. Using mice overexpressing transgene-driven human sPLA2-IIA, we found that the intestinal microbiota was critical for both induction of an immune phenotype and promotion of inflammatory arthritis. The expression of sPLA2-IIA led to alterations of the intestinal microbiota composition, but housing in a more stringent pathogen-free facility revealed that its expression could affect the immune system in the absence of changes to the composition of this flora. In contrast, untargeted lipidomic analysis focusing on bacteria-derived lipid mediators revealed that sPLA2-IIA could profoundly alter the fecal lipidome. The data suggest that a singular protein, sPLA2-IIA, produces systemic effects on the immune system through its activity on the microbiota and its lipidome.

Phospholipases A 2 (PLA 2 s) are found in almost every venomous snake family. In snakebites, some PLA 2 s can quickly cause local myonecrosis, which may lead to permanent sequelae if antivenom is administered belatedly. They hydrolyse phospholipids in membranes through a catalytic calcium ions-dependent mechanism. BthTX-II is a basic PLA 2 and the second major component in the venom of Bothrops jararacussu. Herein, using the software SEQUENCE SLIDER, which integrates crystallographic, mass spectrometry and genetic data, we characterized the primary, tertiary and quaternary structure of two BthTX-II variants (called a and b), which diverge in 7 residues. Crystallographic structure BthTX-IIa is in a Tense-state with its distorted calcium binding loop buried in the dimer interface, contrarily, the novel BthTX-IIb structure is a monomer in a Relax-state with a fatty acid in the hydrophobic channel. Structural data in solution reveals that both variants are monomeric in neutral physiological conditions and mostly dimeric in an acidic environment, being catalytic active in both situations. Therefore, we propose two myotoxic mechanisms for BthTX-II, a catalytic one associated with the monomeric assembly, whereas the other has a calcium independent activity related to its C-terminal region, adopting a dimeric conformation similar to PLA 2 -like proteins., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Phospholipase A 2 (PLA 2 ) enzymes were first recognized as an enzyme activity class in 1961. The secreted (sPLA 2 ) enzymes were the first of the five major classes of human PLA 2 s to be identified and now number nine catalytically-active structurally homologous proteins. The best-studied of these, group IIA sPLA 2 , has a clear role in the physiological response to infection and minor injury and acts as an amplifier of pathological inflammation. The enzyme has been a target for anti-inflammatory drug development in multiple disorders where chronic inflammation is a driver of pathology since its cloning in 1989. Despite intensive effort, no clinically approved medicines targeting the enzyme activity have yet been developed. This review catalogues the major discoveries in the human group IIA sPLA 2 field, focusing on features of enzyme function that may explain this lack of success and discusses future research that may assist in realizing the potential benefit of targeting this enzyme. Functionally-selective inhibitors together with isoform-selective inhibitors are necessary to limit the apparent toxicity of previous drugs. There is also a need to define the relevance of the catalytic function of hGIIA to human inflammatory pathology relative to its recently-discovered catalysis-independent function.

Introduction: Maternal metabolism undergoes dynamic changes during pregnancy. A deviation from this physiological metabolic status might be involved in the pathogenesis of pregnancy complications, such as recurrent pregnancy loss (RPL). Decidua is an important uterine tissue, which provides immunological protection as well as nutritional support to the developing embryo during early pregnancy. Previous studies have shown that aberrant metabolism of the decidua is related to the etiology of RPL. However, the metabolic profile of the decidua in RPL has not yet been fully elucidated., Methods: In the current study, decidual samples from RPL patients (n = 23) and normal pregnancy (NP) women (n = 30) were collected, and hydrophilic and hydrophobic metabolites were extracted and measured using a liquid chromatography electrospray ionization tandem mass spectrometry system. Besides, the mRNA expression of several critical enzymes involved in sphingolipid metabolism and glycerophospholipid metabolism was detected., Results: The OSC-PLS-DA scores plot illustrates that metabolic differences are present in the decidual tissue of RPL patients compared with that of NP women. Combining multivariate analysis with univariate statistical analysis, a total of 62 metabolites related to RPL were selected, including carnitine, glycerophospholipids, sphingomyelin (SM), ceramide, organic acids and their derivatives, and amino acid metabolomics. KEGG analysis showed that abnormalities in multiple metabolic pathways occurred in RPL decidua, including vitamin digestion and absorption, tryptophan metabolism, citrate cycle, arginine biosynthesis, glycerophospholipid metabolism, sphingolipid metabolism, and sphingolipid signaling pathway. Increased SM synthase and decreased Phospholipase A2 Group IIE mRNA levels were detected in RPL compared with NP group., Discussion: Disruption of decidual metabolism, especially glycerophospholipid metabolism and sphingolipid metabolism, might be involved in the occurrence of RPL., Competing Interests: Declaration of competing interests The authors declare no competing interests, (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background/aim: This study aimed to evaluate the prognostic significance of PLA2G2A expression in patients with locally advanced gastric cancer (GC)., Patients and Methods: PLA2G2A expression levels in cancerous tissue specimens and adjacent normal mucosa obtained from 134 patients with stage II/III GC who received adjuvant chemotherapy with S-1 after curative resection were measured using real-time quantitative polymerase chain reaction. Subsequently, the associations of PLA2G2A expression with clinicopathological features and survival were evaluated., Results: No association was observed between clinicopathological features and PLA2G2A expression levels. Overall survival was significantly longer in patients with high PLA2G2A expression levels (p=0.022). Multivariate analysis revealed that PLA2G2A expression was a significant, independent prognostic factor (hazard ratio=0.136; 95% confidence interval=0.0185-0.992; p=0.049)., Conclusion: PLA2G2A mRNA expression may serve as a useful prognostic marker in patients with locally advanced GC who receive curative surgery and adjuvant chemotherapy with S-1., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Secreted phospholipase A2s (sPLA2s) are calcium dependent enzymes involved in various biological events such as lipid metabolism and inflammation. We previously identified the second calcium (Ca2) binding site of human sPLA2 Group IIE (hGIIE) by structural study and suggested that Asn21 act as the switch of Ca2 binding to modulate the enzymatic activity, but the detailed Ca2 binding mechanism is still unclear. Combined with enzymatic assay, model analysis and calcium binding affinity data for mutated hGIIE proteins, we herein further demonstrate that the flexibly bound Ca2 is essential for the catalysis of hGIIE, unlike the stable binding of Ca2 in hGIIA that replenishes the calcium in the typical loop during the reaction. The atypical Ca2 binding feature of hGIIE will provide a better understanding on the catalytic mechanism of hGIIE., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Gastroesophageal reflux and Barrett's esophagus are significant risk factors for the development of esophageal adenocarcinoma. Group IIa secretory phospholipase A 2 (sPLA 2 ) catalyzes the production of various proinflammatory metabolites and plays a critical role in promoting reflux-induced inflammatory changes within the distal esophagus. We hypothesized that inhibition of sPLA 2 in human Barrett's cells would attenuate adhesion molecule expression via decreased activation of nuclear factor kappa B (NF-κB) and decrease cell proliferation, possibly mitigating the invasive potential of Barrett's esophagus., Materials and Methods: Normal human esophageal epithelial cells (HET1A) and Barrett's cells (CPB) were assayed for baseline sPLA 2 expression. CPB cells were treated with a specific inhibitor of sPLA 2 followed by tumor necrosis factor-α. Protein expression was evaluated using immunoblotting. Cell proliferation was assessed using an MTS cell proliferation assay kit. Statistical analysis was performed using the Student's t-test or analysis of variance, where appropriate., Results: CPB cells demonstrated higher baseline sPLA 2 expression than HET1A cells (P = 0.0005). Treatment with 30 μM sPLA 2 inhibitor significantly attenuated intercellular adhesion molecule-1 (P = 0.004) and vascular cell adhesion molecule-1 (P < 0.0001) expression as well as decreased NF-κB activation (P = 0.002). sPLA 2 inhibition decreased cell proliferation in a dose-dependent manner (P < 0.001 for 15, 20, and 30 μM doses)., Conclusions: sPLA 2 inhibition in human Barrett's cells decreases cellular adhesive properties and NF-κB activation as well as decreases cell proliferation, signifying downregulation of the inflammatory response and possible attenuation of cellular malignant potential. These findings identify sPLA 2 inhibition as a potential chemopreventive target for premalignant lesions of the esophagus., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system., Competing Interests: The authors have declared that no competing interests exist.

Obesity is believed to be associated with a dysregulated endocannabinoid system which may reflect enhanced inflammation. However, reports of this in human white adipose tissue (WAT) are limited and inconclusive. Marine long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) have anti-inflammatory actions and therefore may improve obesity-associated adipose tissue inflammation. Therefore, fatty acid (FA) concentrations, endocannabinoid concentrations, and gene expression were assessed in subcutaneous WAT (scWAT) biopsies from healthy normal weight individuals (BMI 18.5-25 kg/m2) and individuals living with metabolically healthy obesity (BMI 30-40 kg/m2) prior to and following a 12-week intervention with 3 g fish oil/day (1.1 g eicosapentaenoic acid (EPA) + 0.8 g DHA) or 3 g corn oil/day (placebo). WAT from individuals living with metabolically healthy obesity had higher n-6 PUFAs and EPA, higher concentrations of two endocannabinoids (anandamide (AEA) and eicosapentaenoyl ethanolamide (EPEA)), higher expression of phospholipase A2 Group IID (PLA2G2D) and phospholipase A2 Group IVA (PLA2G4A), and lower expression of CNR1. In response to fish oil intervention, WAT EPA increased to a similar extent in both BMI groups, and WAT DHA increased by a greater extent in normal weight individuals. WAT EPEA and docosahexaenoyl ethanolamide (DHEA) increased in normal weight individuals only and WAT 2-arachidonyl glycerol (2-AG) decreased in individuals living with metabolically healthy obesity only. Altered WAT fatty acid, endocannabinoid, and gene expression profiles in metabolically healthy obesity at baseline may be linked. WAT incorporates n-3 PUFAs when their intake is increased which affects the endocannabinoid system; however, effects appear greater in normal weight individuals than in those living with metabolically healthy obesity., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Arachidonic acid (AA) is a polyunsaturated fatty acid present at high concentrations in the ovarian cancer (OC) microenvironment and associated with a poor clinical outcome. In the present study, we have unraveled a potential link between AA and macrophage functions. Methods: AA-triggered signal transduction was studied in primary monocyte-derived macrophages (MDMs) by phosphoproteomics, transcriptional profiling, measurement of intracellular Ca 2+ accumulation and reactive oxygen species production in conjunction with bioinformatic analyses. Functional effects were investigated by actin filament staining, quantification of macropinocytosis and analysis of extracellular vesicle release. Results: We identified the ASK1 - p38δ/α (MAPK13/14) axis as a central constituent of signal transduction pathways triggered by non-metabolized AA. This pathway was induced by the Ca 2+ -triggered activation of calmodulin kinase II, and to a minor extent by ROS generation in a subset of donors. Activated p38 in turn was linked to a transcriptional stress response associated with a poor relapse-free survival. Consistent with the phosphorylation of the p38 substrate HSP27 and the (de)phosphorylation of multiple regulators of Rho family GTPases, AA impaired actin filament organization and inhibited actin-driven macropinocytosis. AA also affected the phosphorylation of proteins regulating vesicle biogenesis, and consistently, AA enhanced the release of tetraspanin-containing exosome-like vesicles. Finally, we identified phospholipase A 2 group 2A (PLA2G2A) as the clinically most relevant enzyme producing extracellular AA, providing further potentially theranostic options. Conclusion: Our results suggest that AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages. Besides critical AA-regulated signal transduction proteins identified in the present study, PLA2G2A might represent a potential prognostic tool and therapeutic target to interfere with OC progression., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

It is well known that the prevalence of asthma is higher in athletes, including Olympic athletes, than in the general population. In this study, we analyzed the mechanism of exercise-induced bronchoconstriction by using animal models of athlete asthma. Mice were made to exercise on a treadmill for a total duration of 1 week, 3 weeks, or 5 weeks. We analyzed airway responsiveness, BAL fluid, lung homogenates, and tissue histology for each period. In mice that were treated (i.e., the treatment model), treatments were administered from the fourth to the fifth week. We also collected induced sputum from human athletes with asthma and analyzed the supernatants. Airway responsiveness to methacholine was enhanced with repeated exercise stimulation, although the cell composition in BAL fluid did not change. Exercise induced hypertrophy of airway smooth muscle and subepithelial collagen deposition. Cysteinyl-leukotriene (Cys-LT) levels were significantly increased with exercise duration. Montelukast treatment significantly reduced airway hyperresponsiveness (AHR) and airway remodeling. Expression of PLA 2 G4 (phospholipase A 2 group IV) and leukotriene C 4 synthase in the airway epithelium was upregulated in the exercise model, and inhibition of PLA 2 ameliorated AHR and airway remodeling, with associated lower levels of Cys-LTs. The levels of Cys-LTs in sputum from athletes did not differ between those with and without sputum eosinophilia. These data suggest that AHR and airway remodeling were caused by repeated and strenuous exercise. Cys-LTs from the airway epithelium, but not inflammatory cells, may play an important role in this mouse model.

Polyunsaturated fatty acids (PUFAs) confer health benefits by preventing inflammation and obesity and by increasing thermogenesis in brown and beige adipocytes. As well as being supplied exogenously as nutrients, PUFAs are largely stored in membrane glycerophospholipids and released by phospholipase A 2 s (PLA 2 s). However, the molecular identity of the PLA 2 subtype(s) that supplies endogenous PUFAs for metabolic homeostasis remains unclear. Here we show that PLA2G2D, a secreted PLA 2 isoform, is constitutively expressed in M2-type macrophages in white adipose tissue (WAT) and shows a reciprocal correlation with obesity. Studies using global and macrophage-specific Pla2g2d-deficient mice reveal that PLA2G2D increases energy expenditure and thermogenesis by facilitating adipocyte browning, thereby ameliorating diet-induced obesity, insulin resistance, and WAT inflammation. Mechanistically, PLA2G2D constitutively supplies a pool of PUFAs, ω3 in particular, in WAT. Thus, our present findings underscore the contribution of the macrophage-driven PLA2G2D-ω3 PUFA axis to metabolic health., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The intestinal epithelial monolayer forms a mucosal barrier between the gut microbes and the host tissue. The mucosal barrier is composed of mucins and antimicrobial peptides and proteins (AMPs). Several animal studies have reported that Paneth cells, which occupy the base of intestinal crypts, play an important role in the intestinal innate immunity by producing AMPs, such as lysozyme, Reg3 lectins, α-defensins, and group IIA secretory phospholipase A2 (GIIA sPLA2). The house musk shrew (Suncus murinus) has only a few intestinal commensal bacteria and is reported to lack Paneth cells in the intestine. Although the expression of lysozyme was reported in the suncus intestine, the expression of other AMPs has not yet been reported. Therefore, the current study was focused on GIIA sPLA2 expression in Suncus murinus. GIIA sPLA2 mRNA was found to be most abundant in the spleen and also highly expressed in the intestine. Cells expressing GIIA sPLA2 mRNA were distributed not only in the crypt, but also in the villi. In addition, intragastric injection of lipopolysaccharide increased GIIA sPLA2 expression in the small intestine of suncus. These results suggest that suncus may host unique AMP-secreting cells in the intestine., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Tissue stem cells (SCs) and cancer cells proliferation is regulated by many common signalling mechanisms. These mechanisms temporally balance proliferation and differentiation events during normal tissue homeostasis and repair. However, the effect of these aberrant signalling mechanisms on the ultimate fate of SCs and cancer cells remains obscure., Methods: To evaluate the functional effects of Secretory Phospholipase A 2 -IIA (sPLA 2 -IIA) induced abnormal signalling on normal SCs and cancer cells, we have used K14-sPLA 2 -IIA transgenic mice hair follicle stem cells (HFSCs), DMBA/TPA induced mouse skin tumour tissues, human oral squamous cell carcinoma (OSCC) and skin squamous cell carcinoma (SCC) derived cell lines., Findings: Our study demonstrates that sPLA 2 -IIA induces rapid proliferation of HFSCs, thereby altering the proliferation dynamics leading to a complete loss of the slow cycling H2BGFP positive HFSCs. Interestingly, in vivo reversion study by JNK inhibition exhibited a significant delay in post depilation hair growth, confirming that sPLA 2 -IIA promotes HFSCs proliferation through JNK/c-Jun signalling. In a different cellular context, we showed increased expression of sPLA 2 -IIA in human OSCC and mouse skin cancer tissues. Importantly, a xenograft of sPLA 2 -IIA knockdown cells of OSCC and SCC cell lines showed a concomitant reduction of tumour volume in NOD-SCID mice and decreased JNK/c-Jun signalling., Interpretation: This study unravels how an increased proliferation induced by a common proliferation inducer (sPLA 2 -IIA) alters the fate of normal SCs and cancer cells distinctively through common JNK/c-Jun signalling. Thus, sPLA 2 -IIA can be a potential target for various diseases including cancer. FUND: This work was partly supported by the Indian Council of Medical Research (ICMR-3097) and ACTREC (42) grants., (Copyright © 2019. Published by Elsevier B.V.)

Phospholipase A 2 s (PLA 2 ) play a key role in generation of eicosanoids. Cytosolic PLA 2 α (cPLA 2 α) is constitutively expressed in most cells, whereas IIA secreted PLA 2 (sPLA 2 -IIA) is induced during inflammation and is present at high levels in the synovial fluid of rheumatoid arthritis patients. In mice, both cPLA 2 α and sPLA 2 -IIA have been implicated in autoimmune arthritis; however, the respective contribution of these two enzymes to the pathogenesis and production of eicosanoids is unknown. We evaluated the respective role of cPLA 2 α and sPLA 2 -IIA with regard to arthritis and eicosanoid profile in an in vivo model of arthritis. While arthritis was most severe in mice expressing both enzymes, it was abolished when both cPLA 2 α and sPLA 2 -IIA were lacking. cPLA 2 α played a dominant role in the severity of arthritis, although sPLA 2 -IIA sufficed to significantly contribute to the disease. Several eicosanoids were modulated during the course of arthritis and numerous species involved sPLA 2 -IIA expression. This study confirms the critical role of PLA 2 s in arthritis and unveils the distinct contribution of cPLA 2 α and sPLA 2 -IIA to the eicosanoid profile in arthritis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

GIIA secreted phospholipase A 2 (GIIA sPLA 2 ) is a potent target for drug discovery. To distinguish the activity level of the inhibitors of GIIA sPLA 2 , we built 24 classification models by three machine learning algorithms including support vector machine (SVM), decision tree (DT), and random forest (RF) based on 452 compounds. The molecules were represented by CORINA descriptors, MACCS fingerprints, and ECFP4 fingerprints, respectively. The dataset was split into a training set containing 312 compounds and a test set containing 140 compounds by Kohonen's self-organizing map (SOM) strategy and a random strategy. A recursive feature elimination (RFE) method and an information gain (IG) method were used in the selection of molecular descriptors. Three favorable performing models were obtained. They were built by SVM algorithm with CORINA descriptors (Models 1A and 2A) and ECFP4 fingerprints (Model 10A). In the prediction of test set of Model 10A, the accuracy reached 90.71%, and the Matthews correlation coefficient (MCC) values reached 0.82. In addition, the 452 inhibitors were clustered into eight subsets by K-Means algorithm for analyzing their structural features. It was found that highly active inhibitors mainly contained indole scaffold or indolizine scaffold and four side chains., (© 2018 John Wiley & Sons A/S.)

sPLA 2 is released under inflammatory conditions from neutrophils, basophils and T-cells. They cleave the cellular phospholipids leading to the release of arachidonic acid and there by provide intermediates for biosynthesis of inflammatory mediators. The focus of this study is on the interaction of hesperidin, a natural flavonoid with Group IB, IIA, and V and X isozymes of sPLA 2 . Affinity of hesperidin towards PLA 2 isozymes was analyzed through enzymatic studies and molecular modeling. The experiments showed that hesperidin competitively inhibited PLA 2 with IC 50 of 5.1 µM. Molecular modeling studies revealed the association of hesperidin with the docking scores -6.90, -9.53, -5.63 and -8.29 kcal for isozymes Group IB, IIA, V and X of PLA 2 respectively. Their binding energy values were calculated as -20.25, -21.63, -21.66 and -33.43 kcal for the Group IB, IIA, V and X respectively. Structural model for Group V was made by homology modeling since no structural coordinates were available. Molecular dynamics studies were carried out to evaluate the structural stability of protein ligand complex. The analyses showed that hesperidin blocked the entry of the substrate to the active site of PLA 2 and it was indifferent to the differences of the isozymes. Hence, hesperidin might serve as lead for designing highly specific anti-inflammatory drugs directed to the PLA 2 isozyme specific to various diseases, with IC 50 value of therapeutic significance.

Secretory phospholipase A 2 group IIA (PLA2G2A) is a phospholipase which has a role in inflammation, atherogenesis, and host defense. Previously, we found that PLA2G2A protects mice on high-fat diets from weight gain and insulin resistance. Here, we examined the regulation of PLA2G2A and the metabolic changes that occur in response to variations in thyroid status. In particular, the impact of PLA2G2A on the brown adipose tissue (BAT) thermogenic gene expression was explored. We induced hypothyroidism in C57BL/6 and PLA2G2A-overexpressing (IIA+) mice over a 10 wk period or treated them with thyroid hormone (T 3 ) for 5 wk. There were no significant changes in PLA2G2A abundance in response to thyroid status. The energy expenditure of hypothyroid IIA+ mice did not increase; however, the energy expenditure, substrate utilization, insulin sensitivity, and glucose tolerance were all elevated in the IIA+ mice given T 3 . Moreover, white adipocytes from IIA+ mice were much more prone to "beiging," including increased expression of brown adipose thermogenic markers such as uncoupling protein 1 (UCP1), PR domain containing 16, and early B cell factor 2. Finally, the BAT of IIA+ mice had increased UCP1 and other proteins indicative of mitochondrial uncoupling and nonshivering adaptive thermogenesis. These data reveal a novel role for PLA2G2A on adipose tissue thermogenesis depending on thyroid status.-Kuefner, M. S., Deng, X., Stephenson, E. J., Pham, K., Park, E. A. Secretory phospholipase A 2 group IIA enhances the metabolic rate and increases glucose utilization in response to thyroid hormone.

Borage oil (BO) reverses a disrupted epidermal lipid barrier and hyperproliferation in essential fatty acid deficiency (EFAD). However, little is known about its effect on skin pH, which is maintained by epidermal lactate, free fatty acids (FFAs), and free amino acids (FAAs) which is generated by lactate dehydrogenase (LDH), secreted phospholipase A2 (sPLA2), or filaggrin degradation with peptidylarginine deiminase-3 (PADI3). We hypothesized that BO restores skin pH by regulating epidermal lactate, FFA metabolism, or FAA metabolism in EFAD. To test this hypothesis, EFAD was induced in guinea pigs by a hydrogenated coconut oil (HCO) diet for 8 weeks, followed by 2 weeks of a BO diet (group HCO + BO). As controls, groups HCO and BO were fed HCO or BO diets for 10 weeks. In group HCO + BO, skin pH, which was less acidic in group HCO, was restored; and epidermal lactate and total FFAs, including palmitate, stearate, linoleate, arachidate, behenate, and lignocerate, were higher than in group HCO. LDH and sPLA2 (mainly the PLA2G2F isoform) activities and protein expressions were similar between groups HCO + BO and BO. Epidermal acidic FAAs, as well as filaggrin and PADI3 protein and mRNA expressions were higher in group HCO + BO than in group HCO. Oleate, total FAAs including other FAAs, and LDH and sPLA2 mRNA expressions were not altered between groups HCO and HCO + BO. Basic FAAs were not altered among groups. Dietary BO restored acidic skin pH and increased epidermal levels of lactate, most FFAs, and acidic FAAs by up-regulating LDH, sPLA2, filaggrin, and PADI3 activities as well as protein or mRNA expressions in EFAD., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Inflammation-mediated disorders are on the rise and hence, there is an urgent need for the design and synthesis of new anti-inflammatory drugs with higher affinity and specificity for their potential targets. The current study presents an effective and new protocol for the synthesis of thienyl-pyrazoles through 3 + 2 annulations using a recyclable heterogeneous catalyst Amberlyst-15. Chalcones 3(a-g) prepared from 3-methylthiophene-2-carbaldehyde and acetophenones by Claisen-Schmidt approach reacted with semicarbazide hydrochloride 4 in the presence of Amberlyst-15 in acetonitrile at room temperature producing thienyl-pyrazole carboxamides 5(a-h) in good yields. Alternatively, the compounds 5(a-h) were prepared by conventional method using acetic acid (30%) medium. Structures of synthesized new pyrazoles were confirmed by spectral and crystallographic studies. All the new compounds were evaluated for their in vitro inhibition of Phospholipase A2 from Vipera russelli and preliminary studies revealed that, amongst the designed series, compounds 5b, 5c and 5h showed promising inhibition. Further, the compounds exhibited linear mixed-type inhibition behavior for the sPLA 2 enzyme indicating that they bind to an allosteric site distinct from either the calcium or substrate binding site on the enzyme. These kinetic conclusions were further validated by macromolecular rigid-body docking whereby compounds 5c and 5h showed binding to distinct pockets on the protein. These findings present a promising series of lead molecules that can serve as prototypes for the treatment of inflammatory related disorders., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Phospholipases A 2 are a major component of snake venoms. Some of them cause severe muscle necrosis through an unknown mechanism. Phospholipid hydrolysis is a possible explanation of their toxic action, but catalytic and toxic properties of PLA 2 s are not directly connected. In addition, viperid venoms contain PLA 2 -like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. In this work, the PLA 2 -like Bothrops asper myotoxin-II, conjugated with the fluorophore TAMRA, was found to be internalized in mouse myotubes, and in RAW264.7 cells. Through experiments of protein fishing and mass spectrometry analysis, using biotinylated Mt-II as bait, we found fifteen proteins interacting with the toxin and among them nucleolin, a nucleolar protein present also on cell surface. By means of confocal microscopy, Mt-II and nucleolin were shown to colocalise, at 4 °C, on cell membrane where they form Congo-red sensitive assemblies, while at 37 °C, 20 minutes after the intoxication, they colocalise in intracellular spots going from plasmatic membrane to paranuclear and nuclear area. Finally, nucleolin antagonists were found to inhibit the Mt-II internalization and toxic activity and were used to identify the nucleolin regions involved in the interaction with the toxin.

P. gingivalis (Pg) is an oral pathogen with the ability to induce oral dysbiosis and periodontal disease. Nevertheless, the mechanisms by which mucosal responses to the oral microbiota in the presence of specific pathogens such as Pg could abrogate the host-microbe symbiotic relationship leading to periodontitis remain unclear. Herein, we identified the Notch-1/PLA 2 -IIA axis as a new molecular pathway through which Pg could be specifically modulating oral epithelial antimicrobial and inflammatory responses. Pg activated Notch-1, and inhibition or silencing of Notch-1 completely abrogated Pg-induced PLA 2 -IIA in oral epithelial cells (OECs). Activation of Notch-1 and PLA 2 -IIA production were associated with Pg-produced gingipains. Other oral Gram-positive and Gram-negative species failed to induce similar responses. Pg enhanced OEC antimicrobial activity through PLA 2 -IIA. Increased Notch-1 activation correlated with higher PLA 2 -IIA gingival expression and changes in the abundance of specific oral bacteria phyla during periodontal disease. Oral bacterial species exhibited differential antimicrobial susceptibility to PLA 2 -IIA. These findings support previous evidence suggesting an important role for epithelial Notch-1 activation and PLA 2 -IIA production during health and disease at mucosal surfaces, and provide new mechanistic information concerning the regulation of epithelial antimicrobial and pro-inflammatory responses modulated by oral pathogenic bacteria associated with periodontal disease.

Tuberculosis is a disease caused by Mycobacterium tuberculosis (Mtb). Innate immunity is the first line of defense against Mtb and malfunctions in any of its components are associated with the susceptibility to the disease. Epithelial products such as host defense peptides (HDPs) are the first molecules produced to counteract the infection. Although a wide variety of HDPs are produced by epithelial cells only a few of them have been studied during Mtb infection. Here, we assessed the expression and production of the HDPs psoriasin, secreted phospholipases A2 (sPLA2-IIA) and Ribonuclease (RNase) 7 in airway epithelial cells (NCI-H292), type II pneumocytes (A549 cells) and monocyte-derived macrophages from human peripheral blood mononuclear cells and from the human cell line THP1 after Mtb in vitro infection. Results show that psoriasin and sPLA2-IIA were not induced by Mtb in any of the evaluated cells, while RNase 7 was overexpressed in infected airway epithelial cells. Intracellular analysis by flow cytometry demonstrated that the highest levels of RNase 7 were observed 6 h post-infection and the induction was dependent on direct interaction between airway epithelial cells and Mtb. In addition, analysis by electron microscopy showed that RNase 7 was capable of attaching to the cell wall of intracellular mycobacteria. Our studies suggest that the induction of RNase 7 in response to Mtb could have a role in anti-mycobacterial immunity, which needs to be studied as an innate immune mechanism.

Secretory phospholipase A 2 Group-IIA (sPLA 2 -IIA) is involved in lipid catabolism and growth promoting activity. sPLA 2 -IIA is deregulated in many pathological conditions including various cancers. Here, we have studied the role of sPLA 2 -IIA in the development of cyclic alopecia and wound healing response in relation to complete loss of hair follicle stem cells (HFSCs). Our data showed that overexpression of sPLA 2 -IIA in homozygous mice results in hyperproliferation and terminal epidermal differentiation followed by hair follicle cycle being halted at anagen like stage. In addition, sPLA 2 -IIA induced hyperproliferation leads to compl pathological conditions including various cancers. Here ete exhaustion of hair follicle stem cell pool at PD28 (Postnatal day). Importantly, sPLA 2 -IIA overexpression affects the hair shaft differentiation leading to development of cyclic alopecia. Molecular investigation study showed aberrant expression of Sox21, Msx2 and signalling modulators necessary for proper differentiation of inner root sheath (IRS) and hair shaft formation. Further, full-thickness skin wounding on dorsal skin of K14-sPLA 2 -IIA homozygous mice displayed impaired initial healing response. Our results showed the involvement of sPLA 2 -IIA in regulation of matrix cells differentiation, hair shaft formation and complete loss of HFSCs mediated impaired wound healing response. These novel functions of sPLA 2 -IIA may have clinical implications in alopecia, cancer development and ageing.

Secretory phospholipase A 2 group IIA (PLA2G2A) is a member of a family of secretory phospholipases that have been implicated in inflammation, atherogenesis, and antibacterial actions. Here, we evaluated the role of PLA2G2A in the metabolic response to a high fat diet. C57BL/6 (BL/6) mice do not express PLA2g2a due to a frameshift mutation. We fed BL/6 mice expressing the human PLA2G2A gene (IIA+ mice) a fat diet and assessed the physiologic response. After 10 weeks on the high fat diet, the BL/6 mice were obese, but the IIA+ mice did not gain weight or accumulate lipid. The lean mass in chow- and high fat-fed IIA+ mice was constant and similar to the BL/6 mice on a chow diet. Surprisingly, the IIA+ mice had an elevated metabolic rate, which was not due to differences in physical activity. The IIA+ mice were more insulin sensitive and glucose tolerant than the BL/6 mice, even when the IIA+ mice were provided the high fat diet. The IIA+ mice had increased expression of uncoupling protein 1 (UCP1), sirtuin 1 (SIRT1), and PPARγ coactivator 1α (PGC-1α) in brown adipose tissue (BAT), suggesting that PLA2G2A activates mitochondrial uncoupling in BAT. Our data indicate that PLA2G2A has a previously undiscovered impact on insulin sensitivity and metabolism., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

A series of new of furan derivatised [1,4] benzothiazepine analogues were synthesized starting from 1-(furan-2-yl)ethanone. 1-(Furan-2-yl)ethanone was converted into chalcones by its reaction with various aromatic aldehydes, then were reacted with 2-aminobenzenethiol in acidic conditions to obtain the title compounds in good yields. The synthesized new compounds were characterized by 1 H NMR, 13 C NMR, Mass spectral studies and elemental analyses. All the new compounds were evaluated for their in vitro VRV-PL-8a and H + /K + ATPase inhibitor properties. Preliminary studies revealed that, some molecules amongst the designed series showed promising VRV-PL-8a and H + /K + ATPase inhibitor properties. Further, rigid body docking studies were performed to understand possible docking sites of the molecules on the target proteins and the mode of binding. This finding presents a promising series of lead molecules that can serve as prototypes for the treatment of inflammatory related disorder that can mitigate the ulcer inducing side effect shown by other NSAIDs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Arterial blood pressure-induced shear stress causes endothelial cell apoptosis and inflammation in vein grafts after coronary artery bypass grafting. As the inflammatory protein type IIA secretory phospholipase A 2 (sPLA 2 -IIA) has been shown to progress atherosclerosis, we hypothesized a role for sPLA 2 -IIA herein., Methods: The effects of PX-18, an inhibitor of both sPLA 2 -IIA and apoptosis, on residual endothelium and the presence of sPLA 2 -IIA were studied in human saphenous vein segments (n = 6) perfused at arterial blood pressure with autologous blood for 6 hrs., Results: The presence of PX-18 in the perfusion blood induced a significant 20% reduction in endothelial cell loss compared to veins perfused without PX18, coinciding with significantly reduced sPLA 2 -IIA levels in the media of the vein graft wall. In addition, PX-18 significantly attenuated caspase-3 activation in human umbilical vein endothelial cells subjected to shear stress via mechanical stretch independent of sPLA 2 -IIA., Conclusions: In conclusion, PX-18 protects saphenous vein endothelial cells from arterial blood pressure-induced death, possibly also independent of sPLA 2 -IIA inhibition., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Crotoxin B (CB) is a catalytically active group IIA sPLA 2 from Crotalus durissus terrificus snake venom. In contrast to most GIIA sPLA 2 s, CB exhibits anti-inflammatory effects, including the ability to inhibit leukocyte functions. Lipid droplets (LDs) are lipid-rich organelles associated with inflammation and recognized as a site for the synthesis of inflammatory lipid mediators. Here, the ability of CB to induce formation of LDs and the mechanisms involved in this effect were investigated in isolated macrophages. The profile of CB-induced 15-d-PGJ 2 (15-Deoxy-Delta-12,14-prostaglandin J 2 ) production and involvement of LDs in 15-d-PGJ 2 biosynthesis were also investigated. Stimulation of murine macrophages with CB induced increased number of LDs and release of 15-d-PGJ 2 . LDs induced by CB were associated to PLIN2 recruitment and expression and required activation of PKC, PI3K, MEK1/2, JNK, iPLA 2 and PLD. Both 15-d-PGJ 2 and COX-1 were found in CB-induced LDs indicating that LDs contribute to the inhibitory effects of CB by acting as platform for synthesis of 15-d-PGJ 2 , a pro-resolving lipid mediator. Together, our data indicate that an immunomodulatory GIIA sPLA 2 can directly induce LD formation and production of a pro-resolving mediator in an inflammatory cell and afford new insights into the roles of LDs in resolution of inflammatory processes.

Biologically active F- and E/D-type-prostane ring isomers (F 2 -IP and E 2 /D 2 -IP, respectively) are produced in situ by non-enzymatic peroxidation of arachidonic acid esterified to GroPCho (PtdCho-IP) and are universally distributed in tissue lipoproteins and cell membranes. Previous work has shown that platelet-activating factor acetylhydrolases (PAF-AH) are the main endogenous PLA 2 involved in degradation of PtdCho-IP. The present study shows that the PtdCho-IP are also subject to hydrolysis by group IIA, V and X secretory PLA 2 , which also have a wide peripheral tissue distribution. For this demonstration, we compared the LC/MS profiles of PtdCho-IP of auto-oxidized plasma lipoproteins after incubation for 1-4 h (37 °C) in the absence or presence of recombinant human sPLA 2 (1-2.5 µg/ml). In the absence of exogenously added sPLA 2 the total PtdCho-IP level after 4 h incubation reached 15.9, 21.6 and 8.7 nmol/mg protein of LDL, HDL and HDL 3 , respectively. In the presence of group V or group X sPLA 2 (2.5 µg/ml), the PtdCho-IP was completely hydrolyzed in 1 h, while in the presence of group IIA sPLA 2 (2.5 µg/ml) the hydrolysis was less than 25% in 4 h, although it was complete after 8-24 h incubation. This report provides the first demonstration that PtdCho-IP are readily hydrolyzed by group IIA, V and X sPLA 2 . A co-location of sPLA 2 and the substrates in various tissues has been recorded. Thus, the initiation of interaction and production of isoprostanes in situ are highly probable.

Snake venoms from the Viperidae and Elapidae families often have several phospholipases A 2 (PLA 2 s), which may display different functions despite having a similar structural scaffold. These proteins are considered an important target for the development of drugs against local myotoxic damage because they are not efficiently neutralized by conventional serum therapy. PLA 2 s from these venoms are generally divided into two classes: (i) catalytic PLA 2 s (or Asp49-PLA 2 s) and (ii) non-catalytic PLA 2 -like toxins (or Lys49-PLA 2 s). In many Viperidae venoms, a subset of the basic Asp49-PLA 2 s displays some functional and structural characteristics of PLA 2 -like proteins and group within the same phylogenetic clade, but their myotoxic mechanism is still largely unknown. In the present study, we have crystallized and solved the structure of myotoxin I (MT-I), a basic myotoxic Asp49-PLA 2 isolated from Bothrops asper venom. The structure presents a dimeric conformation that is compatible with that of previous dimers found for basic myotoxic Asp49-PLA 2 s and Lys49-PLA 2 s and has been confirmed by other biophysical and bioinformatics techniques. This arrangement suggests a possible cooperative action between both monomers to exert myotoxicity via two different sites forming a putative membrane-docking site (MDoS) and a putative membrane disruption site (MDiS). This mechanism would resemble that proposed for Lys49-PLA 2 s, but the sites involved appear to be situated in a different region. Thus, as both sites are close to one another, they form a "myotoxic cluster", which is also found in two other basic myotoxic Asp49-PLA 2 s from Viperidae venoms. Such arrangement may represent a novel structural strategy for the mechanism of muscle damage exerted by the group of basic, Asp49-PLA 2 s found in viperid snake venoms., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Secretory phospholipase A2 group IIA (sPLA2-IIA) is an enzyme that hydrolyzes the sn-2 ester bond in glyceroacyl phospholipids present in lipoproteins and cell membranes. As many immunohistochemical studies reveal that the high expression of sPLA2-IIA in the tumorous tissue or plasma of cancer patients, though low expression in other cases, the enzyme is considered highly relevant with cancer development. Effort has been made to establish the mechanism of how sPLA2- IIA is involved in various cancers in order to understand its pathogenic role and to utilize it as a target for cancer diagnosis and therapy. This article specifically reviews recent studies regarding the relevance of sPLA2-IIA with various cancers and reported inhibitors of the protein., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA 2 , proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA 2 and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2μg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30μg/site) and significantly inhibited by both ratios. Venom (10-300μg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai., (Copyright © 2016. Published by Elsevier Inc.)

Human serum amyloid A (SAA) has been demonstrated as a chemoattractant and proinflammatory mediator of lethal systemic inflammatory diseases. In the circulation, it can be sequestered by a high-density lipoprotein, HDL, which carries cholesterol, triglycerides, phospholipids and apolipoproteins (Apo-AI). The capture of SAA by HDL results in the displacement of Apo-AI, and the consequent inhibition of SAA's chemoattractant activities. It was previously unknown whether HDL similarly inhibits SAA-induced sPLA2 expression, as well as the resultant HMGB1 release, nitric oxide (NO) production and autophagy activation. Here we provided compelling evidence that human SAA effectively upregulated the expression and secretion of both sPLA2-IIE and sPLA2-V in murine macrophages, which were attenuated by HDL in a dose-dependent fashion. Similarly, HDL dose-dependently suppressed SAA-induced HMGB1 release, NO production, and autophagy activation. In both RAW 264.7 cells and primary macrophages, HDL inhibited SAA-induced secretion of several cytokines (e.g., IL-6) and chemokines (e.g., MCP-1 and RANTES) that were likely dependent on functional TLR4 signaling. Collectively, these findings suggest that HDL counter-regulates SAA-induced upregulation and secretion of sPLA2-IIE/V in addition to other TLR4-dependent cytokines and chemokines in macrophage cultures., Competing Interests: The authors have declared that no competing interests exist.

Elevated production of arachidonic acid (AA)-derived pro-inflammatory eicosanoids due to the concerted action of secretory phospholipase A 2 group IIA (sPLA 2 IIA), 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) is a common feature of many inflammatory disorders. Hence, modulation of the bioactivity of these 3 enzymes is an important strategy to control inflammation. However, the failure of drugs specific for an individual enzyme (sPLA 2 IIA-, 5-LOX- or COX-2) and the success of 5-LOX/COX-2 dual inhibitors in effectively controlling inflammation in clinical trials prompted us to evaluate a common inhibitor for sPLA 2 IIA, 5-LOX and COX-2 enzymes. Celastrol, a quinone methide triterpene, was selected in this regard through molecular docking studies. We provide the first evidence for celastrol's ability to inhibit the catalytic activity of sPLA 2 IIA, 5-LOX and COX-2 enzymes. Celastrol significantly inhibited the catalytic activity of sPLA 2 IIA (IC 50 =6μM) in vitro, which is independent of substrate and calcium concentration. In addition, celastrol inhibited the catalytic activities of 5-LOX (IC 50 =5μM) and COX-2 (IC 50 =20μM) in vitro; sPLA 2 IIA-induced edema and carrageenan-induced edema in mice; and lipopolysaccharide-stimulated production of PGE 2 in human neutrophils. Thus, celastrol modulates inflammatory responses by targeting multiple enzymes of AA pathway., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The translationally controlled tumor protein (TCTP), initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF). TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12) cells. Treatment with recombinant TCTP (rTCTP) enhanced both basal and depolarization (50 mM KCl)-evoked [³H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca 2+ ] i ), the rTCTP-driven effect on dopamine release was mediated by a Ca 2+ -independent pathway, as evidenced by the fact that Ca 2+ -modulating agents such as Ca 2+ chelators and a voltage-gated L-type Ca 2+ -channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A₂ (PLA₂) in rTCTP-induced dopamine release, the inhibitor for Ca 2+ -independent PLA₂ (iPLA₂) produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca 2+ -dependent cytosolic PLA₂ (cPLA₂) and secretory PLA₂ (sPLA₂) inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca 2+ -independent mechanism that involved PLA₂ in the process, suggesting the regulatory role of TCTP in the neuronal functions., Competing Interests: The authors declare no conflict of interest.

Ovarian endometrioma (OMA) and deep infiltrating endometriosis (DIE) are the most severe forms of endometriosis, but different pathogenetic mechanisms and clinical symptoms distinguish these two forms. Corticotrophin-releasing hormone (CRH) and urocortin (Ucn) are endometrial neuropeptides involved in tissue differentiation and inflammation. The expression of CRH, Ucn, Ucn2, CRH-receptors (type-1 and type-2) and inflammatory enzymes phospholipase-A2 group IIA (PLA2G2A) and cycloxygenase-2 (COX2) were evaluated in OMA (n = 22) and DIE (n = 26). The effect of CRH or Ucn on COX2 mRNA expression was evaluated in cultured human endometrial stromal cells. In DIE lesions, CRH, Ucn and CRH-R2 mRNA levels were significantly higher than in OMA (P < 0.01, P < 0.001 and P < 0.05, respectively); DIE lesions showed a higher expression of COX2 (P < 0.01) and PLA2G2A (P < 0.05) mRNA than OMA, which was positively correlated with CRH-R2 mRNA expression (P < 0.05). Intense immunostaining for CRH and Ucn was shown in DIE. Treatment of cultured endometrial stromal cells with Ucn significantly increased COX2 mRNA expression (P < 0.01); this effect was reversed by the CRH-R2 antagonist astressin-2B. In DIE, DIE lesions highly express neuropeptide and enzyme mRNAs, supporting a strong activation of inflammatory pathways., (Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Secretory phospholipase A2 Group-IIA (sPLA2 -IIA) catalyzes the hydrolysis of the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. sPLA2 -IIA is deregulated in various cancers; however, its role in hair follicle stem cell (HFSC) regulation is obscure. Here we report a transgenic mice overexpressing sPLA2 -IIA (K14-sPLA2 -IIA) showed depletion of HFSC pool. This was accompanied with increased differentiation, loss of ortho-parakeratotic organization and enlargement of sebaceous gland, infundibulum and junctional zone. The colony forming efficiency of keratinocytes was significantly reduced. Microarray profiling of HFSCs revealed enhanced level of epithelial mitogens and transcription factors, c-Jun and FosB that may be involved in proliferation and differentiation. Moreover, K14-sPLA2 -IIA keratinocytes showed enhanced activation of EGFR and JNK1/2 that led to c-Jun activation, which co-related with enhanced differentiation. Further, depletion of stem cells in bulge is associated with high levels of chromatin silencing mark, H3K27me3 and low levels of an activator mark, H3K9ac suggestive of alteration in gene expression contributing toward stem cells differentiation. Our results, first time uncovered that overexpression of sPLA2 -IIA lead to depletion of HFSCs and differentiation associated with altered histone modification. Thus involvement of sPLA2 -IIA in stem cells regulation and disease pathogenesis suggest its prospective clinical implications. Stem Cells 2016;34:2407-2417., (© 2016 AlphaMed Press.)

The intestinal stem cell niche provides cues that actively maintain gut homeostasis. Dysregulation of these cues may compromise intestinal regeneration upon tissue insult and/or promote tumor growth. Here, we identify secreted phospholipases A2 (sPLA2s) as stem cell niche factors with context-dependent functions in the digestive tract. We show that group IIA sPLA2, a known genetic modifier of mouse intestinal tumorigenesis, is expressed by Paneth cells in the small intestine, while group X sPLA2 is expressed by Paneth/goblet-like cells in the colon. During homeostasis, group IIA/X sPLA2s inhibit Wnt signaling through intracellular activation of Yap1. However, upon inflammation they are secreted into the intestinal lumen, where they promote prostaglandin synthesis and Wnt signaling. Genetic ablation of both sPLA2s improves recovery from inflammation but increases colon cancer susceptibility due to release of their homeostatic Wnt-inhibitory role. This "trade-off" effect suggests sPLA2s have important functions as genetic modifiers of inflammation and colon cancer., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Inhibition of group IIA secreted phospholipase A2 (GIIA sPLA2) has been an important objective for medicinal chemists. We have previously shown that inhibitors incorporating the 2-oxoamide functionality may inhibit human and mouse GIIA sPLA2s. Herein, the development of new potent inhibitors by molecular docking calculations using the structure of the known inhibitor 7 as scaffold, are described. Synthesis and biological evaluation of the new compounds revealed that the long chain 2-oxoamide based on (S)-valine GK241 led to improved activity (IC50=143 nM and 68 nM against human and mouse GIIA sPLA2, respectively). In addition, molecular dynamics simulations were employed to shed light on GK241 potent and selective inhibitory activity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The expression of secretory group IIA phospholipase A2 (sPLA2-IIA) is enhanced by development of inflammatory disorders. In this study, sPLA2-IIA expression was induced in the lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells and mice to evaluate the effect of polyozellin. Polyozellin, a major constituent of a Korea edible mushroom Polyozellus multiplex, has been known to exhibit the biological activities such as anti-oxidative and anti-inflammatory effects. Polyozellin remarkably suppressed the LPS-mediated protein expression and activity of sPLA2-IIA via inhibition of phosphorylation of cytosolic phospholipase A2 and extracellular signal-regulated kinase 1/2. These results demonstrated that polyozellin might play an important role in the modulation of sPLA2-IIA expression and activity in response to the inflammatory diseases.

Mitogenesis of Vascular Smooth Muscle Cells (VSMC) plays an important role in atherogenesis. Until recently, the effect of lipid subfractions has not been clarified. Secretory phospholipases A2 (sPLA2's) hydrolyse glycerophospholipids and release pro-inflammatory lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes. They localize in the vascular wall. We hypothesized that structurally similar sPLA2's may exert different impact on VSMC. The influence of sPLA2's, IIA, V, X, HDL, LDL, and hydrolysis products was tested on mitogenesis of VSMC, i.e., the early effect on the cell membrane phospholipids, and on PGE2 and LTB4 release, i.e., late effect of Cyclooxygenase and 5-lipooxygenase activity in VSMC. Mitogenesis was significantly enhanced by HDL and LDL, and by products of sPLA2 hydrolysis. Hydrolysis of HDL or LDL enhanced mitogenic activity in order V>X>IIA. The release of PGE2 was enhanced by group X sPLA2 and by HDL hydrolyzed by groups V and X. LDL and its hydrolysis products enhanced the release of PGE2 in order X>V>IIA. The release of LTB4 was markedly increased by LDL and HDL, and by hydrolytic products of group V and X, but not group IIA sPLA2. Our study demonstrates a diverse interaction of pro-inflammatory sPLA2's with HDL and LDL affecting both mitogenesis and eicosanoid release from VSMC, therefore potentially enhancing their pro-atherogenic activity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Listeria monocytogenes is a bacterial pathogen which establishes intracellular parasitism in various cells, including macrophages and nonhematopoietic cells, such as hepatocytes. It has been reported that several proinflammatory cytokines have pivotal roles in innate protection against L. monocytogenes infection. We found that a proinflammatory cytokine, interleukin 22 (IL-22), was expressed by CD3(+) CD4(+) T cells at an early stage of L. monocytogenes infection in mice. To assess the influence of IL-22 on L. monocytogenes infection in hepatocytes, cells of a human hepatocellular carcinoma line, HepG2, were treated with IL-22 before L. monocytogenes infection in vitro. Gene expression analysis of the IL-22-treated HepG2 cells identified phospholipase A2 group IIA (PLA2G2A) as an upregulated antimicrobial molecule. Addition of recombinant PLA2G2A to the HepG2 culture significantly suppressed L. monocytogenes infection. Culture supernatant of the IL-22-treated HepG2 cells contained bactericidal activity against L. monocytogenes, and the activity was abrogated by a specific PLA2G2A inhibitor, demonstrating that HepG2 cells secreted PLA2G2A, which killed extracellular L. monocytogenes. Furthermore, colocalization of PLA2G2A and L. monocytogenes was detected in the IL-22-treated infected HepG2 cells, which suggests involvement of PLA2G2A in the mechanism of intracellular killing of L. monocytogenes by HepG2 cells. These results suggest that IL-22 induced at an early stage of L. monocytogenes infection enhances innate immunity against L. monocytogenes in the liver by stimulating hepatocytes to produce an antimicrobial molecule, PLA2G2A., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)