High-affinity aptamers selectively inhibit human nonpancreatic secretory phospholipase A2 (hnps-PLA2)

Evidence is growing that reactive oxygen species (ROS), by-products of (normal) cellular aerobic metabolism, are involved in the pathogenesis of neurodegenerative diseases. One of these diseases is amyotrophic lateral sclerosis (ALS), in which motoneurons die, leading to paralysis and death. It remains uncertain whether ROS are the cause of (apoptotic) motoneuron death in ALS. To further understand the role of ROS in motoneuron death, we investigated the effects of ROS on isolated spinal rat motoneurons in culture. ROS were generated with a combination of iron(III) and ascorbate, or with hydrogen peroxide. Both toxic treatments resulted in a dose-dependent motoneuron death. Iron(III)/ascorbate toxicity was completely prevented with the hydrogen peroxide detoxifying enzyme catalase and partially prevented with the antioxidant vitamin E. SOD1, the enzyme that removes superoxide, did not protect against iron(III)/ascorbate toxicity. ROS treatment caused apoptotic motoneuron death: low doses of iron(III)/ ascorbate or hydrogen peroxide resulted in complete apoptosis ending in nuclear fragmentation, while high doses of ROS resulted in incomplete apoptosis (nuclear condensation). Thus, depending on the dose of ROS, the motoneurons complete the apoptotic pathway (low dose) or are stopped somewhere during this route (high dose).     

Microtubule depolymerization selectively down-regulates the synthesis of proinflammatory secretory nonpancreatic phospholipase A2

Reports which analyzed the effects of secondary cytoreductive surgery at second-look laparotomy have often included small numbers of patients who have been treated with a variety of first-line chemotherapy regimens and those who may have progressed on first-line therapy. The purpose of this study was to analyze survival following secondary cytoreductive surgery at second-look laparotomy in patients with advanced ovarian cancer. Review of the surgical data of 153 patients allowed classification of tumor size found at second-look laparotomy and tumor size remaining after cytoreduction. Multivariate analysis evaluated multiple risk factors for survival. Of 153 patients, 124 had macroscopic tumor at second-look laparotomy and 29 had microscopic disease only. Fifteen of 69 (22%) patients were found to have tumor > 1 cm in diameter and were cytoreduced to microscopic residual and 18/69 (26%) were left with 1 cm tumor. Twenty-one of 55 (38%) patients with < or = 1 cm tumor were debulked to microscopic residual. The shortest survival relative to patients found to have microscopic disease at second-look laparotomy was observed among patients whose maximum tumor size remained > 1 cm following second-look laparotomy (relative risk = 3.1, P = 0.0004). No difference in survival was seen between patients found to have microscopic disease and those cytoreduced to microscopic disease (P = 0.24). The risk of death was lower among patients debulked to a lower category (< or = 1 cm debulked to microscopic, relative risk = 0.48, P = 0.02; > 1 cm reduced to < or = 1 cm, relative risk = 0.49, P = 0.02; > 1 cm reduced to microscopic, relative risk = 0.44, P = 0.01). Whether this apparent beneficial effect of cytoreductive surgery at second-look laparotomy reflects the biology of the tumor which allows surgical cytoreduction or the effects of cytoreduction can only be addressed in a randomized prospective trial.     

Insulin-like growth factors counteract the effect of interleukin 1 beta on type II phospholipase A2 expression and arachidonic acid release by rabbit articular chondrocytes

Interleukin 1 beta was found to stimulate arachidonic acid release, and the synthesis and secretion of type II phospholipase A2 by rabbit articular chondrocytes in vitro. Interleukin 1 beta had no effect on the level of cytosolic phospholipase A2 mRNA. Insulin-like growth factors, which help stabilize the cartilage matrix, reduced the effect of interleukin 1 beta on type II phospholipase A2 activity and mRNA level, and decreased the Interleukin 1 beta-stimulated arachidonic acid release to the basal values. This suggests that type II phospholipase A2 plays a key role in arachidonic acid release from rabbit articular chondrocytes and that insulin-like growth factors counteract the effect of interleukin 1 beta. They may therefore be considered as potential antiinflammatory agents.     

Increased expression of human type IIa secretory phospholipase A2 antigen in arthritic synovium

OBJECTIVE: To determine the localisation and level of expression of human type IIa secretory phospholipase A2 (sPLA2) in the synovium of rheumatoid arthritis (RA), osteoarthritis (OA), and non-arthritic (NA) patients and to examine the relation between sPLA2 and histological features of inflammation. METHODS: Immunoperoxidase staining using the anti-sPLA2 monoclonal antibody 9C1 was performed on frozen sections of knee synovium of 10 RA, 10 OA, and 10 NA patients. sPLA2 positive cells were scored on a scale of 0-3 in 10 fields of a representative tissue section from each case. Double labelling immunofluorescence confocal microscopy with antibodies to CD14 or CD45 and 9C1 was used to determine cell type specificity. Inflammation was assessed by semiquantitative scoring of lining layer thickness and mononuclear cell infiltrates (MC) and a cumulative inflammation score, generated by summing the two parameters. Scores in each group were compared using non-parametric statistical analysis. RESULTS: sPLA2 was localised to endothelium (EC), vascular smooth muscle (VSM), and mast cells (M) in all tissue sections. In RA and OA sections, staining was seen in both macrophage-like and fibroblast-like cells in the synovial lining layer (LL) and subsynovial lining layer (SLL). Perineural cells stained positively. Subintimal lymphoid aggregates (LA) were negative in all sections. The RA group showed significantly greater staining in extravascular synovial tissue (median 3.6, range 1.5-6.0) than the OA (median 1.95, range 0-5.3) or NA (median 0, range 0-5.9) groups (p < 0.05). LL staining was significantly higher in RA than both OA and NA sections (p < 0.05). The OA group showed a trend to higher staining scores than the NA group that did not reach significance. There was a significant correlation between the sPLA2 staining score and inflammation score within the RA patient group (p < 0.05). CONCLUSIONS: The synovium is a site of increased expression of sPLA2 antigen in both RA and OA relative to NA. Its presence in both fibroblast and macrophage-like cells in the LL and SLL of synovial tissue in RA and OA, but not NA, indicates that the enzyme is specifically induced in these regions in both conditions with expression in the LL being particularly characteristic of RA. The widespread expression of sPLA2 in synovium suggests it is likely to play a significant part in synovial pathology.     

Inhibition of prothrombinase by human secretory phospholipase A2 involves binding to factor Xa

Human group II secretory phospholipase A2 (hsPLA2) exhibits significant anticoagulant activity that does not require its enzymatic activity. We examined which coagulation factor was targeted by hsPLA2 and analyzed which region of the protein may be involved in this inhibition. Prothrombin time coagulation assays indicated that hsPLA2 did not inhibit activated factor V (FVa) activity, whereas activated factor X (FXa) one-stage coagulation assays suggested that FXa was inhibited. The inhibitory effect of hsPLA2 on prothrombinase activity of FXa, FV, phospholipids, and Ca2+ complex was markedly enhanced upon preincubation of hsPLA2 with FXa but not with FV. Prothrombinase activity was also strongly inhibited by hsPLA2 in the absence of PL. High concentrations of FVa in the prothrombinase generation assay reversed the inhibitory effect of hsPLA2. By using isothermal titration calorimetry, we demonstrated that hsPLA2 binds to FXa in solution with a 1:1 stoichiometry and a Kd of 230 nM. By using surface plasmon resonance we determined the rate constants, kon and koff, of the FXa/hsPLA2 interaction and analyzed the Ca2+ effect on these constants. When preincubated with FXa, synthetic peptides comprising residues 51-74 and 51-62 of hsPLA2 inhibited prothrombinase assays, providing evidence that this part of the molecule, which shares similarities with a region of FVa that binds to FXa, is likely involved in the anticoagulant interaction of hsPLA2 with FXa. In conclusion, we propose that residues 51-62 of hsPLA2 bind to FXa at a FVa-binding site and that hsPLA2 decreases the prothrombinase generation by preventing FXa.FVa complex formation.     

Differential activation of human neutrophil cytosolic phospholipase A2 and secretory phospholipase A2 during priming by 1,2-diacyl- and 1-O-alkyl-2-acylglycerols

We have shown previously that both 1,2-diacylglycerol (AAG) and 1-O-alkyl-2-acylglycerol (EAG) prime neutrophil release of arachidonic acid via uncharacterized phospholipases A2. Therefore, we investigated the actions of EAG and AAG specifically on neutrophil cytosolic (cPLA2) and secretory (sPLA2) phospholipase A2s. We hypothesized that AAG as a protein kinase activator would activate cPLA2 via phosphorylation events. EAG is antagonistic to the AAG activation of PKC, thus it was not expected to act via phosphorylation of cPLA2. Neutrophils were primed with either AAG or EAG and then stimulated with fMLP. When neutrophils were primed with 5-20 microM 1,2-diacylglycerol, a shift was observed in cPLA2 migration on SDS-PAGE gels, consistent with phosphorylation of the protein. This gel shift was not seen after exposure to EAG. AAG also caused a parallel increase in enzymatic activity of cPLA2 that was not seen with EAG. We also investigated whether either diglyceride would cause similar priming or direct secretion of sPLA2. Both AAG and EAG directly caused significant secretion of neutrophil sPLA2. EAG also increased the release of sPLA2 in cells subsequently stimulated with fMLP. Thus, AAG activated cPLA2 and stimulated secretion of sPLA2. In contrast, EAG did not activate cPLA2, but directly activated secretion of sPLA2. We also demonstrated that human synovial fluid sPLA2 increased AA release from resting and fMLP-stimulated neutrophils. Given that diglycerides prime for release of AA, PAF, and LTB4, these current data support the hypothesis that such priming may be mediated by phosphorylation dependent (cPLA2) or phosphorylation independent (e.g. secretion of sPLA2) events.     

A new cacospongionolide inhibitor of human secretory phospholipase A2 from the Tyrrhenian sponge Fasciospongia cavernosa and absolute configuration of cacospongionolides

A new inhibitor of human secretory phospholipase A2 (PLA2), cacospongionolide E (4a), has been isolated from the Tyrrhenian sponge Fasciospongia cavernosa. The structure was proposed on the basis of spectroscopic data and by chemical transformations. The absolute configuration of cacospongionolides 2a-4a was established using the modified Mosher's method. Cacospongionolide E was the most potent inhibitor toward human synovial PLA2, showing higher potency than the reference compound manoalide and exerting no signs of toxicity on human neutrophils. It showed high activity in the Artemia salina bioassay and moderate toxicity in the fish (Gambusia affinis) lethality assay.     

The functions of five distinct mammalian phospholipase A2S in regulating arachidonic acid release. Type IIa and type V secretory phospholipase A2S are functionally redundant and act in concert with cytosolic phospholipase A2

We examined the relative contributions of five distinct mammalian phospholipase A2 (PLA2) enzymes (cytosolic PLA2 (cPLA2; type IV), secretory PLA2s (sPLA2s; types IIA, V, and IIC), and Ca2+-independent PLA2 (iPLA2; type VI)) to arachidonic acid (AA) metabolism by overexpressing them in human embryonic kidney 293 fibroblasts and Chinese hamster ovary cells. Analyses using these transfectants revealed that cPLA2 was a prerequisite for both the calcium ionophore-stimulated immediate and the interleukin (IL)-1- and serum-induced delayed phases of AA release. Type IIA sPLA2 (sPLA2-IIA) mediated delayed AA release and, when expressed in larger amounts, also participated in immediate AA release. sPLA2-V, but not sPLA2-IIC, behaved in a manner similar to sPLA2-IIA. Both sPLA2s-IIA and -V, but not sPLA2-IIC, were heparin-binding PLA2s that exhibited significant affinity for cell-surface proteoglycans, and site-directed mutations in residues responsible for their membrane association or catalytic activity markedly reduced their ability to release AA from activated cells. Pharmacological studies using selective inhibitors as well as co-expression experiments supported the proposal that cPLA2 is crucial for these sPLA2s to act properly. The AA-releasing effects of these sPLA2s were independent of the expression of the M-type sPLA2 receptor. Both cPLA2, sPLA2s-IIA, and -V were able to supply AA to downstream cyclooxygenase-2 for IL-1-induced prostaglandin E2 biosynthesis. iPLA2 increased the spontaneous release of fatty acids, and this was further augmented by serum but not by IL-1. Finally, iPLA2-derived AA was not metabolized to prostaglandin E2. These observations provide evidence for the functional cross-talk or segregation of distinct PLA2s in mammalian cells in regulating AA metabolism and phospholipid turnover.     

Phosphorylation of cytosolic phospholipase A2 and the release of arachidonic acid in human neutrophils

Kinases mediating phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in intact cells remain to be fully characterized. Platelet-activating factor stimulation of human neutrophils increases cPLA2 phosphorylation. This increase is inhibited by PD 98059, a mitogen-activated protein (MAP)/extracellular signal-regulating kinase (erk) 1 inhibitor, but not by SB 203580, a p38 MAP kinase inhibitor, indicating that this action is mediated through activation of the p42 MAP kinase (erk2). However, platelet-activating factor-induced arachidonic acid release is inhibited by both PD 98059 and SB 203580. Stimulation by TNF-alpha increases cPLA2 phosphorylation, which is inhibited by SB 203580, but not PD 98059, suggesting a role for p38 MAP kinase. LPS increases cPLA2 phosphorylation and arachidonic acid release. However, neither of these actions is inhibited by either PD 98059 or SB 203580. PMA increases cPLA2 phosphorylation. This action is inhibited by PD 98059 but not SB 203580. Finally, FMLP increases cPLA2 phosphorylation and arachidonic acid release. Interestingly, while the FMLP-induced phosphorylation of cPLA2 is not affected by the inhibitors of the p38 MAP kinase or erk cascades, both inhibitors significantly decrease arachidonic acid release stimulated by FMLP. SB 203580 or PD 98059 has no inhibitory effects on the activity of coenzyme A-independent transacylase.     

Antiphospholipid antibodies inhibit prostaglandin release by decidual cells of early pregnancy: possible involvement of extracellular secretory phospholipase A2

OBJECTIVE: To investigate the effect of antiphospholipid antibodies on eicosanoid production by human decidual cells and the in vitro interaction between antiphospholipid antibodies and secretory phospholipase A2. DESIGN: Cultures of human decidual cells from early pregnancy. SETTING: All decidual specimens were obtained from the Obstetrics and Gynecology Department of the Catholic University, Rome, Italy. PATIENT(S): Patients were undergoing operative laparoscopy for extrauterine pregnancy, with a period of amenorrhea ranging from 6 to 9 weeks. INTERVENTION(S): Decidual samples were collected at laparoscopy by routine uterine curettage. MAIN OUTCOME MEASURE(S): Decidual cells were incubated with antiphospholipid antibodies, and eicosanoids (prostaglandin [PG] E2, PGF2alpha, and thromboxane B2) were assayed by RIA after 24 hours of culture. In vitro interactions between antiphospholipid antibodies and secretory phospholipase A2 were investigated with use of a modified ELISA for phospholipase A2. RESULT(S): Antiphospholipid antibodies reduced eicosanoid release from decidual cells in a dose-dependent fashion. In vitro assays showed that antiphospholipid antibodies bound secretory phospholipase A2 and that a competition occurred between antiphospholipid antibodies and secretory phospholipase A2 for the common substrate cardiolipin. CONCLUSION(S): In light of the critical role played by eicosanoids in decidual function, we suggest that an interaction between antiphospholipid antibodies and secretory phospholipase A2 occurring in vivo might impair important cellular communications at the decidual level in the antiphospholipid antibody syndrome.     

Lipoproteins are substrates for human secretory group IIA phospholipase A2: preferential hydrolysis of acute phase HDL

Group IIA secretory phospholipase A2 is an acute phase enzyme, co-expressed with serum amyloid A protein. Both are present in atherosclerotic lesions. We report that human normal and acute phase high density lipoproteins and low density lipoprotein are effective substrates for human group IIA phospholipase A2. The enzyme hydrolyzed choline and ethanolamine glycerophospholipids at the sn -2 position resulting in an accumulation of the corresponding lysophospholipids, including the unhydrolyzed alkyl and alkenyl ether derivatives. The hydrolysis of acute phase high density lipoprotein was 2- to 3-fold more rapid and intensive than of normal high density lipoprotein. The hydrolysis of lipoproteins was noted at enzyme concentration as low as 0.05 microgram/mg protein, which was within the range observed in the circulation in acute and chronic inflammatory diseases. The enzyme hydrolyzed the different molecular species of the residual glycerophospholipids in proportion to their mass, showing no preference for the release of arachidonic acid. Group IIA phospholipase A2 preferentially attacked the hydroxy and hydroperoxy linoleates and possibly other oxygenated fatty acids, which were released from the glycerophospholipids at early times of incubation. There was no effect on the content or molecular species composition of the sphingomyelins or neutral lipids of the lipoproteins. In conclusion, human plasma lipoproteins are the first reported natural biological substrates for human group IIA phospholipase A2. The enhanced hydrolysis of acute phase high density lipoproteins is probably due to its association with serum amyloid A protein, which enhances the activity of the enzyme and may promote its penetration to the lipid monolayer. As sPLA2-induced hydrolysis of the lipoproteins leads to accumulation of lysophosphatidylcholine and potentially toxic oxygenated fatty acids, overexpression of this enzyme may be proatherogenic.     

Melittin binds to secretory phospholipase A2 and inhibits its enzymatic activity

Synthetic melittin inhibited the enzymatic activity of secretory phospholipase A2 (PLA2) from various sources, including bee and snake venoms, bovine pancreas, and synovial fluid from rheumatoid arthritis patients, irrespective of substrate (e.g., [14C]-phosphatidylcholine or phosphatidylethanolamine vesicles and [3H]-oleic acid-labeled E.coli). A Lineweaver-Burk analysis showed that melittin was a noncompetitive inhibitor of bee venom PLA2, causing a change in Vmax from 200 to 50 units/min/mg of protein. The Km remained unchanged (0.75 nmole). Melittin inhibited approximately 50% of purified bee venom PLA2 activity in a 30:1 molar ratio (melittin:enzyme). Because the enzyme kinetics indicated a PLA2-melittin interaction, a melittin-sepharose affinity column was used to purify a PLA2 from human serum. Further, an enzyme-linked assay was developed to quantitate PLA2 activity in biological fluids using avidin-peroxidase and ELISA plates coated with biotinylated melittin. These observations may have potential therapeutic significance, as well as provide a convenient basis for the isolation and quantitation of PLA2.     

In vivo cartilage formation from growth factor modulated articular chondrocytes

Recent procedures for autologous repair of cartilage defects may be difficult in elderly patients because of the loss of stem cells and chondrocytes that occurs with age and the slow in vitro proliferation of chondrocytes from aged cartilage. In this study secondary chondroprogenitor cells were obtained by modulating the phenotype of articular chondrocytes with growth factors and stimulating the proliferation of these cells in culture. Chondrocytes isolated from the articular cartilage of mature New Zealand White rabbits were exposed to a combination of transforming growth factor beta and basic fibroblast growth factor treatment. These cells ceased the production of Collagen II (a marker for the chondrocyte phenotype) and underwent a 136-fold increase in cell number. Next, the cells were placed in high density culture and reexpressed the chondrocyte phenotype in vitro and formed hyaline cartilage in an in vivo assay. Primary chondrocytes obtained from articular cartilage of elderly humans could be manipulated in a similar fashion in vitro. These human secondary chondroprogenitor cells formed only cartilage tissue when assayed in vivo and in tissue bioreactors. This approach may be essential for autologous repair of degenerated articular cartilage in elderly patients with osteoarthritis.     

Design and synthesis of new secretory phospholipase A2 inhibitor of a phospholipid analog

All stereoisomers of N-acyl-4,5-disubstituted oxazolidinone phospholipid analogs were synthesized by regio and stereoselective epoxide ring opening accompanied by introduction of an amino group. The (4R,5S)-derivative showed stronger inhibitory activity toward type II phospholipase A2 than the 4-substituted oxazolidinone phospholipid analog previously reported.     

Type IB secretory phospholipase A2 is contained in insulin secretory granules of pancreatic islet beta-cells and is co-secreted with insulin from glucose-stimulated islets

People, now in their fifties and sixties, who were children during the Nazi Holocaust in WWII, endured persecution, massive traumatization, the constant risk of being killed, as well as the violent loss of (most of) their family members. They have internalized the resulting ongoing confusion and conflicts as to whether they should be alive or dead. This is maintained as an integral part of the child component of their compound personality, described in this paper. During the three years of the psychotherapy group, on which we focused here, these issues were expressed in different ways, such as suicide threats, occasional intolerance to physically remaining in the group, and outbursts of annihilating rage at the therapists. The confusion and conflicts about the legitimacy and risks of their survival came to a head during the termination process we insisted upon. Much attention has also been given to the intricacies of our countertransference--further complicated by our own connection to the Holocaust. We learned, and described, just how essential it is to acknowledge and process this countertransference in order to both contain the intense affects of anxiety, rage, and mourning in the groups, and enable them to be safely expressed. We imagine similar dynamics can be expected in group therapy with other populations who suffered massive, man-made traumatization.     

Prostaglandin E2 amplifies cytosolic phospholipase A2- and cyclooxygenase-2-dependent delayed prostaglandin E2 generation in mouse osteoblastic cells. Enhancement by secretory phospholipase A2

We used the MC3T3-E1 cell line, which originates from C57BL/6J mouse that is genetically type IIA secretory phospholipase A2 (sPLA2)-deficient, to reveal the type IIA sPLA2-independent route of the prostanglandin (PG) biosynthetic pathway. Kinetic and pharmacological studies showed that delayed PGE2 generation by this cell line in response to interleukin (IL)-1beta and tumor necrosis factor alpha (TNFalpha) was dependent upon cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2. Expression of these two enzymes was reduced by cPLA2 or COX-2 inhibitors and restored by adding exogenous arachidonic acid or PGE2, indicating that PGE2 produced by these cells acted as an autocrine amplifier of delayed PGE2 generation through enhanced cPLA2 and COX-2 expression. Exogenous addition or enforced expression of type IIA sPLA2 significantly increased IL-1beta/TNFalpha-initiated PGE2 generation, which was accompanied by increased expression of both cPLA2 and COX-2 and suppressed by inhibitors of these enzymes. Thus, our results revealed a particular cross-talk between the two PLA2 enzymes and COX-2 for delayed PGE2 biosynthesis by a type IIA sPLA2-deficient cell line. cPLA2 is responsible for initiating COX-2-dependent delayed PGE2 generation, and sPLA2, if introduced, enhances PGE2 generation by increasing cPLA2 and COX-2 expression via endogenous PGE2.