Deletion Of mPGES-1 Research Articles

Myeloid Cell mPGES-1 Deletion Attenuates Calcium Phosphate-induced Abdominal Aortic Aneurysm in Male Mice.

Microsomal PGE2 synthase (mPGES)-1 is the key enzyme responsible for synthesizing inflammatory prostaglandin E2 (PGE2). Our previous studies have shown that deletion mPGES-1 in myeloid cells hinders atherogenesis, suppresses vascular proliferative response to injury and enhances survival after myocardial infarction.Here we aimed to further explore the influence of myeloid cell mPGES-1 deletion in abdominal aortic aneurysm (AAA) formation. The AAA was triggered by applying 0.5M calcium phosphate (CaPO4) to the infrarenal aorta of both myeloid mPGES-1 knockout (Mac-mPGES-1-KO) and their littermate control Mac-mPGES-1-WT mice. AAA induction was assessed by calculating the expansion of the infrarenal aortic diameter 4weeks after CaPO4 application. The maximum diameters of the aortas were measured by morphometry and the mean maximal diameters were calculated. Paraffin sections of the infrarenal aortas were examined for morphological analysis and immunohistochemical staining. The results showed that myeloid cell mPGES-1 deletion significantly mitigated AAA formation, including reducing expansion of the infrarenal aorta, preventing elastic lamellar degradation, and decreasing aortic calcium deposition. Immunohistochemical staining further indicated that macrophage infiltration and matrix metalloproteinase 2 (MMP2) expression was attenuated in the Mac-mPGES-1-KO aortas. Consistently, in vitro experiments showed that expression of pro-inflammatory cytokines and MMPs was significantly reduced when mPGES-1 was lacking in the primary cultured peritoneal macrophages. These data altogether demonstrated that deletion of mPGES-1 in myeloid cells may attenuate AAA formation and targeting myeloid cell mPGES-1 could potentially offer an effective strategy for the treatment and prevention of vascular inflammatory diseases.

In vitro and in vivo neuroprotective effect of novel mPGES-1 inhibitor in animal model of Parkinson’s disease

mPGES-1 is found to be up-regulated in the dopaminergic neurons of the substantia nigra pars compacta (SNpc) of postmortem brain tissue from Parkinson’s disease (PD) patients and neurotoxin 6-hydroxydopamine (6-OHDA)-induced PD mice. Since the genetic deletion of mPGES-1 abolished 6-OHDA-induced PGE2 production and 6-OHDA-induced dopaminergic neurodegeneration in vitro and in vivo models, mPGES-1 enzyme has the potential to be an important target for PD therapy. In the present work, we investigated whether a small organic molecule as mPGES-1 inhibitor could exhibit the neuroprotective effects against 6-OHDA-induced neurotoxicity in in vitro and in vivo models. For this research goal, a new series of arylsulfonyl hydrazide derivatives was prepared and investigated whether these compounds may protect neurons against 6-OHDA-induced neurotoxicity in both in vitro and in vivo studies. Among them, compound 7s (MPO-0144) as a mPGES-1 inhibitor (PGE2 IC50 = 41.77 nM; mPGES-1 IC50 = 1.16 nM) exhibited a potent neuroprotection (ED50 = 3.0 nM) against 6-OHDA-induced in PC12 cells without its own neurotoxicity (IC50 = >10 μM). In a 6-OHDA-induced mouse model of PD, administration of compound 7s (1 mg/kg/day, for 7 days, i.p.) ameliorated motor impairments and dopaminergic neuronal damage. These significant biological effects of compound 7s provided the first pharmacological evidence that mPGES-1 inhibitor could be a promising therapeutic agent for PD patients.

Facilitation of colonic T cell immune responses is associated with an exacerbation of dextran sodium sulfate\u2013induced colitis in mice lacking microsomal prostaglandin E synthase-1

BackgroundMicrosomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme that acts downstream of cyclooxygenase and plays a major role in inflammation by converting prostaglandin (PG) H2 to PGE2. The present study investigated the effect of genetic deletion of mPGES-1 on the development of immunologic responses to experimental colitis induced by dextran sodium sulfate (DSS), a well-established model of inflammatory bowel disease (IBD).MethodsColitis was induced in mice lacking mPGES-1 (mPGES-1−/− mice) and wild-type (WT) mice by administering DSS for 7 days. Colitis was assessed by body weight loss, diarrhea, fecal bleeding, and histological features. The colonic expression of mPGES-1 was determined by real-time PCR, western blotting, and immunohistochemistry. The impact of mPGES-1 deficiency on T cell immunity was determined by flow cytometry and T cell depletion in vivo.ResultsAfter administration of DSS, mPGES-1−/− mice exhibited more severe weight loss, diarrhea, and fecal bleeding than WT mice. Histological analysis further showed significant exacerbation of colonic inflammation in mPGES-1−/− mice. In WT mice, the colonic expression of mPGES-1 was highly induced on both mRNA and protein levels and colonic PGE2 increased significantly after DSS administration. Additionally, mPGES-1 protein was localized in the colonic mucosal epithelium and infiltrated inflammatory cells in underlying connective tissues and the lamina propria. The abnormalities consistent with colitis in mPGES-1−/− mice were associated with higher expression of colonic T-helper (Th)17 and Th1 cytokines, including interleukin 17A and interferon-γ. Furthermore, lack of mPGES-1 increased the numbers of Th17 and Th1 cells in the lamina propria mononuclear cells within the colon, even though the number of suppressive regulatory T cells also increased. CD4+ T cell depletion effectively reduced symptoms of colitis as well as colonic expression of Th17 and Th1 cytokines in mPGES-1−/− mice, suggesting the requirement of CD4+ T cells in the exacerbation of DSS-induced colitis under mPGES-1 deficiency.ConclusionsThese results demonstrate that mPGES-1 is the main enzyme responsible for colonic PGE2 production and deficiency of mPGES-1 facilitates the development of colitis by affecting the development of colonic T cell–mediated immunity. mPGES-1 might therefore impact both the intestinal inflammation and T cell–mediated immunity associated with IBD.

Sex-dependent compensatory mechanisms preserve blood pressure homeostasis in prostacyclin receptor–deficient mice

Inhibitors of microsomal prostaglandin E synthase 1 (mPGES-1) are in the early phase of clinical development. Deletion of mPges-1 in mice confers analgesia, restrains atherogenesis, and fails to accelerate thrombogenesis, while suppressing prostaglandin E2 (PGE2), but increasing the biosynthesis of prostacyclin (PGI2). In low-density lipoprotein receptor-deficient (Ldlr-/-) mice, this last effect represents the dominant mechanism by which mPges-1 deletion restrains thrombogenesis, while suppression of PGE2 accounts for its antiatherogenic effect. However, the effect of mPges-1 depletion on blood pressure (BP) in this setting remains unknown. Here, we show that mPges-1 depletion significantly increased the BP response to salt loading in male Ldlr-/- mice, whereas, despite the direct vasodilator properties of PGI2, deletion of the I prostanoid receptor (Ipr) suppressed this response. Furthermore, combined deletion of the Ipr abrogated the exaggerated BP response in male mPges-1-/- mice. Interestingly, these unexpected BP phenotypes were not observed in female mice fed a high-salt diet (HSD). This is attributable to the protective effect of estrogen in Ldlr-/- mice and in Ipr-/- Ldlr-/- mice. Thus, estrogen compensates for a deficiency in PGI2 to maintain BP homeostasis in response to high salt in hyperlipidemic female mice. In male mice, by contrast, the augmented formation of atrial natriuretic peptide (ANP) plays a similar compensatory role, restraining hypertension and oxidant stress in the setting of Ipr depletion. Hence, men with hyperlipidemia on a HSD might be at risk of a hypertensive response to mPGES-1 inhibitors.

Microsomal Prostaglandin E2 Synthase-1 Deletion Attenuates Isoproterenol-Induced Myocardial Fibrosis in Mice.

Deletion of microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibits inflammation and protects against atherosclerotic vascular diseases but displayed variable influence on pathologic cardiac remodeling. Overactivation of β-adrenergic receptors (β-ARs) causes heart dysfunction and cardiac remodeling, whereas the role of mPGES-1 in β-AR-induced cardiac remodeling is unknown. Here we addressed this question using mPGES-1 knockout mice, subjecting them to isoproterenol, a synthetic nonselective agonist for β-ARs, at 5 or 15 mg/kg per day to induce different degrees of cardiac remodeling in vivo. Cardiac structure and function were assessed by echocardiography 24 hours after the last of seven consecutive daily injections of isoproterenol, and cardiac fibrosis was examined by Masson trichrome stain in morphology and by real-time polymerase chain reaction for the expression of fibrosis-related genes. The results showed that deletion of mPGES-1 had no significant effect on isoproterenol-induced cardiac dysfunction or hypertrophy. However, the cardiac fibrosis was dramatically attenuated in the mPGES-1 knockout mice after either low-dose or high-dose isoproterenol exposure. Furthermore, in vitro study revealed that overexpression of mPGES-1 in cultured cardiac fibroblasts increased isoproterenol-induced fibrosis, whereas knocking down mPGES-1 in cardiac myocytes decreased the fibrogenesis of fibroblasts. In conclusion, mPGES-1 deletion protects against isoproterenol-induced cardiac fibrosis in mice, and targeting mPGES-1 may represent a novel strategy to attenuate pathologic cardiac fibrosis, induced by β-AR agonists. SIGNIFICANCE STATEMENT: Inhibitors of microsomal prostaglandin E2 synthase-1 (mPGES-1) are being developed as alternative analgesics that are less likely to elicit cardiovascular hazards than cyclooxygenase-2 selective nonsteroidal anti-inflammatory drugs. We have demonstrated that deletion of mPGES-1 protects inflammatory vascular diseases and promotes post-myocardial infarction survival. The role of mPGES-1 in β-adrenergic receptor-induced cardiomyopathy is unknown. Here we illustrated that deletion of mPGES-1 alleviated isoproterenol-induced cardiac fibrosis without deteriorating cardiac dysfunction. These results illustrated that targeting mPGES-1 may represent an efficacious approach to the treatment of inflammatory cardiovascular diseases.

The cyclooxygenase-1/mPGES-1/endothelial prostaglandin EP4 receptor pathway constrains myocardial ischemia-reperfusion injury

The use of nonsteroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX)-1 and COX-2, increases heart failure risk. It is unknown whether microsomal (m) prostaglandin (PG) E synthase (S)-1, a target downstream of COX, regulates myocardial (M) ischemia/reperfusion (I/R) injury, a key determinant of heart failure. Here we report that COX-1 and mPGES-1 mediate production of substantial amounts of PGE2 and confer cardiac protection in MI/R. Deletion of mPges-1 impairs cardiac microvascular perfusion and increases inflammatory cell infiltration in mouse MI/R. Consistently, mPges-1 deletion depresses the arteriolar dilatory response to I/R in vivo and to acetylcholine ex vivo, and enhances leukocyte-endothelial cell interaction, which is mediated via PGE receptor-4 (EP4). Furthermore, endothelium-restricted Ep4 deletion impairs microcirculation, and exacerbates MI/R injury, irrespective of EP4 agonism. Treatment with misoprostol, a clinically available PGE analogue, improves microcirculation and reduces MI/R injury. Thus, mPGES-1, a key microcirculation protector, constrains MI/R injury and this beneficial effect is partially mediated via endothelial EP4.

Myeloid Cell mPges-1 Deletion Attenuates Mortality Without Affecting Remodeling After Acute Myocardial Infarction in Mice.

Selective deletion of microsomal prostaglandin E2 synthase-1 (mPges-1) in myeloid cells retards atherogenesis and suppresses the vascular proliferative response to injury, while it does not predispose to thrombogenesis or hypertension. However, studies using bone marrow transplants from irradiated mice suggest that myeloid cell mPGES-1 facilitates cardiac remodeling and prolongs survival after experimental myocardial infarction (MI). Here, we addressed this question using mice lacking mPges-1 in myeloid cells, particularly macrophages [Mac-mPges-1-knockout (KO)], generated by crossing mPges-1 floxed mice with LysMCre mice and subjecting them to coronary artery ligation. Cardiac structure and function were assessed by morphometric analysis, echocardiography, and invasive hemodynamics 3, 7, and 28 days after MI. Despite a similar infarct size, in contrast to the prior report, the post-MI survival rate was markedly improved in the Mac-mPges-1-KO mice compared with wild-type controls. Left ventricular systolic (reflected by ejection fraction, fractional shortness end systolic volume, and +dP/dt) and diastolic function (reflected by end diastolic volume, -dP/dt, and Tau), cardiac hypertrophy (reflected by left ventricular dimensions), and staining for fibrosis did not differ between the groups. In conclusion, we found that Cre-loxP-mediated deletion of mPges-1 in myeloid cells has favorable effects on post-MI survival, with no detectable adverse influence on post-MI remodeling. These results add to evidence that targeting macrophage mPGES-1 may represent a safe and efficacious approach to the treatment and prevention of cardiovascular inflammatory disease.

Microsomal prostaglandin E synthase-1 is a critical factor in dopaminergic neurodegeneration in Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder of uncertain pathogenesis characterized by the loss of nigrostriatal dopaminergic neurons. Although increased production of prostaglandin E2 (PGE2) has been implicated in tissue damage in several pathological settings, the role of microsomal prostaglandin E synthase-1 (mPGES-1), an inducible terminal enzyme for PGE2 synthesis, in dopaminergic neurodegeneration remains unclear. Here we show that mPGES-1 is up-regulated in the dopaminergic neurons of the substantia nigra of postmortem brain tissue from PD patients and in neurotoxin 6-hydroxydopamine (6-OHDA)-induced PD mice. The expression of mPGES-1 was also up-regulated in cultured dopaminergic neurons stimulated with 6-OHDA. The genetic deletion of mPGES-1 not only abolished 6-OHDA-induced PGE2 production but also inhibited 6-OHDA-induced dopaminergic neurodegeneration both in vitro and in vivo. Nigrostriatal projections, striatal dopamine content, and neurological functions were significantly impaired by 6-OHDA administration in wild-type (WT) mice, but not in mPGES-1 knockout (KO) mice. Furthermore, in cultured primary mesencephalic neurons, addition of PGE2 to compensate for the deficiency of 6-OHDA-induced PGE2 production in mPGES-1 KO neurons recovered 6-OHDA toxicity to almost the same extent as that seen in WT neurons. These results suggest that induction of mPGES-1 enhances 6-OHDA-induced dopaminergic neuronal death through excessive PGE2 production. Thus, mPGES-1 may be a valuable therapeutic target for treatment of PD.

MPGES-1 (Microsomal Prostaglandin E Synthase-1) Mediates Vascular Dysfunction in Hypertension Through Oxidative Stress.

mPGES-1 (microsomal prostaglandin E synthase-1), the downstream enzyme responsible for PGE2 (prostaglandin E2) synthesis in inflammatory conditions and oxidative stress are increased in vessels from hypertensive animals. We evaluated the role of mPGES-1-derived PGE2 in the vascular dysfunction and remodeling in hypertension and the possible contribution of oxidative stress. We used human peripheral blood mononuclear cells from asymptomatic patients, arteries from untreated and Ang II (angiotensin II)-infused mPGES-1-/- and mPGES-1+/+ mice, and vascular smooth muscle cells exposed to PGE2 In human cells, we found a positive correlation between mPGES-1 mRNA and carotid intima-media thickness (r=0.637; P<0.001) and with NADPH oxidase-dependent superoxide production (r=0.417; P<0.001). In Ang II-infused mice, mPGES-1 deletion prevented all of the following: (1) the augmented wall:lumen ratio, vascular stiffness, and altered elastin structure; (2) the increased gene expression of profibrotic and proinflammatory markers; (3) the increased vasoconstrictor responses and endothelial dysfunction; (4) the increased NADPH oxidase activity and the diminished mitochondrial membrane potential; and (5) the increased reactive oxygen species generation and reduced NO bioavailability. In vascular smooth muscle cells or aortic segments, PGE2 increased NADPH oxidase expression and activity and reduced mitochondrial membrane potential, effects that were abolished by antagonists of the PGE2 receptors (EP), EP1 and EP3, and by JNK (c-Jun N-terminal kinase) and ERK1/2 (extracellular-signal-regulated kinases 1/2) inhibition. Deletion of mPGES-1 augmented vascular production of PGI2 suggesting rediversion of the accumulated PGH2 substrate. In conclusion, mPGES-1-derived PGE2 is involved in vascular remodeling, stiffness, and endothelial dysfunction in hypertension likely through an increase of oxidative stress produced by NADPH oxidase and mitochondria.

Abstract 270: Microsomal Prostaglandin E Synthase-1 and Endothelial EP4 Receptor Reduces Myocardial Ischemia-reperfusion Injury via Improving Microcirculatory Perfusion

Objective: Myocardial (M) ischemia/reperfusion (I/R) injury limits the efficacy of reperfusion therapy in patients with myocardial infarction (MI) and contributes to the development of heart failure. Nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit both cyclooxygenase (COX)-1 and COX-2, or those selective for COX-2 inhibition, are associated with an increased risk of heart failure. Although suppression of COX-2-derived prostaglandin (PG) I 2 predisposes cardiovascular risks, it remains unknown whether microsomal (m) PGE synthase (S)-1, a therapeutic target alternative to COX-2, might participate in MI/R injury. Approach and Results: Mice deficient in COX-1 or -2, mPGES-1, or endothelial PGE receptor-4 (EP4), together with pharmacological interventions, were utilized in a mouse model of MI/R. Microvascular perfusion was assessed in vivo using laser Doppler flow technique. COX-1 deficiency reduced biosynthesis of PGE 2 and increased infarct size in the heart after MI/R. Deletion of mPGES-1 also depressed PGE 2 while exacerbated MI/R injury. Cardiac perfusion was impaired by mPGES-1 deletion following reperfusion, without change in baseline flow. Consistently, mPGES-1 deletion abolished arteriolar dilation in I/R, and mPGES-1-derived PGE 2 acting on EP4 receptor restrains myeloid cell adhesion to endothelial cells in vitro and limits leukocyte adhesion to vasculature in I/R in vivo. Endothelium-restricted deletion of EP4 receptor impaired microcirculatory perfusion and exacerbated cardiac injury in MI/R. By contrast, treatment with misoprostol, a clinically available PGE 2 analogue, improved cardiac microcirculation post MI and protected against I/R injury. Conclusions: mPGES-1-derived PGE 2 and endothelial EP4 receptor protect against MI/R injury via preserving cardiac microcirculation. mPGES-1 inhibitors may harm microcirculation in patients with acute MI undergoing revascularization. Inhibition of COX-1-derived PGE 2 may contribute to the cardiovascular side effects of NSAIDs in the setting of I/R. Key Words: myocardial infarction, ischemia reperfusion, cyclooxygenase, prostaglandin E synthase-1, PGE 2 , EP4, microcirculation, endothelium

Deletion of mPGES-1 affects platelet functions in mice.

Microsomal prostaglandin E2 synthase-1 (mPGES-1) constitutes an essential player in inflammation and is involved in the pathogenesis of rheumatoid arthritis. Platelets participate in the regulation of inflammatory processes by the release of proinflammatory mediators and platelet-derived microparticles (PMPs). However, the role of the inducible mPGES-1/PGE2 pathway in platelet functions has not been investigated. In the present study we report a significant impact of mPGES-1 on platelet functions during inflammation. Wild-type (WT) and mPGES-1-/- knockout (KO) mice were stimulated with lipopolysaccharide (LPS) for 24h. Platelet counts and activation were assessed by flow cytometry analysing CD62P-CD154 expression, PMP numbers, platelet-leukocyte aggregates and platelet aggregation. The accumulation of platelets and fibrinogen in the liver was analysed by immunofluorescent staining. In native platelets from WT and mPGES-1 KO mice, there were no differences among the investigated functions. After LPS treatment, the number of platelets was significantly decreased in WT, but not in KO mice. Platelet activation, platelet-leukocyte aggregates and PMP numbers were all significantly lower in KO mice compared with WT mice after LPS treatment. In addition, KO mice displayed a significant reduction in platelet aggregation ex vivo In the liver of LPS-stimulated WT and KO mice, there were no differences in platelet accumulation, although the percentage of total vessel area in the KO liver was significantly lower compared with the WT one. Our results demonstrate that systemic inhibition of mPGES-1 prevents platelet activation, which should have important implications with regard to the cardiovascular safety of mPGES-1 inhibitors.

Abstract 654: Impact of epithelial cell-specific deletion of mPGES-1 on colon carcinogenesis

Abstract Activation of the COX-2/mPGES-1/PGE2 signaling axis is a hallmark of many cancers, including colorectal cancer (CRC), prompting the implementation of cancer prevention strategies targeting COX-2 activity. We have shown that targeting the downstream terminal PGE2 synthase, mPGES-1, specifically reduces inducible PGE2 formation without disrupting synthesis of other essential prostanoids. Additionally, global deletion of mPGES-1 (gKO) confers dramatic protection against intestinal cancer development in several mouse models, including the AOM+DSS inflammation-associated colon cancer model. Understanding the role of PGE2 under inflammatory conditions, however, is complicated by the diverse cellular sources and context-dependence of inducible PGE2. To clarify the relative contribution of inducible PGE2 synthesis within distinct cell compartments in colon, we have recently created an mPGES-1 conditional knockout mouse (cKO), where exon 3 of the mPGES-1 gene (mPtges) is floxed for tissue-specific Cre-mediated deletion. To test the role of epithelial-derived PGE2 synthesis on inflammation-associated cancer, we crossed the cKO mice with carbonic anhydrase 1 (Car1)-controlled Cre mice (cKO:Car1), thereby inactivating mPGES-1 function specifically within colonic epithelial cells. Based on our recent observation that global inactivation mPGES-1 sensitizes the colonic mucosa to direct environmental injury, we first examined the sensitivity of the cKO:Car1 mice to DSS-induced acute colitis. Two days after a five-day treatment with 0, 0.5 or 1% DSS, clinical signs of colitis, including the extent of colonic ulceration were determined. Both cKO:Car1 and cKO:WT control mice showed a comparable extent of colonic ulceration, covering ∼60% of the epithelial surface. We next examined the impact of epithelial-specific deletion of mPGES-1 on DSS-induced colon carcinogenesis. KO:Car1 and cKO:WT control mice were treated with a single injection of AOM (10 mg/kg bw) followed by five days of 0, 0.5 or 1% DSS, and colons were examined five weeks after the end of DSS treatment. Surprisingly, epithelial deletion of mPGES-1 in the cKO:Car1 mice failed to provide against cancer development in comparison to the cKO:WT mice. This is the first study to show that genetic inactivation of inducible PGE2 formation within a specific cell lineage (e.g. epithelial cells) does not affect both the extent of acute colonic inflammation nor subsequent inflammation-associated carcinogenesis. Citation Format: Masako Nakanishi, Daniel W. Rosenberg. Impact of epithelial cell-specific deletion of mPGES-1 on colon carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 654.

The involvement of prostaglandin E2 in interleukin-1β evoked anorexia is strain dependent

From experiments in mice in which the prostaglandin E2 (PGE2) synthesizing enzyme mPGES-1 was genetically deleted, as well as from experiments in which PGE2 was injected directly into the brain, PGE2 has been implicated as a mediator of inflammatory induced anorexia. Here we aimed at examining which PGE2 receptor (EP1–4) that was critical for the anorexic response to peripherally injected interleukin-1β (IL-1β). However, deletion of neither EP receptor in mice, either globally (for EP1, EP2, and EP3) or selectively in the nervous system (EP4), had any effect on the IL-1β induced anorexia. Because these mice were all on a C57BL/6 background, whereas previous observations demonstrating a role for induced PGE2 in IL-1β evoked anorexia had been carried out on mice on a DBA/1 background, we examined the anorexic response to IL-1β in mice with deletion of mPGES-1 on a C57BL/6 background and a DBA/1 background, respectively. We confirmed previous findings that mPGES-1 knock-out mice on a DBA/1 background displayed attenuated anorexia to IL-1β; however, mice on a C57BL/6 background showed the same profound anorexia as wild type mice when carrying deletion of mPGES-1, while displaying almost normal food intake after pretreatment with a cyclooxygenase-2 inhibitor. We conclude that the involvement of induced PGE2 in IL-1β evoked anorexia is strain dependent and we suggest that different routes that probably involve distinct prostanoids exist by which inflammatory stimuli may evoke an anorexic response and that these routes may be of different importance in different strains of mice.

Increased lethality and defective pulmonary clearance of Streptococcus pneumoniae in microsomal prostaglandin E synthase-1-knockout mice.

The production of prostaglandin E2 (PGE2) increases dramatically during pneumococcal pneumonia, and this lipid mediator impairs alveolar macrophage (AM)-mediated innate immune responses. Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme involved in the synthesis of PGE2, and its expression is enhanced during bacterial infections. Genetic deletion of mPGES-1 in mice results in diminished PGE2 production and elevated levels of other prostaglandins after infection. Since PGE2 plays an important immunoregulatory role during bacterial pneumonia we assessed the impact of mPGES-1 deletion in the host defense against pneumococcal pneumonia in vivo and in AMs in vitro. Wild-type (WT) and mPGES-1 knockout (KO) mice were challenged with Streptococcus pneumoniae via the intratracheal route. Compared with WT animals, we observed reduced survival and increased lung and spleen bacterial burdens in mPGES-1 KO mice 24 and 48 h after S. pneumoniae infection. While we found modest differences between WT and mPGES-1 KO mice in pulmonary cytokines, AMs from mPGES-1 KO mice exhibited defective killing of ingested bacteria in vitro that was associated with diminished inducible nitric oxide synthase expression and reduced nitric oxide (NO) synthesis. Treatment of AMs from mPGES-1 KO mice with an NO donor restored bacterial killing in vitro. These results suggest that mPGES-1 plays a critical role in bacterial pneumonia and that genetic ablation of this enzyme results in diminished pulmonary host defense in vivo and in vitro. These results suggest that specific inhibition of PGE2 synthesis by targeting mPGES-1 may weaken host defense against bacterial infections.

Abstract 14122: Myeloid Cell mPGES-1 Depletion Attenuates Mortality After Myocardial Infarction in Mice

Selective depletion of microsomal PGE2 synthase (mPGES) -1 in myeloid cells retards atherogenesis and suppresses the vascular proliferative response to injury, while it does not predispose to thrombogenesis or hypertension. However, studies using bone marrow transplants from irradiated mice suggest that myeloid cell mPGES-1 facilitates cardiac remodeling and prolongs survival after experimental myocardial infarction (MI). Here we addressed this question using mice lacking mPGES-1 in myeloid cells, particularly macrophages (Mac-mPGES-1 KO), generated by crossing mPGES-1 floxed mice with LysMCre mice, and subjecting them to coronary artery ligation. Cardiac structure and function were assessed by morphometric analysis, echocardiography, and invasive hemodynamics 7 and 28 days after MI. Despite a similar infarct size, in contrast to the prior report, the post-MI survival rate was markedly improved in the Mac-mPGES-1 KO mice compared to WT controls (92.9% vs. 55.6%, p=0.03). Left ventricular systolic (reflected by ejection fraction, fractional shortening, end systolic pressure, and +dP/dt) and diastolic function (reflected by end diastolic pressure, -dP/dt, and Tau), cardiac hypertrophy (reflected by LV dimensions) and staining for fibrosis did not differ between the groups. In conclusion, Cre-loxP mediated deletion of mPGES-1 in myeloid cells has favorable effects on post-MI survival, with no detectable adverse influence on post-MI remodeling. These results add to evidence that targeting macrophage mPGES-1 may represent a safe and efficacious approach to the treatment and prevention of cardiovascular inflammatory disease.

Paradoxical Increase in Mortality and Rupture of Intracranial Aneurysms in Microsomal Prostaglandin E2 Synthase Type 1-Deficient Mice: Attenuation by Aspirin.

Inflammation plays an important role in formation and rupture of intracranial aneurysms. Expression of microsomal prostaglandin E2 (PGE2) synthase type 1 (mPGES-1) is increased in the wall of intracranial aneurysms in humans. PGE2, a by-product of mPGES-1, is associated with inflammation and cerebrovascular dysfunction. To test the hypothesis that deletion of mPGES-1 decreases the formation and rupture of intracranial aneurysms in a murine model. Intracranial aneurysms were induced in wild-type and mPGES-1 knockout (mPGES-1 KO) mice by using a combination of deoxycorticosterone acetate-salt-induced hypertension and intracranial injection of elastase in the basal cistern. Prevalence of aneurysms, subarachnoid hemorrhage, and mortality were assessed. We also tested the effects of administration of aspirin (6 mg/kg/d) by gavage and PGE2 (1 mg/kg/d) by subcutaneous infusion. Systolic blood pressure and prevalence of aneurysm were similar in wild-type and mPGES-1 KO mice. However, mortality and the prevalence of subarachnoid hemorrhage were markedly increased in mPGES-1 KO mice (P < .05). Bone marrow reconstitution studies suggest that mPGES-1 derived from leukocytes does not appear to increase rupture of intracranial aneurysms. Aspirin, but not PGE2, attenuated the increased mortality in mPGES-1 KO mice (P < .05). Vascular mPGES-1 plays a protective role in blood vessels and attenuates rupture of cerebral aneurysms. In contrast to effects on abdominal aneurysms, mPGES-1 deficiency is associated with an increase in rupture of cerebral aneurysms and mortality, which are attenuated by low-dose aspirin.

Abstract 682: Pharmacological Inhibition of COX and LOX Pathways Leads to Substrate Rediversion: A Novel Plasma Lipidomics Assay to Screen Drugs in Human Blood

Cardiovascular complications from cyclooxygenase (COX)-2-selective nonsteroidal anti-inflammatory drugs (NSAIDs) prompted interest in microsomal prostaglandin synthase 1 (mPGES-1), downstream of COX-2, as an alternative drug target. Unlike COX-2, global deletion of mPGES-1 in mice suppresses prostaglandin E2 (PGE2) and augments prostacyclin PGI2 through substrate rediversion, and does not predispose to thrombogenesis and hypertension. However, after cell-specific deletions of mPGES-1, the predominant substrate rediversion product amongst the prostaglandins varies by cell type. We hypothesized that inhibition of mPGES-1 leads to changes in bioactive lipids beyond the prostaglandin pathway, and that substrate rediversion associated with a more favorable cardiovascular profile after mPGES-1 vs COX-2 inhibition can be translated from mouse to human studies. In this project, we aimed 1) to develop a mass spectrometry-based, broad-spectrum lipidomics approach to measure arachidonate-derived products additional to prostanoids; and 2) to work out an in vitro human whole blood assay (hWBA) to study substrate rediversion upon mPGES-1 vs COX-2 inhibition. We have developed a novel, reproducible analytical method to scan the impact of drugs on the bioactive lipidome. Using an in vitro hWBA and HPLC-MS/MS lipidomics analysis, we observed substrate rediversion in both COX and LOX pathways after mPGES-1 inhibition, which was distinct from inhibiting COX-1/2. We further optimized this assay to screen combinations of inhibitors from COX and lipoxygenase (LOX) cascades. Overall, we have shown that a clear signature of inhibiting diverse targets - mPGES-1, COX and LOX enzymes - can be readily detected from experiments of small samples sizes, and discovered novel aspects of drug effect due to substrate rediversion. Our findings contribute to the comparative cardiovascular safety of inhibiting mPGES-1 vs COX-2 and demonstrate the impact of combining mPGES-1 with LOX inhibitions on the hemostatic system

Regulatory T cell differentiation and Th17 T cell function are regulated by mPGES-1-dependent PGE2 (IRC11P.424)

Abstract Prostaglandin E2 (PGE2) is a lipid mediator that regulates inflammatory and immune responses. PGE2 has pleiotropic effects on many cell types, but it is still unclear how it affects T cell differentiation and function. The mPGES1 enzyme is part of the PGE2 biosynthetic pathway and, due to its inducible nature, it controls PGE2 concentrations during activation of the inflammatory program. Absence of mPGES1 causes shunting to alternate prostaglandins and changes the characteristics of the inflammatory response. We identified that, compared to WT, naïve T cells from mice with genetic deletion of mPGES1 express a 4-fold lower level of TFGbR1 and 8-fold lower level of the EP4 PGE receptor. Since TGF-b is a key cytokine for the generation of both regulatory and Th17 cells we addressed the effect of mPGES1 deficiency during in vitro T cell polarization. We observed that mPGES1-/- naïve CD4+ T cells generate less iTreg cells, while Th17 commitment is unaffected. However, when stimulating T cells with anti-CD3/CD28 from mice following immunization with type II collagen (CII), mPGES1-/- CD4+ T cells were unable to secrete IL-17A compared to its WT T cell counterparts. Furthermore, in-vivo antigen-specific Th17 responses from CII immunized mice were also impaired, showing a decrease in IL17A production in mPGES1-/- T cells compared to WT. Hence, endogenous production and sensing of PGE2 modulates T cell differentiation possibly by altering TGF-b responses.

Abstract 928: Deletion of mammary epithelial mPGES1 suppresses tumor development in mice: a possible effect of substrate re-diversion

Abstract Microsomal prostaglandin E2 synthase (mPGES1) is the terminal enzyme in cyclooxygenase (COX)-2-mediated prostaglandin (PG) E2 biosynthesis. The pro-tumor contributions of COX-2 and PGE2 are established as is the clinical efficacy of pharmacological COX-2 inhibition. However, in addition to the desired suppression of tumor PGE2, collateral loss of vascular endothelial COX-2-derived PGI2, an endogenous anti-platelet mediator, imposes a cardiovascular hazard that limits clinical use of COX-2 inhibitors. mPGES1 is a promising alternative target to interrupt COX-2-driven events in tumors without elevating cardiovascular risk. We engineered mice transgenic for an activated HER2/neu oncogene to lack mPGES1 in mammary epithelial cells (MEC; mPGES1 KOMEC ). mPGES1 KOMEC mice and their wild type (WT) littermates were sacrificed at 22 weeks and mammary glands harvested. Glands were sectioned, every 10th slide was H&amp;E stained and scanned to obtain whole slide images. For each mouse, multiplicity was calculated using the slide containing the greatest number of lesions. Adjacent sections were immunostained for Ki67, caspase 3, and Factor VIII, to assess proliferation, apoptosis, and angiogenesis. Tumor multiplicity was significantly higher in WT compared to mPGES1 KOMEC mice (4.17 ± 1.05 [n=6] vs 0.29 ± 0.19 [n=7], respectively; p=0.002). In addition, in a second cohort of mice followed until tumors were palpable, mPGES1 KOMEC mice trended toward a longer tumor free period. Concordant with these observations, shRNA knock down (KD) of mPGES1 in mammary tumor cells dramatically suppressed their growth as orthotopic tumors in syngeneic immune competent WT hosts - 5 out of 6 non-target shRNA control tumors grew successfully while, in all cases, mPGES1 KD cells failed to grow. By immunohistochemistry, no difference was observed in proliferation or apoptosis between WT and mPGES1 KOMEC spontaneous tumors. Interestingly, and in contrast to our previous study of neu-driven mammary tumors in mice lacking MEC COX-2 (COX-2 KOMEC), vascularization was not different between mPEGS-1 KOMEC and WT spontaneous tumors suggesting divergence in the anti-tumorigenic mechanisms in mPEGS-1 KOMEC and COX-2 KOMEC tumors. Multiple studies of mPGES-1 deletion report re-diversion of COX-2-derived substrate to other prostanoid synthetic pathways, in particular PGD2. By Q-PCR, other than reduced mPGES1 mRNA, terminal prostanoid enzymes were not altered in mPGES1 KOMEC tumors. Interestingly, while the expression of PGE2 receptors EP2 and EP4 trended downwards in mPGES1 KOMEC tumors, the DP2 receptor for PGD2 was significantly increased. Taken together, these data establish mPGES1 as a promising therapeutic target in breast cancer. Further, we propose that, in addition to loss of PGE2, re-diversion of COX-2-derived substrate to the PGD2-DP2 pathway may be involved in delayed tumorigenesis. Citation Format: Nune Markosyan, Emer M. Smyth. Deletion of mammary epithelial mPGES1 suppresses tumor development in mice: a possible effect of substrate re-diversion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 928. doi:10.1158/1538-7445.AM2014-928

Deletion of prostaglandin E2 synthesizing enzymes in brain endothelial cells attenuates inflammatory fever.

Fever is a hallmark of inflammatory and infectious diseases. The febrile response is triggered by prostaglandin E2 synthesis mediated by induced expression of the enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1). The cellular source for pyrogenic PGE2 remains a subject of debate; several hypotheses have been forwarded, including immune cells in the periphery and in the brain, as well as the brain endothelium. Here we generated mice with selective deletion of COX-2 and mPGES1 in brain endothelial cells. These mice displayed strongly attenuated febrile responses to peripheral immune challenge. In contrast, inflammation-induced hypoactivity was unaffected, demonstrating the physiological selectivity of the response to the targeted gene deletions. These findings demonstrate that PGE2 synthesis in brain endothelial cells is critical for inflammation-induced fever.