Molecular clocks and the human condition: approaching their characterization in human physiology and disease.

Molecular clockworks knit together diverse biological networks and compelling evidence from model systems infers their importance in metabolism, immunological and cardiovascular function. Despite this and the diurnal variation in many aspects of human physiology and the phenotypic expression of disease, our understanding of the role and importance of clock function and dysfunction in humans is modest. There are tantalizing hints of connection across the translational divide and some correlative evidence of gene variation and human disease but most of what we know derives from forced desynchrony protocols in controlled environments. We now have the ability to monitor quantitatively ex vivo or in vivo the genome, metabolome, proteome and microbiome of humans in the wild. Combining this capability, with the power of mobile telephony and the evolution of remote sensing, affords a new opportunity for deep phenotyping, including the characterization of diurnal behaviour and the assessment of the impact of the clock on approved drug function.

A metabolomic study of adipose tissue in mice with a disruption of the circadian system.

Adipose tissue functions in terms of energy homeostasis as a rheostat for blood triglyceride, regulating its concentration, in response to external stimuli. In addition it acts as a barometer to inform the central nervous system of energy levels which can vary dramatically between meals and according to energy demand. Here a metabolomic approach, combining both Mass Spectrometry and Nuclear Magnetic Resonance spectroscopy, was used to analyse both white and brown adipose tissue in mice with adipocyte-specific deletion of Arntl (also known as Bmal1), a gene encoding a core molecular clock component. The results are consistent with a peripheral circadian clock playing a central role in metabolic regulation of both brown and white adipose tissue in rodents and show that Arntl induced global changes in both tissues which were distinct for the two types. In particular, anterior subcutaneous white adipose tissue (ASWAT) tissue was effected by a reduction in the degree of unsaturation of fatty acids, while brown adipose tissue (BAT) changes were associated with a reduction in chain length. In addition the aqueous fraction of metabolites in BAT were profoundly affected by Arntl disruption, consistent with the dynamic role of this tissue in maintaining body temperature across the day-night cycle and an upregulation in fatty acid oxidation and citric acid cycle activity to generate heat during the day when rats are inactive (increases in 3-hydroxybutyrate and glutamate), and increased synthesis and storage of lipids during the night when rats feed more (increased concentrations of glycerol, choline and glycerophosphocholine).

Vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials.

BACKGROUND: The vascular and gastrointestinal effects of non-steroidal anti-inflammatory drugs (NSAIDs), including selective COX-2 inhibitors (coxibs) and traditional non-steroidal anti-inflammatory drugs (tNSAIDs), are not well characterised, particularly in patients at increased risk of vascular disease. We aimed to provide such information through meta-analyses of randomised trials. METHODS: We undertook meta-analyses of 280 trials of NSAIDs versus placebo (124,513 participants, 68,342 person-years) and 474 trials of one NSAID versus another NSAID (229,296 participants, 165,456 person-years). The main outcomes were major vascular events (non-fatal myocardial infarction, non-fatal stroke, or vascular death); major coronary events (non-fatal myocardial infarction or coronary death); stroke; mortality; heart failure; and upper gastrointestinal complications (perforation, obstruction, or bleed). FINDINGS: Major vascular events were increased by about a third by a coxib (rate ratio [RR] 1·37, 95% CI 1·14-1·66; p=0·0009) or diclofenac (1·41, 1·12-1·78; p=0·0036), chiefly due to an increase in major coronary events (coxibs 1·76, 1·31-2·37; p=0·0001; diclofenac 1·70, 1·19-2·41; p=0·0032). Ibuprofen also significantly increased major coronary events (2·22, 1·10-4·48; p=0·0253), but not major vascular events (1·44, 0·89-2·33). Compared with placebo, of 1000 patients allocated to a coxib or diclofenac for a year, three more had major vascular events, one of which was fatal. Naproxen did not significantly increase major vascular events (0·93, 0·69-1·27). Vascular death was increased significantly by coxibs (1·58, 99% CI 1·00-2·49; p=0·0103) and diclofenac (1·65, 0·95-2·85, p=0·0187), non-significantly by ibuprofen (1·90, 0·56-6·41; p=0·17), but not by naproxen (1·08, 0·48-2·47, p=0·80). The proportional effects on major vascular events were independent of baseline characteristics, including vascular risk. Heart failure risk was roughly doubled by all NSAIDs. All NSAID regimens increased upper gastrointestinal complications (coxibs 1·81, 1·17-2·81, p=0·0070; diclofenac 1·89, 1·16-3·09, p=0·0106; ibuprofen 3·97, 2·22-7·10, p<0·0001; and naproxen 4·22, 2·71-6·56, p<0·0001). INTERPRETATION: The vascular risks of high-dose diclofenac, and possibly ibuprofen, are comparable to coxibs, whereas high-dose naproxen is associated with less vascular risk than other NSAIDs. Although NSAIDs increase vascular and gastrointestinal risks, the size of these risks can be predicted, which could help guide clinical decision making. FUNDING: UK Medical Research Council and British Heart Foundation.

Effect of assay specificity on the association of urine 11-dehydro thromboxane B2 determination with cardiovascular risk.

BACKGROUND: Elevated urine 11-dehydro TXB(2), an indicator of persistent thromboxane generation in aspirin-treated patients, correlates with adverse cardiovascular outcome and has recently been identified as an independent risk factor for vein graft thrombosis after cardiac bypass surgery in the Reduction in Graft Occlusion Rates (RIGOR) study. The polyclonal antibody-based ELISA used to measure 11-dehydro TXB(2) in these previous studies is no longer clinically available and has been supplanted by a Food and Drug Administration (FDA)-cleared second-generation monoclonal antibody-based ELISA. OBJECTIVES: To compare the laboratory and clinical performance of the first- and second-generation assays in a well-defined study population. METHODS: 11-dehydro TXB(2) was quantified in 451 urine samples from 229 Reduction in Graft Occlusion Rates (RIGOR) subjects using both ELISA. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and spiking studies were used to investigate discordant assay results. The association of 11-dehydro TXB(2) to clinical outcome was assessed for each assay using multivariate modeling. RESULTS: Median 11-dehydro TXB(2) levels were higher by monoclonal antibody- compared with polyclonal antibody-based ELISA (856 vs. 399 pg mg(-1) creatinine, P < 0.000001), with the latter providing values similar to UPLC-MS/MS. This discrepancy was predominantly as a result of cross-reactivity of the monoclonal antibody with 11-dehydro-2,3-dinor TXB(2), a thromboxane metabolite present in a similar concentration but with a poor direct correlation with 11-dehydro TXB(2). In contrast to the first-generation ELISA, 11-dehydro TXB(2) measured by the monoclonal antibody-based ELISA failed to associate with the risk of vein graft occlusion. CONCLUSION: Quantification of urine 11-dehydro TXB(2) by monoclonal antibody-based ELISA was confounded by interference from 11-dehydro-2,3-dinor TXB(2) which reduced the accuracy and clinical utility of this second-generation assay.

Comparative impact on prostanoid biosynthesis of celecoxib and the novel nonsteroidal anti-inflammatory drug CG100649.

Nonsteroidal anti-inflammatory drugs (NSAIDs) elevate cardiovascular risk by disrupting cyclooxygenase-2 (COX-2)-dependent biosynthesis of prostacyclin (PGI(2)). CG100649 is a novel NSAID proposed to inhibit both COX-2 and carbonic anhydrase (CA)-I/-II. We compared its impact on prostanoid biosynthesis with that of celecoxib, an NSAID purposefully designed to selectively inhibit COX-2. In a controlled, double-blind randomized trial, single oral doses of 2 or 8 mg CG100649, 200 mg celecoxib, or placebo were well tolerated by healthy volunteers (n = 23). Both CG100649 and celecoxib had the effect of depressing urinary excretion of 2,3-dinor-6-keto-PGF(1α) (PGI-M); the effect of CG100649 was dose-dependent and more sustained (up to 240 h after the dose) than that of celecoxib. Neither CG100649 nor celecoxib significantly inhibited COX-1-dependent prostanoid formation. CA inhibition was not detected after administration of CG100649, despite its partitioning asymmetrically into erythrocytes. CG100649 and celecoxib are both relatively selective inhibitors of COX-2, but they differ in duration of action. Whether they have similar impact on cardiovascular events remains to be determined.

Ablation of cyclooxygenase-2 in forebrain neurons is neuroprotective and dampens brain inflammation after status epilepticus.

Cyclooxygenase-2 (COX-2), a source of inflammatory mediators and a multifunctional neuronal modulator, is rapidly induced in select populations of cortical neurons after status epilepticus. The consequences of rapid activity-triggered induction of COX-2 in neurons have been the subject of much study and speculation. To address this issue directly, we created a mouse in which COX-2 is conditionally ablated in selected forebrain neurons. Results following pilocarpine-induced status epilepticus indicate that neuronal COX-2 promotes early neuroprotection and then delayed neurodegeneration of CA1 pyramidal neurons, promotes neurodegeneration of nearby somatostatin interneurons in the CA1 stratum oriens and dentate hilus (which themselves do not express COX-2), intensifies a broad inflammatory reaction involving numerous cytokines and other inflammatory mediators in the hippocampus, and is essential for development of a leaky blood-brain barrier after seizures. These findings point to a profound role of seizure-induced neuronal COX-2 expression in neuropathologies that accompany epileptogenesis.

Effects of celecoxib on major prostaglandins in asthma.

BACKGROUND: Prostaglandin (PG) D(2) is a pro-inflammatory and bronchoconstrictive mediator released from mast cells, and is currently evaluated as a new target for treatment of asthma and rhinitis. It is not known which cyclooxygenase (COX) isoenzyme catalyses its biosynthesis in subjects with asthma. OBJECTIVES: Primarily, to assess whether treatment with the COX-2 selective inhibitor celecoxib inhibited biosynthesis of PGD(2) , monitored as urinary excretion of its major tetranor metabolite (PGDM). Secondarily, to determine the effects of the treatment on biosynthesis of PGE(2) , thromboxane A(2) and PGI(2) , also measured as major urinary metabolites. METHODS: Eighteen subjects with asthma participated in a cross-over study where celecoxib 200mg or placebo were given b.i.d. on 3 consecutive days following 2 untreated baseline days. Six healthy controls received active treatment with the same protocol. Urinary excretion of the eicosanoid metabolites was determined by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Lung function was followed as FEV(1) and airway inflammation as fraction of exhaled nitric oxide (F(E) NO). RESULTS: Celecoxib treatment inhibited urinary excretion of PGEM by 50% or more in subjects with asthma and healthy controls, whereas there was no significant change in the excretion of PGDM. In comparison with the healthy controls, the subjects with asthma had higher baseline levels of urinary PGDM but not of PGEM. The 3-day treatment did not cause significant changes in FEV(1) or F(E) NO. CONCLUSION AND CLINICAL RELEVANCE: Biosynthesis of PGD(2) was increased in subjects with asthma and its formation is catalysed predominantly by COX-1. By contrast, COX-2 contributes substantially to the biosynthesis of PGE(2) . The asymmetric impact of celecoxib on prostanoid formation raises the possibility of long-term adverse consequences of COX-2 inhibition on airway homeostasis by the decreased formation of bronchodilator PGs and maintained production of increased levels of bronchoconstrictor PGs in asthmatics.

The translational therapeutics of prostaglandin inhibition in atherothrombosis.

Prostaglandins, products of the cyclo-oxygenase (COX) enzymes, can both promote and restrain atherothrombosis. While non-steroidal anti-inflammatory drugs (NSAIDs) selective for inhibition of COX-2 predispose to myocardial infarction, heart failure, hypertension and stroke, suppression of products of COX-1, such as thromboxane (Tx) A2, underlie cardioprotection from low-dose aspirin. Data from clinical pharmacology, rodent models, human genetics, observational studies and randomized trials provide insight into the implications of inhibiting COX product synthesis or function. Many lines of evidence afford a mechanistic explanation for the cardiovascular (CV) hazard from NSAIDs. Elucidation of the biology of this pathway using diversified approaches is also relevant to understanding the implications of substrate rediversion following inhibition of enzymes downstream of COXs, such as the microsomal PGE synthase (mPGES)-1 and of combining D prostanoid antagonism with niacin to attenuate facial flushing.

Aspirin reduces the prothrombotic activity of C-reactive protein.

AIM: C-reactive protein (CRP) is a risk marker and a potential modulator of vascular disease. Previous studies support a prothrombotic activity of CRP, with impaired thromboregulation. The present study examined the antithrombotic effect of aspirin in mice transgenic for human CRP (CRPtg mice). Mechanistic investigations further elucidated the effect of CRP on prostanoid metabolism in vivo and in vitro. METHODS AND RESULTS: Administration of aspirin (30 mg kg(-1) day(-1)) to CRPtg mice slowed the accelerated thrombosis after photochemical injury to the carotid (99 +/- 32 vs. 45 +/- 24 min and 75 +/- 23 vs. 82 +/- 26 min in wild-type mice vs. CRPtg mice, without and following aspirin treatment, respectively). Vascular injury modulated the expression of key pathways in prostanoid metabolism differently in CRPtg mice and wild-type mice. Suppression of cyclo-oxygenase 2 (COX-2)-derived metabolism with suppression of prostaglandin I2 (PGI2) synthase and PGI2 metabolism was recorded in the injured artery with increased thromboxane receptor expression. Aspirin therapy reduced the difference in PGI2 biosynthesis between CRPtg mice and wild-type mice. In vitro studies in human-derived cells further supported these findings. Incubation of human umbilical vein endothelial cells (HUVECs) with human recombinant CRP (5 microg mL(-1)) suppressed PGI2 synthase expression and significantly increased thromboxane receptor levels. Incubation of smooth muscle cells with CRP did not affect prostanoid expression. CONCLUSIONS: CRP modulates prostanoid metabolism to favor vascular occlusion. Elevated CRP levels might predispose to the cardiovascular hazard conferred by selective COX-2 inhibitors, and the risk mediated by CRP may be limited by aspirin.

Microsomal prostaglandin E synthase-1 inhibition in cardiovascular inflammatory disease.

Prostaglandins (PGs), particularly PGE2 and prostacyclin (PGI2), are potent mediators of pain and inflammation. Both atherosclerosis and aortic aneurysm exhibit the hallmarks of inflammation. However, randomized trials of inhibitors of PG synthesis--nonsteroidal anti-inflammatory drugs--reveal that they predispose to cardiovascular risk. This appears to be consequent to inhibition of PGI2 and PGE2 formed by cyclooxygenase-2 (COX-2). Inhibitors of microsomal PGE synthase-1 (mPGES-1) are being developed for relief of pain and interest has focused on their potential impact on the cardiovascular system. Deletion of mPGES-1 retards atherogenesis and limits aortic aneurysm formation in hyperlipidaemic mice. However, it does not predispose to thrombogenesis and has a limited impact on blood pressure compared to inhibition of COX-2. This occurs despite the potential of the suppressed PGE2 in affording cardioprotection via its EP2 and EP4 receptors. However, deletion of mPGES-1 permits rediversion of the PGH2 substrate to other PG synthases and augmented formation of PGI2 and PGD2 mitigates this effect. However, increased PGI2 may also attenuate relief of pain. Pain relief seems likely to be a nuanced indication for mPGES-1 inhibitors, but they have therapeutic potential in syndromes of cardiovascular inflammation, cancer and perhaps in neurodegenerative disease. However, as the products of substrate rediversion vary according to cell type, these drugs may have contrasting impact amongst individuals at varied stages of disease evolution.

Eicosanoids and the vascular endothelium.

Cyclooxygenase (COX) enzymes catalyse the biotransformation of arachidonic acid to prostaglandins which subserve important functions in cardiovascular homeostasis. Prostacyclin (PGI2) and prostaglandin (PG)E2, dominant products of COX activityin macro- and microvascular endothelial cells, respectively, in vitro, modulate the interaction of blood cells with the vasculature and contribute to the regulation of blood pressure. COXs are the target for inhibition by nonsteroidal anti-inflammatory drugs (NSAIDs--which include those selective for COX-2) and for aspirin. Modulation of the interaction between COX products of the vasculature and platelets underlies both the cardioprotection afforded by aspirin and the cardiovascular hazard which characterises specific inhibitors of COX-2.

Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

Biomarkers of oxidative stress study III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning.

Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.

Enhanced monocyte expression of tissue factor by oxidative stress in patients with antiphospholipid antibodies: effect of antioxidant treatment.

In a first study, we performed a cross-sectional analysis of urinary excretion of isoprostanes, IPF(2alpha-III) and (VI), and monocyte tissue factor (TF) antigen and activity between 11 antiphospholipid (APL) antibody-positive patients and 13 APL negative subjects. In a second study, 11 APL positive patients were randomly supplemented either with (n = 6) or without (n = 5) antioxidants (vitamin E at 900 IU day(-1), vitamin C at 2000 mg day(-1)) for 6 weeks. In a third study, TF and superoxide anion were measured in human monocytes incubated with anti-beta(2) glycoprotein 1 (beta(2)GP(1)) or control IgG, either with or without vitamin E. APL-positive patients had higher values of isoprostanes (P < 0.05) and monocyte TF antigen (P = 0.001) and activity (P = 0.0001) than APL-negative subjects. Only in APL positive patients did monocyte TF antigen correlate significantly with IPF(2alpha-III) (rho 0.79; P < 0.003) and IPF(2alpha-VI) (rho = 0.87; P < 0.0001). In patients who received antioxidant supplementation, we found a significant decrease of isoprostanes (P < 0.05) and monocyte TF antigen (P < 0.01) and activity (P < 0.007). In vitro experiments demonstrated that anti-beta(2)GP(1) antibodies dose-dependently enhanced the monocyte production of the superoxide anion and TF, which were significantly inhibited by vitamin E. This study demonstrates that in APL-positive patients, oxidative stress contributes to activate the clotting system via over-expression of monocyte TF. We suggest that anti-beta(2)GP(1) antibodies could play a pivotal role by enhancing the monocyte production of oxygen free radicals.

Cyclooxygenase inhibitors and the antiplatelet effects of aspirin.

BACKGROUND: Patients with arthritis and vascular disease may receive both low-dose aspirin and other nonsteroidal antiinflammatory drugs. We therefore investigated potential interactions between aspirin and commonly prescribed arthritis therapies METHODS: We administered the following combinations of drugs for six days: aspirin (81 mg every morning) two hours before ibuprofen (400 mg every morning) and the same medications in the reverse order; aspirin two hours before acetaminophen (1000 mg every morning) and the same medications in the reverse order; aspirin two hours before the cyclooxygenase-2 inhibitor rofecoxib (25 mg every morning) and the same medications in the reverse order; enteric-coated aspirin two hours before ibuprofen (400 mg three times a day); and enteric-coated aspirin two hours before delayed-release diclofenac (75 mg twice daily) RESULTS: Serum thromboxane B(2) levels (an index of cyclooxygenase-1 activity in platelets) and platelet aggregation were maximally inhibited 24 hours after the administration of aspirin on day 6 in the subjects who took aspirin before a single daily dose of any other drug, as well as in those who took rofecoxib or acetaminophen before taking aspirin. In contrast, inhibition of serum thromboxane B(2) formation and platelet aggregation by aspirin was blocked when a single daily dose of ibuprofen was given before aspirin, as well as when multiple daily doses of ibuprofen were given. The concomitant administration of rofecoxib, acetaminophen, or diclofenac did not affect the pharmacodynamics of aspirin CONCLUSIONS: The concomitant administration of ibuprofen but not rofecoxib, acetaminophen, or diclofenac antagonizes the irreversible platelet inhibition induced by aspirin. Treatment with ibuprofen in patients with increased cardiovascular risk may limit the cardioprotective effects of aspirin.

COX-2 inhibitors and the cardiovascular system.

Cyclooxygenase-2 selective inhibitors (coxibs) represent a new class of non steroidal anti-inflammatory drugs that exhibit preference for inhibition of cyclooxygenase-2 (COX-2), the COX isoform thought to account largely for prostanoid formation in inflammation. We review the divergent incidence of cardiovascular events derived from the two large clinical trials of coxibs, the Vioxx Gastrointestinal Outcomes Research Trial (VIGOR) and the Celecoxib Long-term Arthritis Safety Study (CLASS), in the context of current understanding of relevant clinical and basic pharmacology. The incidence of cardiovascular events was higher in patients receiving rofecoxib than in those receiving naproxen in VIGOR and did not differ between the groups in CLASS. By contrast, while the primary gastrointestinal (GI) endpoint comparison favored rofecoxib in VIGOR, no significant difference in the incidence of the primary GI endpoint was evident between celecoxib and two NSAID comparators not attained in CLASS. The cardiovascular results in VIGOR may have resulted from chance, a cardioprotective effect of naproxen, or suppression of prostacyclin but not thromboxane on rofecoxib. Differences in cardiovascular outcome between the two trials may also have resulted either from chance, or from aspects of the trial design (such as the use of aspirin by roughly one-fifth of the participants in CLASS), or from differences in the COX-2 selectivity or other pharmacology of the coxibs. Individuals who warrant low-dose aspirin for cardioprotection may have less likelihood of a GI event if they combine aspirin with rofecoxib, rather than a traditional NSAID. However, evidence addressing directly this hypothesis is currently unavailable. On the other hand, coxib consumption alone does not currently warrant initiation of a cardioprotective regimen, such as low-dose aspirin.