Search

Resistance to immunotherapy is one of the biggest problems of current oncotherapeutics. WhileT cell abundance is essential for tumor responsiveness to immunotherapy, factors that define the T cell inflamed tumor microenvironment are not fully understood. We conducted an unbiased approach to identify tumor-intrinsic mechanisms shaping the immune tumor microenvironment(TME), focusing on pancreatic adenocarcinoma because it is refractory to immunotherapy and excludes T cells from the TME. From human tumors, we identified EPHA2 as a candidate tumor intrinsic driver of immunosuppression. Epha2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy. We found that PTGS2, the gene encoding cyclooxygenase-2, lies downstream of EPHA2 signaling through TGFβ and is associated with poor patient survival. Ptgs2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy; pharmacological inhibition of PTGS2 was similarly effective. Thus, EPHA2-PTGS2 signaling in tumor cells regulates tumor immune phenotypes; blockade may represent a novel therapeutic avenue for immunotherapy-refractory cancers. Our findings warrant clinical trials testing the effectiveness of therapies combining EPHA2-TGFβ-PTGS2 pathway inhibitors with anti-tumor immunotherapy, and may change the treatment of notoriously therapy-resistant pancreatic adenocarcinoma.

Background: Large-scale, placebo-controlled trials established that nonsteroidal anti-inflammatory drugs confer a cardiovascular hazard: this has been attributed to depression of cardioprotective products of cyclooxygenase (COX)-2, especially prostacyclin. An alternative mechanism by which nonsteroidal anti-inflammatory drugs might constrain cardioprotection is by enhancing the formation of methylarginines in the kidney that would limit the action of nitric oxide throughout the vasculature., Methods: Targeted and untargeted metabolomics were used to investigate the effect of COX-2 deletion or inhibition in mice and in osteoarthritis patients exposed to nonsteroidal anti-inflammatory drugs on the l-arginine/nitric oxide pathway., Results: Analysis of the plasma and renal metabolome was performed in postnatal tamoxifen-inducible Cox-2 knockout mice, which exhibit normal renal function and blood pressure. This revealed no changes in arginine and methylarginines compared with their wild-type controls. Moreover, the expression of genes in the l-arginine/nitric oxide pathway was not altered in the renal medulla or cortex of tamoxifen inducible Cox-2 knockout mice. Therapeutic concentrations of the selective COX-2 inhibitors, rofecoxib, celecoxib, and parecoxib, none of which altered basal blood pressure or renal function as reflected by plasma creatinine, failed to elevate plasma arginine and methylarginines in mice. Finally, plasma arginine or methylarginines were not altered in osteoarthritis patients with confirmed exposure to nonsteroidal anti-inflammatory drugs that inhibit COX-1 and COX-2. By contrast, plasma asymmetrical dimethylarginine was increased in mice infused with angiotensin II sufficient to elevate blood pressure and impair renal function. Four weeks later, blood pressure, plasma creatinine, and asymmetrical dimethylarginine were restored to normal levels. The increase in asymmetrical dimethylarginine in response to infusion with angiotensin II in celecoxib-treated mice was also related to transient impairment of renal function., Conclusions: Plasma methylarginines are not altered by COX-2 deletion or inhibition but rather are elevated coincident with renal compromise.

An inducible gene expression program is a hallmark of the host inflammatory response. Recently, long intergenic non-coding RNAs (lincRNAs) have been shown to regulate the magnitude, duration, and resolution of these responses. Among these is lincRNA-Cox2, a dynamically regulated gene that broadly controls immune gene expression. To evaluate the in vivo functions of this lincRNA, we characterized multiple models of lincRNA-Cox2-deficient mice. LincRNA-Cox2-deficient macrophages and murine tissues had altered expression of inflammatory genes. Transcriptomic studies from various tissues revealed that deletion of the lincRNA-Cox2 locus also strongly impaired the basal and inducible expression of the neighboring gene prostaglandin-endoperoxide synthase (Ptgs2), encoding cyclooxygenase-2, a key enzyme in the prostaglandin biosynthesis pathway. By utilizing different genetic manipulations in vitro and in vivo, we found that lincRNA-Cox2 functions through an enhancer RNA mechanism to regulate Ptgs2. More importantly, lincRNA-Cox2 also functions in trans, independently of Ptgs2, to regulate critical innate immune genes in vivo., (Published by Elsevier Inc.)

The distinct functions of each cyclooxygenase (COX) isoform in renal homeostasis have been the subject of intense investigation for many years. We took the novel approach of using 3 characterized mouse lines, where the prostaglandin (PG)-endoperoxide synthase genes 1 and 2 ( Ptgs1 and Ptgs2) substitute for one another to delineate distinct roles and the potential for COX isoform substitution. Flipped Ptgs genes generate a reversed COX-expression pattern in the kidney, where the knockin COX-2 is highly expressed. Normal nephrogenesis was sustained in all 3 strains at the postnatal stage d 8 (P8). Knockin COX-1 can temporally restore renal function and delay but not prevent renal pathology consequent to COX-2 deletion. Loss of COX-2 in adult COX-1 > COX-2 mice results in severe nephropathy, which leads to impaired renal function. These defects are partially rescued by the knockin COX-2 in Reversa mice, whereas COX-2 can compensate for the loss of COX-1 in COX-2 > COX-1 mice. Intriguingly, the highly expressed knockin COX-2 enzyme barely makes any PGs or thromboxane in neonatal P8 or adult mice, demonstrating that prostanoid biosynthesis requires native COX-1 and cannot be rescued by the knockin COX-2. In summary, the 2 COX isoforms can preferentially compensate for some renal functions, which appears to be independent of the PG-synthetic capacity.-Li, X., Mazaleuskaya, L. L., Ballantyne, L. L., Meng, H., FitzGerald, G. A., Funk, C. D. Differential compensation of two cyclooxygenases in renal homeostasis is independent of prostaglandin-synthetic capacity under basal conditions.

The participation of cyclooxygenase (COX) in embryo implantation and parturition has been studied extensively. However, the distinct role of the two COX isoforms in these processes still remains unclear. Using three characterized mouse lines where the Ptgs1 and Ptgs2 genes substitute for one another, this study focused on the reproductive significance of their distinct roles and potential biological substitution. In both non-gravid and gravid uteri, the knock-in COX-2 is expressed constitutively, whereas the knock-in COX-1 is slightly induced in early implantation. The delayed onset of parturition previously found in COX-1 null mice was corrected by COX-2 exchange in COX-2>COX-1 mice, with normal term pregnancy, gestation length and litter size. In contrast, loss of native COX-2 in COX-1>COX-2 mice resulted in severely impaired reproductive functions. Knock-in COX-1 failed to substitute for the loss of COX-2 in COX-1>COX-2 mice during implantation, indicating that COX-1 may be replaced by COX-2, but not vice versa. A panel of prostaglandins detected in uterus and ovary demonstrates that prostaglandin biosynthesis preferentially depends on native COX-1, but not COX-2. More interestingly, preferential compensations by the COX isoforms were sustained despite weak dependency on their role in prostaglandin biosynthesis in the uterus and ovary.

Previous studies using genetic mouse models have implicated COX-2 in the browning of white adipose tissues (WATs) in mice during cold exposure. However, COX-2 is important during development, and conventional knockouts (KOs) exhibit many defects, conditioned by genetic background. Similarly, the physiological relevance of transgenic overexpression of COX-2 is questionable. In the present study, we utilized mice in which COX-2 was deleted postnatally, bypassing the consequences of enzyme deficiency during development. Despite activation of thermogenesis and browning of inguinal WAT, cold exposure failed to increase COX-2 expression in the adipose tissues of mice with different genetic backgrounds, and the body temperature response to cold was unaltered in postnatal global COX-2 KOs. Selective disruption of COX-2 in adipose tissues also failed detectably to impact systemic prostaglandin biosynthesis. Browning of inguinal WATs induced by exposure to cold is independent of adipose tissue COX-2., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Platelet-activating factor (PAF) stimulates numerous cell types via activation of the G protein-coupled PAF receptor (PAFR). PAFR activation not only induces acute proinflammatory responses, but it also induces delayed systemic immunosuppressive effects by modulating host immunity. Although enzymatic synthesis and degradation of PAF are tightly regulated, oxidative stressors, such as UVB, chemotherapy, and cigarette smoke, can generate PAF and PAF-like molecules in an unregulated fashion via the oxidation of membrane phospholipids. Recent studies have demonstrated the relevance of the mast cell (MC) PAFR in PAFR-induced systemic immunosuppression. The current study was designed to determine the exact mechanisms and mediators involved in MC PAFR-mediated systemic immunosuppression. By using a contact hypersensitivity model, the MC PAFR was not only found to be necessary, but also sufficient to mediate the immunosuppressive effects of systemic PAF. Furthermore, activation of the MC PAFR induces MC-derived histamine and PGE 2 release. Importantly, PAFR-mediated systemic immunosuppression was defective in mice that lacked MCs, or in MC-deficient mice transplanted with histidine decarboxylase- or cyclooxygenase-2-deficient MCs. Lastly, it was found that PGs could modulate MC migration to draining lymph nodes. These results support the hypothesis that MC PAFR activation promotes the immunosuppressive effects of PAF in part through histamine- and PGE 2 -dependent mechanisms., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Cyclooxygenase-2 (COX-2) expression is associated with poor prognosis across a range of human cancers, including breast cancer. The contribution of tumor cell-derived COX-2 to tumorigenesis has been examined in numerous studies; however, the role of stromal-derived COX-2 is ill-defined. Here, we examined how COX-2 in myeloid cells, an immune cell subset that includes macrophages, influences mammary tumor progression. In mice engineered to selectively lack myeloid cell COX-2 [myeloid-COX-2 knockout (KO) mice], spontaneous neu oncogene-induced tumor onset was delayed, tumor burden reduced, and tumor growth slowed compared with wild-type (WT). Similarly, growth of neu-transformed mammary tumor cells as orthotopic tumors in immune competent syngeneic myeloid-COX-2 KO host mice was reduced compared with WT. By flow cytometric analysis, orthotopic myeloid-COX-2 KO tumors had lower tumor-associated macrophage (TAM) infiltration consistent with impaired colony stimulating factor-1-dependent chemotaxis by COX-2 deficient macrophages in vitro. Further, in both spontaneous and orthotopic tumors, COX-2-deficient TAM displayed lower immunosuppressive M2 markers and this was coincident with less suppression of CD8(+) cytotoxic T lymphocytes (CTLs) in myeloid-COX-2 KO tumors. These studies suggest that reduced tumor growth in myeloid-COX-2 KO mice resulted from disruption of M2-like TAM function, thereby enhancing T-cell survival and immune surveillance. Antibody-mediated depletion of CD8(+), but not CD4(+) cells, restored tumor growth in myeloid-COX-2 KO to WT levels, indicating that CD8(+) CTLs are dominant antitumor effectors in myeloid-COX-2 KO mice. Our studies suggest that inhibition of myeloid cell COX-2 can potentiate CTL-mediated tumor cytotoxicity and may provide a novel therapeutic approach in breast cancer therapy., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Placebo-controlled trials of nonsteroidal anti-inflammatory drugs selective for inhibition of cyclooxygenase-2 (COX-2) reveal an emergent cardiovascular hazard in patients selected for low risk of heart disease. Postnatal global deletion of COX-2 accelerates atherogenesis in hyperlipidemic mice, a process delayed by selective enzyme deletion in macrophages., Methods and Results: In the present study, selective depletion of COX-2 in vascular smooth muscle cells and endothelial cells depressed biosynthesis of prostaglandin I2 and prostaglandin E2, elevated blood pressure, and accelerated atherogenesis in Ldlr knockout mice. Deletion of COX-2 in vascular smooth muscle cells and endothelial cells coincided with an increase in COX-2 expression in lesional macrophages and increased biosynthesis of thromboxane. Increased accumulation of less organized intimal collagen, laminin, α-smooth muscle actin, and matrix-rich fibrosis was also apparent in lesions of the mutants., Conclusions: Although atherogenesis is accelerated in global COX-2 knockouts, consistent with evidence of risk transformation during chronic nonsteroidal anti-inflammatory drug administration, this masks the contrasting effects of enzyme depletion in macrophages versus vascular smooth muscle cells and endothelial cells. Targeting delivery of COX-2 inhibitors to macrophages may conserve their efficacy while limiting cardiovascular risk.

Prostacyclin (PGI(2)) is a vasodilator and platelet inhibitor, properties consistent with cardioprotection. More than a decade ago, inhibition of cyclooxygenase-2 (COX-2) by the nonsteroidal anti-inflammatory drugs (NSAIDs) rofecoxib and celecoxib was found to reduce the amount of the major metabolite of PGI(2) (PGI-M) in the urine of healthy volunteers. This suggested that NSAIDs might cause adverse cardiovascular events by reducing production of cardioprotective PGI(2). This prediction was based on the assumption that the concentration of PGI-M in urine likely reflected vascular production of PGI(2) and that other cardioprotective mediators, especially nitric oxide (NO), were not able to compensate for the loss of PGI(2). Subsequently, eight placebo-controlled clinical trials showed that NSAIDs that block COX-2 increase adverse cardiovascular events. We connect tissue-specific effects of NSAID action and functional correlates in mice with clinical outcomes in humans by showing that deletion of COX-2 in the mouse vasculature reduces excretion of PGI-M in urine and predisposes the animals to both hypertension and thrombosis. Furthermore, vascular disruption of COX-2 depressed expression of endothelial NO synthase and the consequent release and function of NO. Thus, suppression of PGI(2) formation resulting from deletion of vascular COX-2 is sufficient to explain the cardiovascular hazard from NSAIDs, which is likely to be augmented by secondary mechanisms such as suppression of NO production.

Suppression of cyclooxygenase 2 (COX-2)-derived prostacyclin (PGI(2)) is sufficient to explain most elements of the cardiovascular hazard from nonsteroidal antinflammatory drugs (NSAIDs). However, randomized trials are consistent with the emergence of cardiovascular risk during chronic dosing with NSAIDs. Although deletion of the PGI(2) receptor fosters atherogenesis, the importance of COX-2 during development has constrained the use of conventional knockout (KO) mice to address this question. We developed mice in which COX-2 was deleted postnatally, bypassing cardiorenal defects exhibited by conventional KOs. When crossed into ApoE-deficient hyperlipidemic mice, COX-2 deletion accelerated atherogenesis in both genders, with lesions exhibiting leukocyte infiltration and phenotypic modulation of vascular smooth muscle cells, as reflected by loss of α-smooth muscle cell actin and up-regulation of vascular cell adhesion molecule-1. Stimulated peritoneal macrophages revealed suppression of COX-2-derived prostanoids and augmented 5-lipoxygenase product formation, consistent with COX-2 substrate rediversion. Although deletion of the 5-lipoxygenase activating protein (FLAP) did not influence atherogenesis, it attenuated the proatherogeneic impact of COX-2 deletion in hyperlipidemic mice. Chronic administration of NSAIDs may increasingly confer a cardiovascular hazard on patients at low initial risk. Promotion of atherogenesis by postnatal COX-2 deletion affords a mechanistic explanation for this observation. Coincident inhibition of FLAP may offer an approach to attenuating such a risk from NSAIDs.

Inhibition of cyclooxygenase (COX) 2, which is associated with >40% of breast cancers, decreases the risk of tumorigenesis and breast cancer recurrence. To study the role of COX-2 in breast cancer, we engineered mice that lack selectively mammary epithelial cell (MEC) COX-2 (COX-2 KO(MEC)). Compared with wild type (WT), MEC from COX-2 KO(MEC) mice expressed >90% less COX-2 messenger RNA (mRNA) and protein and produced 90% less of the dominant pro-oncogenic COX-2 product, prostaglandin (PG) E(2). We confirmed COX-2 as the principle source of PGE(2) in MEC treated with selective COX-2 and COX-1 inhibitors. Tumors were induced in mice using medroxyprogesterone acetate and 7,12-dimethylbenz[a]anthracene. Breast cancer onset was significantly delayed in COX-2 KO(MEC) compared with WT (P = 0.03), equivalent to the delay following systemic COX-2 inhibition with rofecoxib. Compared with WT, COX-2 KO(MEC) tumors showed increased mRNA for Caspase-3, Ki-67 and common markers for leukocytes (CD45) and macrophages (F4/80). Analysis of multiple markers/cytokines, namely CD86, inducible nitric oxide synthase (iNOS), interleukin-6, tumor necrosis factor α (TNFα) and Tim-3 indicated a shift toward antitumorigenic type 1 immune responses in COX-2 KO(MEC) tumors. Immunohistochemical analysis confirmed elevated expression of CD45, F4/80 and CD86 in COX-2 KO(MEC) tumors. Concordant with a role for COX-2 in restraining M1 macrophage polarization, CD86 and TNFα expression were offset by exogenous PGE(2) in bone marrow-derived macrophages polarized in vitro to the M1 phenotype. Our data reveal the importance of epithelial COX-2 in tumor promotion and indicate that deletion of epithelial COX-2 may skew tumor immunity toward type 1 responses, coincident with delayed tumor development.

Background: Although the dominant product of vascular Cyclooxygenase-2 (COX-2), prostacyclin (PGI(2)), restrains atherogenesis, inhibition and deletion of COX-2 have yielded conflicting results in mouse models of atherosclerosis. Floxed mice were used to parse distinct cellular contributions of COX-2 in macrophages and T cells (TCs) to atherogenesis., Methods and Results: Deletion of macrophage-COX-2 (Mac-COX-2KOs) was attained with LysMCre mice and completely suppressed lipopolysaccharide-stimulated macrophage prostaglandin (PG) formation and lipopolysaccharide-evoked systemic PG biosynthesis by approximately 30%. Lipopolysaccharide-stimulated COX-2 expression was suppressed in polymorphonuclear leukocytes isolated from MacKOs, but PG formation was not even detected in polymorphonuclear leukocyte supernatants from control mice. Atherogenesis was attenuated when MacKOs were crossed into hyperlipidemic low-density lipoprotein receptor knockouts. Deletion of Mac-COX-2 appeared to remove a restraint on COX-2 expression in lesional nonleukocyte (CD45- and CD11b-negative) vascular cells that express vascular cell adhesion molecule and variably alpha-smooth muscle actin and vimentin, portending a shift in PG profile and consequent atheroprotection. Basal expression of COX-2 was minimal in TCs, but use of CD4Cre to generate TC knockouts depressed its modest upregulation by anti-CD3epsilon. However, biosynthesis of PGs, TC composition in lymphatic organs, and atherogenesis in low-density lipoprotein receptor knockouts were unaltered in TC knockouts., Conclusions: Macrophage-COX-2, primarily a source of thromboxane A(2) and prostaglandin (PG)E(2), promotes atherogenesis and exerts a restraint on enzyme expression by lesional cells suggestive of vascular smooth muscle cells, a prominent source of atheroprotective prostacyclin. TC COX-2 does not detectably influence TC development or function or atherogenesis in mice.

Rationale: Cyclooxygenase (COX)-derived prostanoids (PGs) are involved in blood pressure homeostasis. Both traditional nonsteroidal antiinflammatory drugs (NSAIDs) that inhibit COX-1 and COX-2 and NSAIDs designed to be selective for inhibition of COX-2 cause sodium retention and elevate blood pressure., Objective: To elucidate the role of COX-2 in blood pressure homeostasis using COX-1>COX-2 mice, in which the COX-1 expression is controlled by COX-2 regulatory elements., Methods and Results: COX-1>COX-2 mice developed systolic hypertension relative to wild types (WTs) on a high-salt diet (HSD); this was attenuated by a PGI(2) receptor agonist. HSD increased expression of COX-2 in WT mice and of COX-1 in COX-1>COX-2 mice in the inner renal medulla. The HSD augmented in all strains urinary prostanoid metabolite excretion, with the exception of the major PGI(2) metabolite that was suppressed on regular chow and unaltered by the HSD in both mutants. Furthermore, inner renal medullary expression of the receptor for PGI(2), but not for other prostanoids, was depressed by HSD in WT and even more so in both mutant strains. Increasing osmolarity augmented expression of COX-2 in WT renal medullary interstitial cells and again the increase in formation of PGI(2) observed in WTs was suppressed in cells derived from both mutants. Intramedullary infusion of the PGI(2) receptor agonist increased urine volume and sodium excretion in mice., Conclusions: These studies suggest that dysregulated expression of the COX-2 dependent, PGI(2) biosynthesis/response pathway in the renal inner renal medulla undermines the homeostatic response to a HSD. Inhibition of this pathway may contribute directly to the hypertensive response to NSAIDs.

Nonsteroidal antinflammatory drugs (NSAIDs) inhibit prostaglandin formation by cyclooxygenases (COX) 1 and 2. NSAIDs selective for inhibition of COX-2 are less likely than traditional drugs to cause serious gastrointestinal adverse effects, but predispose to adverse cardiovascular events, such as heart failure, myocardial infarction, and stroke. Evidence from human pharmacology and genetics, genetically manipulated rodents, and other animal models and randomized trials indicates that this is consequent to suppression of COX-2-dependent cardioprotective prostagladins, particularly prostacyclin. Lessons drawn from how this saga unfolded are relevant to how we approach drug surveillance and regulation, integrate diversifed forms of information and might pursue a more personalized approach to drug efficacy and risk.

Nonsteroidal anti-inflammatory drugs selective for inhibition of COX-2 increase heart failure and elevate blood pressure. The COX-2 gene was floxed and crossed into merCremer mice under the alpha-myosin heavy-chain promoter. Tamoxifen induced selective deletion of COX-2 in cardiomyocytes depressed cardiac output, and resulted in weight loss, diminished exercise tolerance, and enhanced susceptibility to induced arrhythmogenesis. The cardiac dysfunction subsequent to pressure overload recovered progressively in the knockouts coincident with increasing cardiomyocyte hypertrophy and interstitial and perivascular fibrosis. Inhibition of COX-2 in cardiomyocytes may contribute to heart failure in patients receiving nonsteroidal anti-inflammatory drugs specific for inhibition of COX-2.

A cardinal feature of peripheral inflammation is pain. The most common way of managing inflammatory pain is to use nonsteroidal antiinflammatory agents (NSAIDs) that reduce prostanoid production, for example, selective inhibitors of COX2. Prostaglandins produced after induction of COX2 in immune cells in inflamed tissue contribute both to the inflammation itself and to pain hypersensitivity, acting on peripheral terminals of nociceptors. COX2 is also induced after peripheral inflammation in neurons in the CNS, where it aids in developing a central component of inflammatory pain hypersensitivity by increasing neuronal excitation and reducing inhibition. We engineered mice with conditional deletion of Cox2 in neurons and glial cells to determine the relative contribution of peripheral and central COX2 to inflammatory pain hypersensitivity. In these mice, basal nociceptive pain was unchanged, as was the extent of peripheral inflammation, inflammatory thermal pain hypersensitivity, and fever induced by lipopolysaccharide. By contrast, peripheral inflammation-induced COX2 expression in the spinal cord was reduced, and mechanical hypersensitivity after both peripheral soft tissue and periarticular inflammation was abolished. Mechanical pain is a major symptom of most inflammatory conditions, such as postoperative pain and arthritis, and induction of COX2 in neural cells in the CNS seems to contribute to this.

Objective: Prostaglandins (PGs) are found in high levels in the synovial fluid of patients with rheumatoid arthritis, and nonsteroidal blockade of these bioactive lipids plays a role in patient care. The aim of this study was to explore the relative contribution of cyclooxygenase (COX) isoforms and PG species in the autoantibody-driven K/BxN serum-transfer arthritis., Methods: The prostanoid content of arthritic ankles was assessed in ankle homogenates, and the importance of this pathway was confirmed with pharmacologic blockade. The presence of COX isoforms was assessed by Western blotting and their functional contribution was compared using COX-1-/- and COX-2-/- mice as well as isoform-specific inhibitors. The relative importance of PGE2 and PGI2 (prostacyclin) was determined using mice deficient in microsomal PGE synthase 1 (mPGES-1) and in the receptors for PGI2., Results: High levels of PGE2 and 6-keto-PGF1alpha (a stable metabolite of PGI2) were detected in arthritic joint tissues, correlating strongly with the intensity of synovitis. Pharmacologic inhibition of PG synthesis prevented arthritis and ameliorated active disease. While both COX isoforms were found in inflamed joint tissues, only COX-1 contributed substantially to clinical disease; COX-1-/- mice were fully resistant to disease, whereas COX-2-/- mice remained susceptible. These findings were confirmed by isoform-specific pharmacologic inhibition. Mice lacking mPGES-1 (and therefore PGE2) developed arthritis normally, whereas mice incapable of responding to PGI2 exhibited a significantly attenuated arthritis course, confirming a role of PGI2 in this arthritis model., Conclusion: These findings challenge previous paradigms of distinct "housekeeping" versus inflammatory functions of the COX isoforms and highlight the potential pathogenic contribution of prostanoids synthesized via COX-1, in particular PGI2, to inflammatory arthritis.

The prostaglandin G/H synthase enzymes, commonly termed COX-1 and COX-2, differ markedly in their responses to regulatory stimuli and their tissue expression patterns. COX-1 is the dominant source of "housekeeping" prostaglandins, whereas COX-2 synthesizes prostaglandins of relevance to pain, inflammation, and mitogenesis. Despite these distinctions, the two enzymes are remarkably conserved, and their subcellular distributions overlap considerably. To address the functional interchangeability of the two isozymes, mice in which COX-1 is expressed under COX-2 regulatory elements were created by a gene targeting "knock-in" strategy. In macrophages from these mice, COX-1 was shown to be lipopolysaccharide-inducible in a manner analogous to COX-2 in wild-type macrophages. However, COX-1 failed to substitute effectively for COX-2 in lipopolysaccharide-induced prostaglandin E2 synthesis at low concentrations of substrate and in the metabolism of the endocannabinoid 2-arachidonylglycerol. The marked depression of the major urinary metabolite of prostacyclin in COX-2 null mice was only partially rescued by COX-1 knock-in, whereas the main urinary metabolite of prostaglandin E2 was rescued totally. Replacement with COX-1 partially rescued the impact of COX-2 deletion on reproductive function. The renal pathology consequent to COX-2 deletion was delayed but not prevented, whereas the corresponding peritonitis was unaltered. Insertion of COX-1 under the regulatory sequences that drive COX-2 expression indicated that COX-1 can substitute for some COX-2 actions and rescue only some of the consequences of gene disruption. Manipulation of COX-2 also revealed a preference for coupling with distinct downstream prostaglandin synthases in vivo. These mice will provide a valuable reagent with which to elucidate the distinct roles of the COX enzymes in mammalian biology.

Prostacyclin is one of a number of lipid mediators elaborated from the metabolism of arachidonic acid by the cyclooxygenase (COX) enzymes. This prostanoid is a potent inhibitor of platelet aggregation, and its production by endothelial cells and protective role in the vasculature are well established. In contrast, much less is known regarding the function of this prostanoid in other disease processes. We show here that COX-2-dependent production of prostacyclin plays an important role in the development of fibrotic lung disease, limiting both the development of fibrosis and the consequential alterations in lung mechanics. In stark contrast, loss of prostaglandin E(2) synthesis and signaling through the G(s)-coupled EP2 and EP4 receptors had no effect on the development of disease. These findings suggest that prostacyclin analogs will protect against bleomycin-induced pulmonary fibrosis in COX-2(-/-) mice. If such protection is observed, investigation of these agents as a novel therapeutic approach to pulmonary fibrosis in humans may be warranted.

Selective inhibitors of cyclooxygenase-2 (COX2) have attracted widespread media attention because of evidence of an elevated risk of cardiovascular complications in placebo-controlled trials, resulting in the market withdrawal of some members of this class. These drugs block the cyclooxygenase activity of prostaglandin H synthase-2 (PGHS2), but do not affect the associated peroxidase function. They were developed with the rationale of conserving the anti-inflammatory and analgesic actions of traditional nonsteroidal anti-inflammatory drugs (tNSAIDs) while sparing the ability of PGHS1-derived prostaglandins to afford gastric cytoprotection. PGHS1 and PGHS2 coexist in the vasculature and in macrophages, and are upregulated together in inflammatory tissues such as rheumatoid synovia and atherosclerotic plaque. They are each believed to function as homodimers. Here, we developed a new genetic mouse model of selective COX2 inhibition using a gene-targeted point mutation, resulting in a Y385F substitution. Structural modeling and biochemical assays showed the ability of PGHS1 and PGHS2 to heterodimerize and form prostaglandins. The heterodimerization of PGHS1-PGHS2 may explain how the ductus arteriosus closes normally at birth in mice expressing PGHS2 Y385F, but not in PGHS2-null mice.

We investigated the mechanisms by which inhibitors of prostaglandin G/H synthase-2 (PGHS-2; known colloquially as COX-2) increase the incidence of myocardial infarction and stroke. These inhibitors are believed to exert both their beneficial and their adverse effects by suppression of PGHS-2-derived prostacyclin (PGI(2)) and PGE(2). Therefore, the challenge remains to identify a mechanism whereby PGI(2) and PGE(2) expression can be suppressed while avoiding adverse cardiovascular events. Here, selective inhibition, knockout, or mutation of PGHS-2, or deletion of the receptor for PGHS-2-derived PGI(2), was shown to accelerate thrombogenesis and elevate blood pressure in mice. These responses were attenuated by COX-1 knock down, which mimics the beneficial effects of low-dose aspirin. PGE(2) biosynthesis is catalyzed by the coordinate actions of COX enzymes and microsomal PGE synthase-1 (mPGES-1). We show that deletion of mPGES-1 depressed PGE(2) expression, augmented PGI(2) expression, and had no effect on thromboxane biosynthesis in vivo. Most importantly, mPGES-1 deletion affected neither thrombogenesis nor blood pressure. These results suggest that inhibitors of mPGES-1 may retain their antiinflammatory efficacy by depressing PGE(2), while avoiding the adverse cardiovascular consequences associated with PGHS-2-mediated PGI(2) suppression.

Inhibitors selective for prostaglandin G/H synthase-2 (PGHS-2) (known colloquially as COX-2) were designed to minimize gastrointestinal complications of traditional NSAIDs--adverse effects attributed to suppression of COX-1-derived PGE2 and prostacyclin (PGI2). Evidence from 2 randomized controlled-outcome trials (RCTs) of 2 structurally distinct selective inhibitors of COX-2 supports this hypothesis. However, 5 RCTs of 3 structurally distinct inhibitors also indicate that such compounds elevate the risk of myocardial infarction and stroke. The clinical information is biologically plausible, as it is compatible with evidence that inhibition of COX-2-derived PGI2 removes a protective constraint on thrombogenesis, hypertension, and atherogenesis in vivo. However, the concept of simply tipping a "balance" between COX-2-derived PGI2 and COX-1-derived platelet thromboxane is misplaced. Among the questions that remain to be addressed are the following: (a) whether this hazard extends to all or some of the traditional NSAIDs; (b) whether adjuvant therapies, such as low-dose aspirin, will mitigate the hazard and if so, at what cost; (c) whether COX-2 inhibitors result in cardiovascular risk transformation during chronic dosing; and (d) how we might identify individuals most likely to benefit or suffer from such drugs in the future.

Suppression of cyclooxygenase 2 (COX-2)-derived prostacyclin (PGI2) is sufficient to explain most elements of the cardiovascular hazard from nonsteroidal antinflammatory drugs (NSAIDs). However, randomized trials are consistent with the emergence of cardiovascular risk during chronic dosing with NSAIDs. Although deletion of the PGI2 receptor fosters atherogenesis, theimportance of COX-2 during development has constrained the use of conventional knockout (KO) mice to address this question. We developed mice in which COX-2 was deleted postnatally, bypassing cardiorenal defects exhibited by conventional KOs. When crossed into ApoE-deficient hyperlipidemic mice, COX-2 deletion accelerated atherogenesis in both genders, with lesions exhibiting leukocyte infiltration and phenotypic modulation of vascular smooth muscle cells, as reflected by loss of a-smooth muscle cell actin and up-regulation of vascular cell adhesion molecule-1. Stimulated peritoneal macrophages revealed suppression of COX-2-derived prostanoids and augmented 5-lipoxygenase product formation, consistent with COX-2 substrate rediversion. Although deletion of the 5-lipoxygenase activating protein (FLAP) did not influence atherogenesis, it attenuated the proather. ogeneic impact of COX-2 deletion in hyperlipidemic mice. Chronic administration of NSAIDs may increasingly confer a cardiovascular hazard on patients at low initial risk. Promotion of atherogenesis by postnatal COX-2 deletion affords a mechanistic explanation for this observation. Coincident inhibition of FLAP may offer an approach to attenuating such a risk from NSAIDs. [ABSTRACT FROM AUTHOR]

The article presents a study on the effect of cyclooxygenase (COX)-derived prostanoids (PGs) in blood pressure homeostasis with the use of COX-1>COX-2 mice models wherein COX-2 regulatory elements control COX-1 expression. The study measured systolic blood and analyzed renal medullar perfusion and urinary sodium excretion. Results show that COX-1>COX-2 mice exhibited systolic hypertension on a high-salt diet (HSD), which in turn depressed renal medullary expression of PGI2 receptor.

The inhibition of cyclooxygenase (COX)-2 confers a small but absolute risk of cardiovascular events on patients taking nonsteroidal anti-inflammatory drugs (NSAIDs). This risk has been established by placebo-controlled trials of NSAIDs selective for COX-2; traditional NSAIDs seem to be heterogeneous with respect to cardiovascular risk. The American Heart Association (AHA) has proposed a ‘stepped-care’ approach to the use of NSAIDs in patients with cardiovascular disease, whereas the Food and Drug Administration (FDA) has failed to approve the COX-2-selective NSAID etoricoxib. In this article, these actions of the AHA and the FDA are interpreted in the light of current knowledge, prompting the formulation of scientific questions that might be addressed. [Copyright &y& Elsevier]

Selective inhibitors of cyclooxygenase-2 (COX2) have attracted widespread media attention because of evidence of an elevated risk of cardiovascular complications in placebo-controlled trials, resulting in the market withdrawal of some members of this class. These drugs block the cyclooxygenase activity of prostaglandin H synthase-2 (PGHS2), but do not affect the associated peroxidase function. They were developed with the rationale of conserving the anti-inflammatory and analgesic actions of traditional nonsteroidal anti-inflammatory drugs (tNSAIDs) while sparing the ability of PGHS1-derived prostaglandins to afford gastric cytoprotection. PGHS1 and PGHS2 coexist in the vasculature and in macrophages, and are upregulated together in inflammatory tissues such as rheumatoid synovia and atherosclerotic plaque. They are each believed to function as homodimers. Here, we developed a new genetic mouse model of selective COX2 inhibition using a gene-targeted point mutation, resulting in a Y385F substitution. Structural modeling and biochemical assays showed the ability of PGHS1 and PGHS2 to heterodimerize and form prostaglandins. The heterodimerization of PGHS1-PGHS2 may explain how the ductus arteriosus closes normally at birth in mice expressing PGHS2 Y385F, but not in PGHS2-null mice. [ABSTRACT FROM AUTHOR]

Background & Aims: Variability in response to drugs may influence both efficacy and safety. Cyclooxygenase (COX)-2 inhibitors pose a cardiovascular risk by potentially increasing the likelihood of thrombosis, hypertension, and atherogenesis. Differences between individuals in the response to COX-2 inhibitors would be expected to influence their susceptibility to cardiovascular complications. We examined the variability in degree and selectivity of COX-2 inhibition in humans in response to celecoxib and rofecoxib. Methods: Fifty healthy volunteers received placebo, rofecoxib (25 mg), and celecoxib (200 mg), randomized by order. COX-1 and COX-2 inhibition was determined using ex vivo and in vivo indices of enzymatic activity. A subset of 5 individuals underwent 5 replicate studies to estimate variability in drug response both within and between subjects. Results: Despite the higher COX-2 selectivity of rofecoxib in vitro, the average selectivity attained by 25 mg rofecoxib and 200 mg celecoxib in vivo were not different. However, there was considerable variability at an individual level in the degree of COX-2 inhibition and selectivity attained by both drugs. Approximately one third of the variability was attributable to differences between individuals, suggesting the contribution of genetic sources of variance, such as candidate polymorphisms detected in COX-1 and CYP2C9. Conclusions: The actual degree of selectivity for inhibition of COX-2 achieved by the coxibs relates both to chemical properties of the drug and to factors within an individual that modulate drug response. These sources of variability might be exploited to identify patients uniquely susceptible to benefit or at developing risk of cardiovascular complications. [Copyright &y& Elsevier]

Details a medical case report of a woman presented with pulmonary embolism and who had been taking oral contraceptives and Valdecoxib. Effect of Coxibs, selective inhibitors of cyclooxygenase-2 on patients with cardiovascular disease; Risk factors for spontaneous thrombosis; Genetic risk factors documented.

The article comments on the withdrawal of Vioxx from the American market. Explanation of how the COX-2 inhibitor drugs work and what actions might be responsible for the increased risk of heart attacks in patients undergoing treatment for benign sporadic colonic adenomas; Clinical implications of the trial; Suggestion that other COX-2 drugs may be used in gastroenterology patients with a low cardiovascular risk; How the incident speaks poorly of the drug approval process; Call for a follow up of patients in the colonic trial to see how rapidly the risk evaporates; Need to determine if the cardiovascular risk is a class problem.

Presents a correction to the article "COX-2 and Beyond: Approaches to Prostaglandin Inhibition in Human Disease".

Background: Inhibitors of cyclooxygenase-2 alleviate pain and reduce fever and inflammation by suppressing the biosynthesis of prostacyclin (PGI2) and prostaglandin E2. However, suppression of these prostaglandins, particularly PGI2, by cyclooxygenase-2 inhibition or deletion of its I prostanoid receptor also predisposes to accelerated atherogenesis and thrombosis in mice. By contrast, deletion of microsomal prostaglandin E synthase 1 (mPGES-1) confers analgesia, attenuates atherogenesis, and fails to accelerate thrombogenesis, while suppressing prostaglandin E2, but increasing biosynthesis of PGI2.Methods: To address the cardioprotective contribution of PGI2, we generated mice lacking the I prostanoid receptor together with mPges-1 on a hyperlipidemic background (low-density lipoprotein receptor knockouts).Results: mPges-1 depletion modestly increased thrombogenesis, but this response was markedly further augmented by coincident deletion of the I prostanoid receptor (n=10-18). By contrast, deletion of the I prostanoid receptor had no effect on the attenuation of atherogenesis by mPGES-1 deletion in the low-density lipoprotein receptor knockout mice (n=17-21).Conclusions: Although suppression of prostaglandin E2 accounts for the protective effect of mPGES-1 deletion in atherosclerosis, augmentation of PGI2 is the dominant contributor to its favorable thrombogenic profile. The divergent effects on these prostaglandins suggest that inhibitors of mPGES-1 may be less likely to cause cardiovascular adverse effects than nonsteroidal anti-inflammatory drugs specific for inhibition of cyclooxygenase-2. [ABSTRACT FROM AUTHOR]

Background--Placebo-controlled trials of nonsteroidal anti-inflammatory drugs selective for inhibition of cyclooxygenase-2 (COX-2) reveal an emergent cardiovascular hazard in patients selected for low risk of heart disease. Postnatal global deletion of COX-2 accelerates atherogenesis in hyperlipidemic mice, a process delayed by selective enzyme deletion in macrophages. Methods and Results--In the present study, selective depletion of COX-2 in vascular smooth muscle cells and endothelial cells depressed biosynthesis of prostaglandin I2 and prostaglandin E2, elevated blood pressure, and accelerated atherogenesis in Ldlr knockout mice. Deletion of COX-2 in vascular smooth muscle cells and endothelial cells coincided with an increase in COX-2 expression in lesional macrophages and increased biosynthesis of thromboxane. Increased accumulation of less organized intimal collagen, laminin, α-smooth muscle actin, and matrix-rich fibrosis was also apparent in lesions of the mutants. Conclusions--Although atherogenesis is accelerated in global COX-2 knockouts, consistent with evidence of risk transformation during chronic nonsteroidal anti-inflammatory drug administration, this masks the contrasting effects of enzyme depletion in macrophages versus vascular smooth muscle cells and endothelial cells. Targeting delivery of COX-2 inhibitors to macrophages may conserve their efficacy while limiting cardiovascular risk. [ABSTRACT FROM AUTHOR]

Selective inhibitors of cyclooxygenase (COX)-2 depress prostacyclin (PGI2) without a concomitant inhibition of platelet COX-1-derived thromboxane (Tx)A2. Experiments in gene-deleted mice have shown that ablation of the PGI2 receptor (the IP) predisposes to an exaggerated response to agonists which elevate blood pressure, accelerate atherogenesis and induce thrombosis. Such a class-based effect would be expected to be modulated by the underlying risk of cardiovascular disease in patients, elements of drug exposure, such as dose, duration of action and duration of dosing, and inter-individual variability of drug response. Five placebo-controlled trials of three structurally distinct selective inhibitors of COX-2 have revealed an increased hazard of myocardial infarction and stroke consistent with a mechanism-based class-specific cardiovascular hazard. Sustained inhibition of platelet TxA2 by aspirin affords cardiovascular benefit, despite concomitant inhibition of PGI2. Although there is no information from randomized placebo-controlled trials, traditional non-steroidal anti-inflammatory drugs, such as naproxen, dicofenac and ibuprofen, might differ in their effects of cardiovascular biology. [Copyright &y& Elsevier]