Activation of cytosolic phospholipase A2 and 15-lipoxygenase by oxidized low-density lipoproteins in cultured human lung fibroblasts

In cell cultures of human lung fibroblasts, we found that oxidized LDL (oxLDL), after 24-h treatment, stimulated arachidonic acid release. A putative role for phospholipases A(2) and MAPK activities in this process was postulated. Consequently, we studied the contribution of either Ca(2+)-dependent, cytosolic phospholipase A(2) (cPLA(2)) or Ca(2+)-independent phospholipase A(2) (iPLA(2)), and the role of the MAP kinase family in oxLDL toxicity to fibroblastic cells in vitro. Activation of extracellular signal-regulated kinases ERK1/2, p38 and c-Jun NH(2)-terminal kinase (JNK) was also assessed with Western blotting. Compared with cellular samples untreated or treated with native LDL, treatment with oxLDL (50-100 microM hydroperoxides) for 24 h significantly increased the levels of either cPLA(2) protein expression or constitutively phosphorylated cPLA(2) protein; in addition we observed enzyme translocation to membranes. iPLA(2) activity was not stimulated by oxLDL. Arachidonic acid release appeared to be associated with phosphorylation of ERK1/2 which was significantly enhanced in a dose-dependent manner whereas no activation of p38 and JNKs was found, indicating that these MAPKs are not involved in mediating the maximal oxLDL response. Western blotting on subcellular fractions and confocal microscopy analyses confirmed an increase in 15-lipoxygenase (15-LO) protein expression and translocation upon activation. A significant increase of cyclooxygenase-2 expression into membrane fraction was also found. Collectively, the data presented link the stimulation of ERK-cPLA(2)-15-LO pathway by oxLDL to the prooxidant mechanism of the lipoprotein complex. It may initially stimulate the fibroblast reaction against the oxidation challenge as well as metabolic repair, such as during lung inflammation and pulmonary fibrosis.     

Endothelial cell-pericyte cocultures induce PLA2 protein expression through activation of PKCalpha and the MAPK/ERK cascade

Little is known about the regulatory mechanisms of endothelial cell (EC) proliferation by retinal pericytes and vice versa. In a model of coculture with bovine retinal pericytes lasting for 24 h, rat brain ECs showed an increase in arachidonic acid (AA) release, whereas Western blot and RT-PCR analyses revealed that ECs activated the protein expression of cytosolic phospholipase A(2) (cPLA(2)) and its phosphorylated form and calcium-independent intracellular phospholipase A(2) (iPLA(2)). No activation of the same enzymes was seen in companion pericytes. In ECs, the protein level of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 was also enhanced significantly, a finding not observed in cocultured pericytes. The expression of protein kinase C-alpha (PKCalpha) and its phosphorylated form was also enhanced in ECs. Wortmannin, LY294002, and PD98059, used as inhibitors of upstream kinases (the PI3-kinase/Akt/PDK1 or MEK-1 pathway) in cultures, markedly attenuated AA release and the expression of phosphorylated forms of endothelial cPLA(2), PKCalpha, and ERK1/2. By confocal microscopy, activation of PKCalpha in perinuclear regions of ECs grown in coculture as well as strong activation of cPLA(2) in ECs taken from a model of mixed culture were clearly observed. However, no increased expression of both enzymes was found in cocultured pericytes. Our findings indicate that a sequential activation of PKCalpha contributes to endothelial ERK1/2 and cPLA(2) phosphorylation induced by either soluble factors or direct cell-to-cell contact, and that the PKCalpha-cPLA(2) pathway appears to play a key role in the early phase of EC-pericyte interactions regulating blood retina or blood-brain barrier maturation.     

High glucose and advanced glycation end products induce phospholipid hydrolysis and phospholipid enzyme inhibition in bovine retinal pericytes

In the present study, we investigated the possible role of oxidative stress and the modulation of phospholipid turnover in two related models of pericyte injury, i.e., treatment with high glucose or advanced glycation end products (AGEs). Growing microcapillary pericytes from bovine retinas in culture were incubated, for 3 weeks, with 20-50 mM glucose or 2-20 microM AGEs, and peroxidation parameters (malondialdehyde, conjugated diene, hydroperoxide, glutathione (GSH) levels and lactate dehydrogenase (LDH) release) were evaluated. Arachidonate (AA) and choline release from membrane phospholipids was determined in pericytes prelabeled with [1-(14)C]arachidonate and [Me-(3)H]choline, respectively, and stimulated with elevated glucose or AGEs for 30 min or 2 h. [1-(14)C]arachidonate and [Me-(3)H]choline incorporation into phospholipids, for 2 h and 3 h respectively, was also studied in conditioned and serum-starved cultures. Finally, lysates of treated and control cells were assayed for cytosolic phospholipase A(2) (cPLA(2)), acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (AT), CTP:phosphocholine cytidylyltransferase (CT) and microsomal choline phosphotransferase (CPT) enzyme activities. We found that high glucose and AGEs caused neither significant production of reactive oxygen species nor cell toxicity or death, unlike other cell types. Both agents had no significant effect on the cellular ultrastructure, evaluated by light and electron microscopy, AA incorporation and release, cytosolic phospholipase A(2) (cPLA(2)) and AT activities. On the contrary, choline incorporation into phosphatidylcholine, CT and CPT activities were significantly reduced either by 50 mM glucose or 20 microM AGEs. Simultaneously, [Me-(3)H]choline release was significantly stimulated by both agents. We conclude that prolonged treatments with high glucose or AGEs are not able to induce oxidative injury in bovine retinal capillary pericytes. Nevertheless, they do induce phospholipid hydrolysis and phospholipid enzyme activity inhibition.     

Modification of LDL with human secretory phospholipase A(2) or sphingomyelinase promotes its arachidonic acid-releasing propensity

Oxidation and lipolytic remodeling of LDL are believed to stimulate LDL entrapment in the arterial wall, expanding the inflammatory response and promoting atherosclerosis. However, the cellular responses and molecular mechanisms underlying the atherogenic effects of lipolytically modified LDL are incompletely understood. Human THP-1 monocytes were prelabeled with [(3)H]arachidonic acid (AA) before incubation with LDL or LDL lipolytically modified by secretory PLA(2) (sPLA(2)) or bacterial sphingomyelinase (SMase). LDL elicited rapid and dose-dependent extracellular release of AA in monocytes. Interestingly, LDL modified by sPLA(2) or SMase displayed a marked increase in AA mobilization relative to native LDL, and this increase correlated with enhanced activity of cytosolic PLA(2) (cPLA(2)) assayed in vitro as well as increased monocyte tumor necrosis factor-alpha secretion. The AA liberation was attenuated by inhibitors toward cPLA(2) and sPLA(2), indicating that both PLA(2) enzymes participate in LDL-induced AA release. In conclusion, these results demonstrate that LDL lipolytically modified by sPLA(2) or SMase potentiates cellular AA release and cPLA(2) activation in human monocytes. From our results, we suggest novel atherogenic properties for LDL modified by sPLA(2) and SMase in AA release and signaling, which could contribute to the inflammatory gene expression observed in atherosclerosis.     

Oxidized LDL activates phospholipase A2 to supply fatty acids required for cholesterol esterification

We examined the roles of phospholipase A2 (PLA2) in oxidized LDL (oxLDL)-induced cholesteryl ester formation in macrophages. In [3H]oleic acid-labeled RAW264.7 cells and mouse peritoneal macrophages, oxLDL induced [3H]cholesteryl oleate formation with an increase in free [3H]oleic acid and a decrease in [3H]phosphatidylcholine. The changes in these lipids were suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cytosolic PLA2 (cPLA2) inhibitor. However, MAFP had no effect on the ACAT activity or the binding and/or uptake of oxLDL. Stimulation with oxLDL in the presence of [3H]cholesterol increased [3H]cholesteryl ester bearing fatty acyl chains derived from cellular and/or exogenous (oxLDL) lipids. The formation of cholesteryl ester under this condition was also inhibited by MAFP, and the inhibitory effect was reversed by adding oleic acid. While oxLDL did not affect the activity or amounts of cPLA2, preincubation with oxLDL enhanced the release of oleic acid and arachidonic acid induced by ionomycin in RAW264.7 cells. 13(S)-hydroxyoctadecadienoic acid, but not 7-ketocholesterol, also enhanced ionomycin-induced oleic acid release. These results suggest that oxLDL induces cPLA2 activation, which contributes, at least in part, to the supply of fatty acids required for the cholesteryl esterification, probably through the acceleration by oxidized lipids of the catalytic action of cPLA2 in macrophages.     

Differential participation of phospholipase A(2) isoforms during iron-induced retinal toxicity. Implications for age-related macular degeneration

Both elevated iron concentrations and the resulting oxidative stress condition are common signs in retinas of patients with age-related macular degeneration (AMD). The role of phospholipase A(2) (PLA(2)) during iron-induced retinal toxicity was investigated. To this end, isolated retinas were exposed to increasing Fe(2+) concentrations (25, 200 or 800μM) or to the vehicle, and lipid peroxidation levels, mitochondrial function, and the activities of cytosolic PLA(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) were studied. Incubation with Fe(2+) led to a time- and concentration-dependent increase in retinal lipid peroxidation levels whereas retinal cell viability was only affected after 60min of oxidative injury. A differential release of arachidonic acid (AA) and palmitic acid (PAL) catalyzed by cPLA(2) and iPLA(2) activities, respectively, was also observed in microsomal and cytosolic fractions obtained from retinas incubated with iron. AA release diminished as the association of cyclooxigenase-2 increased in microsomes from retinas exposed to iron. Retinal lipid peroxidation and cell viability were also analyzed in the presence of cPLA(2) inhibitor, arachidonoyl trifluoromethyl ketone (ATK), and in the presence of iPLA(2) inhibitor, bromoenol lactone (BEL). ATK decreased lipid peroxidation levels and also ERK1/2 activation without affecting cell viability. BEL showed the opposite effect on lipid peroxidation. Our results demonstrate that iPLA(2) and cPLA(2) are differentially regulated and that they selectively participate in retinal signaling in an experimental model resembling AMD.     

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.     

Activation of cytosolic phospholipase A2 downstream of the Src-phospholipase D1 (PLD1)-protein kinase C γ (PKCγ) signaling axis is required for hypoxia-induced pathological retinal angiogenesis

In view of understanding the mechanisms of retinal neovascularization, we had reported previously that vascular endothelial growth factor (VEGF)-induced pathological retinal angiogenesis requires the activation of Src-PLD1-PKCγ signaling. In the present work, we have identified cytosolic phospholipase A(2) (cPLA(2)) as an effector molecule of Src-PLD1-PKCγ signaling in the mediation of VEGF-induced pathological retinal angiogenesis based on the following observations. VEGF induced cPLA(2) phosphorylation in a time-dependent manner in human retinal microvascular endothelial cells (HRMVECs). VEGF also induced arachidonic acid (AA) release in a dose-, time-, and cPLA(2)-dependent manner. Depletion of cPLA(2) levels inhibited VEGF-induced HRMVEC DNA synthesis, migration, and tube formation. In addition, the exogenous addition of AA rescued VEGF-induced HRMVEC DNA synthesis, migration, and tube formation from inhibition by down-regulation of cPLA(2). Inhibition of Src, PLD1, or PKCγ attenuated VEGF-induced cPLA(2) phosphorylation and AA release. Consistent with these findings, hypoxia induced cPLA(2) phosphorylation and activity in VEGF-Src-PLD1-PKCγ-dependent manner in a mouse model of oxygen-induced retinopathy. In addition, siRNA-mediated down-regulation of cPLA(2) levels in the retina abrogated hypoxia-induced retinal endothelial cell proliferation and neovascularization. These observations suggest that cPLA(2)-dependent AA release is required for VEGF-induced Src-PLD1-PKCγ-mediated pathological retinal angiogenesis.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Antioxidant alpha-keto-carboxylate pyruvate protects low-density lipoprotein and atherogenic macrophages

Oxidative modification of low-density lipoprotein (oxLDL) plays a pathogenic role in atherogenesis. Classical antioxidants such as L-ascorbic acid can inhibit formation of oxLDL. Alpha-Keto-carboxylates such as pyruvate and congeners also display antioxidant properties in some cell-free and intact cell systems. We tested the hypothesis that pyruvate or alpha-keto-glutarate may function as antioxidants with respect to LDL incubated with 5 or 10 microM Cu2+ alone or in combination with THP-1-derived macrophages. alpha-Hydroxy-carboxylates (L-lactate), linear aliphatic monocarboxylates (acetate/caprylate) and L-ascorbic acid served as controls. The oxLDL formation was ascertained by electrophoretic mobility and oxLDL cytotoxicity was judged by macrophage viability and thiobarbituric acid reactive substances (TBARS) formation. Cu2+ alone was not cytotoxic but increased electrophoretic mobility of cell-free LDL, stimulating TBARS. Millimolar pyruvate, alpha-ketoglutarate, or micromolar L-ascorbic acid partially inhibited oxLDL formation, while alpha-hydroxy-carboxylate or the aliphatic mono-carboxylates had no measurable antioxidant properties in cell-free LDL. Co-culture of LDL with macrophages and Cu2+ augmented TBARS release and resulted in 95% macrophage death. Pyruvate improved macrophage viability with 5 microM Cu2+ up to 60%. L-Ascorbic acid (> or = 100 microM) protected macrophages up to 80%. When > or = 100 microM L-ascorbic acid was combined with pyruvate, oxLDL formation and macrophage death were fully prevented. Thus, alpha-keto-carboxylates, but not physiological alpha-hydroxy-carboxylates or aliphatic monocarboxylates qualify as antioxidants in LDL systems. Since alpha-keto-carboxylates enhanced the antioxidant power of L-ascorbic acid, our findings may have implications for strategies attenuating atherosclerosis.     

17Beta-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells.

Increase of intracellular reactive oxygen species (ROS) has been proposed to cause endothelial injury, and oxidized LDL (oxLDL) actions are associated with an early increase of ROS. Estrogen protects vascular cells partly via its antioxidant effects and by preventing lipid peroxidation. However, whether it can inhibit oxLDL-induced stimulation of ROS generation in endothelial cells is unknown. We utilized the fluorescent dye (DCFH-DA) to measure ROS generation and compared the stimulant effect of tert-butylhydroperoxide (TBH) and oxLDL in human umbilical vein endothelial cells (HUVECs). We found that TBH, H2O2, and oxLDL rapidly stimulated ROS generation, and in a dose-dependent manner with TBH. A concentration of estrogen effective in preventing lipid peroxidation was employed either by pretreatment of cells 18 h prior to or by direct co-incubation (30 min) with HUVEC and oxLDL. Estrogen (54 microM) pretreatment significantly suppressed both TBH- and oxLDL- induced stimulation of ROS generation. Both 1 and 54 microM concentration of estrogen could directly inhibit oxLDL-induced ROS production in HUVECs. Thus, either 18 h pretreatment or 30 min co-incubation with estrogen reduced stimulated ROS generation, suggesting that both cellular and direct actions of estrogen may be involved.     

Phosphatidylinositide 3-kinase regulates angiotensin II-induced cytosolic phospholipase A2 activity and growth in vascular smooth muscle cells

Angiotensin (Ang) II via the AT(1) receptor acts as a mitogen in vascular smooth muscle cells (VSMC) through stimulation of multiple signaling mechanisms, including tyrosine kinases and mitogen-activated protein kinase (MAPK). In addition, cytosolic phospholipase A(2)(cPLA(2))-dependent release of arachidonic acid (AA) is linked to VSMC growth and we have reported that Ang II stimulates cPLA(2) activity via the AT(1) receptor. The coupling of Ang II to the activation of cPLA(2) appears to involve mechanisms both upstream and downstream of MAPK such that AA stimulates MAPK activity which phosphorylates cPLA(2) to further enhance AA release. However, the upstream mechanisms responsible for activation of cPLA(2) are not well-defined. One possibility includes phosphatidylinositide 3-kinase (PI3K), since PI3K has been reported to participate in the upstream signaling events linked to activation of MAPK. However, it is not known whether PI3K is involved in the Ang II-induced activation of cPLA(2) or if this mechanism is associated with the Ang II-mediated growth of VSMC. Therefore, we used cultured rat VSMC to examine the role of PI3K in the Ang II-dependent phosphorylation of cPLA(2), release of AA, and growth induced by Ang II. Exposure of VSMC to Ang II (100 nM) increased [(3)H]thymidine incorporation, cell number, and the release of [(3)H]AA. Also, using Western analysis, Ang II increased the phosphorylation of MAPK and cPLA(2) which were blocked by the MAPK kinase inhibitor PD98059 (10 microM/L). Similarly, the PI3K inhibitor LY294002 (10 microM/L) abolished the Ang II-mediated increase in MAPK phosphorylation, as well as phosphoserine-PLA(2). Further, inhibition of PI3K blocked the Ang II-induced release of AA and VSMC mitogenesis. However, exogenous AA was able to restore VSMC growth in the presence of LY294002, as well as reverse the inhibition of MAPK and cPLA(2) phosphorylation by LY294002. Thus, it appears from these data that Ang II stimulates the PI3K-sensitive release of AA which stimulates MAPK to phosphorylate cPLA(2) and enhance AA release. This mechanism may play an important role in the Ang II-induced growth of VSMC.     

Arachidonate metabolism and the signaling pathway of induction of apoptosis by oxidized LDL/oxysterol

Owing at least in part to oxysterol components that can induce apoptosis, oxidized LDL (oxLDL) is cytotoxic to mammalian cells with receptors that can internalize it. Vascular cells possess such receptors, and it appears that the apoptotic response of vascular cells to the oxysterols borne by oxLDL is an important part of the atherogenic effects of oxLDL. Thus, an analysis of the signaling pathway of apoptotic induction by oxysterols is of value in understanding the development of atherosclerotic plaque. In a prior study, we demonstrated an induction of calcium ion flux into cells treated with 25-hydroxycholesterol (25-OHC) and showed that this response is essential for 25-OHC-induced apoptosis. One possible signal transduction pathway initiated by calcium ion fluxes is the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we demonstrate that activation of cPLA2 does occur in both macrophages and fibroblasts treated with 25-OHC or oxLDL. Activation is evidenced by 25-OHC-induced relocalization of cPLA2 to the nuclear envelope and arachidonic acid release. Loss of cPLA2 activity, either through genetic knockout in mice, or by treatment with a cPLA2 inhibitor, results in an attenuation of arachidonic acid release as well as of the apoptotic response to oxLDL in peritoneal macrophages or to 25-OHC in cultured fibroblast and macrophage cell lines.     

Anti-apoptotic defense of bcl-2 gene against hydroperoxide-induced cytotoxicity together with suppressed lipid peroxidation,enhanced ascorbate uptake,and upregulated Bcl-2 protein

Although it is well known that Bcl-2 can prevent apoptosis, the Bcl-2's anti-apoptotic mechanism is not fully understood. Here, we investigate the mechanism of oxidant-induced cell death and to investigate the role of Bcl-2 in the tert-butyl hydroperoxide (t-BuOOH)-induced oxidant injury in Rat-1 fibroblasts and their bcl-2 transfected counterparts, b5 cells. Treatment with t-BuOOH causes mitochondrial disfunction and induced morphological features consistent with apoptosis more markedly in Rat-1 cells than in b5 cells. The hydroperoxide t-BuOOH at concentrations less than 100 nM for as long as 48 h or with higher concentrations (up to 100 microM) for only 3 h induces death in Rat-1 cells, whereas their bcl-2 transfectants were significantly resistant to cytotoxicity by both time and all concentration other than 100 microM. The similar results were obtained also for DNA strand cleavages as detected by TUNEL stain. The bcl-2 transfectants significantly suppressed t-BuOOH-induced increases in both lipid peroxidation and caspase-3 activation 3 and 1 h after t-BuOOH exposure, respectively, but failed to suppress either caspase-1 activation or an enhanced production of the intracellular reactive oxygen species (ROS). Intracellular uptake of [1-(14)C] ascorbic acid (Asc) into the bcl-2 transfectants was superior to that into the non-transfectants always under examined conditions regardless of serum addition to culture medium and cell density. Upregulation of Bcl-2 proteins was rapidly induced after t-BuOOH exposure in the transfectants, but not in non-transfectants, and restored till 24 h to the normal Bcl-2 level. Thus suppressions of both lipid peroxidation and the subsequent cell death events such as caspase-3 activation and DNA cleavage were concerned with the inhibitory effects of Bcl-2 on the t-BuOOH-induced cytotoxicity. And some of these events may correlate with Bcl-2 expression-induced partial enhanced anti-oxidant cellular ability including enrichment of intracellular Asc and oxidative stress-induced upregulation of Bcl-2 protein. On the other hand, ROS production and caspase-1 activation were not related to cytoprotection by Bcl-2.     

Effect of acetaminophen on the myeloperoxidase-hydrogen peroxide-nitrite mediated oxidation of LDL

Myeloperoxidase, in the presence of hydrogen peroxide and nitrite, promotes the lipid peroxidation of low density lipoprotein (LDL); the modified lipoprotein is then capable of being readily endocytosed by macrophages. Since acetaminophen has been shown to inhibit the leukocyte myeloperoxidase antimicrobial system and is, under certain experimental conditions, an antioxidant, the effect of acetaminophen on the myeloperoxidase-hydrogen peroxide-nitrite mediated oxidation of LDL was examined. The content of LDL lipid hydroperoxides after incubation with 50 nM myeloperoxidase, 100 microM nitrite and a hydrogen peroxide generating system for 6 h was reduced by approx. 80% in the presence of 25-250 microM acetaminophen. The production of thiobarbituric acid-reactive substances was also inhibited by acetaminophen to a similar extent. Acetylsalicylic acid (25-100 microM) did not inhibit LDL lipid peroxidation mediated by the myeloperoxidase enzyme system. LDL, treated with myeloperoxidase, hydrogen peroxide and nitrite for 14 h, was metabolized by macrophages to a much greater extent than native LDL. The presence of acetaminophen prevented the modification of LDL; the lipoprotein was metabolized by macrophages to the same extent as was native LDL. These results demonstrate that acetaminophen is a potent inhibitor of the myeloperoxidase-hydrogen peroxide-nitrite mediated modification of LDL.     

Synergism between platelet-activating factor-like phospholipids and peroxisome proliferator-activated receptor gamma agonists generated during low density lipoprotein oxidation that induces lipid body formation in leukocytes

Oxidized low density lipoprotein (LDL) has an important proinflammatory role in atherogenesis. In this study, we investigated the ability of oxidized LDL (oxLDL) and its phospholipid components to induce lipid body formation in leukocytes. Incubation of mouse peritoneal macrophages with oxidized, but not with native LDL led to lipid body formation within 1 h. This was blocked by platelet-activating factor (PAF) receptor antagonists or by preincubation of oxLDL with rPAF acetylhydrolase. HPLC fractions of phospholipids purified from oxLDL induced calcium flux in neutrophils as well as lipid body formation in macrophages. Injection of the bioactive phospholipid fractions or butanoyl and butenoyl PAF, a phospholipid previously shown to be present in oxLDL, into the pleural cavity of mice induced lipid body formation in leukocytes recovered after 3 h. The 5-lipoxygenase and cyclooxygenase-2 colocalized within lipid bodies formed after stimulation with oxLDL, bioactive phospholipid fractions, or butanoyl and butenoyl PAF. Lipid body formation was inhibited by 5-lipoxygenase antagonists, but not by cyclooxygenase-2 inhibitors. Azelaoyl-phosphatidylcholine, a peroxisome proliferator-activated receptor-gamma agonist in oxLDL phospholipid fractions, induced formation of lipid bodies at late time points (6 h) and synergized with suboptimal concentrations of oxLDL. We conclude that lipid body formation is an important proinflammatory effect of oxLDL and that PAF-like phospholipids and peroxisome proliferator-activated receptor-gamma agonists generated during LDL oxidation are important mediators in this phenomenon.     

Inhibition of nitric-oxide synthase-I (NOS-I)-dependent nitric oxide production by lipopolysaccharide plus interferon-gamma is mediated by arachidonic acid. Effects on NFkappaB activation and late inducible NOS expression

Previous results have indicated that lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma) inhibits nitric-oxide synthase (NOS)-I activity in glial cells. We report here that arachidonic acid (AA) plays a pivotal role in this response, which was consistently reproduced in different glial cell lines and in primary rat astrocytes. This notion was established using pharmacological inhibitors of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2) antisense oligonucleotides, and AA add-back experiments. This approach not only allowed the demonstration that AA promotes inhibition of NOS-I activity but also produced novel experimental evidence that LPS/IFNgamma itself is a potential stimulus for NOS-I. Indeed, LPS/IFNgamma fails to generate nitric oxide (NO) via NOS-I activation simply because it activates the AA-dependent signal that impedes NOS-I activity. Otherwise, LPS/IFNgamma promotes NO formation, sensitive to exogenous AA, in cells in which cPLA2 is pharmacologically inhibited or genetically depleted. Because NO suppresses the NFkappaB-dependent NOS-II expression, inactivation of NOS-I by the LPS/IFNgamma-induced AA pathway provides optimal conditions for NFkappaB activation and subsequent NOS-II expression. Inhibition of cPLA2 activity, while reducing the availability of AA, consistently inhibited NFkappaB activation and NOS-II mRNA induction and delayed NO formation. These responses were promptly reestablished by addition of exogenous AA. Finally, we have demonstrated that the LPS/IFNgamma-dependent tyrosine phosphorylation of NOS-I and inhibition of its activity are mediated by endogenous AA.     

Translocation of phospholipase A2 to membranes by oxidized LDL and hydroxyoctadecadienoic acid to contribute to cholesteryl ester formation

We examined the mechanisms underlying the activation of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) contributing to the supply of fatty acids required for the formation of cholesteryl ester in oxidized low-density lipoprotein (oxLDL)-stimulated macrophages. The possible involvement of oxidized lipids was also examined. In [(3)H]arachidonic acid-labeled mouse peritoneal macrophages, oxLDL stimulated the release of arachidonic acid, which was suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cPLA(2)alpha inhibitor. oxLDL induced an increase in PLA(2)alpha levels in the membrane fraction without affecting those in whole cells or the activity in the lysate. Among 13-hydroxyoctadecadienoic acid (13-HODE), 7-ketocholesterol, and 25-hydroxycholesterol, oxidized lipids present in oxLDL particles, only 13-HODE induced the release of arachidonic acid, which was also sensitive to MAFP. Under conditions where addition of Ca(2+) to the cell lysate induced an increase in cPLA(2)alpha protein in the membrane fraction, preincubation with 13-HODE facilitated the Ca(2+)-dependent translocation of cPLA(2)alpha. Furthermore, 13-HODE increased cholesteryl ester formation in the presence of [(3)H]cholesterol. These results suggest that 13-HODE mediates the oxLDL-induced activation of cPLA(2)alpha through an increase in cPLA(2)alpha protein in the membranes, thus contributing, in part, to the supply of fatty acids required for the esterification of cholesterol in macrophages.