Recent progress in phospholipase A 2 research: From cells to animals to humans

F2-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F2-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F2-isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F2-isoprostanes. However, the rate of esterified F2-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F2-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F2-isoprostanes esterified on phospholipids in vivo.

Apolipoprotein A-I (apoA-I), the major protein of HDL, is probably among the most intensively studied of all proteins. It functions in HDL assembly, in the removal of excess cholesterol from cells, and in the transport of cholesterol from peripheral tissues to the liver, a process known as “reverse cholesterol transport.” It is a cofactor for lecithin:cholesterol acyl transferase (LCAT), the enzyme responsible for cholesterol esterification in HDL, and it stabilizes certain antioxidant enzymes, such as serum paraoxonase, that are carried on HDL.1,2 Rare null mutations of apoA-I in human populations are associated with early coronary heart disease,3 and overexpression of apoA-I in transgenic mice protects against atherogenesis.4 An exciting recent development was the 4-angstrom-resolution x-ray structure for the helical part of apoA-I, encompassing residues 44 to 243 (reviewed by Segrest et al5). See page 1977 In contrast, apolipoprotein A-II (apoA-II), the second most abundant protein of HDL, has no known function, and it has been the subject of few detailed studies. Its presence in HDL has been reported to decrease,6 or have no influence on, cholesterol efflux from cells,7,8 nor is it a cofactor for any known enzyme. Rare individuals lacking apoA-II appear to have normal plasma lipids.9 In fact, all of the known effects of apoA-II are deleterious. In studies with mice and humans, apoA-II levels have been associated with increased susceptibility to atherosclerosis,10–13 increased free fatty acid levels,14,15 increased body fat,15,16 and increased insulin resistance.15,17 Undoubtedly, apoA-II has some unknown beneficial functions, possibly related to fatty acid metabolism or host defense. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , …

Phospholipase A 2 enzymes

A new era of secreted phospholipase A₂

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate arachidonic acid (AA), a precursor of eicosanoids including prostaglandins (PGs) and leukotrienes (LTs). The same reaction also produces lysophosholipids, which represent another class of lipid mediators. So far, at least 19 enzymes that possess PLA2 activity have been identified in mammals. The secretory PLA2 (sPLA2) family, in which 10 isozymes have been identified, consists of low-molecular-weight, Ca2+-requiring, secretory enzymes that have been implicated in a number of biological processes, such as modification of eicosanoid generation, inflammation, host defense, and atherosclerosis. The cytosolic PLA2 (cPLA2) family consists of 3 enzymes, among which cPLA2alpha plays an essential role in the initiation of AA metabolism. Intracellular activation of cPLA2alpha is tightly regulated by Ca2+ and phosphorylation. The Ca2+-independent PLA2 (iPLA2) family contains 2 enzymes and may play a major role in membrane phospholipid remodeling. The platelet-activating factor (PAF) acetylhydrolase (PAF-AH) family represents a unique group of PLA2 that contains 4 enzymes exhibiting unusual substrate specificity toward PAF and/or oxidized phospholipids. In this review, we will overview current understanding of the properties and functions of each enzyme belonging to the sPLA2, cPLA2, and iPLA2 families, which have been implicated in signal transduction.

Chapter 4 - Current understanding of phospholipase A2s based on knockout/transgenic mice and human diseases

Transgenic mice overexpressing human apolipoprotein A-II (huapoA-II) display high VLDL and low HDL levels. To evaluate the antioxidant potential of huapoA-II enriched HDL, we measured the activities of paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH). Both activities decreased up to 43% in the serum of transgenic mice compared with controls, varied in parallel to HDL levels, but decreased less than HDL levels. The major part of PON and PAF-AH was associated with HDL, except in fed high huapoA-II-expressing mice, in which 20% of PAF-AH and 9% of PON activities were associated with VLDL. PON mRNA levels in the liver, its major site of synthesis, were similar in transgenic and control animals, indicating normal enzyme synthesis. In transgenic mice, the basal oxidation of lipoproteins was not increased, whereas their VLDL were more susceptible to oxidation than VLDL of controls. Interestingly, HDL of transgenic mice protected VLDL from oxidation more efficiently than HDL of controls. In conclusion, the decrease in both PON and PAF-AH activities in huapoA-II transgenic mice is best explained by their lower plasma HDL levels. However, the unchanged basal lipoprotein oxidation in transgenic mice suggests that huapoA-II-rich HDL may maintain adequate antioxidant potential.—Boisfer, E., D. Stengel, D. Pastier, P. M. Laplaud, N. Dousset, E. Ninio, and A-D. Kalopissis. Antioxidant properties of HDL in transgenic mice overexpressing human apolipoprotein A-II. J. Lipid Res. 2002. 43: 732–741.

Various mechanisms may contribute to the antiatherogenic potential of apolipoprotein A-I (apo A-I) and high density lipoproteins (HDLs). Therefore, the effect of adenovirus-mediated human apo A-I gene transfer or human apo A-I transgenesis on platelet-activating factor acetylhydrolase (PAF-AH) and arylesterase/paraoxonase (PON1) was studied in C57BL/6 and C57BL/6 apo E(-/-) mice. Human apo A-I transgenesis in C57BL/6 mice resulted in a 4.2-fold (P<0.0001) increase of PAF-AH and a 1.7-fold (P=0.0012) increase of PON1 activity. The apo E deficiency was associated with a 1.6-fold (P=0.008) lower PAF-AH and a 2.0-fold (P=0.012) lower PON1 activity. Human apo A-I transgenesis in C57BL/6 apo E(-/-)mice increased PAF-AH and PON1 activity by 2.1-fold (P=0.01) and 2.5-fold (P=0.029), respectively. After adenovirus-mediated gene transfer of human apo A-I into C57BL/6 apo E(-/-)mice, a strong correlation between human apo A-I plasma levels and PAF-AH activity was observed at day 6 (r=0.92, P<0.0001). However, PON1 activity failed to increase, probably as a result of cytokine-mediated inhibition of PON 1 expression. In conclusion, this study indicates that overexpression of human apo A-I increases HDL-associated PAF-AH activity. PON1 activity was also increased in human apo A-I transgenic mice, but not after human apo A-I gene transfer, a result that was probably related to cytokine production induced in the liver by the adenoviral vectors. Increased levels of these HDL-associated enzymes may contribute to the anti-inflammatory and antioxidative potential of HDL and thereby to the protection conferred by HDL against atherothrombosis.

Phospholipase A2s (PLA2s) are a group of enzymes that hydrolyze the sn-2 position of phospholipids to generate fatty acids and lysophospholipids, which serve as lipid mediators or their precursors. Mammalian genomes encode genes for more than 30 PLA2s or related enzymes, which are subdivided into several groups on the basis of their structures, enzymatic properties, and evolutional relationships. Among them, the Ca2+-dependent cytosolic PLA2 (cPLA2), Ca2+-independent PLA2 (iPLA2), and secreted PLA2 (sPLA2) families are regarded as the “big three.” From a general point of view, cPLA2α (the prototypic cPLA2) plays a major role in the initiation of arachidonic acid (AA) metabolism, the iPLA2 family contributes to membrane homeostasis or energy metabolism, and the sPLA2 family affects various biological events by modulating extracellular phospholipid milieus in response to given microenvironmental cues. In this chapter, we overview current understanding of the biological functions of PLA2s as revealed by gene-manipulated mice and human diseases.

Lack of Protection against Oxidative Modification of LDL by Avian HDL

Cysteinyl leukotriene receptor 1 (CYSLTR1) is observed to increase in psoriatic skin lesions. Montelukast, a CYSLTR1 antagonist, effectively treats inflammatory disorders, such as rheumatoid arthritis, multiple sclerosis, and atopic dermatitis. Thus, blocking CYSLTR1 may be a promising strategy for psoriasis immunotherapy. We prepared a montelukast sodium cream and solution and investigated their effects on psoriasis-like skin lesions induced by imiquimod (IMQ). After the treatment, serum, skin, and spleen samples were collected for evaluation. We treated human T helper (Th) 17 cells with montelukast in vitro to study its effect on Th17 differentiation and nuclear factor kappa-B (NF-κB) signaling. We also created a keratinocyte proliferation model induced by M5 cytokines and assessed the influence of montelukast on key psoriasis-related genes. We induced psoriasis in CYSLTR1 knockout (KO) mice using IMQ to explore the role of CYSLTR1 in psoriasis development. Montelukast sodium cream and solution effectively reduced the psoriasis area and severity index (PASI) and alleviated disease symptoms in IMQ-induced mice. Furthermore, reduced infiltration of inflammatory cells (Th1, Th17, and T follicular helper [Tfh] cells), decreased mRNA expression of cytokines in the skin (interleukin [IL]-17/F and IL-23), and lower serum concentrations of various cytokines (IL-2, IL-6, IL-13, and IL-17A/F) were observed. Montelukast cream and solution also decreased spleen size and the proportion of Th17 and Tfh cells, and significantly inhibited NF-κB signaling-related genes after application. Moreover, montelukast inhibited Th17 cell differentiation and suppressed NF-κB signaling in vitro. CYSLTR1 KO mice induced with IMQ showed improvement in PASI scores, serum IL-17A/F levels, and lower Th1 and Th17 cells in the spleen and skin compared to wild-type mice. Montelukast also suppressed the proliferation and inflammatory response of keratinocytes by regulating NF-κB signaling. Collectively, our results strongly indicate that inhibition of CYSLTR1 signaling to target the Th17 response holds significant promise as a therapeutic approach to manage psoriasis.

We have previously demonstrated that thrombin stimulation of endothelial cells results in increased membrane-associated, Ca(2+)-independent phospholipase A2 (iPLA2) activity, accelerated hydrolysis of membrane plasmalogen phospholipids, and production of several biologically active phospholipid metabolites, including prostacyclin and platelet-activating factor (PAF) that is abolished by pretreatment with the iPLA2-selective inhibitor bromoenol lactone. This study was designed to further investigate the role of alternative PLA2 inhibitors, including methyl arachidonyl fluorophosphonate (MAFP, an inhibitor of cytosolic PLA2 isoforms), on phospholipid turnover and PAF production from thrombin-stimulated human coronary artery endothelial cells (HCAECs). Paradoxically, pretreatment of HCAEC with MAFP (5-25 microM) resulted in a significant increase in PAF production in both unstimulated and thrombin-stimulated cells that was found to be a direct result of inhibition of PAF acetylhydrolase (PAF-AH) activity. Pretreatment with MAFP did not significantly inhibit HCAEC PLA2 activity, possibly due to the localization of PLA2 activity in the membrane fraction rather than the cytosol. Bromoenol lactone did not inhibit PAF-AH activity, even at concentrations as high as 20 microM. We conclude that MAFP augments thrombin-stimulated PAF production by inhibition of PAF catabolism without affecting membrane-associated iPLA2 activity.

Characterization of the Cysteinyl Leukotriene 2 Receptor in Novel Expression Sites of the Gastrointestinal Tract

IntroductionThe cysteinyl leukotriene receptor 1 (CysLTR1) is known as a potent lipid mediator with a well-established role in inflammatory regulation and lung disease. While its involvement in immune cell recruitment has been previously reported, its broader impact on pulmonary metabolism remains poorly understood.ObjectivesThe study aims to investigate the metabolic consequences of a CysLTR1 deletion in mice to elucidate its role in pulmonary metabolic homeostasis.MethodsBronchoalveolar lavage fluid (BALF) was collected from CysLTR1 knockout (KO) and wild-type (WT) mice and analysed using standardized untargeted gas chromatography–time-of-flight mass spectrometry (GC-TOFMS) metabolomics.ResultsMetabolomics analyses of the BALF collected from the CysLTR1 KO mice presented significantly reduced levels of glucose, glucosamine, and glyceric acid, indicating the role of the CysLTR in lung glucose uptake and consequently lung glycolysis and gluconeogenesis. This is further supported by reductions in myo-inositol and D-chiro-inositol, also supporting previous findings that this occurs due to insulin resistance. Consequential disruption of various glucose-dependent pathways, including the pentose phosphate pathway (reduced gluconic acid, sedoheptulose and xylose) and purine metabolism (reduced 1-methylinosine) indicates a consequential altered nucleotide turnover, and the significantly reduced concentrations of butanoic acid, decan-2-ol, and 1-hexadecanol, indicate changes to fatty acid metabolism in the lung, as a compensatory response to the initial glucose deficiency induced by the CysLTR1 KO. Lastly, the changes to mandelic acid, glutaric acid, tricarballylic acid, and decan-2-ol, furthermore, indicate the role of CysLTR1 in the composition/metabolism of the microbiome.ConclusionThis study expands our knowledge on the role of CysLTR1 beyond its role in immune regulation, that may later serve towards a better understanding of CysLTR1 associated lung diseases and in the development of improved therapeutic strategies.

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.