sPLA2 Activity Research Articles

Surfactant and Varespladib Co-Administration in Stimulated Rat Alveolar Macrophages Culture

Acute lung injury is a life-threatening condition characterized by surfactant dysfunction and raised secretory phospholipase A2 (sPLA2) activity. Varespladib is a sPLA2 inhibitor shown to be effective in animal models of acute lung injury. We aimed at investigating the effect of co-administration of surfactant and varespladib on sPLA2 activity. Alveolar macrophages were cultured and stimulated with lipopolysaccharide and then treated with either varespladib, surfactant, varespladib followed by surfactant or nothing. sPLA2 activity, free fatty acids, tumour necrosis factor-α (TNF-α) and protein concentrations were measured in culture supernatants. Treatment with varespladib (p=0.019) and varespladib + surfactant (p=0.013), reduced the enzyme activity by approximately 15% from the basal level measured in the untreated cultures. Surfactant, varespladib and varespladib + surfactant, respectively decreased free fatty acids by -45% (p=0.045), - 62% (p=0.009) and -48% (p=0.015), from the baseline concentration of the untreated cultures. Varespladib and poractant- α co-administration reduces sPLA2 activity and free fatty acids release in cultured rat alveolar macrophages, although a clear drug synergy was not evident. Since co-administration may be useful to reduce inflammation and surfactant inactivation in acute lung injury, further in vivo studies are warranted to verify its clinical usefulness.

Oral administration of anti‐sPLA2‐IB antibodies reduces sepsis mortality in murine endotoxemia

Annually 750,000 people develop severe sepsis and 215,000 die at a cost of more than $28 billion. Evidence suggests sepsis progresses when bacteria translocate across a compromised intestinal epithelial barrier. Secretory intestinal phospholipase (sPLA2‐IB) may play an important role in regulation of gutmediated inflammation. The objective of this study was to demonstrate reduced sepsis mortality by oral administration of antibodies against sPLA2‐IB. We made peptide antibodies to ten 8 amino acid sequences of the sPLA2‐IB molecule and tested their efficacy in inhibiting sPLA2 enzyme activity and in reducing endotoxin (LPS) induced TNF production in RAW264.7 macrophages in vitro. Three antibodies inhibited sPLA2 activity and four antibodies decreased LPS induced TNFα by more than 50%. We orally administered these antibodies to female Balb/c mice given 10mg/kg LPS. Mice fed antibody that inhibited TNF (anti‐ TYSYSCSG) had 100% endotoxin‐induced mortality while antibody that inhibited PLA2 activity (anti‐VPYNKEYK) had 0% mortality and mice receiving no oral antibody had 45% mortality. Antibody #6 is so specific that changing the primary sequence target by 4 amino acids in either the amino or carboxyl direction makes it ineffective. In summary, oral administration of an antibody that inhibits sPLA2‐IB enzyme activity prevents sepsis related mortality caused by endotoxemia.Research supported by a Draper Technology Innovation Fund Grant.

Role of endogenous psychosine accumulation in oligodendrocyte differentiation and survival: Implication for Krabbe disease

Krabbe disease is a lethal, demyelinating condition caused by genetic deficiency of galactocerebrosidase (GALC) and resultant accumulation of its cytotoxic substrate, psychosine (galactosylsphingosine), primarily in oligodendrocytes (OLs). Psychosine is generated by galactosylation of sphingosine by UDP-galactose:ceramide galactosyltransferase (CGT), a galactosylceramide synthesizing enzyme which is primarily expressed in OLs. The expression of CGT and the synthesis of galactosyl-sphingolipids are associated with the terminal differentiation of OL, but little is known about the participation of endogenous psychosine accumulation in OL differentiation under GALC deficient conditions. In this study, we report that accumulation of endogenous psychosine under GALC deficient Krabbe conditions impedes OL differentiation process both by decreasing the expression of myelin lipids and protein and by inducing the cell death of maturating OLs. The psychosine pathology under GALC deficient conditions involves participation of secretory phospholipase A2 (sPLA2) activation and increase in its metabolites, as evidenced by attenuation of psychosine-induced pathology by treatment with pharmacological inhibitor of sPLA2 7,7-dimethyleicosadienoic acid (DEDA). These observations suggest for potential therapeutic efficacy of sPLA2 inhibitor in Krabbe disease.

Secretory Phospholipase A2 in Dromedary Tears: a Host Defense Against Staphylococci and Other Gram-Positive Bacteria

The best known physiologic function of secreted phospholipase A2 (sPLA2) group IIA (sPLA2-IIA) is defense against bacterial infection through hydrolytic degradation of bacterial membrane phospholipids. In fact, sPLA2-IIA effectively kills Gram-positive bacteria and to a lesser extent Gram-negative bacteria and is considered a major component of the eye's innate immune defense system. The antibacterial properties of sPLA2 have been demonstrated in rabbit and human tears. In this report, we have analyzed the bactericidal activity of dromedary tears and the subsequently purified sPLA2 on several Gram-positive bacteria. Our results showed that the sPLA2 displays a potent bactericidal activity against all the tested bacteria particularly against the Staphylococcus strains when tested in the ionic environment of tears. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2. Interestingly, lysozyme purified from dromedary tears showed a significant bactericidal activity against Listeria monocytogene and Staphylococcus epidermidis, whereas the one purified from human tears displayed no activity against these two strains. We have also demonstrated that Ca(2+) is crucial for the activity of dromedary tear sPLA2 and to a less extent Mg(2+) ions. Given the presence of sPLA2 in tears and intestinal secretions, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against Gram-positive bacteria.

Effect of Some Divalent Metal Ions on Enzymatic Activity of Secreted Phospholipase A2 (sPLA2) Isolated from Bulgarian Vipera Ammodytes Meridionalis

ABSTRACTThe effect of Ca2+ and some divalent metal ions related to it (Mg, Sr, Ba, Cd) on the hydrolysis of natural and artificial substrates catalyzed by vipoxin secreted phospholipase A2 subunit (sPLA2) was evaluated. The results showed that the hydrolysis of natural glycerophospholipids proceeds at a highest rate when the enzyme is activated by calcium ions. The catalytic activity of sPLA2 decreased in the presence of other metal ions possibly due to their lower coordination ability to the head group of lipids. Oppositely, the hydrolysis of artificial substrates was facilitated by metal ions with ionic radii larger than Ca2+, such as Sr2+ and Ba2+, suggesting that not only the interaction with metal cations, but also the origin of the head group should be considered. The fluorescence assay revealed that conformational changes occur during the coordination of metal ions into the catalytic site of sPLA2 prior to subsequent hydrolysis. It could be concluded that the rate-limiting step in the catalytic cycle (e.g. the chemical interaction) is not completely adequate to evaluate all factors affecting the hydrolytic activity of vipoxin sPLA2.

Atividades de fosfolipase A2 secretada e glutationa peroxidase em filés PSE (pale, soft, exudative) de frango

The excessive release of calcium from the sarcoplasmic reticulum during the installation of PSE (Pale, Soft, Exudative) meat, leads to increase of phospholipase A2 (PLA2) activity and consequently causes a higher lipid oxidation. In contrast, glutathione peroxidase (GSH-Px) is a selenium-dependent antioxidant enzyme that prevents the oxidative damage. The aim of this work was to investigate the secreted PLA2 (sPLA2) and GSH-PX activities in PSE poultry meat. Breast meat samples (Pectoralis major m.) (n=24) were obtained from commercial slaughterhouse. Samples were classified as PSE and Control Meat based on pH and L* values, fillets with pH1h30? 6.0 and L24h* ?53.0 as PSE and fillets with pH1h30> 6.0 and L24h*<53.0 with Control. sPLA2 was analyzed using enzymatic assay kit and GSH-Px was measured a coupled assay procedure recording the NADPH (nicotinamide adenine dinucleotide phosphate reduced) oxidation. The sPLA2 average was 0.295 Umg-1 of protein for PSE meat and 0.380 Umg-1 of protein for Control meat, did not differ significantly (p >0,05). The GSH-Px activity was approximately 24,4% higher (p?0,05) for Control meat when compared to PSE meat. The sPLA2 activity of PSE fillets did not changed, however PSE fillets present the enzymatic system of antioxidant defense compromised with lower GSH-PX activity.

Atividades de fosfolipase A2 secretada e glutationa peroxidase em filés PSE (pale, soft, exudative) de frango

The excessive release of calcium from the sarcoplasmic reticulum during the installation of PSE (Pale, Soft, Exudative) meat, leads to increase of phospholipase A2 (PLA2) activity and consequently causes a higher lipid oxidation. In contrast, glutathione peroxidase (GSH-Px) is a selenium-dependent antioxidant enzyme that prevents the oxidative damage. The aim of this work was to investigate the secreted PLA2 (sPLA2) and GSH-PX activities in PSE poultry meat. Breast meat samples ( Pectoralis major m. ) (n=24) were obtained from commercial slaughterhouse. Samples were classified as PSE and Control Meat based on pH and L* values, fillets with pH1h30? 6.0 and L24h* ?53.0 as PSE and fillets with pH1h30> 6.0 and L24h* 0,05). The GSH-Px activity was approximately 24,4% higher (p?0,05) for Control meat when compared to PSE meat. The sPLA2 activity of PSE fillets did not changed, however PSE fillets present the enzymatic system of antioxidant defense compromised with lower GSH-PX activity.

Ammodytoxins efficiently release arachidonic acid and induce apoptosis in a motoneuronal cell line in an enzymatic activity-dependent manner

Secreted phospholipases A2 (sPLA2s) are phospholipolytic enzymes and receptor ligands whose action affects cell death and survival. We have previously shown that ammodytoxin A (AtxA), a snake venom sPLA2, is rapidly internalized into motoneuronal NSC34 cells, inducing characteristic neurotoxic sPLA2 cell damage and apoptosis. In this study, we have analyzed the role of sPLA2 enzymatic activity, including arachidonic acid (AA) release, in the induction of motoneuronal apoptosis by AtxA and homologous recombinant sPLA2s with different enzymatic properties: an AtxA mutant (V31W) with very high enzymatic activity, enzymatically inactive S49-sPLA2 (ammodytin L, AtnL), its mutant (LW) with restored enzymatic activity, and non-toxic, enzymatically active sPLA2 (AtnI2). Addition of AA, AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, to NSC34 cells resulted in caspase-3 activation, DNA fragmentation and disruption of mitochondrial membrane potential, leading to a significant and rapid decrease in motoneuronal cell viability that was not observed in C2C12 myoblasts and HEK293 cells. AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, also liberated large amounts of AA specifically from motoneuronal cells, and this ability correlated well with the ability to induce apoptotic changes and decrease cell viability. The enzymatic activity of AtxA and similar sPLA2s is thus necessary, but not sufficient, for inducing motoneuronal apoptosis. This suggests that specific binding to the motoneuronal cell surface, followed by internalization and enzymatic activity-dependent induction of apoptosis, possibly as a consequence of extensive extra- and intracellular AA release, is necessary for Atx-induced motoneuronal cell death.

Genetic ontogeny of pancreatic enzymes in Labrus bergylta larvae and the effect of feed type on enzyme activities and gene expression

A newly cultivated wrasse species, Labrus bergylta, have shown great potential for use in Atlantic salmon (Salmo salar) farms in the battle against sea lice (Lepeoptheirus salmonis) infections. Hatchery reared L. bergylta were studied from 2 to 55 DPH to examine the molecular basis of digestive ontogeny related to the pancreas. An isolated feeding trial was performed on 27-34 DPH larvae to compare the effect of diet on enzyme activity and the possible exogenous contribution by live feed. The following genes coding for key pancreatic enzymes were analyzed by qPCR: trypsin, Cyp7 A1, BAL, sPLA(2) 1B, amylase and pancreatic chitinase. Enzyme activity was measured on trypsin, neutral lipase, sPLA(2), amylase and chitinase in fed and unfed larvae. We did not observe any effects of the formulated diet v.s. rotifers on enzyme activities of neutral lipase, chitinase and sPLA(2). However, a probable feed-dependency was observed at a transcriptional level, where rotifers seem to stimulate upregulation. The regulation of BAL was the only exception, where an upregulation was observed after weaning both in the ontogeny series and the experimental part. Our data on pancreatic chitinase and amylase mRNA levels suggest the importance of carbohydrates in the diet of early larval and juvenile L. bergylta.

Ex Vivo Effect of Varespladib on Secretory Phospholipase A2 Alveolar Activity in Infants with ARDS

BackgroundSecretory phospholipase A2 (sPLA2) plays a pivotal role in acute respiratory distress syndrome (ARDS). This enzyme seems an interesting target to reduce surfactant catabolism and lung tissue inflammation. Varespladib is a specifically designed indolic sPLA2 inhibitor, which has shown promising results in animals and adults. No specific data in pediatric ARDS patients are yet available.MethodsWe studied varespladib in broncho-alveolar lavage (BAL) fluids obtained ex vivo from pediatric ARDS patients. Clinical data and worst gas exchange values during the ARDS course were recorded. Samples were treated with saline or 10–40–100 µM varespladib and incubated at 37°C. Total sPLA2 activity was measured by non-radioactive method. BAL samples were subjected to western blotting to identify the main sPLA isotypes with different sensitivity to varespladib. Results was corrected for lavage dilution using the serum-to-BAL urea ratio and for varespladib absorbance.ResultsVarespladib reduces sPLA2 activity (p<0.0001) at 10,40 and 100 µM; both sPLA2 activity reduction and its ratio to total proteins significantly raise with increasing varespladib concentrations (p<0.001). IC50 was 80 µM. Western blotting revealed the presence of sPLA2-IIA and –IB isotypes in BAL samples. Significant correlations exist between the sPLA2 activity reduction/proteins ratio and PaO2 (rho = 0.63;p<0.001), PaO2/FiO2 (rho = 0.7; p<0.001), oxygenation (rho = −0.6; p<0.001) and ventilation (rho = −0.4;p = 0.038) indexes.ConclusionsVarespladib significantly inhibits sPLA2 in BAL of infants affected by post-neonatal ARDS. Inhibition seems to be inversely related to the severity of gas exchange impairment.

58 Effect of Varespladib-Protected Surfactant in Cultured Rat Alveolar Macrophages Stimulated with LPS

BackgroundSecretory phospholipase A2 (sPLA2) is a crucial enzyme for inflammatory response and surfactant catabolism. Acute lung injury (ALI) is a life-threating syndrome characterized by surfactant dysfunction and raised levels of...

402 Effect of Antenatal Corticosteroids in Activity and Expression of Secretory Phospholipase A2 and TNF Alfa in Lung of Newborn Rats

IntroductionThe sPLA2 plays an important role in the development of acute respiratory distress syndrome. It is regulated by many factors including steroids and TNFa. Antenatal corticosteroids are recommended for preventing...

Cloning and functional expression of secreted phospholipases A2 from Bothrops diporus (Yarará Chica)

Bothrops diporus is a very common viper in Argentina. At present, no complete sequence of secreted phospholipase A2 (sPLA2) from this snake has been reported. We have cloned two sPLA2 isoenzymes as well as a putative sPLA2-like myotoxin from venom gland. The two sPLA2 were expressed as inclusion bodies in Escherichia coli with an N-terminal tag of ubiquitin. After in vitro renaturation and cleavage step, using an ubiquitin specific peptidase, the recombinants exhibited sPLA2 activity when analyzed by means of Langmuir dilauroylphosphatidylcholine monolayers as substrate. Both enzymes have a similar surface pressure-activity profile when compared with non-recombinant purified isoforms. To our knowledge, this is the first time that analysis of optimal lateral pressure of substrate monolayers by using the surface barostat technique is performed on recombinant sPLA2s.

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Inhibition of cPLA2 and sPLA2 Activities in Primary Cultures of Rat Cortical Neurons by Centella asiatica Water Extract

Leaf extract of Centella asiatica has been used as an alternative medicine for memory improvement in the Indian Ayurvedic system of medicine for a long time. Although several studies have revealed its effect in ameliorating the cognitive impairment in rat models of Alzheimer's disease, the molecular mechanism of C. asiatica on neuroprotection still remains unexplained. In this study, we investigated the effects of C. asiatica water extract on activity of subtypes of phospholipase A2 (PLA2) in primary cultures of rat cortical neurons and quantified by HPLC a possible molecule responsible for the activity. The cPLA2 and sPLA2 activities were inhibited in vitro by asiaticoside present in the water extract of C. asiatica. This extract may be a candidate for the treatment of neurodegenerative processes because of its pharmacological activity in the brain and its low toxicity, as attested by its long popular use as a natural product.

Circulating levels of secretory- and lipoprotein-associated phospholipase A2 activities: relation to atherosclerotic plaques and future all-cause mortality

Secretory- and lipoprotein-associated phospholipases A2 (sPLA2 and Lp-PLA2) are enzymes both suggested to be of importance for atherosclerosis. We investigated relationships between the activities of these enzymes in the circulation and atherosclerosis as well as future clinical events. The population-based Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study included 1016 randomly selected subjects, all aged 70. The prevalence of carotid artery plaques was recorded by ultrasound (n= 954), and arterial stenosis was assessed by whole-body magnetic resonance angiography (WBMRA, n= 302). Secretory-associated phospholipase A2 [odds ratio 1.23 for 1 SD increase, 95% confidence interval (CI): 1.05-1.44, P= 0.007], but not Lp-PLA2 (P= 0.26), activity was significantly related to carotid atherosclerosis and to the amount of stenosis at WBMRA (P= 0.006) following adjustment for multiple risk factors (waist circumference, serum triglycerides, body mass index, C-reactive protein, high density lipoprotein-C, low density lipoprotein-C, triglycerides, GFR, fasting glucose, blood pressure, statin use, and exercise habits). Secretory-associated phospholipase A2 [hazard ratio (HR) 1.45 for 1 SD increase, 95% CI: 1.15-1.84, P= 0.001], but not Lp-PLA2 (HR 0.95, P= 0.55), activity was a significant risk factor for all-cause mortality (114 had died) during 7.0 years follow-up after adjustment for the risk factors described above. In a sample of 1029 post-myocardial infarction (MI) patients (French registry of Acute ST-elevation and non-ST-elevation Myocardial Infarction), sPLA2 (adjusted HR 1.32 for 1 unit increase, 95% CI: 1.02-1.71, P= 0.036), but not Lp-PLA2 (HR 1.03, P= 0.90), activity predicted death or recurrent MI during 1-year follow-up (n= 136 cases). sPLA2 activity was related to atherosclerosis and predicted all-cause mortality in a sample of elderly subjects, as well as death or MI in post-MI patients.

Acute impact of apheresis on oxidized phospholipids in patients with familial hypercholesterolemia

We measured oxidized phospholipids (OxPL), lipoprotein (a) [Lp(a)], and lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) pre- and postapheresis in 18 patients with familial hypercholesterolemia (FH) and with low(∼10 mg/dl; range 10-11 mg/dl), intermediate (∼50 mg/dl; range 30-61 mg/dl), or high (>100 mg/dl; range 78-128 mg/dl) Lp(a) levels. By using enzymatic and immunoassays, the content of OxPL and Lp-PLA(2) mass and activity were quantitated in lipoprotein density fractions plated in microtiter wells, as well as directly on apoB-100, Lp(a), and apoA-I immunocaptured within each fraction (i.e., OxPL/apoB and Lp-PLA(2)/apoB). In whole fractions, OxPL was primarily detected in the Lp(a)-containing fractions, whereas Lp-PLA(2) was primarily detected in the small, dense LDL and light Lp(a) range. In lipoprotein capture assays, OxPL/apoB and OxPL/apo(a) increased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apoB and Lp-PLA(2)/apoA-I levels were highest in the low Lp(a) group but decreased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apo(a) was lowest in patients with low Lp(a) levels and increased proportionally with increasing Lp(a) levels. Apheresis significantly reduced levels of OxPL and Lp-PLA(2) on apoB and Lp(a) (50-75%), particularly in patients with intermediate and high Lp(a) levels. In contrast, apheresis increased Lp-PLA(2)-specific activity (activity/mass ratio) in buoyant LDL fractions. The impact of apheresis on Lp(a), OxPL, and Lp-PLA(2) provides insights into its therapeutic benefits beyond lowering apoB-containing lipoproteins.

Antioxidant and gastric cytoprotective prostaglandins properties of Cassia sieberiana roots bark extract as an anti-ulcerogenic agent

BackgroundCassia sieberiana is a savannah tree with a wide phytotherapeutic application including the use of its roots in the management of various stomach disorders including gastric ulcer, stomach pains and indigestion. The aim of the study is to evaluate the antioxidant, gastric cytoprotective prostaglandins, secretory phospholipase A2, phytochemical and acute toxicity properties of Cassia sieberiana roots bark extract in a bid to justify its phytotherapeutic applications in gastric ulcer.MethodsAntioxidant and radical scavenging activities of the roots bark extract of Cassia sieberiana were assayed. Serum secretory phospholipase A2 (sPLA2) concentration and activity and the formation of gastric mucosal prostaglandins E2 (PGE2) and I2 (PGI2) were also assessed. Comparisons between means were performed using analysis of variance (ANOVA) followed by Students Standard Newman-Keuls post hoc analysis to determine statistical significance. P < 0.05 was considered significant.ResultsThe extract was found to possess significant ferric reducing antioxidant power and can scavenge hydroxyl radicals. The extract also possesses DPPH scavenging activity, can chelate ferrous ion and a dose-dependent protective effect against lipid peroxidation and free radical generation. Prostaglandin studies showed that the roots bark extract dose dependently increased gastric mucosal PGE2 and PGI2 levels and also decreased serum sPLA2 activity. Phytochemical analyses suggest that the roots extract contains polyhydroxyl/phenolic substances. Acute toxicity test showed no sign of toxicity up to a dose level of 2000 mg/kg body weight p.o.ConclusionsC. sieberiana roots extract possesses significant antioxidant and gastric cytoprotective prostaglandin properties as well as serum secretory phospholipase A2 inhibitory activity which could be due to its content of polyhydroxy and/or phenolic substances. This may justify its use as an anti-ulcerogenic agent in traditional medicine in West Africa.

Varespladib Inhibits Secretory Phospholipase A2 in Bronchoalveolar Lavage of Different Types of Neonatal Lung Injury

Secretory phospholipase A2 (sPLA2), which links surfactant catabolism and lung inflammation, is associated with lung stiffness, surfactant dysfunction, and degree of respiratory support in acute respiratory distress syndrome and in some forms of neonatal lung injury. Varespladib potently inhibits sPLA2 in animal models. The authors investigate varespladib ex vivo efficacy in different forms of neonatal lung injury. Bronchoalveolar lavage fluid was obtained from 40 neonates affected by hyaline membrane disease, infections, or meconium aspiration and divided in 4 aliquots added with increasing varespladib or saline. sPLA2 activity, proteins, and albumin were measured. Dilution was corrected with the urea ratio. Varespladib was also tested in vitro against pancreatic sPLA2 mixed with different albumin concentration. Varespladib was able to inhibit sPLA2 in the types of neonatal lung injury investigated. sPLA2 activity was reduced in hyaline membrane disease (P < .0001), infections (P = .003), and meconium aspiration (P = .04) using 40 µM varespladib; 10 µM was able to lower enzyme activity (P = .001), with an IC(50) of 87 µM. An inverse relationship existed between protein level and activity reduction (r = 0.5; P = .029). The activity reduction/protein ratio tended to be higher in hyaline membrane disease. Varespladib efficacy was higher in vitro than in lavage fluids obtained from neonates (P < .001).

Escin attenuates cell migration induced with extracellular nucleotides by reducing sPLA2 activity

Escin attenuates cell migration induced with extracellular nucleotides by reducing sPLA2 activity