A2 Family Research Articles

Varespladib: targeting the inflammatory face of atherosclerosis

This editorial refers to ‘Randomized trial of an inhibitor of secretory phospholipase A2 on atherogenic lipoprotein subclasses in statin-treated patients with coronary heart disease’, by R.S. Rosenson et al., doi:10.1093/eurheartj/ehq374 Atherosclerosis is a complex disease and in its pathogenesis depends on the interaction between lipoproteins and inflammatory mediators.1 This interaction orchestrates the progression of atherosclerosis from its earliest stage when oxidized lipoproteins and leucocytes penetrate dysfunctional endothelium and enter the subendothelial space, right until the end of the process when the plaque becomes unstable and ruptures, leading to a clinical event. Whereas our pharmacological armatorium that targets the lipid component of the risk factor spectrum has been tremendously succesful with the clinical introduction of statins in the middle of the 1990s, drugs that target the inflammatory face of atherosclerosis have thus far been scarce. Among the wide range of chemokines, adhesion molecules, and pro-oxidative substances that affect macrophage recruitment into the arterial wall, members of the phospholipase A2 (PLA2) family have received considerable attention since their purification from human plasma lipoproteins in 1987.2 PLA2s are small and relatively stable ubiquitous enzymes that hydrolyse the sn -2-acyl bond of phospholipids in cell membranes as well as in lipoproteins. This enzymatic activity yields non-esterified fatty acids and lysophospholipids, which are precursors of various proinflammatory mediators including leukotrienes, eicosanoids, prostaglandins, and platelet-activating factors ( Figure 1A ).3 In addition, both circulating and lipoprotein-bound PLA2s play a role in lipoprotein remodelling by high-affinity binding to specific domains on LDLs and apolipoprotein B. Such modified or oxidized LDL particles bind with higher affinity to endothelial proteoglycans, facilitating the penetration of these atherogenic particles into the subendothelial matrix where they accumulate and trigger additional proinflammatory cascades. The recent large-scale meta-analysis of genome-wide association studies for plasma lipids …

The measurement of lysosomal phospholipase A2 activity in plasma

A deficiency of lysosomal phospholipase A2 (LPLA2) causes macrophage-associated phospholipidosis, suggesting that the enzyme is important in the lipid catabolism. Because LPLA2 is secreted by macrophages, extracellular LPLA2 activity may potentially reflect a change in macrophage activation. In this report, the detection of LPLA2 activity in plasma was established by the measurement of the transacylase activity of LPLA2 under acidic conditions. No transacylase activity of LPLA2 was detected in normal human plasma when the plasma was incubated with liposomes consisting of 1,2-dioleoylphosphatidylcholine/sulfatide/N-acetylsphingosine (NAS) at pH 4.5. However, the transacylase activity in the plasma was detected when liposomes consisting of 1,2-dioleoylphosphatidylglycerol/NAS were used as a substrate. To establish the specificity of the assay, ceramide transacylase activity was detected in the plasma of wild-type mice. By contrast, the plasma obtained from LPLA2-knockout mice had no measurable transacylase activity under the same conditions. The enzymatic activity of recombinant LPLA2 was inhibited by treatment with methylarachidonylfluorophosphonate. The inhibitor also suppressed the transacylase activity observed in both normal human and wild-type mouse plasma, establishing that the transacylase activity observed in plasma is due to LPLA2. Plasma LPLA2 activity may be a useful bioassay marker for the identification of LPLA2-related disorders.

Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice

Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3-/- mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3-/- mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3-/- mice. Moreover, the gonads of Pla2g3-/- mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction.

Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice

Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization.

A Joint Estimate of the Precipitation Climate Signal in Europe Using Eight Regional Models and Five Observational Datasets

Abstract This paper presents an analysis of the precipitation climate signal in Europe emerging from a simulation of heterogeneous regional climate models (RCMs) using five observational datasets as the reference for present day climate conditions. Current climate simulations, as well as those from the A2 family of scenarios from the Intergovernmental Panel on Climate Change Special Report on Emissions Scenarios (SRES-A2), from eight RCMs involved in the Prediction of Regional Scenarios and Uncertainties for Defining European Climate Change Risks and Effects (PRUDENCE) project have been cross-compared with data from the Climate Research Unit (CRU), the Global Precipitation Climatology Project (GPCP), the Global Precipitation Climatology Centre (GPCC), the Climate Prediction Center (CPC), and the CPC Merged Analysis of Precipitation (CMAP) databases for Europe. The RCMs used are HIRHAM, the Climate High Resolution Model (CHRM), the Rossby Centre Atmosphere–Ocean (RCAO) model, the GKSS Climate Version of the Local Model (CLM), the Hadley Center RCM (HadRM3H), the Atmospheric Hydrostatic Regional Model (REMO), the Prognostic Model at the Mesoscale (PROMES), and the regional coupled ocean–atmosphere–ice model (RACMO). The comparison shows that the climate signal has to be interpreted depending on the reference data used. Although each validation dataset has its own relative merits and shortcomings, it is known that all of the datasets present variable uncertainties and error sources, which impedes consideration of a single dataset as the only valid representation of actual precipitation. Hence, it is suggested that a robust joint estimate of changes in future precipitation might include the uncertainties of both the RCMs and those of the observational datasets. After accounting for the difference between observed and simulated precipitation in the present climate, the analysis of such joint estimates reveals significant agreement in the climate signal for most of Europe. This lends confidence to the idea that the RCMs are able to correctly simulate future changes in precipitation.

Genetic association between the polymorphism of cytosolic PLA2 gene family and schizophrenia

Abnormal phospholipid metabolism in the brain plays an important role in neuropsychiatric diseases. Phospholipase A2 (PLA2) is a crucial element for normal neuro-physiological function. This study aims to investigate the genetic association between the polymorphism of cytosolic phospholipase A2 (cPLA2) family genes and schizophrenia among Han Chinese in the northern part of China. The polymerase chain reaction-based ligase detection reaction (PCR-LDR) was applied to detect the genotype ten single nucleotide polymorphisms (SNPs) of cPLA2 family genes among 201 pedigrees consisting of fathers, mothers and affected offsprings with schizophrenia. The pedigrees were collected from 2000 to 2006. Haplotype relative risk (HRR) test, transmission disequilibrium test (TDT), haplotype transmission analysis and multiple locus analysis were conducted to analyze the genotyping data. The genotypic frequency of cPLA2 gene did not deviate from Hardy-Weinberg equilibrium either in case or control group. HRR and TDT showed that the ten SNPs were not associated with schizophrenia (P > 0.05). Analysis for haplotype transmission showed that no haplotype system was associated with schizophrenia (P > 0.05). The conditioning on allele (COA) and conditioning on gene (COG) tests showed disease associations with the haplotype of rs2162886-rs1668589, rs891014-rs1668589 and rs2307279-rs7542180 (χ2 = 6.913, P = 0.032; χ2 = 8.393, P = 0.015; χ2= 8.447, P = 0.038). Our data suggest that many loci in the cPLA2 family genes may be associated with schizophrenia.

Characterization of an human leucocyte antigen A2‐restricted Epstein–Barr virus nuclear antigen‐1‐derived cytotoxic T‐lymphocyte epitope

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is regularly expressed in all proliferating virus-infected cells and is therefore an interesting target for immunotherapy. Alleles of the human leucocyte antigen (HLA) -A2 family are dominantly expressed in Caucasians so we sought to identify EBNA1-specific cytotoxic T-lymphocyte (CTL) responses restricted through this allele. We report on the characterization of the LQTHIFAEV (LQT) epitope. LQT-specific memory CTL responses were reactivated in three of 14 healthy EBV seropositive donors (21%) whereas responses to HLA-A2-restricted epitopes, two derived from LMP2 and one from EBNA3A, were detected in 93%, 71% and 42% of the donors, respectively. The LQT-specific CTL clones did not lyse EBV-carrying lymphoblastoid cell lines and Burkitt's lymphoma cell lines nor EBNA1-transfected Burkitt's lymphoma cells but specifically released interferon-gamma upon stimulation with HLA-matched EBNA1-expressing cells and this response was enhanced by deletion of the Gly-Ala repeat domain that inhibits proteasomal degradation. The poor presentation of the endogenously expressed LQT epitope was not affected by inhibition of peptidases that trim antigenic peptides in the cytosol but full presentation was achieved in cells expressing a trojan antigen construct that releases the epitope directly into the endoplasmic reticulum. Hence, inefficient proteasomal processing appears to be mainly responsible for the poor presentation of this epitope.

Cerebrospinal Fluid Secretory Ca2+-Dependent Phospholipase A2 Activity Is Increased in Alzheimer Disease

The phospholipase A(2) (PLA2) family comprises multiple isoenzymes that vary in their physicochemical properties, cellular localizations, calcium sensitivities, and substrate specificities. Despite these differences, PLA2s share the ability to catalyze the synthesis of the precursors of the proinflammatory mediators. To investigate the potential of PLA2 as a biomarker in screening neuroinflammatory disorders in both clinical and research settings, we developed a PLA2 assay and determined the predominant types of PLA2 activity in cerebrospinal fluid (CSF). We used liposomes composed of a fluorescent probe (bis-Bodipy FL C11-PC [1,2-bis-(4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine]) and 1,2-dioleoyl-l-alpha-phosphatidylcholine as a substrate to measure CSF PLA2 activity in a 96-well microtiter plate format. We established the type of CSF PLA2 activity using type-specific inhibitors of PLA2. Using 5 microL CSF per assay, our PLA2 activity assay was reproducible with CVs <15% in 2 CSF samples and for recombinant secretory Ca(2+)-dependent PLA2 (sPLA2) in concentrations ranging from 0.25 to 1 micromol/L. This PLA2 assay allowed identification of sPLA2 activity in lumbar CSF from healthy individuals 20-77 years old that did not depend on either sex or age. Additionally, CSF sPLA2 activity was found to be increased (P = 0.0008) in patients with Alzheimer disease. Adult human CSF has sPLA2 activity that can be measured reliably with the assay described. This enzyme activity in the CSF is independent of both sex and age and might serve as a valuable biomarker of neuroinflammation, as we demonstrated in Alzheimer disease.

Multiple-Docking and Affinity Fingerprint Methods for Protein Classification and Inhibitors Selection

The function-based protein classification holds tremendous promise for molecular recognition and the structure-based design process. We describe here a new strategy combined with multiple-docking tools and "affinity fingerprint" analysis technology to detect functional relationships among proteins based on the substrate binding features and protein-ligand interaction matrix and apply it successfully for the family of phospholipase A2 to investigate protein-ligand binding, function-based protein classification, and inhibitor selection, evaluation. Binding data and matrix were generated by multiple versus multiple-docking among 12 PLA2s and 84 PLA2 inhibitors. Three kinds of statistic techniques, principal component analysis, multidimensional scaling, and cluster algorithms, were chosen to distinguish the groups with similar binding characteristics. The 12 PLA2s were automatically categorized into reasonable subfamilies on the basis of the protein-ligand binding matrix, and the classifying problem of cPLA2 (PDB ID: 1CJY ) with relatively low homology was successfully dealt with. This approach was also used to identify and group the selective inhibitors against human nonpancreatic sPLA2. A sound pharmacophore has been defined from these selective inhibitors. It shows that the method is quite robust against individual data deviation, especially false positive, which makes it possible to be used in virtual screening with large enzyme families to generate selective inhibitors of targets on the basis of limited structural/function information.

Varespladib (A-002), a Secretory Phospholipase A2 Inhibitor, Reduces Atherosclerosis and Aneurysm Formation in ApoE− / − Mice

The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE mouse models.

Pepsin homologues in bacteria.

BackgroundPeptidase family A1, to which pepsin belongs, had been assumed to be restricted to eukaryotes. The tertiary structure of pepsin shows two lobes with similar folds and it has been suggested that the gene has arisen from an ancient duplication and fusion event. The only sequence similarity between the lobes is restricted to the motif around the active site aspartate and a hydrophobic-hydrophobic-Gly motif. Together, these contribute to an essential structural feature known as a psi-loop. There is one such psi-loop in each lobe, and so each lobe presents an active Asp. The human immunodeficiency virus peptidase, retropepsin, from peptidase family A2 also has a similar fold but consists of one lobe only and has to dimerize to be active. All known members of family A1 show the bilobed structure, but it is unclear if the ancestor of family A1 was similar to an A2 peptidase, or if the ancestral retropepsin was derived from a half-pepsin gene. The presence of a pepsin homologue in a prokaryote might give insights into the evolution of the pepsin family.ResultsHomologues of the aspartic peptidase pepsin have been found in the completed genomic sequences from seven species of bacteria. The bacterial homologues, unlike those from eukaryotes, do not possess signal peptides, and would therefore be intracellular acting at neutral pH. The bacterial homologues have Thr218 replaced by Asp, a change which in renin has been shown to confer activity at neutral pH. No pepsin homologues could be detected in any archaean genome.ConclusionThe peptidase family A1 is found in some species of bacteria as well as eukaryotes. The bacterial homologues fall into two groups, one from oceanic bacteria and one from plant symbionts. The bacterial homologues are all predicted to be intracellular proteins, unlike the eukaryotic enzymes. The bacterial homologues are bilobed like pepsin, implying that if no horizontal gene transfer has occurred the duplication and fusion event might be very ancient indeed, preceding the divergence of bacteria and eukaryotes. It is unclear whether all the bacterial homologues are derived from horizontal gene transfer, but those from the plant symbionts probably are. The homologues from oceanic bacteria are most closely related to memapsins (or BACE-1 and BACE-2), but are so divergent that they are close to the root of the phylogenetic tree and to the division of the A1 family into two subfamilies.

Biology of Secretory Phospholipase A2

IntroductionThe secretory phospholipase A2 (sPLA2) family provides a seemingly endless array of potential biological functions that is only beginning to be appreciated. In humans, this family comprises 9 different members that vary in their tissue distribution, hydrolytic activity, and phospholipid substrate specificity. Through their lipase activity, these enzymes trigger various cell-signaling events to regulate cellular functions, directly kill bacteria, or modulate inflammatory responses. In addition, some sPLA2’s are high affinity ligands for cellular receptors.ObjectiveThis review merely scratches the surface of some of the actions of sPLA2s in innate immunity, inflammation, and atherosclerosis. The goal is to provide an overview of recent findings involving sPLA2s and to point to potential pathophysiologic mechanisms that may become targets for therapy.

Intracellular phospholipase A2 group IVA and group VIA play important roles in Wallerian degeneration and axon regeneration after peripheral nerve injury

We provide evidence that two members of the intracellular phospholipase A(2) family, namely calcium-dependent group IVA (cPLA(2) GIVA) and calcium-independent group VIA (iPLA(2) GVIA) may play important roles in Wallerian degeneration in the mouse sciatic nerve. We assessed the roles of these PLA(2)s in cPLA(2) GIVA(-/-) mice, and mice treated with a selective inhibitor of iPLA(2) GVIA (FKGK11). Additionally, the effects of both these PLA(2)s were assessed by treating cPLA(2) GIVA(-/-) mice with the iPLA(2) inhibitor. Our data suggest that iPLA(2) GVIA may play more of a role in the early stages of myelin breakdown, while cPLA(2) GIVA may play a greater role in myelin clearance by macrophages. Our results also show that the delayed myelin clearance and Wallerian degeneration after sciatic nerve crush injury in mice lacking cPLA(2) and iPLA(2) activities is accompanied by a delay in axon regeneration, target re-innervation and functional recovery. These results indicate that the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) contribute significantly to various aspects of Wallerian degeneration in injured peripheral nerves, which is then essential for successful axon regeneration. This work has implications for injury responses and recovery after peripheral nerve injuries in humans, as well as for understanding the slow clearance of myelin after CNS injury and its potential consequences for axon regeneration.

CGI-58 facilitates lipolysis on lipid droplets but is not involved in the vesiculation of lipid droplets caused by hormonal stimulation

A lipid droplet (LD)-associated protein, perilipin, is a critical regulator of lipolysis in adipocytes. We previously showed that Comparative Gene Identification-58 (CGI-58), a product of the causal gene of Chanarin-Dorfman syndrome, interacts with perilipin on LDs. In this study, we investigated the function of CGI-58 using RNA interference. Notably, CGI-58 knockdown caused an abnormal accumulation of LDs in both 3T3-L1 preadipocytes and Hepa1 hepatoma cells. CGI-58 knockdown did not influence the differentiation of 3T3-L1 adipocytes but reduced the activity of both basal and cAMP-dependent protein kinase-stimulated lipolysis. In vitro studies showed that CGI-58 itself does not have lipase/esterase activity, but it enhanced the activity of adipose triglyceride lipase. Upon lipolytic stimulation, endogenous CGI-58 was rapidly dispersed from LDs into the cytosol along with small particulate structures. This shift in localization depends on the phosphorylation of perilipin, because phosphorylated perilipin lost the ability to bind CGI-58. During lipolytic activation, LDs in adipocytes vesiculate into micro-LDs. Using coherent anti-Stokes Raman scattering microscopy, we pursued the formation of micro-LDs in single cells, which seemed to occur in cytoplasmic regions distant from the large central LDs. CGI-58 is not required for this process. Thus, CGI-58 facilitates lipolysis in cooperation with perilipin and other factors, including lipases.

Obesity and Atherogenic Dyslipidemia

Obesity is associated with an increased risk of coronary heart disease, in part due to its strong association with atherogenic dyslipidemia, characterized by high triglycerides and low high-density lipoprotein (HDL) cholesterol. There has been substantial research effort focused on the mechanisms of the link between obesity and atherogenic dyslipidemia, both in the absence and presence of insulin resistance. After a brief overview of the epidemiology of atherogenic dyslipidemia, this article details the known molecular mechanisms of adipocyte function and its relationship to apoB-containing lipoprotein assembly and metabolism, both in the healthy as well as in the obese states. We also discuss the pathophysiology of low HDL cholesterol in obesity and the implications for cardiovascular disease risk.

PLA 2 -V

An important early step in atherosclerosis is binding of apolipoprotein B-100 (apoB-100)–containing lipoproteins (VLDL, IDL, and LDL) to the proteoglycan component of the extracellular matrix of the arterial intima.1,2 When oxidative agents or enzymes attack the proteoglycan-bound lipoproteins, they become oxidatively modified, and when various intimal proteases or lipases hydrolyze them, they become nonoxidatively modified. Whereas oxidation of proteoglycan-bound apoB-100–containing particles triggers their release and subsequent uptake by macrophages, the nonoxidative modifications do the opposite: they enhance the strength of particle binding to proteoglycans.3 In addition, such nonoxidatively modified apoB-100–containing lipoprotein particles tend to aggregate and fuse, which allows progressive extracellular accumulation of lipoprotein lipids, a key phenomenon in atherogenesis. The nonoxidative modifications can also lead to intracellular lipid accumulation and so induce foam cell formation, a hallmark of early atherogenesis.4 Thus, local modification of apoB-100–containing lipoproteins leads to both extra- and intracellular accumulation of lipids in the arterial intima. See page 600 In general, the nonoxidative modifications are caused by enzymes that are hydrolytic in nature, either proteolytic or lipolytic. Indeed, the arterial intima contains many different types of hydrolytic enzymes capable of lipoprotein modification. Unfortunately, however, it is currently not known which hydrolytic enzymes of the human arterial intima are actually responsible for turning the intimal lipoproteins into atherogenic particles. Among the lipolytic enzymes potentially involved in such atherogenic conversion of lipoproteins are several members of the secretory phospholipase A2 (sPLA2) family. The hydrolytic action of these enzymes on phosphatidylcholine, the major glycerophospholipid in lipoprotein particles, leads to the generation of lysophosphatidylcholine (lysoPC) and a free fatty acid (FFA). In the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Bostrom et al5 have examined in vivo the role of Group V sPLA2 (sPLA2-V) in the context of atherogenesis. …

2602: Low Dose of Radiation (3 Gy) Initiates an Immediate Signal Transduction Through Lysophosphatidylcholine (LPC), a Novel Second Messenger Generated by Cytosolic Phospholipase A2, Which Regulates Response in Vascular Endothelium

Ionizing radiation is a widely used and effective treatment of cancer. However, clinically relevant low doses of radiation trigger activation of survival pathways in tumor vasculature, which could cause decreased therapeutic ratio of ionizing radiation. Therefore, combination of radiotherapy with the drugs targeting vascular endothelium is an effective way to enhance sensitivity to ionizing radiation. The purpose of this research is to identify the initial signal transduction pathway that is triggered by 3 Gy within vascular endothelium. Growth factor-independent activation of cell viability occurs within endothelial cells treated with 1–3 Gy. We identified the immediate signaling events that could prevent this activation and provide novel molecular therapeutic targets for tumor sensitization to radiotherapy. Human Umbilical Vein Endothelial Cells (HUVEC), employed as a model cell system for vascular endothelium, were irradiated with 3 Gy. Inhibitors of different phospholipase A2 (PLA2) family members (cytosolic, Ca2+-independent and secretory) were used to study effect of radiation on PLA2 activity and subsequent cellular responses. Enzymatic activity of PLA2 was measured using PLA2 activity kit (Cayman). To evaluate PLA2 involvement in radiation-dependent activation of PI3K/Akt and ERK1/2 pathways, cells were lysed 2–30 minutes after beginning of irradiation and subjected to Western blot analysis with phospho-specific antibodies to Akt (Thr308/Ser473) and ERK1/2 (Thr202/Tyr204). Assays for clonogenic survival, apoptosis, tubule formation in matrigel and endothelial cell migration were used to study the role of cytosolic PLA2 in endothelial cell function in response to low doses of irradiation. In HUVEC, Akt and ERK1/2 are phosphorylated as early as 3 minutes after irradiation with 3 Gy. We detected that shortly before this phosphorylation, cytosolic phospholipase A2 (cPLA2) is activated and the second messenger LPC is mobilized. Inhibitors of cytosolic phospholipase A2, but not the secretory PLA2, added prior to irradiation markedly decreased activation of Akt and ERK1/2, suggesting cPLA2 involvement in radiation-induced activation of these kinases. Moreover, inhibition of cPLA2 significantly enhanced radiation-induced cell death in HUVEC as shown by clonogenic survival assay. This enhancement appears to be due to increased apoptosis and mitotic catastrophe. We also demonstrated that inhibition cPLA2 activity affected endothelial functions in irradiated HUVEC. When compared to radiation alone, in cells treated with cPLA2 inhibitors before irradiation, we observed decreased endothelial tubule formation in matrigel and reduced cell migration. We identified activation of cytosolic PLA2 as one of the immediate molecular events occurring in irradiated vascular endothelium. We also demonstrated that following ionizing radiation exposure, cPLA2 contributes to endothelial cell survival and functions through LPC production and consecutive regulation of Akt and ERK1/2 pathways. Therefore, cPLA2 signaling pathway could present new molecular targets for the tumor sensitization to radiotherapy.

Inflammation and Atherosclerosis

The secretory phospholipase A2 (sPLA2) family hydrolyzes glycerophospholipids at the sn-2 position to generate lysophospholipids and free fatty acids. Ten members have been described in mammals. These enzymes must be clearly distinguished from the intracellular phospholipase A2s that are involved in intracellular signaling. They are also distinct from lipoprotein-associated phospholipase A2 (Lp-PLA2), which has been shown to be bound predominantly to LDL particles in human plasma, and hydrolyzes oxidized LDL to generate lysophosphatidylcholine and oxidized nonesterified fatty acids.1 Group IIa sPLA2, which was originally isolated from rheumatoid arthritis fluids, is considered to be the prototypic inflammatory sPLA2. Its expression is markedly elevated in many cell types in response to proinflammatory stimuli. The concentration of Group IIa sPLA2 in sera or exudating fluids correlates well with the severity of inflammatory disease. In humans, Group IIa and Group V sPLA2 are tightly linked on chromosome 1 and their gene regulation and functions have largely been viewed as overlapping. Indeed, cross-reactivity of antibodies and molecular probes for these related enzymes has led to confusion in older literature. The study by Rosengren et al, reported in this issue, provides new insights into the expression and functional activity of Group IIa and Group V sPLA2 in the context of atherosclerosis.2 See page 1579 A role for sPLA2s in atherogenic processes has been suggested by epidemiological studies showing that in humans, serum concentrations of Group IIa …

Inactivation of NADPH oxidase organizer 1 Results in Severe Imbalance

Otoconia are biominerals of the vestibular system that are indispensable for the perception of gravity. Despite their importance, the process of otoconia genesis is largely unknown. Reactive oxygen species (ROS) have been recognized for their toxic effects in antimicrobial host defense as well as in aging and carcinogenesis. Enzymes evolved for ROS production belong to the recently discovered NADPH oxidase (Nox) enzyme family . Here we show that the inactivation of a regulatory subunit, NADPH oxidase organizer 1 (Noxo1), resulted in the severe balance deficit seen in the spontaneous mutant "head slant" (hslt) mice whose phenotype was rescued by Noxo1 transgenes. Wild-type Noxo1 was expressed in the vestibular and cochlear epithelia and was required for ROS production by an oxidase complex. In contrast, the hslt mutation of Noxo1 was biochemically inactive and led to an arrest of otoconia genesis, characterized by a complete lack of calcium carbonate mineralization and an accumulation of otoconial protein, otoconin-90/95 (OC-90/95). These results suggest that ROS generated by a Noxo1-dependent vestibular oxidase are critical for otoconia formation and may be required for interactions among otoconial components. Noxo1 mutants implicate a constructive developmental role for ROS, in contrast to their previously described toxic effects.

Possible roles of S···O and S···N interactions in the functions and evolution of phospholipase A2.

To investigate possible roles of S···X (X= O, N, S) interactions in the functions and evolution of a protein, two types of database analyses were carried out for a vertebrate phospholipase A2 (PLA2) family. A comprehensive search for close S···X contacts in the structures retrieved from protein data bank (PDB) revealed that there are four common S···O interactions and one common S···N interaction for the PLA2 domain group (PLA2-DG), while an additional three S···O interactions were found for the snake PLA2 domain group (sPLA2-DG). On the other hand, a phylogenetic analysis on the conservation of the observed S···O and S···N interactions over various amino acid sequences of sPLA2-DG demonstrated probable clustering of the interactions on the dendrogram. Most of the interactions characterized for PLA2 were found to reside in the vicinity of the active site and to be able to tolerate the conformational changes due to the substrate binding. These observations suggested that the S···X interactions play some role in the functions and evolution of the PLA2 family.