Emanuela Ricciotti

Prostaglandins and Inflammation

Prostaglandins are lipid autacoids derived from arachidonic acid. They both sustain homeostatic functions and mediate pathogenic mechanisms, including the inflammatory response. They are generated from arachidonate by the action of cyclooxygenase isoenzymes, and their biosynthesis is blocked by nonsteroidal antiinflammatory drugs, including those selective for inhibition of cyclooxygenase-2. Despite the clinical efficacy of nonsteroidal antiinflammatory drugs, prostaglandins may function in both the promotion and resolution of inflammation. This review summarizes insights into the mechanisms of prostaglandin generation and the roles of individual mediators and their receptors in modulating the inflammatory response. Prostaglandin biology has potential clinical relevance for atherosclerosis, the response to vascular injury and aortic aneurysm.

Understanding multicellular function and disease with human tissue-specific networks

Tissue and cell-type identity lie at the core of human physiology and disease. Understanding the genetic underpinnings of complex tissues and individual cell lineages is crucial for developing improved diagnostics and therapeutics. We present genome-wide functional interaction networks for 144 human tissues and cell types developed using a data-driven Bayesian methodology that integrates thousands of diverse experiments spanning tissue and disease states. Tissue-specific networks predict lineage-specific responses to perturbation, identify the changing functional roles of genes across tissues and illuminate relationships among diseases. We introduce NetWAS, which combines genes with nominally significant genome-wide association study (GWAS) P values and tissue-specific networks to identify disease-gene associations more accurately than GWAS alone. Our webserver, GIANT, provides an interface to human tissue networks through multi-gene queries, network visualization, analysis tools including NetWAS and downloadable networks. GIANT enables systematic exploration of the landscape of interacting genes that shape specialized cellular functions across more than a hundred human tissues and cell types.

Clinical Pharmacology of Platelet, Monocyte, and Vascular Cyclooxygenase Inhibition by Naproxen and Low-Dose Aspirin in Healthy Subjects

Background—The current controversy on the potential cardioprotective effect of naproxen prompted us to evaluate the extent and duration of platelet, monocyte, and vascular cyclooxygenase (COX) inhibition by naproxen compared with low-dose aspirin. Methods and Results—We performed a crossover, open-label study of low-dose aspirin (100 mg/d) or naproxen (500 mg BID) administered to 9 healthy subjects for 6 days. The effects on thromboxane (TX) and prostacyclin biosynthesis were assessed up to 24 hours after oral dosing. Serum TXB2, plasma prostaglandin (PG) E2, and urinary 11-dehydro-TXB2 and 2,3-dinor-6-keto-PGF1&agr; were measured by previously validated radioimmunoassays. The administration of naproxen or aspirin caused a similar suppression of whole-blood TXB2 production, an index of platelet COX-1 activity ex vivo, by 94±3% and 99±0.3% (mean±SD), respectively, and of the urinary excretion of 11-dehydro-TXB2, an index of systemic biosynthesis of TXA2 in vivo, by 85±8% and 78±7%, respectively, that persisted throughout the dosing interval. Naproxen, in contrast to aspirin, significantly reduced systemic prostacyclin biosynthesis by 77±19%, consistent with differential inhibition of monocyte COX-2 activity measured ex vivo. Conclusions—The regular administration of naproxen 500 mg BID can mimic the antiplatelet COX-1 effect of low-dose aspirin. Naproxen, unlike aspirin, decreased prostacyclin biosynthesis in vivo.

Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis

Prostaglandin (PG) E2 is formed from PGH2 by a series of PGE synthase (PGES) enzymes. Microsomal PGES-1−/− (mPGES-1−/−) mice were crossed into low-density lipoprotein receptor knockout (LDLR−/−) mice to generate mPGES-1−/− LDLR−/−s. Urinary 11α-hydroxy-9, 15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M) was depressed by mPGES-1 deletion. Vascular mPGES-1 was augmented during atherogenesis in LDLR−/−s. Deletion of mPGES-1 reduced plaque burden in fat-fed LDLR−/−s but did not alter blood pressure. mPGES-1−/− LDLR−/− plaques were enriched with fibrillar collagens relative to LDLR−/−, which also contained small and intermediate-sized collagens. Macrophage foam cells were depleted in mPGES-1−/− LDLR−/− lesions, whereas the total areas rich in vascular smooth muscle cell (VSMC) and matrix were unaltered. mPGES-1 deletion augmented expression of both prostacyclin (PGI2) and thromboxane (Tx) synthases in endothelial cells, and VSMCs expressing PGI synthase were enriched in mPGES-1−/− LDLR−/− lesions. Stimulation of mPGES-1−/− VSMC and macrophages with bacterial LPS increased PGI2 and thromboxane A2 to varied extents. Urinary PGE-M was depressed, whereas urinary 2,3-dinor 6-keto PGF1α, but not 2,3-dinor-TxB2, was increased in mPGES-1−/− LDLR−/−s. mPGES-1-derived PGE2 accelerates atherogenesis in LDLR−/− mice. Disruption of this enzyme retards atherogenesis, without an attendant impact on blood pressure. This may reflect, in part, rediversion of accumulated PGH2 to augment formation of PGI2. Inhibitors of mPGES-1 may be less likely than those selective for cyclooxygenase 2 to result in cardiovascular complications because of a divergent impact on the biosynthesis of PGI2.

Vascular COX-2 Modulates Blood Pressure and Thrombosis in Mice

Deletion of vascular COX-2 predisposes mice to thrombosis and hypertension. Chronicle of Some Deaths Foretold Nonsteroidal anti-inflammatory drugs (NSAIDs) relieve pain and inflammation by blocking the formation of prostaglandins due to the action of the enzymes cyclooxygenase-1 (COX-1) and COX-2. NSAIDs that inhibit COX-2 selectively are less likely to cause gastrointestinal side effects. More than a decade ago, it was discovered that such drugs depressed biosynthesis of prostaglandin I2 (PGI2, prostacyclin) in healthy human volunteers, as reflected by a decrease in its major metabolite PGI-M in urine. It was suggested that hemodynamic shear induced COX-2 expression in endothelial cells in vivo, accounting for the impact of the drug on PGI-M in urine. Based on the potentially cardioprotective effects of PGI2, a powerful platelet inhibitor and vasodilator in vitro, it was proposed that inhibition of COX-2 might confer a cardiovascular hazard. Subsequently, eight placebo-controlled trials of three structurally distinct COX-2 inhibitors established that there was an increased risk of myocardial infarction, stroke, hypertension, and heart failure in a subpopulation of patients taking these drugs. Despite these findings, there has been debate about the mechanistic underpinnings of this cardiovascular risk. In a study that helps to put this debate to rest, Yu et al. show that selective deletion of COX-2 in the vasculature of mice depresses PGI-M in mouse urine and predisposes them to both hypertension and thrombosis. Furthermore, expression of endothelial nitric oxide (NO) synthase and consequent release of NO are depressed if vascular COX-2 is deleted. This study provides clear evidence for a link between selective disruption of COX-2 in the vasculature and clinical outcomes in humans. Suppression of PGI2 formation due to deletion of vascular COX-2 is sufficient to explain the cardiovascular hazard from NSAIDs, which may be augmented by secondary mechanisms such as suppression of NO production. Prostacyclin (PGI2) 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 PGI2 (PGI-M) in the urine of healthy volunteers. This suggested that NSAIDs might cause adverse cardiovascular events by reducing production of cardioprotective PGI2. This prediction was based on the assumption that the concentration of PGI-M in urine likely reflected vascular production of PGI2 and that other cardioprotective mediators, especially nitric oxide (NO), were not able to compensate for the loss of PGI2. 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 PGI2 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.

Microsomal Prostaglandin E Synthase-1 Deletion Suppresses Oxidative Stress and Angiotensin II–Induced Abdominal Aortic Aneurysm Formation

Background— Microsomal prostaglandin (PG) E2 synthase-1 (mPGES-1) catalyzes isomerization of the cyclooxygenase product PGH2 into PGE2. Deletion of mPGES-1 modulates experimentally evoked pain and inflammation and retards atherogenesis. The role of mPGES-1 in abdominal aortic aneurysm is unknown. Methods and Results— The impact of mPGES-1 deletion on formation of angiotensin II–induced abdominal aortic aneurysm was studied in mice lacking low-density lipoprotein receptor (LDLR−/−). Male mice deficient in both mPGES-1 and LDLR (mPGES-1−/− LDLR−/−) and littermate LDLR−/− mice were initiated on a high-fat diet at 6 months of age, followed 1 week later by continuous infusion of angiotensin II (1 &mgr;g/kg per minute) for an additional 4 weeks. Angiotensin II infusion upregulated aortic expression of cyclooxygenase-2 and mPGES-1, increased aortic macrophage recruitment and vascular nitrotyrosine staining (which reflects local oxidative stress), and augmented urinary excretion of the isoprostane 8,12-iso-iPF2&agr;-VI (which reflects lipid peroxidation in vivo) and the major metabolite of PGE2 (PGE-M). Deletion of mPGES-1 decreased both the incidence (87.5% versus 27.3%; P=0.02) and the severity of abdominal aortic aneurysm and depressed the aortic and systemic indices of oxidative stress. Deletion of mPGES-1 also depressed urinary PGE-M, whereas it augmented excretion of PGD2 and PGI2 metabolites, reflecting rediversion of the accumulated PGH2 substrate in the double knockouts. Conclusions— Deletion of mPGES-1 protects against abdominal aortic aneurysm formation induced by angiotensin II in hyperlipidemic mice, coincident with a reduction in oxidative stress. The potential efficacy of selective inhibition of mPGES-1 in preventing or retarding aneurysm formation warrants further investigation.

Cardiomyocyte cyclooxygenase-2 influences cardiac rhythm and function

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 α-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.

The biochemical selectivity of novel COX-2 inhibitors in whole blood assays of COX-isozyme activity.

Summary We have evaluated the biochemical selectivity of novel cyclo-oxygenase (COX)-2 inhibitors, etoricoxib, valdecoxib, DFU and DFP, vs rofecoxib and celecoxib, using the human whole blood assays of COX-isozyme activity, in vitro. Compounds were incubated with human whole blood samples, allowed to clot for 1 h at 37°C, or stimulated with lipopolysaccharide (10|ig/ml) for 24 h at 37°C. Serum thromboxane (TX) B2 and plasma prostaglandin (PG) E2 levels were measured by specific radioimmunoassays as indices of platelet COX-1 and monocyte COX-2 activity, respectively. Valdecoxib, etoricoxib, DFU and DFP inhibited platelet COX-1 and monocyte COX-2 with the following COX-1/COX-2 IC50 ratios: 61.5, 344, 660 and 1918, respectively. The reference compounds, celecoxib and rofecoxib had corresponding values of 29.6 and 272. In conclusion, a second wave of COX-2 inhibitors with higher biochemical selectivity than the existing coxibs has been developed. Whether their administration will be associated with improved clinical efficacy and/or safety visà-vis celecoxib and rofecoxib remains to be established.

Tetranor PGDM, an Abundant Urinary Metabolite Reflects Biosynthesis of Prostaglandin D2 in Mice and Humans

Prostaglandin D2 (PGD2) is a cyclooxygenase (COX) product of arachidonic acid that activates D prostanoid receptors to modulate vascular, platelet, and leukocyte function in vitro. However, little is known about its enzymatic origin or its formation in vivo in cardiovascular or inflammatory disease. 11,15-Dioxo-9α-hydroxy-2,3,4,5-tetranorprostan-1,20-dioic acid (tetranor PGDM) was identified by mass spectrometry as a metabolite of infused PGD2 that is detectable in mouse and human urine. Using liquid chromatography-tandem mass spectrometry, tetranor PGDM was much more abundant than the PGD2 metabolites, 11β-PGF2α and 2,3-dinor-11β-PGF2α, in human urine and was the only endogenous metabolite detectable in mouse urine. Infusion of PGD2 dose dependently increased urinary tetranor PGDM > 2,3-dinor-11β-PGF2α > 11β-PGF2α in mice. Deletion of either lipocalin-type or hemopoietic PGD synthase enzymes decreased urinary tetranor PGDM. Deletion or knockdown of COX-1, but not deletion of COX-2, decreased urinary tetranor PGDM in mice. Correspondingly, both PGDM and 2,3-dinor-11β-PGF2α were suppressed by inhibition of COX-1 and COX-2, but not by selective inhibition of COX-2 in humans. PGD2 has been implicated in both the development and resolution of inflammation. Administration of bacterial lipopolysaccharide coordinately elevated tetranor PGDM and 2,3-dinor-11β-PGF2α in volunteers, coincident with a pyrexial and systemic inflammatory response, but both metabolites fell during the resolution phase. Niacin increased tetranor PGDM and 2,3-dinor-11β-PGF2α in humans coincident with facial flushing. Tetranor PGDM is an abundant metabolite in urine that reflects modulated biosynthesis of PGD2 in humans and mice.

Targeted deletions of cyclooxygenase-2 and atherogenesis in mice.

Background— Although the dominant product of vascular Cyclooxygenase-2 (COX-2), prostacyclin (PGI2), 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 ≈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 &agr;-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-CD3&egr;. 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 A2 and prostaglandin (PG)E2, 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.