Macrophage polarization is linked to Ca2+-independent phospholipase A2ß-derived lipids and cross-cell signaling in mice.

Phospholipases A2 (PLA2s) catalyze hydrolysis of the sn-2 substituent from glycerophospholipids to yield a free fatty acid (i.e., arachidonic acid), which can be metabolized to pro- or anti-inflammatory eicosanoids. Macrophages modulate inflammatory responses and are affected by Ca2+-independent phospholipase A2 (PLA2)ß (iPLA2ß). Here, we assessed the link between iPLA2ß-derived lipids (iDLs) and macrophage polarization. Macrophages from WT and KO (iPLA2ß-/-) mice were classically M1 pro-inflammatory phenotype activated or alternatively M2 anti-inflammatory phenotype activated, and eicosanoid production was determined by ultra-performance LC ESI-MS/MS. As a genotypic control, we performed similar analyses on macrophages from RIP.iPLA2ß.Tg mice with selective iPLA2ß overexpression in ß-cells. Compared with WT, generation of select pro-inflammatory prostaglandins (PGs) was lower in iPLA2ß-/- , and that of a specialized pro-resolving lipid mediator (SPM), resolvin D2, was higher; both changes are consistent with the M2 phenotype. Conversely, macrophages from RIP.iPLA2ß.Tg mice exhibited an opposite landscape, one associated with the M1 phenotype: namely, increased production of pro-inflammatory eicosanoids (6-keto PGF1α, PGE2, leukotriene B4) and decreased ability to generate resolvin D2. These changes were not linked with secretory PLA2 or cytosolic PLA2α or with leakage of the transgene. Thus, we report previously unidentified links between select iPLA2ß-derived eicosanoids, an SPM, and macrophage polarization. Importantly, our findings reveal for the first time that ß-cell iPLA2ß-derived signaling can predispose macrophage responses. These findings suggest that iDLs play critical roles in macrophage polarization, and we posit that they could be targeted therapeutically to counter inflammation-based disorders.

Genetic Ablation of Calcium-independent Phospholipase A2γ Exacerbates Glomerular Injury in Adriamycin Nephrosis in Mice.

Genetic ablation of calcium-independent phospholipase A2γ (iPLA2γ) in mice results in marked damage of mitochondria and enhanced autophagy in glomerular visceral epithelial cells (GECs) or podocytes. The present study addresses the role of iPLA2γ in glomerular injury. In adriamycin nephrosis, deletion of iPLA2γ exacerbated albuminuria and reduced podocyte number. Glomerular LC3-II increased and p62 decreased in adriamycin-treated iPLA2γ knockout (KO) mice, compared with treated control, in keeping with increased autophagy in KO. iPLA2γ KO GECs in culture also demonstrated increased autophagy, compared with control GECs. iPLA2γ KO GECs showed a reduced oxygen consumption rate and increased phosphorylation of AMP kinase (pAMPK), consistent with mitochondrial dysfunction. Adriamycin further stimulated pAMPK and autophagy. After co-transfection of GECs with mito-YFP (to label mitochondria) and RFP-LC3 (to label autophagosomes), or RFP-LAMP1 (to label lysosomes), there was greater colocalization of mito-YFP with RFP-LC3-II and with RFP-LAMP1 in iPLA2γ KO GECs, compared with WT, indicating enhanced mitophagy in KO. Adriamycin increased mitophagy in WT cells. Thus, iPLA2γ has a cytoprotective function in the normal glomerulus and in glomerulopathy, as deletion of iPLA2γ leads to mitochondrial damage and impaired energy homeostasis, as well as autophagy and mitophagy.

Bone marrow-derived cPLA2α contributes to renal fibrosis progression.

The group IVA calcium-dependent cytosolic phospholipase A2 (cPLA2α) enzyme directs a complex "eicosanoid storm" that accompanies the tissue response to injury. cPLA2α and its downstream eicosanoid mediators are also implicated in the pathogenesis of fibrosis in many organs, including the kidney. We aimed to determine the role of cPLA2α in bone marrow-derived cells in a murine model of renal fibrosis, unilateral ureteral obstruction (UUO). WT C57BL/6J mice were irradiated and engrafted with donor bone marrow from either WT mice [WT-bone marrow transplant (BMT)] or mice deficient in cPLA2α (KO-BMT). After full engraftment, mice underwent UUO and kidneys were collected 3, 7, and 14 days after injury. Using picrosirius red, collagen-3, and smooth muscle α actin staining, we determined that renal fibrosis was significantly attenuated in KO-BMT animals as compared with WT-BMT animals. Lipidomic analysis of homogenized kidneys demonstrated a time-dependent upregulation of pro-inflammatory eicosanoids after UUO; KO-BMT animals had lower levels of many of these eicosanoids. KO-BMT animals also had fewer infiltrating pro-inflammatory CD45+CD11b+Ly6Chi macrophages and reduced message levels of pro-inflammatory cytokines. Our results indicate that cPLA2α and/or its downstream mediators, produced by bone marrow-derived cells, play a major role in eicosanoid production after renal injury and in renal fibrinogenesis.

Cytosolic Phospholipase A2α Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection.

Pulmonary infection by Streptococcus pneumoniae is characterized by a robust alveolar infiltration of neutrophils (polymorphonuclear cells [PMNs]) that can promote systemic spread of the infection if not resolved. We previously showed that 12-lipoxygenase (12-LOX), which is required to generate the PMN chemoattractant hepoxilin A3 (HXA3) from arachidonic acid (AA), promotes acute pulmonary inflammation and systemic infection after lung challenge with S. pneumoniae As phospholipase A2 (PLA2) promotes the release of AA, we investigated the role of PLA2 in local and systemic disease during S. pneumoniae infection. The group IVA cytosolic isoform of PLA2 (cPLA2α) was activated upon S. pneumoniae infection of cultured lung epithelial cells and was critical for AA release from membrane phospholipids. Pharmacological inhibition of this enzyme blocked S. pneumoniae-induced PMN transepithelial migration in vitro Genetic ablation of the cPLA2 isoform cPLA2α dramatically reduced lung inflammation in mice upon high-dose pulmonary challenge with S. pneumoniae The cPLA2α-deficient mice also suffered no bacteremia and survived a pulmonary challenge that was lethal to wild-type mice. Our data suggest that cPLA2α plays a crucial role in eliciting pulmonary inflammation during pneumococcal infection and is required for lethal systemic infection following S. pneumoniae lung challenge.

Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H2O2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A2 and α/ß-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/ß-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2.

Heterozygous knockout of cytosolic phospholipase A2α attenuates Alzheimer's disease pathology in APP/PS1 transgenic mice.

Cytosolic phospholipase A2α (cPLA2α) is a key enzyme in regulation of inflammation process and neuromembrane homeostasis, both of which are critical in pathogenesis of Alzheimer's diseases. By hybride APP/PS1 Tg-AD mice with cPLA2α knockout mice, three lines of APP/PS1 Tg-AD mice were produced with genotypes of cPLA2α+/+, cPLA2α+/- and cPLA2α-/-. Compared to cPLA2α+/+ Tg-AD mice, the amyloid plaque formation was significantly downregulated in the brain of cPLA2α+/- Tg-AD mice, but not in cPLA2α-/- Tg-AD mice. The reactive gliosis were also significantly downregulated in both cPLA2α+/- and cPLA2α-/- Tg-AD mouse lines. The paradoxical effects of cPLA2α on the amyloid plaques reveal a complex role of cPLA2α in pathogenesis of AD and could be a potential target for prevention and treatment of AD.

Cytosolic phospholipase A2α increases proliferation and de-differentiation of human renal tubular epithelial cells.

The group IVA calcium-dependent cytosolic phospholipase A2 (cPLA2α) enzyme controls the release of arachidonic acid from membrane bound phospholipids and is the rate-limiting step in production of eicosanoids. A variety of different kidney injuries activate cPLA2α, therefore we hypothesized that cPLA2α activity would regulate pathologic processes in HK-2 cells, a human renal tubular epithelial cell line, by regulating cell phenotype and proliferation. In two lentiviral cPLA2α-silenced knockdowns, we observed decreased proliferation and increased apoptosis compared to control HK-2 cells. cPLA2α-silenced cells also demonstrated an altered morphology, had increased expression E-cadherin, and decreased expression of Ncadherin. Increased levels of E-cadherin were associated with increased promoter activity and decreased levels of SNAIL1, SNAIL2, and ZEB1, transcriptional repressors of E-cadherin expression. Addition of exogenous arachidonic acid, but not PGE2, reversed the phenotypic changes in cPLA2α-silenced cells. These data suggest that cPLA2α may play a key role in renal repair after injury through a PGE2-independent mechanism.

Prostaglandins from Cytosolic Phospholipase A2α/Cyclooxygenase-1 Pathway and Mitogen-activated Protein Kinases Regulate Gene Expression in Candida albicans-infected Macrophages.

In Candida albicans-infected resident peritoneal macrophages, activation of group IVA cytosolic phospholipase A2(cPLA2α) by calcium- and mitogen-activated protein kinases triggers the rapid production of prostaglandins I2 and E2 through cyclooxygenase (COX)-1 and regulates gene expression by increasing cAMP. InC. albicans-infected cPLA2α(-/-)or COX-1(-/-)macrophages, expression ofI l10,Nr4a2, and Ptgs2 was lower, and expression ofTnfα was higher, than in wild type macrophages. Expression was reconstituted with 8-bromo-cAMP, the PKA activator 6-benzoyl-cAMP, and agonists for prostaglandin receptors IP, EP2, and EP4 in infected but not uninfected cPLA2α(-/-)or COX-1(-/-)macrophages. InC. albicans-infected cPLA2α(+/+)macrophages, COX-2 expression was blocked by IP, EP2, and EP4 receptor antagonists, indicating a role for both prostaglandin I2 and E2 Activation of ERKs and p38, but not JNKs, by C. albicansacted synergistically with prostaglandins to induce expression of Il10,Nr4a2, and Ptgs2. Tnfα expression required activation of ERKs and p38 but was suppressed by cAMP. Results using cAMP analogues that activate PKA or Epacs suggested that cAMP regulates gene expression through PKA. However, phosphorylation of cAMP-response element-binding protein (CREB), the cAMP-regulated transcription factor involved inIl10,Nr4a2,Ptgs2, andTnfα expression, was not mediated by cAMP/PKA because it was similar inC. albicans-infected wild type and cPLA2α(-/-)or COX-1(-/-)macrophages. CREB phosphorylation was blocked by p38 inhibitors and induced by the p38 activator anisomycin but not by the PKA activator 6-benzoyl-cAMP. Therefore, MAPK activation inC. albicans-infected macrophages plays a dual role by promoting the cPLA2α/prostaglandin/cAMP/PKA pathway and CREB phosphorylation that coordinately regulate immediate early gene expression.

Bleeding diathesis and gastro-duodenal ulcers in inherited cytosolic phospholipase-A2 alpha deficiency.

Arachidonic acid (AA), when cleaved from phospholipids by cytosolic phospholipase A2 alpha (cPLA2a), generates eicosanoids, with pro-hemostatic, pro-inflammatory, vasoactive and gastro-protective functions. We describe a patient (27-year-old man) and his twin-sister with early-onset bleeding diathesis and recurrent gastro-intestinal (GI) ulcers. Platelet aggregation/δ-granules secretion by collagen was impaired, but normal by AA; serum levels of thromboxane (Tx) B2 and 12-hydroxyeicosatetraenoic acid, and urinary levels of 11-dehydro-TxB2 were extremely low. Patients were homozygous for 1723G>C transition in PLA2G4A gene, which changed the codon for Asp575 to His. GI ulcers affected 5/14 heterozygous (< 40 years) and 1/16 wild-type homozygous (> 60 years) family members; none had bleeding diathesis. The proband, his sister and mother also had mildly reduced factor XI levels. Platelet messenger RNA expression did not differ among subjects with different PLA2G4A genotypes. Conversely, platelet cPLA2a was undetectable by Western Blotting in the proband and his sister, and decreased in 1723G>C heterozygous subjects, suggesting that the variant is transcribed, but not translated or translated into an unstable protein. We described a syndromic form of deficiency of cPLA2a , characterised by recurrent GI ulcers and bleeding diathesis, associated with mild inherited deficiency of factor XI. Unlike other reported patients with cPLA2a deficiency, these patients had extremely low levels of platelet TxA2 biosynthesis.

Cytosolic phospholipase A2 protein as a novel therapeutic target for spinal cord injury.

OBJECTIVE: The objective of this study was to investigate whether cytosolic phospholipase A2 (cPLA2 ), an important isoform of PLA2 that mediates the release of arachidonic acid, plays a role in the pathogenesis of spinal cord injury (SCI). METHODS: A combination of molecular, histological, immunohistochemical, and behavioral assessments were used to test whether blocking cPLA2 activation pharmacologically or genetically reduced cell death, protected spinal cord tissue, and improved behavioral recovery after a contusive SCI performed at the 10th thoracic level in adult mice. RESULTS: SCI significantly increased cPLA2 expression and activation. Activated cPLA2 was localized mainly in neurons and oligodendrocytes. Notably, the SCI-induced cPLA2 activation was mediated by the extracellular signal-regulated kinase signaling pathway. In vitro, activation of cPLA2 by ceramide-1-phosphate or A23187 induced spinal neuronal death, which was substantially reversed by arachidonyl trifluoromethyl ketone, a cPLA2 inhibitor. Remarkably, blocking cPLA2 pharmacologically at 30 minutes postinjury or genetically deleting cPLA2 in mice ameliorated motor deficits, and reduced cell loss and tissue damage after SCI. INTERPRETATION: cPLA2 may play a key role in the pathogenesis of SCI, at least in the C57BL/6 mouse, and as such could be an attractive therapeutic target for ameliorating secondary tissue damage and promoting recovery of function after SCI.

Micromolar changes in lysophosphatidylcholine concentration cause minor effects on mitochondrial permeability but major alterations in function.

Mice deficient in group 1b phospholipase A2 have decreased plasma lysophosphatidylcholine and increased hepatic oxidation that is inhibited by intraperitoneal lysophosphatidylcholine injection. This study sought to identify a mechanism for lysophosphatidylcholine-mediated inhibition of hepatic oxidative function. Results showed that in vitro incubation of isolated mitochondria with 40-200µM lysophosphatidylcholine caused cyclosporine A-resistant swelling in a concentration-dependent manner. However, when mitochondria were challenged with 220µM CaCl2, cyclosporine A protected against permeability transition induced by 40µM, but not 80µM lysophosphatidylcholine. Incubation with 40-120µM lysophosphatidylcholine also increased mitochondrial permeability to 75µM CaCl2 in a concentration-dependent manner. Interestingly, despite incubation with 80µM lysophosphatidylcholine, the mitochondrial membrane potential was steady in the presence of succinate, and oxidation rates and respiratory control indices were similar to controls in the presence of succinate, glutamate/malate, and palmitoyl-carnitine. However, mitochondrial oxidation rates were inhibited by 30-50% at 100µM lysophosphatidylcholine. Finally, while 40µM lysophosphatidylcholine has no effect on fatty acid oxidation and mitochondria remained impermeable in intact hepatocytes, 100µM lysophosphatidylcholine inhibited fatty acid stimulated oxidation and caused intracellular mitochondrial permeability. Taken together, these present data demonstrated that LPC concentration dependently modulates mitochondrial microenvironment, with low micromolar concentrations of lysophosphatidylcholine sufficient to change hepatic oxidation rate whereas higher concentrations are required to disrupt mitochondrial integrity.

Retrograde cPLA2α/arachidonic acid/2-AG signaling is essential for cerebellar depolarization-induced suppression of excitation and long-term potentiation.

Cytosolic phospholipase A2 alpha (cPLA2α) responds to micromolar intracellular Ca(2+) and produces arachidonic acid, which regulates cellular homeostasis, neurotoxicity, and inflammation. Endocannabinoids are the derivates of arachidonic acid and widely distributed in the cerebellum. However, the role of cPLA2α/arachidonic acid pathway in cerebellar synaptic transmission and plasticity is unknown. We utilized cPLA2α knockout mice and slice whole-cell patch clamp to study the action of cPLA2α/arachidonic acid signaling on the depolarization-induced suppression of excitation (DSE) and long-term potentiation at parallel fiber-Purkinje cell synapses. Our data showed that DSE was significantly inhibited but rescued by arachidonic acid in cPLA2α knockout mice. The degradation enzyme of 2-arachidonoylglycerol (2-AG), monoacylglycerol lipase, blocked DSE, while another catabolism enzyme for N-arachidonoylethanolamine, fatty acid amide hydrolase, did not, suggesting that 2-AG is responsible for DSE in Purkinje cells. Co-application of paxilline reversed the blockade of DSE by internal K(+), indicating that large-conductance Ca(2+)-activated potassium channel is sufficient to inhibit cPLA2α/arachidonic acid-mediated DSE. On the other hand, we found that 1 Hz parallel fiber stimuli-triggered long-term potentiation (LTP) was deficient in cPLA2α knockout mice. LTP was also inhibited when AACOCF3, an inhibitor of cPLA2α, was given. Arachidonic acid was necessary for the LTP induction. Therefore, these data showed that cPLA2α/arachidonic acid/2-AG signaling pathway mediates DSE and LTP at parallel fiber-Purkinje cell synapse.

cPLA2α knockout mice exhibit abnormalities in the architecture and synapses of cortical neurons.

Cytosolic phospholipase A2α (cPLA2α) affects membrane fluidity and permeability by catalyzing the hydrolysis of membrane phospholipids. We hypothesize that cPLA2α deficiency induces rigidity and architectural changes in cell membranes, especially in large cortical neurons. These membrane changes are discernible using light and electron microscopy. Through careful comparison with wild-type counterparts, we observed significant morphological changes in cortical neurons of cPLA2α knockout mice. These changes included the following: (1) increased numbers of nucleoli and enlarged nuclei, (2) narrower spaces between the inner and outer nuclear membranes, (3) reduced numbers of nuclear pores and altered nuclear pore structure, and (4) morphological changes in synaptic clefts. These results further suggest that cPLA2α and its cleaved arachidonic acids play important roles in cortical neuronal maturation and in normal neurochemical processes.

Cytosolic phospholipase A2 alpha/arachidonic acid signaling mediates depolarization-induced suppression of excitation in the cerebellum.

BACKGROUND: Depolarization-induced suppression of excitation (DSE) at parallel fiber-Purkinje cell synapse is an endocannabinoid-mediated short-term retrograde plasticity. Intracellular Ca(2+) elevation is critical for the endocannabinoid production and DSE. Nevertheless, how elevated Ca(2+) leads to DSE is unclear. METHODOLOGY/PRINCIPAL FINDINGS: We utilized cytosolic phospholipase A(2) alpha (cPLA(2)α) knock-out mice and whole-cell patch clamp in cerebellar slices to observed the action of cPLA(2)α/arachidonic acid signaling on DSE at parallel fiber-Purkinje cell synapse. Our data showed that DSE was significantly inhibited in cPLA(2)α knock-out mice, which was rescued by arachidonic acid. The degradation enzyme of 2-arachidonoylglycerol (2-AG), monoacylglycerol lipase (MAGL), blocked DSE, while another catabolism enzyme for N-arachidonoylethanolamine (AEA), fatty acid amide hydrolase (FAAH), did not affect DSE. These results suggested that 2-AG is responsible for DSE in Purkinje cells. Co-application of paxilline reversed the blockade of DSE by internal K(+), indicating that large conductance Ca(2+)-activated potassium channel (BK) is sufficient to inhibit cPLA(2)α/arachidonic acid-mediated DSE. In addition, we showed that the release of 2-AG was independent of soluble NSF attachment protein receptor (SNARE), protein kinase C and protein kinase A. CONCLUSIONS/SIGNIFICANCE: Our data first showed that cPLA(2)α/arachidonic acid/2-AG signaling pathway mediates DSE at parallel fiber-Purkinje cell synapse.

Inhibition of cytosolic phospholipase A(2) alpha protects against focal ischemic brain damage in mice.

It is postulated that inhibition of cytosolic phospholipase A(2) alpha (cPLA(2)α) can reduce severity of stroke injury. This is supported by the finding that cPLA(2)α-deficient mice are partially protected from transient, focal cerebral ischemia. The object of this study was to determine the effect of cPLA(2)α inhibition with arachidonyl trifluoromethyl ketone (ATK) on stroke injury in mice. Male C57BL/6 mice were subjected to 1h of focal cerebral ischemia followed by 24 or 72 h of reperfusion. Mice were treated with ATK or vehicle by intermittent intraperitoneal injection or continuous infusion via an implanted infusion pump. ATK injections 1h before and then 1 and 6h after the start of reperfusion significantly reduced infarction volumes in striatum and hemisphere after 24h of reperfusion. ATK did not reduce injury if it was not administered before onset of ischemia or was not administered after 6h of reperfusion. Intermittent doses of ATK failed to reduce infarct volume after 72 h of reperfusion. Continuous infusion with ATK throughout 72h of reperfusion significantly reduced cortical and whole hemispheric infarct volume compared to vehicle treatment. Following ischemia and reperfusion, ATK treatment significantly reduced brain PLA(2) activity. These results are the first to demonstrate a therapeutic effect of cPLA(2)α inhibition on ischemia and reperfusion injury and define a therapeutic time window. cPLA(2)α activity augments injury in the acute and delayed phases of cerebral ischemia and reperfusion injury. We conclude that cPLA(2)α inhibition may be clinically useful if started before initiation of cerebral ischemia.

Disruption of group IVA cytosolic phospholipase A(2) attenuates myocardial ischemia-reperfusion injury partly through inhibition of TNF-α-mediated pathway.

Group IVA cytosolic phospholipase A(2) (cPLA(2)α), which preferentially cleaves arachidonic acid from phospholipids, plays a role in apoptosis and tissue injury. Downstream signals in response to tumor necrosis factor (TNF)-α, a mediator of myocardial ischemia-reperfusion (I/R) injury, involve cPLA(2)α activation. This study examined the potential role of cPLA(2)α and its mechanistic link with TNF-α in myocardial I/R injury using cPLA(2)α knockout (cPLA(2)α(-/-)) mice. Myocardial I/R was created with 10-wk-old male mice by 1 h ligation of the left anterior descending coronary artery, followed by 24 h of reperfusion. As a result, compared with wild-type (cPLA(2)α(+/+)) mice, cPLA(2)α(-/-) mice had a 47% decrease in myocardial infarct size, preservation of echocardiographic left ventricle (LV) function (%fractional shortening: 14 vs. 21%, respectively), and lower content of leukotriene B(4) and thromboxane B(2) (62 and 50% lower, respectively) in the ischemic myocardium after I/R. Treatment with the TNF-α inhibitor (soluble TNF receptor II/IgG1 Fc fusion protein, sTNFR:Fc) decreased myocardial I/R injury and LV dysfunction in cPLA(2)α(+/+) mice but not cPLA(2)α(-/-) mice. sTNFR:Fc also suppressed cPLA(2)α phosphorylation in the ischemic myocardium after I/R of cPLA(2)α(+/+) mice. Similarly, sTNFR:Fc exerted protective effects against hypoxia-reoxygenation (H/R)-induced injury in the cultured cardiomyocytes from cPLA(2)α(+/+) mice but not cPLA(2)α(-/-) cardiomyocytes. H/R and TNF-α induced cPLA(2)α phosphorylation in cPLA(2)α(+/+) cardiomyocytes, which was reversible by sTNFR:Fc. In cPLA(2)α(-/-) cardiomyocytes, TNF-α induced apoptosis and release of arachidonic acid to a lesser extent than in cPLA(2)α(+/+) cardiomyocytes. In conclusion, disruption of cPLA(2)α attenuates myocardial I/R injury partly through inhibition of TNF-α-mediated pathways.

Mitochondrial dysfunction and ß-cell failure in type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is the most common human endocrine disease and is characterized by peripheral insulin resistance and pancreatic islet ß-cell failure. Accumulating evidence indicates that mitochondrial dysfunction is a central contributor to ß-cell failure in the evolution of T2DM. As reviewed elsewhere, reactive oxygen species (ROS) produced by ß-cell mitochondria as a result of metabolic stress activate several stress-response pathways. This paper focuses on mechanisms whereby ROS affect mitochondrial structure and function and lead to ß-cell failure. ROS activate UCP2, which results in proton leak across the mitochondrial inner membrane, and this leads to reduced ß-cell ATP synthesis and content, which is a critical parameter in regulating glucose-stimulated insulin secretion. In addition, ROS oxidize polyunsaturated fatty acids in mitochondrial cardiolipin and other phospholipids, and this impairs membrane integrity and leads to cytochrome c release into cytosol and apoptosis. Group VIA phospholipase A2 (iPLA2ß) appears to be a component of a mechanism for repairing mitochondrial phospholipids that contain oxidized fatty acid substituents, and genetic or acquired iPLA2ß-deficiency increases ß-cell mitochondrial susceptibility to injury from ROS and predisposes to developing T2DM. Interventions that attenuate ROS effects on ß-cell mitochondrial phospholipids might prevent or retard development of T2DM.

Cytosolic phospholipase A(2)α protects against ischemia/reperfusion injury in the heart.

Studies with sPLA(2) Group X, and cPLA(2) α gene-targeted mice suggest that absence of sPLA(2) Group X results in protection from ischemia/reperfusion (I/R) injury in the heart, and absence of cPLA(2) α Group IV is protective in the brain. Although latter studies might suggest a similar deleterious role for cPLA(2) α in I/R injury in the heart, the pathophysiology of stroke is intricately related to excitotoxicity and cannot necessarily be extrapolated to the heart. We report here that unlike findings in the brain, cPLA(2) α((-/-)) mice have exaggerated injury following I/R in vivo. In contrast, there is no difference in injury induced by simulated ischemia in cardiomyocytes isolated from cPLA(2) α((-/-)) versus cPLA(2) α((+/+)) mice. This suggests that cPLA(2) α does not have an important cardiomyocyte autonomous effect on ischemic injury. Prostaglandin E(2) (PGE(2) ) levels are significantly reduced in the hearts of the cPLA(2) α((-/-)) mice, and the enhanced injury is ameliorated by treatment with the PGE analog, misoprostol. We demonstrate that cPLA(2) α is cardioprotective in vivo, and this is likely via cPLA(2) α-mediated production of cardioprotective eicosanoids. These studies are the first to identify a protective role for cPLA(2) in I/R injury in any organ and raise concerns over long-term inhibition of cPLA(2).

Phospholipase A2 superfamily members play divergent roles after spinal cord injury.

Spinal cord injury (SCI) results in permanent loss of motor functions. A significant aspect of the tissue damage and functional loss may be preventable as it occurs, secondary to the trauma. We show that the phospholipase A(2) (PLA(2)) superfamily plays important roles in SCI. PLA(2) enzymes hydrolyze membrane glycerophospholipids to yield a free fatty acid and lysophospholipid. Some free fatty acids (arachidonic acid) give rise to eicosanoids that promote inflammation, while some lysophospholipids (lysophosphatidylcholine) cause demyelination. We show in a mouse model of SCI that two cytosolic forms [calcium-dependent PLA(2) group IVA (cPLA(2) GIVA) and calcium-independent PLA(2) group VIA (iPLA(2) GVIA)], and a secreted form [secreted PLA(2) group IIA (sPLA(2) GIIA)] are up-regulated. Using selective inhibitors and null mice, we show that these PLA(2)s play differing roles. cPLA(2) GIVA mediates protection, whereas sPLA(2) GIIA and, to a lesser extent, iPLA(2) GVIA are detrimental. Furthermore, completely blocking all three PLA(2)s worsens outcome, while the most beneficial effects are seen by partial inhibition of all three. The partial inhibitor enhances expression of cPLA(2) and mediates its beneficial effects via the prostaglandin EP1 receptor. These findings indicate that drugs that inhibit detrimental forms of PLA(2) (sPLA(2) and iPLA2) and up-regulate the protective form (cPLA2) may be useful for the treatment of SCI.