Cyclooxygenase-1 mediates neuroinflammation and neurotoxicity in a mouse model of retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa (RP) is a group of inherited eye disorders with progressive degeneration of photoreceptors in the retina, ultimately leading to partial or complete blindness. The mechanisms underlying photoreceptor degeneration are not yet completely understood. Neuroinflammation is reported to play a pathological role in RP. However, the mechanisms that trigger neuroinflammation remain largely unknown. To address this question, we investigated the role of cyclooxygenase-1 (COX-1), a key enzyme in the conversion of arachidonic acid to proinflammatory prostaglandins, in the rd10 mouse model of RP. METHODS: We backcrossed COX-1 knockout mice (COX-1-/-) onto the rd10 mouse model of RP and investigated the impact of COX-1 deletion on neuroinflammation in the resulting COX-1-/-/rd10 mouse line, using a combination of immunocytochemistry, flow cytometry, qPCR, ELISA, and a series of simple visual tests. RESULTS: We found that genetic ablation or pharmacological inhibition of COX-1 alleviated neuroinflammation and subsequently preserved retinal photoreceptor and function and visual performance in rd10 mice. Moreover, we observed that the pharmacological inhibition of the prostaglandin E2 (PGE2) EP2 receptors largely replicated the beneficial effects of COX-1 deletion, suggesting that EP2 receptor was a critical downstream effector of COX-1-mediated neurotoxicity in rd10 mice. CONCLUSION: Our data suggest that the COX-1/PGE2/EP2 signaling pathway was partly responsible for significantly increased neuroinflammation and disease progression in rd10 mice, and that EP2 receptor could be targeted therapeutically to block the pathological activity of COX-1 without inducing any potential side effects in treating RP patients.

Cell-Specific Gene Deletion Reveals the Antithrombotic Function of COX1 and Explains the Vascular COX1/Prostacyclin Paradox.

RATIONALE: Endothelial cells (ECs) and platelets, which respectively produce antithrombotic prostacyclin and prothrombotic thromboxane A2, both express COX1 (cyclooxygenase1). Consequently, there has been no way to delineate any antithrombotic role for COX1-derived prostacyclin from the prothrombotic effects of platelet COX1. By contrast, an antithrombotic role for COX2, which is absent in platelets, is straightforward to demonstrate. This has resulted in an incomplete understanding of the relative importance of COX1 versus COX2 in prostacyclin production and antithrombotic protection in vivo. OBJECTIVE: We sought to identify the role, if any, of COX1-derived prostacyclin in antithrombotic protection in vivo and compare this to the established protective role of COX2. METHODS AND RESULTS: We developed vascular-specific COX1 knockout mice and studied them alongside endothelial-specific COX2 knockout mice. COX1 immunoreactivity and prostacyclin production were primarily associated with the endothelial layer of aortae; freshly isolated aortic ECs released >10-fold more prostacyclin than smooth muscle cells. Moreover, aortic prostacyclin production, the ability of aortic rings to inhibit platelet aggregation and plasma prostacyclin levels were reduced when COX1 was knocked out in ECs but not in smooth muscle cells. When thrombosis was measured in vivo after FeCl3 carotid artery injury, endothelial COX1 deletion accelerated thrombosis to a similar extent as prostacyclin receptor blockade. However, this effect was lost when COX1 was deleted from both ECs and platelets. Deletion of COX2 from ECs also resulted in a prothrombotic phenotype that was independent of local vascular prostacyclin production. CONCLUSIONS: These data demonstrate for the first time that, in healthy animals, endothelial COX1 provides an essential antithrombotic tone, which is masked when COX1 activity is lost in both ECs and platelets. These results help us define a new 2-component paradigm wherein thrombotic tone is regulated by both COX1 and COX2 through complementary but mechanistically distinct pathways.

Loss of hypothermic and anti-pyretic action of paracetamol in cyclooxygenase-1 knockout mice is indicative of inhibition of cyclooxygenase-1 variant enzymes.

Paracetamol (acetaminophen), is a centrally-acting antipyretic analgesic drug, which can also lower body temperature. Despite a century of clinical use, its mechanism of pharmacological action has not been completely elucidated. Previously, we demonstrated significant attenuation in the paracetamol induced hypothermia in parallel with its inhibitory action on the synthesis of brain prostaglandin E2 (PGE2) in cyclooxygenase-1 (COX-1) knockout mice in comparison to wild-type mice. The above reported pharmacological actions by paracetamol were completely retained in COX-2 knockout mice. We thus concluded that the mechanism of hypothermic action of paracetamol is dependent on inhibition of a COX-1 gene-derived enzyme. In the current investigation, we provide further support for this notion by demonstrating that the paracetamol-induced hypothermia is not mediated through inhibition of COX-1 as neither the COX-1 selective inhibitor, SC560, nor the COX-1/COX-2 dual inhibitor, indomethacin, induced hypothermia at pharmacologically active doses in mice. In addition, using a COX-2-dependent and PGE2-mediated model of endotoxin-induced fever, paracetamol induced anti-pyretic and hypothermic actions in COX-1 wild-type mice. These effects were fully or partially attenuated in COX-1 knockout mice after prophylactic or therapeutic administration, respectively. Therapeutically-administered paracetamol also reduced hypothalamic PGE2 biosynthesis in febrile COX-1 wild-type mice, but not in febrile COX-1 knockout mice. In conclusion, we provide further evidence which suggests that the hypothermic and now anti-pyretic actions of paracetamol are mediated through inhibition of a COX-1 variant enzyme.

Platelet-Specific Deletion of Cyclooxygenase-1 Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice.

Inflammatory bowel disease (IBD) is associated with an increased risk for thromboembolism, platelet activation, and abnormalities in platelet number and size. In colitis, platelets can extravasate into the colonic interstitium. We generated a mouse with a specific deletion of cyclooxygenase (COX)-1 in megakaryocytes/platelets [(COX-1 conditional knockout (cKO)] to clarify the role of platelet activation in the development of inflammation and fibrosis in dextran sodium sulfate (DSS)-induced colitis. The disease activity index was assessed, and colonic specimens were evaluated for histologic features of epithelial barrier damage, inflammation, and fibrosis. Cocultures of platelets and myofibroblasts were performed. We found that the specific deletion of COX-1 in platelets, which recapitulated the human pharmacodynamics of low-dose aspirin, that is, suppression of platelet thromboxane (TX)A2 production associated with substantial sparing of the systemic production of prostacyclin, resulted in milder symptoms of colitis, in the acute phase, and almost complete recovery from the disease after DSS withdrawal. Reduced colonic accumulation of macrophages and myofibroblasts and collagen deposition was found. Platelet-derived TXA2 enhanced the ability of myofibroblasts to proliferate and migrate in vitro, and these effects were prevented by platelet COX-1 inhibition or antagonism of the TXA2 receptor. Our findings allow a significant advance in the knowledge of the role of platelet-derived TXA2 in the development of colitis and fibrosis in response to intestinal damage and provide the rationale to investigate the potential efficacy of the antiplatelet agent low-dose aspirin in limiting the inflammatory response and fibrosis associated with IBD. SIGNIFICANCE STATEMENT: Inflammatory bowel disease (IBD) is characterized by the development of a chronic inflammatory response, which can lead to intestinal fibrosis for which currently there is no medical treatment. Through the generation of a mouse with specific deletion of cyclooxygenase-1 in megakaryocytes/platelets, which recapitulates the human pharmacodynamics of low-dose aspirin, we demonstrate the important role of platelet-derived thromboxane A2 in the development of experimental colitis and fibrosis, thus providing the rationale to investigate the potential efficacy of low-dose aspirin in limiting the inflammation and tissue damage associated with IBD.

TRPV4 Channel Signaling in Macrophages Promotes Gastrointestinal Motility via Direct Effects on Smooth Muscle Cells.

Intestinal macrophages are critical for gastrointestinal (GI) homeostasis, but our understanding of their role in regulating intestinal motility is incomplete. Here, we report that CX3C chemokine receptor 1-expressing muscularis macrophages (MMs) were required to maintain normal GI motility. MMs expressed the transient receptor potential vanilloid 4 (TRPV4) channel, which senses thermal, mechanical, and chemical cues. Selective pharmacologic inhibition of TRPV4 or conditional deletion of TRPV4 from macrophages decreased intestinal motility and was sufficient to reverse the GI hypermotility that is associated with chemotherapy treatment. Mechanistically, stimulation of MMs via TRPV4 promoted the release of prostaglandin E2 and elicited colon contraction in a paracrine manner via prostaglandin E receptor signaling in intestinal smooth muscle cells without input from the enteric nervous system. Collectively, our data identify TRPV4-expressing MMs as an essential component required for maintaining normal GI motility and provide potential drug targets for GI motility disorders.

Proteomic Analysis Shows Constitutive Secretion of MIF and p53-associated Activity of COX-2-/- Lung Fibroblasts.

The differential expression of two closelyassociated cyclooxygenase isozymes, COX-1 and COX-2, exhibited functions beyond eicosanoid metabolism. We hypothesized that COX-1 or COX-2 knockout lung fibroblasts may display altered protein profiles which may allow us to further differentiate the functional roles of these isozymes at the molecular level. Proteomic analysis shows constitutive production of macrophage migration inhibitory factor (MIF) in lung fibroblasts derived from COX-2-/- but not wild-type (WT) or COX-1-/- mice. MIF was spontaneously released in high levels into the extracellular milieu of COX2-/- fibroblasts seemingly from the preformed intracellular stores, with no change in the basal gene expression of MIF. The secretion and regulation of MIF in COX-2-/- was "prostaglandin-independent." GO analysis showed that concurrent with upregulation of MIF, there is a significant surge in expression of genes related to fibroblast growth, FK506 binding proteins, and isomerase activity in COX-2-/- cells. Furthermore, COX-2-/- fibroblasts also exhibit a significant increase in transcriptional activity of various regulators, antagonists, and co-modulators of p53, as well as in the expression of oncogenes and related transcripts. Integrative Oncogenomics Cancer Browser (IntroGen) analysis shows downregulation of COX-2 and amplification of MIF and/or p53 activity during development of glioblastomas, ependymoma, and colon adenomas. These data indicate the functional role of the MIF-COX-p53 axis in inflammation and cancer at the genomic and proteomic levels in COX-2-ablated cells. This systematic analysis not only shows the proinflammatory state but also unveils a molecular signature of a pro-oncogenic state of COX-1 in COX-2 ablated cells.

Genomic, Lipidomic and Metabolomic Analysis of Cyclooxygenase-null Cells: Eicosanoid Storm, Cross Talk, and Compensation by COX-1.

The constitutively-expressed cyclooxygenase 1 (COX-1) and the inducible COX-2 are both involved in the conversion of arachidonic acid (AA) to prostaglandins (PGs). However, the functional roles of COX-1 at the cellular level remain unclear. We hypothesized that by comparing differential gene expression and eicosanoid metabolism in lung fibroblasts from wild-type (WT) mice and COX-2(-/-) or COX-1(-/-) mice may help address the functional roles of COX-1 in inflammation and other cellular functions. Compared to WT, the number of specifically-induced transcripts were altered descendingly as follows: COX-2(-/-)>COX-1(-/-)>WT+IL-1ß. COX-1(-/-) or COX-2(-/-) cells shared about 50% of the induced transcripts with WT cells treated with IL-1ß, respectively. An interactive "anti-inflammatory, proinflammatory, and redox-activated" signature in the protein-protein interactome map was observed in COX-2(-/-) cells. The augmented COX-1 mRNA (in COX-2(-/-) cells) was associated with the upregulation of mRNAs for glutathione S-transferase (GST), superoxide dismutase (SOD), NAD(P)H dehydrogenase quinone 1 (NQO1), aryl hydrocarbon receptor (AhR), peroxiredoxin, phospholipase, prostacyclin synthase, and prostaglandin E synthase, resulting in a significant increase in the levels of PGE2, PGD2, leukotriene B4 (LTB4), PGF1α, thromboxane B2 (TXB2), and PGF2α. The COX-1 plays a dominant role in shifting AA toward the LTB4 pathway and anti-inflammatory activities. Compared to WT, the upregulated COX-1 mRNA in COX-2(-/-) cells generated an "eicosanoid storm". The genomic characteristics of COX-2(-/-) is similar to that of proinflammatory cells as observed in IL-1ß induced WT cells. COX-1(-/-) and COX-2(-/-) cells exhibited compensation of various eicosanoids at the genomic and metabolic levels.

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.

Ibuprofen slows migration and inhibits bowel colonization by enteric nervous system precursors in zebrafish, chick and mouse.

Hirschsprung Disease (HSCR) is a potentially deadly birth defect characterized by the absence of the enteric nervous system (ENS) in distal bowel. Although HSCR has clear genetic causes, no HSCR-associated mutation is 100% penetrant, suggesting gene-gene and gene-environment interactions determine HSCR occurrence. To test the hypothesis that certain medicines might alter HSCR risk we treated zebrafish with medications commonly used during early human pregnancy and discovered that ibuprofen caused HSCR-like absence of enteric neurons in distal bowel. Using fetal CF-1 mouse gut slice cultures, we found that ibuprofen treated enteric neural crest-derived cells (ENCDC) had reduced migration, fewer lamellipodia and lower levels of active RAC1/CDC42. Additionally, inhibiting ROCK, a RHOA effector and known RAC1 antagonist, reversed ibuprofen effects on migrating mouse ENCDC in culture. Ibuprofen also inhibited colonization of Ret+/- mouse bowel by ENCDC in vivo and dramatically reduced bowel colonization by chick ENCDC in culture. Interestingly, ibuprofen did not affect ENCDC migration until after at least three hours of exposure. Furthermore, mice deficient in Ptgs1 (COX 1) and Ptgs2 (COX 2) had normal bowel colonization by ENCDC and normal ENCDC migration in vitro suggesting COX-independent effects. Consistent with selective and strain specific effects on ENCDC, ibuprofen did not affect migration of gut mesenchymal cells, NIH3T3, or WT C57BL/6 ENCDC, and did not affect dorsal root ganglion cell precursor migration in zebrafish. Thus, ibuprofen inhibits ENCDC migration in vitro and bowel colonization by ENCDC in vivo in zebrafish, mouse and chick, but there are cell type and strain specific responses. These data raise concern that ibuprofen may increase Hirschsprung disease risk in some genetically susceptible children.

Role of cyclooxygenase-1 and -2 in endothelium-dependent contraction of atherosclerotic mouse abdominal aortas.

The objective of this study was to determine the role of cyclooxygenase (COX)-1 or -2 in endothelium-dependent contraction under atherosclerotic conditions. Atherosclerosis was induced in apoE knockout (apoE(-/-)) mice and those with COX-1(-/-) (apoE(-/-)-COX-1(-/-)) by feeding with high fat and cholesterol food. Aortas (abdominal or the whole section) were isolated for functional and/or biochemical analyses. As in non-atherosclerotic conditions, the muscarinic receptor agonist acetylcholine (ACh) evoked an endothelium-dependent, COX-mediated contraction following NO synthase (NOS) inhibition in abdominal aortic rings from atherosclerotic apoE(-/-) mice. Interestingly, COX-1 inhibition not only abolished such a contraction in rings showing normal appearance, but also diminished that in rings with plaques. Accordingly, only a minor contraction (<30% that of apoE(-/-) counterparts) was evoked by ACh (following NOS inhibition) in abdominal aortic rings of atherosclerotic apoE(-/-)-COX-1(-/-) mice with plaques, and none was evoked in those showing normal appearance. Also, the contraction evoked by ACh in apoE(-/-)-COX-1(-/-) abdominal aortic rings with plaques was abolished by non-selective COX inhibition, thromboxane-prostanoid (TP) receptor antagonism, or endothelial denudation. Moreover, it was noted that ACh evoked a predominant production of the prostacyclin (PGI2, which mediates abdominal aortic contraction via TP receptors in mice) metabolite 6-keto-PGF1α, which was again sensitive to COX-1 inhibition or COX-1(-/-). Therefore, in atherosclerotic mouse abdominal aortas, COX-1 can still be the major isoform mediating endothelium-dependent contraction, which probably results largely from PGI2 synthesis as in non-atherosclerotic conditions. In contrast, COX-2 may have only a minor role in such response limited to areas of plaques under the same pathological condition.

Cyclooxygenase-1 serves a vital hepato-protective function in chemically induced acute liver injury.

Cyclooxygenase-1 (COX-1) is the constitutive form of the COX enzyme family, which produces bioactive lipids called prostanoids. Although the role of COX-2 in liver diseases has been studied, little is known about the function of COX-1 in liver injury. We aimed to investigate the role and mechanism of COX-1 in acute liver injury. Carbon tetrachloride (CCl(4)) was administered to induce acute liver injury in wild-type or COX-1-deficient mice. Both genetic (partially or completely) deletion of COX-1 expression and pharmacological inhibition of COX-1 activity in mice exacerbated acute liver injury induced by CCl(4), revealing the (1) histopathological changes and increased serum levels of aminotransferases; (2) oxidative stress in the liver partly through the action of cytochrome P450 2E1-dependent pathway; (3) enhanced inflammatory and chemoattractive responses with increased number of activated macrophages; and (4) increased apoptosis through both intrinsic and extrinsic apoptotic pathways. These pathological changes were partly through the modulation of transcription factor-dependent pathways (eg, NF-κB and C/EBP-α). Pre-treatment with prostaglandin E2 (PGE(2)) or 5-lipoxygenase (5-LO) inhibitor in homozygous COX-1 knockout mice significantly ameliorated CCl(4)-induced hepatic injury. In addition, level of hepato-protective molecules (eg, OSM and OSMR) and associated liver regeneration pathway were significantly inhibited by the deficiency of COX-1 but restored by the addition of PGE(2) or the inhibition of 5-LO. Furthermore, the alternative arachidonic acid metabolism pathway of 5-LO, which induced additional inflammation in the liver, was activated in response to the deficiency of COX-1. In conclusion, basal expression of COX-1 is essential for the protection of liver against chemical-induced hepatotoxicity and required for hepatic homeostatic maintenance.

Vasoconstrictor role of cyclooxygenase-1-mediated prostacyclin synthesis in non-insulin-dependent diabetic mice induced by high-fat diet and streptozotocin.

This study tested the hypothesis that in diabetic arteries, cyclooxygenase (COX)-1 mediates endothelial prostacyclin (PGI2) synthesis, which evokes vasoconstrictor activity under the pathological condition. Non-insulin-dependent diabetes was induced to C57BL/6 mice and those with COX-1 deficiency (COX-1(-/-) mice) using a high-fat diet in combination with streptozotocin injection. In vitro analyses were performed 3 mo after. Results showed that in diabetic aortas, the endothelial muscarinic receptor agonist ACh evoked an endothelium-dependent production of the PGI2 metabolite 6-keto-PGF1α, which was abolished in COX-1(-/-) mice. Meanwhile, COX-1 deficiency or COX-1 inhibition prevented vasoconstrictor activity in diabetic abdominal aortas, resulting in enhanced relaxation evoked by ACh. In a similar manner, COX-1 deficiency increased the relaxation evoked by ACh in nitric oxide synthase-inhibited diabetic renal arteries. Also, in diabetic abdominal aortas and/or renal arteries, both PGI2 and the COX substrate arachidonic acid evoked contractions similar to those of nondiabetic mice. However, the contraction to arachidonic acid, but not that to PGI2, was abolished in vessels from COX-1(-/-) mice. Moreover, we found that 3 mo after streptozotocin injection, systemic blood pressure increased in diabetic C57BL/6 mice but not in diabetic COX-1(-/-) mice. These results explicitly demonstrate that in the given arteries from non-insulin-dependent diabetic mice, COX-1 remains a major contributor to the endothelial PGI2 synthesis that evokes vasoconstrictor activity under the pathological condition. Also, our data suggest that COX-1 deficiency prevents or attenuates diabetic hypertension in mice, although this could be related to the loss of COX-1-mediated activities derived from both vascular and nonvascular tissues.

Late postoperative hemorrhage in a patient with undiagnosed COX-1 deficiency after third molar extractions.

Oral maxillofacial surgeons direct invasive procedures that often cause significant bleeding. Uncontrolled hemorrhage is a rare, yet serious, complication that can be seen in patients with thrombocytopathy. Platelets have 3 distinct roles in coagulation: initial adhesion, phospholipid externalization, and platelet aggregation.(1) Several types of platelet deficiencies, including defects of adhesion (Bernard-Soulier syndrome), defects of aggregation (Glanzmann thrombasthenia), and disorders of platelet secretion due to a deficiency of adenosine diphosphate (ADP) or cyclooxygenase-1 (COX-1).(2-4) COX has 2 isoforms: COX-1 and COX-2.(5,6) COX-1 is expressed constitutively in most tissues, and COX-2 is induced primarily by inflammatory mediators.(7,8) Although both isoforms are present in platelets, COX-1 is the major isoform that contributes to coagulation, because it is critically important in the formation of thromboxane A2 (TXA2) by way of the arachidonic acid (AA) pathway.(9) AA is a potent inducer of platelet aggregation.(1,3,4) When AA is exposed to an activating agent, such as ADP, it undergoes a series of enzymatic reactions that culminates in the production of TXA2.(10) TXA2 is the predominant product of the COX-1 pathway and is a major metabolite of AA in platelets. TXA2 is necessary for normal platelet function. Therefore, the inhibition of, or a deficiency in, COX-1 will compromise the AA pathway, thereby reducing platelet secretion and altering normal platelet aggregatory function.(1,3) COX-1 deficiencies are usually caused by drug interactions with the enzyme itself. In addition, studies have identified genetic mutations that can result in COX-1 deficiency.(2) We present the hospital course, management, and diagnosis of a patient with an undiagnosed COX-1 deficiency who had had third molars removed in a private office. To our knowledge, this is the first case of COX-1 deficiency diagnosed after exodontia documented in English studies. In addition, we reviewed the published data of this rare disorder that has significant clinical implications.

COX-1-derived PGE2 and PGE2 type 1 receptors are vital for angiotensin II-induced formation of reactive oxygen species and Ca(2+) influx in the subfornical organ.

Regulation of blood pressure by angiotensin II (ANG II) is a process that involves the reactive oxygen species (ROS) and calcium. We have shown that ANG-II type 1 receptor (AT1R) and prostaglandin E2 (PGE2) type 1 receptors (EP1R) are required in the subfornical organ (SFO) for ROS-mediated hypertension induced by slow-pressor ANG-II infusion. However, the signaling pathway associated with this process remains unclear. We sought to determine mechanisms underlying the ANG II-induced ROS and calcium influx in mouse SFO cells. Ultrastructural studies showed that cyclooxygenase 1 (COX-1) codistributes with AT1R in the SFO, indicating spatial proximity. Functional studies using SFO cells revealed that ANG II potentiated PGE2 release, an effect dependent on AT1R, phospholipase A2 (PLA2) and COX-1. Furthermore, both ANG II and PGE2 increased ROS formation. While the increase in ROS initiated by ANG II, but not PGE2, required the activation of the AT1R/PLA2/COX-1 pathway, both ANG II and PGE2 were dependent on EP1R and Nox2 as downstream effectors. Finally, ANG II potentiated voltage-gated L-type Ca(2+) currents in SFO neurons via the same signaling pathway required for PGE2 production. Blockade of EP1R and Nox2-derived ROS inhibited ANG II and PGE2-mediated Ca(2+) currents. We propose a mechanism whereby ANG II increases COX-1-derived PGE2 through the AT1R/PLA2 pathway, which promotes ROS production by EP1R/Nox2 signaling in the SFO. ANG II-induced ROS are coupled with Ca(2+) influx in SFO neurons, which may influence SFO-mediated sympathoexcitation. Our findings provide the first evidence of a spatial and functional framework that underlies ANG-II signaling in the SFO and reveal novel targets for antihypertensive therapies.

Impact of hematopoietic cyclooxygenase-1 deficiency on obesity-linked adipose tissue inflammation and metabolic disorders in mice.

OBJECTIVE: Adipose tissue (AT)-specific inflammation is considered to mediate the pathological consequences of obesity and macrophages are known to activate inflammatory pathways in obese AT. Because cyclooxygenases play a central role in regulating the inflammatory processes, we sought to determine the role of hematopoietic cyclooxygenase-1 (COX-1) in modulating AT inflammation in obesity. MATERIALS/METHODS: Bone marrow transplantation was performed to delete COX-1 in hematopoietic cells. Briefly, female wild type (wt) mice were lethally irradiated and injected with bone marrow (BM) cells collected from wild type (COX-1+/+) or COX-1 knock-out (COX-1-/-) donor mice. The mice were fed a high fat diet for 16 weeks. RESULTS: The mice that received COX-1-/- bone marrow (BM-COX-1-/-) exhibited a significant increase in fasting glucose, total cholesterol and triglycerides in the circulation compared to control (BM-COX-1+/+) mice. Markers of AT-inflammation were increased and were associated with increased leptin and decreased adiponectin in plasma. Hepatic inflammation was reduced with a concomitant reduction in TXB2 levels. The hepatic mRNA expression of genes involved in lipogenesis and lipid transport was increased while expression of genes involved in regulating hepatic glucose output was reduced in BM-COX-1-/- mice. Finally, renal inflammation and markers of renal glucose release were increased in BM-COX-1-/- mice. CONCLUSION: Hematopoietic COX-1 deletion results in impairments in metabolic homeostasis which may be partly due to increased AT inflammation and dysregulated adipokine profile. An increase in renal glucose release and hepatic lipogenesis/lipid transport may also play a role, at least in part, in mediating hyperglycemia and dyslipidemia, respectively.

Cultured preadipocytes undergoing stable transfection with cyclooxygenase-1 in the antisense direction accelerate adipogenesis during the maturation phase of adipocytes.

The arachidonate cyclooxygenase (COX) pathway is involved in the generation of several types of endogenous prostaglandins (PGs) with opposite effects on adipogenesis at different life stages of adipocytes. However, the specific role of COX isoforms, the rate-limiting enzymes for the pathway, remains elusive in the regulation of the endogenous synthesis of PGs. This study was aimed at the selective suppression of the constitutive COX-1 in cultured preadipocytes by the isolation of cloned preadipocytes transfected stably with a mammalian expression vector harboring cDNA encoding mouse COX-1 in the antisense direction. The gene expression analysis revealed that the transcript and protein levels of the constitutive COX-1 were substantially suppressed in the isolated cloned transfectants with antisense COX-1. By contrast, the expression of the inducible COX-2 was not affected in the stable transfectants with antisense COX-1. All of the cloned stable transfectants with antisense COX-1 exhibited a significant reduction in the immediate synthesis of PGE2 serving as an anti-adipogenic factor. The sustained expression of COX-1 in the antisense direction induced the appreciable stimulation of fat storage in adipocytes during the maturation phase, which was associated with the higher expression levels of adipocyte-specific genes, indicating the positive regulation of adipogenesis program. Moreover, the up-regulation of adipogenesis is accompanied by a higher production of J2 series PGs including 15-deoxy-Δ(12,14)-PGJ2 and Δ(12)-PGJ2, known as pro-adipogenic factors by the transfectants with antisense COX-1. The results suggest that the inducible COX-2 can contribute to the endogenous synthesis of PGJ2 derivatives acting as autocrine mediators to simulate adipogenesis during the maturation phase by way of compensation for the suppressed expression of the constitutive COX-1.

Neutrophils control the magnitude and spread of the immune response in a thromboxane A2-mediated process.

Neutrophils are obligate cells entering lymph nodes shortly after immunization with protein antigens in adjuvants, starting during the first hour and continuing for several days in two distinct waves. Previously, we demonstrated the strong suppressive effects of neutrophils on CD4 T cell and B cell responses, using either neutrophil-depleting antibodies or genetically neutropenic mice. In this study, we find that neutrophils are the major cells controlling the spread of T cell responses to distal lymph nodes. Although in the presence of neutrophils, ∼75% of the response was restricted to the draining node, in their absence, most of the response was found in distal nodes. Prostanoids were responsible for the rapid entry of neutrophils into the draining nodes, as well as for the two distinct neutrophil effects: the modulation of the magnitude of the cellular response, and in its spread outside the draining nodes. Neutrophil-produced thromboxane A(2) was the key eicosanoid controlling both effects. Adoptive transfer of neutrophils into mice genetically deficient in neutrophils indicated their role in both. These functions of neutrophils are important in infections and vaccinations with adjuvants where neutrophils are abundant in the initial stages.

Rapid induction of inflammatory lipid mediators by the inflammasome in vivo.

Detection of microbial products by host inflammasomes is an important mechanism of innate immune surveillance. Inflammasomes activate the caspase-1 (CASP1) protease, which processes the cytokines interleukin (IL)-1ß and IL-18, and initiates a lytic host cell death called pyroptosis. To identify novel CASP1 functions in vivo, we devised a strategy for cytosolic delivery of bacterial flagellin, a specific ligand for the NAIP5 (NLR family, apoptosis inhibitory protein 5)/NLRC4 (NLR family, CARD-domain-containing 4) inflammasome. Here we show that systemic inflammasome activation by flagellin leads to a loss of vascular fluid into the intestine and peritoneal cavity, resulting in rapid (less than 30 min) death in mice. This unexpected response depends on the inflammasome components NAIP5, NLRC4 and CASP1, but is independent of the production of IL-1ß or IL-18. Instead, inflammasome activation results, within minutes, in an 'eicosanoid storm'--a pathological release of signalling lipids, including prostaglandins and leukotrienes, that rapidly initiate inflammation and vascular fluid loss. Mice deficient in cyclooxygenase-1, a critical enzyme in prostaglandin biosynthesis, are resistant to these rapid pathological effects of systemic inflammasome activation by either flagellin or anthrax lethal toxin. Inflammasome-dependent biosynthesis of eicosanoids is mediated by the activation of cytosolic phospholipase A(2) in resident peritoneal macrophages, which are specifically primed for the production of eicosanoids by high expression of eicosanoid biosynthetic enzymes. Our results therefore identify eicosanoids as a previously unrecognized cell-type-specific signalling output of the inflammasome with marked physiological consequences in vivo.

Concomitant activation of functionally opposing prostacyclin and thromboxane prostanoid receptors by cyclo-oxygenase-1-mediated prostacyclin synthesis in mouse arteries.

This study aimed to determine whether cyclo-oxygenase-1 (COX-1) mediates dilatation of mouse arteries via synthesis of prostacyclin (PGI(2)) and, if so, how PGI(2) (IP) receptors contribute and whether thromboxane prostanoid (TP) receptors are implicated in the process. Mesenteric arteries were isolated from wild-type mice or mice with COX-1 deficiency (COX-1(-/-)). The vasomotor reaction to the COX substrate arachidonic acid (AA) was determined with isometric force measurement, while the in vitro production or the plasma level of the PGI(2) metabolite 6-keto-PGF(1α) was analysed with high-performance liquid chromatography-mass spectroscopy or enzyme immunoassay, respectively. Results showed that AA, which evoked endothelium-dependent 6-keto-PGF(1α) production, elicited relaxation that was inhibited or enhanced by antagonizing IP or TP receptors, respectively. Also, IP receptor blockade resulted in contraction in response to AA (following NO synthase inhibition), which was prevented by a concomitant TP receptor antagonism. Meanwhile, COX-1(-/-) or COX-1 inhibition abolished the in vitro 6-keto-PGF(1α) production and reduced the relaxation or contraction observed with AA. Real-time PCR showed that whereas TP receptor mRNAs were detected at similar levels, IP receptor mRNAs were present at higher levels in the branches than in the main stem of the mesenteric artery. In addition, antagonizing the IP receptors enhanced the contraction evoked by PGI(2) in the carotid artery. Also, we noted that COX-1(-/-) mice had a reduced basal plasma 6-keto-PGF(1α) level. These results demonstrate an explicit vasodilator role for COX-1-mediated endothelial PGI(2) synthesis and suggest that the functionally opposing IP and TP receptors concomitantly mediate the vasomotor reaction to PGI(2), with the dilator activity of IP receptors being compromised by the vasoconstrictor effect of TP receptors and vice versa.