Isozymes inhibited by active site blocking: versatility of calcium indifferent hesperidin binding to phospholipase A2 and its significance.

sPLA2 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 sPLA2. Affinity of hesperidin towards PLA2 isozymes was analyzed through enzymatic studies and molecular modeling. The experiments showed that hesperidin competitively inhibited PLA2 with IC50 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 PLA2 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 PLA2 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 PLA2 isozyme specific to various diseases, with IC50 value of therapeutic significance.

New pyrimidine-benzoxazole/benzimidazole hybrids: Synthesis, antioxidant, cytotoxic activity, in vitro cyclooxygenase and phospholipase A2-V inhibition.

To enhance the cytotoxicity of benzimidazole and/or benzoxazole core, the benzimidazole/benzoxazole azo-pyrimidine were synthesized through diazo-coupling of 3-aminophenybenzimidazole (6a) or 3-aminophenylbenzoxazole (6b) with diethyl malonate. The new azo-molanates 6a&b mixed with urea in sodium ethoxide to afford the benzimidazolo/benzoxazolopyrimidine 7a&b. The structure elucidation of new synthesized targets was proved using spectroscopic techniques NMR, IR and elemental analysis. The cytoxicity screening had been carried out against five cancer cell lines: prostate cancer (PC-3), lung cancer (A-549), breast cancer (MCF-7), pancreas cancer (PaCa-2) and colon cancer (HT-29). Furthermore, the antioxidant activity, phospholipase A2-V and cyclooxygenases inhibitory activities of the target compounds 7a&b were evaluated and the new compounds showed potent activity (cytotoxicity IC50 range from 4.3 to 9.2 µm, antioxidant activity from 40% to 80%, COXs or LOX inhibitory activity from 1.92 µM to 8.21 µM). The docking of 7a&b was made to confirm the mechanism of action.

Specific roles for Group V secretory PLA2 in retinal iron-induced oxidative stress. Implications for age-related macular degeneration.

Iron accumulation and oxidative stress are hallmarks of retinas from patients with age-related macular degeneration (AMD). We have previously demonstrated that iron-overloaded retinas are a good in vitro model for the study of retinal degeneration during iron-induced oxidative stress. In this model we have previously characterized the role of cytosolic phospholipase A2 (cPLA2) and calcium-independent isoform (iPLA2). The aim of the present study was to analyze the implications of Group V secretory PLA2 (sPLA2), another member of PLA2 family, in cyclooxygenase (COX)-2 and nuclear factor kappa B (NF-κB) regulation. We found that sPLA2 is localized in cytosolic fraction in an iron concentration-dependent manner. By immunoprecipitation (IP) assays we also demonstrated an increased association between Group V sPLA2 and COX-2 in retinas exposed to iron overload. However, COX-2 activity in IP assays was observed to decrease in spite of the increased protein levels observed. p65 (RelA) NF-κB levels were increased in nuclear fractions from retinas exposed to iron. In the presence of ATK (cPLA2 inhibitor) and YM 26734 (sPLA2 inhibitor), the nuclear localization of both p65 and p50 NF-κB subunits was restored to control levels in retinas exposed to iron-induced oxidative stress. Membrane repair mechanisms were also analyzed by studying the participation of acyltransferases in phospholipid remodeling during retinal oxidation stress. Acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylserine (PS), were observed to show an inhibited acylation profile in retinas exposed to iron while phosphatidylethanolamine (PE) showed the opposite. The use of PLA2 inhibitors demonstrated that PS is actively deacylated during iron-induced oxidative stress. Results from the present study suggest that Group V sPLA2 has multiple intracellular targets during iron-induced retinal degeneration and that the specific role of sPLA2 could be related to inflammatory responses by its participation in NF-κB and COX-2 regulation.

Coordinate regulation of TLR-mediated arachidonic acid mobilization in macrophages by group IVA and group V phospholipase A2s.

Macrophages can be activated through TLRs for a variety of innate immune responses. In contrast with the wealth of data existing on TLR-dependent gene expression and resultant cytokine production, very little is known on the mechanisms governing TLR-mediated arachidonic acid (AA) mobilization and subsequent eicosanoid production. We have previously reported the involvement of both cytosolic group IVA phospholipase A(2) (cPLA(2)) and secreted group V phospholipase A(2) (sPLA(2)-V) in regulating the AA mobilization response of macrophages exposed to bacterial LPS, a TLR4 agonist. In the present study, we have used multiple TLR agonists to define the role of various PLA(2)s in macrophage AA release via TLRs. Activation of P388D(1) and RAW2647.1 macrophage-like cells via TLR1/2, TLR2, TLR3, TLR4, TLR6/2, and TLR7, but not TLR5 or TLR9, resulted in AA mobilization that appears to involve the activation of both cPLA(2) and sPLA(2) but not of calcium-independent phospholipase A(2). Furthermore, inhibition of sPLA(2)-V by RNA interference or by two cell-permeable compounds, namely scalaradial and manoalide, resulted in a marked reduction of the phosphorylation of ERK1/2 and cPLA(2) via TLR1/2, TLR2, TLR3, and TLR4, leading to attenuated AA mobilization. Collectively, the results suggest a model whereby sPLA(2)-V contributes to the macrophage AA mobilization response via various TLRs by amplifying cPLA(2) activation through the ERK1/2 phosphorylation cascade.

Group V secreted phospholipase A2 contributes to LPS-induced leukocyte recruitment.

Secreted phospholipases A(2) (sPLA(2)s) are well known for their contribution in the biosynthesis of inflammatory eicosanoids. These enzymes also participate in the inflammatory process by regulating chemokine production and protein expression of adhesion molecules. The majority of sPLA(2) isoforms are up-regulated by proinflammatory stimuli such as bacterial lipopolysaccharide (LPS), which predominantly increases the expression of group V sPLA(2) (sPLA(2)-V). Furthermore, it has recently been shown that sPLA(2)-V is a critical messenger in the regulation of cell migration during allergic airway responsiveness. Herein, we investigated the effect of sPLA(2)-V on LPS-mediated leukocyte recruitment and its capacity to modulate adhesion molecule expression. We conducted our study in the murine air pouch model, using sPLA(2)-V null mice (sPLA(2)-V(-/-)) and control wild-type (WT) littermates. We observed that LPS (1 microg/ml)-mediated leukocyte emigration in sPLA(2)-V(-/-) was attenuated by 52% and 86% upon 6 and 12 h of treatment respectively, as compared to WT mice. In WT mice, treatment with the cell-permeable sPLA(2) inhibitor (12-epi-scalaradial; SLD) reduced LPS-mediated leukocyte recruitment by 67%, but had no additional inhibitory effect in sPLA(2)-V(-/-) mice. Protein analyses from the air pouch skin were carried out upon LPS-challenge, and the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were both significantly reduced in sPLA(2)-V(-/-) mice as compared to control WT mice. Together, our data demonstrate the role of sPLA(2)-V in LPS-induced ICAM-1 and VCAM-1 protein overexpression and leukocyte recruitment, supporting the contribution of sPLA(2)-V in the development of inflammatory innate immune responses.

The presence of a secretory phospholipase A2 in the nuclei of neuronal and glial cells of rat brain cortex.

Confocal immunofluorescence analysis indicated a relatively high localization of group V secretory phospholipase A(2) (GV) in the nuclei of cultured PC12 and U251 astrocytoma cells. Here, we report the biochemical evidence for the presence of a secretory PLA(2) in the nuclei of neuronal and glial cells from rat brain cortex. Enzymic activity was determined using [(3)H]oleate labelled Escherichia coli membranes in intact nuclei and in their soluble fractions in which the specific activity was significantly more elevated. The treatment of soluble nuclear fractions with inhibitors of cytosolic Ca(2+)-dependent or Ca(2+)-independent phospholipases A(2) was ineffective whereas DTT or Indoxam, a specific inhibitor of all isoforms of sPLA(2), abolished enzyme activity. The enzyme was identified as group V secretory phospholipase A(2) (GV) by Western blot analysis and its nucleoplasmic localization was demonstrated by CLSM.

Group V secretory phospholipase A2 impairs endothelial protein C receptor-dependent protein C activation and accelerates thrombosis in vivo.

BACKGROUND: Endothelial protein C receptor (EPCR) must be bound to a molecule of phosphatidylcholine (PC) to be fully functional, i.e. to interact with protein C/activated protein C (APC) properly. PC can be replaced with other lipids, such as lysophosphatidylcholine or platelet-activating factor, by the action of group V secretory phospholipase A2 (sPLA2-V), an enzyme that is upregulated in a variety of inflammatory conditions. Studies in purified systems have demonstrated that the substitution of PC notably impairs EPCR function in a process called EPCR encryption. OBJECTIVES: To analyze whether sPLA2-V was able to regulate EPCR-dependent protein C activation in vivo, and its impact on thrombosis and the hemostatic system. METHODS: Mice were transfected with sPLA2-V by hydrodynamic gene delivery. The effects on thrombosis were studied with the laser carotid artery occlusion model, and APC generation capacity was measured with ELISA. Global hemostasis was analyzed with thromboelastometry. RESULTS: We found that sPLA2-V overexpression in mice significantly decreased their ability to generate APC. Furthermore, a murine carotid artery laser thrombosis model revealed that higher sPLA2-V levels were directly associated with faster artery thrombosis. CONCLUSIONS: sPLA2-V plays a thrombogenic role by impairing the ability of EPCR to promote protein C activation.

Group V secretory phospholipase A2 plays a pathogenic role in myocardial ischaemia-reperfusion injury.

AIMS: Group V secretory phospholipase A(2) (sPLA(2)-V) is highly expressed in the heart. This study examined (i) the role of sPLA(2)-V in myocardial ischaemia-reperfusion (I/R) injury and (ii) the cooperative action of sPLA(2)-V and cytosolic PLA(2) (cPLA(2)) in myocardial I/R injury, using sPLA(2)-V knockout (sPLA(2)V(-/-)) mice. METHODS AND RESULTS: Myocardial I/R injury was created by 1 h ligation of the left anterior descending coronary artery, followed by 24 h of reperfusion. The sPLA(2)V(-/-) mice had a 44% decrease in myocardial infarct size, a preservation of echocardiographic LV function (%fractional shortening: 40 ± 3.5 vs. 21 ± 4.6, respectively), and lower content of leucotriene B(4) (LTB(4)) and thromboxane B(2) (TXB(2)) (40 and 37% lower, respectively) in the ischaemic myocardium after I/R compared with wild-type (WT) mice. Intraperitoneal administration of AACOCF3 or MAFP, inhibitors of cPLA(2) activity, decreased myocardial infarct size and myocardial content of LTB(4) and TXB(2) in both genotyped mice. The decrease in myocardial infarct size and content of LTB(4) and TXB(2) after cPLA(2) inhibitor administration was greater in WT mice than in sPLA(2)V(-/-) mice. I/R increased phosphorylation of extracellular signal-related kinase 1/2, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in the ischaemic myocardium in association with cPLA(2) phosphorylation. The I/R-induced increase in the phosphorylation of p38 and cPLA(2) was less in sPLA(2)-V(-/-) mice than in WT mice. Pretreatment with the p38 inhibitor SB202190 suppressed an increase in cPLA(2) phosphorylation after I/R in WT mice. CONCLUSION: sPLA(2)-V plays an important role in the pathogenesis of myocardial I/R injury partly in concert with the activation of cPLA(2).