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Driven by autonomous molecular clocks that are synchronized by a master pacemaker in the suprachiasmatic nucleus, cardiac physiology fluctuates in diurnal rhythms that can be partly or entirely circadian. Cardiac contractility, metabolism, and electrophysiology, all have diurnal rhythms, as does the neurohumoral control of cardiac and kidney function. In this review, we discuss the evidence that circadian biology regulates cardiac function, how molecular clocks may relate to the pathogenesis of heart failure, and how chronotherapeutics might be applied in heart failure. Disrupting molecular clocks can lead to heart failure in animal models, and the myocardial response to injury seems to be conditioned by the time of day. Human studies are consistent with these findings, and they implicate the clock and circadian rhythms in the pathogenesis of heart failure. Certain circadian rhythms are maintained in patients with heart failure, a factor that can guide optimal timing of therapy. Pharmacologic and nonpharmacologic manipulation of circadian rhythms and molecular clocks show promise in the prevention and treatment of heart failure. more...

Circadian omics analyses present investigators with large amounts of data to consider and many choices for methods of analysis. Visualization is crucial as rhythmicity can take many forms and p-values offer an incomplete picture. Yet statically viewing the entirety of high-throughput datasets is impractical, and there is often limited ability to assess the impact of choices, such as significance threshold cutoffs. Nitecap provides an intuitive and unified web-based solution to these problems. Through highly responsive visualizations, Nitecap enables investigators to see dataset-wide behavior. It supports deep analyses, including comparisons of two conditions. Moreover, it focuses upon ease-of-use and enables collaboration through dataset sharing. As an application, we investigated cross talk between peripheral clocks in adipose and liver tissues and determined that adipocyte clock disruption does not substantially modulate the transcriptional rhythmicity of liver but does advance the phase of core clock gene Bmal1 (Arntl) expression in the liver. Nitecap is available at nitecap.org and is free-to-use. more...

This study shows that the supramolecular arrangement of proteins in nanoparticle structures predicts nanoparticle accumulation in neutrophils in acute lung inflammation (ALI). We observed homing to inflamed lungs for a variety of nanoparticles with agglutinated protein (NAPs), defined by arrangement of protein in or on the nanoparticles via hydrophobic interactions, crosslinking and electrostatic interactions. Nanoparticles with symmetric protein arrangement (for example, viral capsids) had no selectivity for inflamed lungs. Flow cytometry and immunohistochemistry showed NAPs have tropism for pulmonary neutrophils. Protein-conjugated liposomes were engineered to recapitulate NAP tropism for pulmonary neutrophils. NAP uptake in neutrophils was shown to depend on complement opsonization. We demonstrate diagnostic imaging of ALI with NAPs; show NAP tropism for inflamed human donor lungs; and show that NAPs can remediate pulmonary oedema in ALI. This work demonstrates that structure-dependent tropism for neutrophils drives NAPs to inflamed lungs and shows NAPs can detect and treat ALI. more...

Treatment failure due to variability in the pharmacologic response to drugs has more commonly been attributed to non-genetic factors than to prototypic pharmacogenetic mechanisms.1 Treatment failure on low-dose aspirin taken to prevent arterial thrombotic events, including myocardial infarction and stroke, is no exception. A closer look at aspirin’s human pharmacology reveals many potential sources of variability that are rooted in platelet biology, from aspirin insensitive mechanisms of activation to accelerated turnover. more...

Cerium oxide nanoparticles (CeONP), having potent antioxidant properties, are highly promising nanomaterials for treatment of diseases in which oxidative stress from excessive reactive oxygen species (ROS) plays a critical role in the pathogenesis and progression. However, most previously reported CeONP formulations were not efficiently cleared from the body, precluding their clinical translation. Herein, we report ultrasmall CeONP that can mitigate activation of macrophages and subsequent acute inflammation. It is found that these CeONP can effectively scavenge reactive species, inhibit macrophage activation, and minimize their recruitment and infiltration to the inflammation site, which lead to alleviation of edema and pain hypersensitivity. Moreover, we demonstrate that CeONP can be effectively excreted from the body within 24 h of systemic administration, minimizing long-term toxicity concerns. Altogether, our findings suggest that CeONP may be explored as both antioxidant and anti-inflammatory agents that can reduce acute inflammation with a better safety profile than existing nanoparticles. more...

The pro-oncogenic kinase PKCε is overexpressed in human prostate cancer and cooperates with loss of the tumor suppressor Pten for the development of prostatic adenocarcinoma. However, the effectors driving PKCε-mediated phenotypes remain poorly defined. Here, using cellular and mouse models, we showed that PKCε overexpression acts synergistically with Pten loss to promote NF-κB activation and induce cyclooxygenase-2 (COX-2) expression, phenotypic traits which are also observed in human prostate tumors. Targeted disruption of PKCε from prostate cancer cells impaired COX-2 induction and PGE2 production. Notably, COX-2 inhibitors selectively killed prostate epithelial cells overexpressing PKCε, and this ability was greatly enhanced by Pten loss. Long-term COX-2 inhibition markedly reduced adenocarcinoma formation, as well as angiogenesis in a mouse model of prostate-specific PKCε expression and Pten loss. Overall, our results provide strong evidence for the involvement of the canonical NF-κB pathway and its target gene COX2 as PKCε effectors, and highlight the potential of PKCε as a useful biomarker for the use of COX inhibition for chemopreventive and/or chemotherapeutic purposes in prostate cancer. more...

Inhibitors of the cyclooxygenases (COXs), the nonsteroidal antiinflammatory drugs (NSAIDs), relieve inflammatory pain, but are associated with gastrointestinal and cardiovascular complications. Given the widespread use of NSAIDs, there has been a longstanding interest in optimizing their risk-benefit ratio, for example by reducing their gastrointestinal risk. More recently, the focus has shifted toward the cardiovascular complications of NSAIDs and very large prospective studies have been performed to compare cardiovascular risk across distinct NSAIDs. Surprisingly, much less attention has been paid to the efficacy side of the risk-benefit ratio. There is marked variability in the degree of pain relief by NSAIDs due to the complex interplay of molecular mechanisms contributing to the pain sensation, variability in the disposition of NSAIDs, and imprecision in the quantification of human pain. Here we discuss how NSAIDs relieve pain, how molecular mechanisms relate to clinical efficacy, and how this may inform our interpretation of clinical trials. more...

The principal molecular mechanisms underlying the cardiovascular (CV) and renal adverse effects of nonsteroidal anti-inflammatory drugs (NSAIDs), such as myocardial infarction and hypertension, are understood in more detail than most side effects of drugs. Less is known, however, about differences in the CV safety profile between chemically distinct NSAIDs and their relative predisposition to complications. In review article, we discuss how heterogeneity in the pharmacokinetics and pharmacodynamics of distinct NSAIDs may be expected to affect their CV risk profile. We consider evidence afforded by studies in model systems, mechanistic clinical trials, a meta-analysis of randomized controlled trials, and two recent large clinical trials, Standard Care vs. Celecoxib Outcome Trial (SCOT) and Prospective Randomized Evaluation of Celecoxib Integrated Safety versus Ibuprofen or Naproxen (PRECISION), designed specifically to compare the CV safety of the cyclooxygenase-2-selective NSAID, celecoxib, with traditional NSAIDs. We conclude that SCOT and PRECISION have apparently not compared equipotent doses and have other limitations that bias them toward underestimation of the relative risk of celecoxib. more...

Nonsteroidal anti-inflammatory drugs (NSAIDs) relieve inflammatory pain by predominant suppression of cyclooxygenase-2 derived prostaglandin (PG) E2. Innate immune cells contribute to inflammatory pain hypersensitivity and may be an attractive target for novel non-addictive approaches to pain management. We studied the contribution of PGE2 produced by myeloid cell microsomal prostaglandin E synthase -1 (mPGES-1) to peripheral inflammation and hyperalgesia in mice. Selective deletion of mPGES-1 in myeloid cells by crossing LysM-Cre mice with mPGES-1flox/flox mice (Mac-mPGES-1-KO) resulted in significantly reduced mechanical and thermal hyperalgesia in complete Freund’s adjuvant (CFA)-evoked hind paw inflammation, zymosan-induced peri-articular inflammation and collagen II antibody-induced arthritis models. Systemic COX-2 inhibition or myeloid cell specific COX-2 deletion (by crossing LysM-Cre with COX-2 flox/flox mice) recapitulated reduction of CFA-induced inflammation and hyperalgesia. In contrast, deletion of mPGES-1 in neurons and glial cells by crossing mPGES-1flox/flox mice with Nestin-Cre mice had no detectable effect on inflammatory pain hypersensitivity. While macrophage recruitment was unaltered, tissue concentrations of PGE2, IL-1β and TNFα were significantly reduced in Mac-mPGES-1-KO paw tissues following CFA induction. Our results demonstrate that myeloid cell mPGES-1 is the dominant source of PGE2 in inflammatory pain hypersensitivity. Targeting myeloid cell mPGES-1 may afford a novel approach to inflammatory pain therapy. more...

Coronavirus disease 2019 (COVID-19) is upending education. Operating schools during the pandemic entails balancing health risks against the consequences of disrupting in-person learning. In the United States, plans differ among states as schools have already reopened or plan to reopen. Scientific understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the cause of COVID-19) should inform how schools reopen. Although school children and adolescents (ages 3 to 18 years) can develop COVID-19, most remain asymptomatic or experience mild [illness][1]. These youngsters may be less susceptible to infection than older individuals but probably spread the infection at similar rates. SARS-CoV-2 infections in children and adolescents are rising faster than in other age groups as restrictions have been [eased][2]. Infections have been imported into schools from the community. But further transmission within schools has been rare when rigorous measures have been implemented to reduce the risk of person-to-person spread. Larger school outbreaks are associated with increased community transmission, insufficient physical distancing, poor ventilation, and lack of [masking][3]. Schools that implemented transmission mitigation measures (including in European countries) seem not to have substantially contributed to increased circulation of the virus among local communities. What can schools do? The evidence thus far points to three mitigation strategies for reopening. Minimizing the import of infections into the school can stem the spread of COVID-19. Daily symptom screening can identify individuals with COVID-19 at first presentation. They should seek diagnostic testing. However, infections can be silent. Approximately 15 to 50% of children and 10 to 30% of adults will either not notice [symptoms][4] while their immune system fights the infection (asymptomatic carriers) or become infectious 1 to 3 days before symptom onset (presymptomatic [carriers][5]). Current diagnostic tests cannot identify silent infections reliably and are not sufficiently fast and inexpensive to make a school-wide testing-based surveillance system [practical][6]. Thus, the most effective tool for minimizing the risk of infections being carried into schools is to restrict in-person learning to when infection in the local community is controlled. Countries with widespread testing began opening schools with rigorous safety measures in place when fewer than 30 to 50 new infections were observed within 7 days per 100,000 residents over a prolonged period. Countries providing in-person schooling with basic mitigation measures (i.e., distancing, face masks worn in hallways but not classrooms, hand hygiene, ventilation, and staying home with minimal symptoms) typically have close to zero community [transmission][3]. The likelihood of further transmission must be minimized if infections are brought into school. COVID-19 is spread through liquid particles containing the virus that are generated by breathing, speaking, shouting, singing, coughing, and [sneezing][4]. The rapid settling rate of large droplets underlies recommendations for physical distancing, surface disinfection, ventilation, and hand hygiene. Because smaller liquid particles dispersed as aerosols stay airborne, it is not only the distance from another person that determines the risk of transmission, but also the duration of [exposure][7]. Limiting room occupancy, avoiding activities such as singing, and improving ventilation are critical in transmission control. Masks reduce spread by droplets and aerosols by limiting release and inhalation. Airborne spread is much less likely outdoors, but sports, where proximity to excessive exhalation is intrinsic to the game, need to be avoided. Large outbreaks in school can be minimized by limiting secondary transmission to the smallest possible number of persons. Cohorts that remain relatively isolated from each other can reduce person-to-person contact and can facilitate contact tracing if outbreaks [occur][8]. Early detection of infected individuals through symptom surveillance and diagnostic testing can limit quarantine measures to the affected cohorts, rather than having to close grades or the entire school. From these three efforts, a layered approach to risk mitigation in schools can be developed where measures with partial effectiveness are combined to reduce the probability of children, teachers, staff, and family members becoming ill with COVID-19.[*][9] The lower the infection rate in the community, the less stringent other risk mitigation measures need to be. If communities prioritize suppressing viral spread in other social gatherings, then children can go to school. [1]: https://doi.org/10.1007%2Fs00431-020-03684-7 [2]: https://downloads.aap.org/AAP/PDF/AAP%20and%20CHA%20-%20Children%20and%20COVID-19%20State%20Data%20Report%208.6.20%20FINAL.pdf [3]: https://www.ecdc.europa.eu/en/publications-data/children-and-school-settings-covid-19-transmission [4]: https://doi.org/10.1126/science.abc6197 [5]: https://doi.org/10.1016/S0140-6736(20)31483-5 [6]: https://doi.org/10.1056/NEJMp2015897 [7]: https://doi.org/10.1073/pnas.2009637117 [8]: https://doi.org/10.1038/s41562-020-0898-6 [9]: #fn-1 more...

Postsurgical dental pain is mainly driven by inflammation, particularly through the generation of prostaglandins via the cyclooxygenase system. Thus, it is no surprise that numerous randomized placebo-controlled trials studying acute pain following the surgical extraction of impacted third molars have demonstrated the remarkable efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, naproxen sodium, etodolac, diclofenac, and ketorolac in this prototypic condition of acute inflammatory pain. Combining an optimal dose of an NSAID with an appropriate dose of acetaminophen appears to further enhance analgesic efficacy and potentially reduce the need for opioids. In addition to being on average inferior to NSAIDs as analgesics in postsurgical dental pain, opioids produce a higher incidence of side effects in dental outpatients, including dizziness, drowsiness, psychomotor impairment, nausea/vomiting, and constipation. Unused opioids are also subject to misuse and diversion, and they may cause addiction. Despite these risks, some dental surgical outpatients may benefit from a 1- or 2-d course of opioids added to their NSAID regimen. NSAID use may carry significant risks in certain patient populations, in which a short course of an acetaminophen/opioid combination may provide a more favorable benefit versus risk ratio than an NSAID regimen. more...

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used analgesics due to their lack of addictive potential. However, NSAIDs have the potential to cause serious gastrointestinal, renal, and cardiovascular adverse events. CYP2C9 polymorphisms influence metabolism and clearance of several drugs in this class, thereby affecting drug exposure and potentially safety. We summarize evidence from the published literature supporting these associations and provide therapeutic recommendations for NSAIDs based on CYP2C9 genotype (updates at www.cpicpgx.org). more...

The mechanisms underlying interindividual variability in analgesic efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) are not well understood. Therefore, we performed pain phenotyping, functional neuroimaging, pharmacokinetic/pharmacodynamic assessments, inflammation biomarkers, and gene expression profiling in healthy subjects who underwent surgical extraction of bony impacted third molars and were treated with ibuprofen (400 mg; N = 19) or placebo (N = 10). Analgesic efficacy was not associated with demographic or clinical characteristics, ibuprofen pharmacokinetics, or the degree of cyclooxygenase inhibition by ibuprofen. Compared with partial responders to ibuprofen (N = 9, required rescue medication within the dosing interval), complete responders (N = 10, no rescue medication) exhibited greater induction of urinary prostaglandin metabolites and serum tumor necrosis factor-α and interleukin 8. Differentially expressed genes in peripheral blood mononuclear cells were enriched for inflammation-related pathways. These findings suggest that a less pronounced activation of the inflammatory prostanoid system is associated with insufficient pain relief on ibuprofen alone and the need for additional therapeutic intervention. more...

The cardiovascular biology of proton radiotherapy is not well understood. We aimed to compare the genomic dose-response to proton and gamma radiation of the mouse aorta to assess whether their vascular effects may diverge. We performed comparative RNA sequencing of the aorta following (4 hrs) total-body proton and gamma irradiation (0.5-200 cGy whole body dose, 10 dose levels) of conscious mice. A trend analysis identified genes that showed a dose response. While fewer genes were dose-responsive to proton than gamma radiation (29 vs. 194 genes; q-value ≤ 0.1), the magnitude of the effect was greater. Highly responsive genes were enriched for radiation response pathways (DNA damage, apoptosis, cellular stress and inflammation; p-value ≤ 0.01). Gamma, but not proton radiation induced additionally genes in vasculature specific pathways. Genes responsive to both radiation types showed almost perfectly superimposable dose-response relationships. Despite the activation of canonical radiation response pathways by both radiation types, we detected marked differences in the genomic response of the murine aorta. Models of cardiovascular risk based on photon radiation may not accurately predict the risk associated with proton radiation. more...

The analgesic efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) has long been recognized to be limited by substantial interindividual variability in pain relief, but the underlying mechanisms are not well understood. We performed pain phenotyping, functional neuroimaging, pharmacokinetic/pharmacodynamic assessments, inflammation biomarkers, and gene expression profiling in healthy subjects who underwent surgical extraction of bony impacted third molars, in order to characterize factors associated with heterogeneity in response to ibuprofen.Subjects were treated with rapid-acting ibuprofen (400 mg; N=19) or placebo (N=10) in a randomized, double-blind design. Compared to placebo, ibuprofen-treated subjects exhibited greater reduction in pain scores, alterations in regional cerebral blood flow in brain regions associated with pain processing, and inhibition of ex vivo cyclooxygenase activity and urinary prostaglandin metabolite excretion as indices of biochemical drug action (p2 metabolite, serum TNFα and IL-8 were higher in patients who did not require rescue medication compared those who did (p < 0.05). RNAseq gene expression analysis in PBMCs collected after surgery and ibuprofen administration showed enrichment of inflammation related pathways among genes differentially expressed (q < 0.2) between complete and partial respondersThese findings suggest that patients who receive substantial pain relief from ibuprofen have a more pronounced activation of the prostanoid biosynthetic pathway and regulation of the inflammatory pain phenotype differs from those patients who are insufficiently treated with ibuprofen alone and may require an opioid or other therapeutic intervention. more...

Purpose. The cardiovascular biology of proton radiotherapy is not well understood. We aimed to compare the genomic dose-response to proton and gamma radiation of the mouse aorta to assess whether their vascular effects may diverge.Materials and methods.We performed comparative RNA sequencing of the aorta following (4 hrs) total-body proton and gamma irradiation (0.5 - 200 cGy whole body dose, 10 dose levels) of conscious mice. A trend analysis identified genes that showed a dose response.Results.While fewer genes were dose-responsive to proton than gamma radiation (29 vs. 194 genes;q-value ≤ 0.1), the magnitude of the effect was greater. Highly responsive genes were enriched for radiation response pathways (DNA damage, apoptosis, cellular stress and inflammation;p-value ≤ 0.01). Gamma, but not proton radiation induced additionally genes in vasculature specific pathways. Genes responsive to both radiation types showed almost perfectly superimposable dose-response relationships.Conclusions.Despite the activation of canonical radiation response pathways by both radiation types, we detected marked differences in the genomic response of the murine aorta. Models of cardiovascular risk based on photon radiation may not accurately predict the risk associated with proton radiation. more...

It is common to measure a large number of features in parallel to identify those differing between two experimental conditions - e.g. the search for differentially expressed genes using microarrays or RNA-Seq. Ranking features by p-value allows for control of the TYPE I error, but p-values are not reliable when there are very few replicates; and investigators typically require features be ranked by "fold change" x¯/y¯ in conjunction with p-values. At first glance the fold change appears to be a natural quantity on which to compare the differential behavior of features. But it is highly sensitive to small values in the denominator and is problematic in how it equates changes in both small and large numbers such as a change from 1 to 2 versus a change from 100 to 200. The strategy of adjusting all values by adding one is a widely used heuristic approach to try to mitigate the problems with fold-change. However, that can be far from optimal. A systematic strategy to determine an optimal value (pseudocount) to adjust by is employed using both real and simulated benchmark data. In RNA-Seq a value of 20 appears to be close to optimal in all cases. Another strategy is to sort by difference y¯ - x¯, but this is problematic for comparing measurements across a wide spectum, as large differences of small values rank below proportionally smaller difference in large values. An abstract mathematical framework is introduced to describe the problem of ranking by differential effect size, enabling us to study the ranking problem in general as opposed to specific contexts such as fold-change or difference. From this framework we discovered a remarkable property of pseudocounts, in that they strike a balance between sorting by fold-change and sorting by difference. Lastly, another fundamentally different type of application is presented, which is to rank di-codons by their differential abundance in the ORFeome of different species. more...

Prostaglandin (PG) D2 is formed by two distinct PGD synthases (PGDS): lipocalin-type PGDS (L-PGDS), which acts as a PGD2-producing enzyme and as extracellular lipophilic transporter, and hematopoietic PGDS (H-PGDS), a σ glutathione-S-transferase. PGD2 plays an important role in the maintenance of vascular function; however, the relative contribution of L-PGDS– and H-PGDS–dependent formation of PGD2 in this setting is unknown. To gain insight into the function played by these distinct PGDS, we assessed systemic blood pressure (BP) and thrombogenesis in L-Pgds and H-Pgds knockout (KO) mice. Deletion of L-Pgds depresses urinary PGD2 metabolite (PGDM) by ∼35%, whereas deletion of H-Pgds does so by ∼90%. Deletion of L-Pgds, but not H-Pgds, elevates BP and accelerates the thrombogenic occlusive response to a photochemical injury to the carotid artery. HQL-79, a H-PGDS inhibitor, further depresses PGDM in L-Pgds KO mice, but has no effect on BP or on the thrombogenic response. Gene expression profiling reveals that pathways relevant to vascular function are dysregulated in the aorta of L-Pgds KOs. These results indicate that the functional impact of L-Pgds deletion on vascular homeostasis may result from an autocrine effect of L-PGDS–dependent PGD2 on the vasculature and/or the L-PGDS function as lipophilic carrier protein. more...

Objective To examine the comparative safety of individual NSAIDs when given concomitantly with clopidogrel. Methods We conducted a retrospective cohort study using Medicaid claims from five US states during 1999-2010, supplemented with Medicare claims for dual-enrollees. The exposure of interest was the first concomitant use of clopidogrel and one of the 10 selected NSAIDs after a 1-year baseline period. The outcomes were: all-cause mortality; acute myocardial infarction (AMI)/ischemic stroke; and gastrointestinal bleeding (GIB)/intracranial hemorrhage (ICH). We calculated the hazard ratio of each NSAID for each outcome, with ibuprofen as the reference drug, using high-dimensional propensity score-adjusted proportional-hazards regression models. Results Of 1,060,412 clopidogrel users, 268,114 concomitant NSAID users met inclusion/exclusion criteria, contributing 48,483 person-years. We observed 2,463 deaths, 2,822 AMI/ischemic stroke outcomes, and 2,620 GIB/ICH outcomes, for unadjusted incidence rates of 50.8, 58.6, and 54.3 per 1,000 person-years, respectively. Compared with ibuprofen and controlling for potential confounders, rofecoxib (hazard ratio [HR] = 1.22; 95% confidence interval [CI]: 1.04, 1.43) and valdecoxib (HR = 0.66; 95% CI: 0.48, 0.92) showed higher and lower hazards of mortality, respectively. Indomethacin showed an increased AMI/ischemic stroke hazard (HR = 1.38; 95% CI: 1.09, 1.74). For GIB/ICH, indomethacin (HR = 2.18; 95% CI: 1.74, 2.73), diclofenac (HR = 1.65; 95% CI: 1.39, 1.97), naproxen (HR = 1.47; 95% CI: 1.28, 1.70), and rofecoxib (HR = 1.26; 95% CI: 1.08, 1.48) showed higher hazards, and valdecoxib (HR = 0.73; 95% CI: 0.55, 0.98) showed a lower hazard. Conclusion The bleeding risks of individual NSAIDs varied more markedly than thrombotic risks when used concomitantly with clopidogrel. Moreover, bleeding risk and thrombotic risk among individual NSAIDs did not appear to be inversely related to each other in the presence of clopidogrel. Further studies are needed to elucidate underlying biological mechanisms and help clinical decision-making for a better NSAID choice in clopidogrel users. more...

Much has been written recently about the prevalence of chronic pain (1), the dramatic increase in opioid prescriptions in the United States over the past 15 years, the concomitant rise in opioid dependency and addiction, and the quadrupling of deaths from opioid abuse. Although indispensable for managing acute severe traumatic pain and pain in a palliative setting, most opioids are prescribed either by dentists or by primary practitioners for chronic nonmalignant pain, and marketed aggressively to consumers for the latter, despite no scientific evidence supporting such treatment beyond 12 weeks (1). On the contrary, chronic opioid use can itself lead to pain. Most abuse (perhaps 70%) involves access to opioids that are prescribed for others—a diversion problem. more...

The prostanoids, which include the prostaglandins (PGs) and thromboxane (Tx)A2, are a family of locally acting lipid mediators. Two isozymes, cyclooxygenase (COX)-1 and COX-2, of which only COX-1 is expressed in mature platelets, form an unstable intermediate product, PGH2, from arachidonic acid released from membrane phospholipids. At least nine PG synthases metabolize PGH2 further to five biologically active prostanoids, which each act through one or more specific G-protein coupled receptor. more...

Current methods of drug screening in human blood focus on the immediate products of the affected pathway and mostly rely on approaches that lack sensitivity and the capacity for multiplex analysis. We have developed a sensitive and selective method based on ultra-performance liquid chromatography–tandem mass spectrometry to scan the effect of drugs on the bioactive eicosanoid lipidome in vitro and ex vivo. Using small sample sizes, we can reproducibly measure a broad spectrum of eicosanoids in human blood and capture drug-induced substrate rediversion and unexpected shifts in product formation. Microsomal prostaglandin E synthase-1 (mPGES-1) is an antiinflammatory drug target alternative to COX-1/-2. Contrasting effects of targeting mPGES-1 versus COX-1/-2, due to differential substrate shifts across the lipidome, were observed and can be used to rationalize and evaluate drug combinations. Finally, the in vitro results were extrapolated to ex vivo studies by administration of the COX-2 inhibitor, celecoxib, to volunteers, illustrating how this approach can be used to integrate preclinical and clinical studies during drug development. more...

The selective COX-2 inhibitor, celecoxib, shows a complex but important pharmacogenomics profile. The use of this highly effective anti-inflammatory and antitumor drug is limited by concerns about its potential for increased cardiovascular risk. Although the mechanism of action of celecoxib is well studied and many large clinical trials have examined its efficacy, we still lack a complete understanding of the sources of variability in the occurrence of adverse effects as well as the differential risk profile for all drugs in the NSAID class. Preliminary work showed that genetic factors influence response to celecoxib and may be useful in selecting individuals at risk for adverse events. However, the existing pharmacogenetic studies of celecoxib toxicity are still exploratory, with limited sample size and limited number of events. NSAIDs are widely used, and the lower gastrointestinal toxicity of the coxibs represents an important potential advantage over nonselective NSAIDs, as long as we can identify those at an increased risk for cardiovascular toxicity. Larger outcome trials and comprehensive analysis of both pharmacokinetic and pharmacodynamic candidate genes are needed to identify genetic markers to predict variations in response and celecoxib-induced adverse events. more...

The PTGS2 gene codes for prostaglandin G/H synthase-2, which catalyses the first two steps in the metabolism of arachadonic acid. Prostaglandin G/H synthase-2 has two active sites, a hydroperoxidase and a cyclooxygenase (COX) site, and is colloquially termed COX-2. The bifunctional enzyme performs the bis-dioxygenation and reduction of arachadonic acid to form prostaglandin (PG)G2 and H2. PGH2 is then converted to other PGs that modulate inflammation, including PGD2, PGE2, PGF2α, PGI2, and thromboxane A2 (This pathway is shown in the Lipid maps database at http://www.lipidmaps.org/data/IntegratedPathways Data/SetupIntegratedPathways.pl? imgsize=730&Mode=RAW2647&DataType=FAEicosanoidsMedia). COX-2 is the target for nonsteroidal anti-inflammatory drugs (NSAIDS) including those that were purposefully designed (pd) to be selective for COX-2 (pdNSAIDs or coxibs). PTGS2 is a paralog of PTGS1, which codes for the COX-1 enzyme. Although both proteins perform the same endogenous reactions, there are dramatic differences in the pattern, regulation, location, and timing of their expression that suggest crucial differences in the function. COX-1 seems to be predominantly responsible for homeostatic functions including the protection of the gastric mucosa. COX-2, in contrast, is responsible for pathogenic and inflammatory responses. These differences led to the development of selective COX-2 inhibitors to treat more...

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. more...

Maternal smoking during pregnancy retards fetal growth and depresses infant birth weight. The magnitude of these effects may be moderated by fetal genotype. The current study investigated maternal smoking, fetal genotype, and fetal growth in a large population sample of dizygotic twins. Maternal smoking retarded fetal growth in a dose-dependent fashion. In a subsample of 497 twin pairs whose mothers smoked during pregnancy, a functional polymorphism in the NAD(P)H:quinone oxidoreductase gene (NQO1 Pro187Ser; rs1800566) was significantly associated with fetal growth within families. The effect was strongest among moderate smokers. This is the first demonstration that fetal genotype for an enzyme involved in tobacco smoke metabolism influences intrauterine growth independent of maternal genotype. Future studies should conduct formal tests of Fetal Genotype x Maternal Smoking interactions. more...

Shotgun proteomic strategies based on the identification of proteolytic peptides by tandem mass spectrometry and sequence database searches have emerged as useful approaches for qualitative and quantitative measurement of proteins in large scale studies. Coincidentally, the automated analysis of the resulting mass spectrometry data has become increasingly challenging. Important steps in the analysis include the statistical validation of peptide and protein identifications, the extraction of quantitative information, and the interpretation of identified peptide and protein lists. This review discusses current informatics methods and software available to analyze shotgun proteomics data. more...

Haematopoietic stem cell (HSC) homeostasis is tightly controlled by growth factors, signalling molecules and transcription factors. Definitive HSCs derived during embryogenesis in the aorta-gonad-mesonephros region subsequently colonize fetal and adult haematopoietic organs1,2. To identify new modulators of HSC formation and homeostasis, a panel of biologically active compounds was screened for effects on stem cell induction in the zebrafish aorta-gonad-mesonephros region. Here, we show that chemicals that enhance prostaglandin (PG) E2 synthesis increased HSC numbers, and those that block prostaglandin synthesis decreased stem cell numbers. The cyclooxygenases responsible for PGE2 synthesis were required for HSC formation. A stable derivative of PGE2 improved kidney marrow recovery following irradiation injury in the adult zebrafish. In murine embryonic stem cell differentiation assays, PGE2 caused amplification of multipotent progenitors. Furthermore, ex vivo exposure to stabilized PGE2 enhanced spleen colony forming units at day 12 post transplant and increased the frequency of long-term repopulating HSCs present in murine bone marrow after limiting dilution competitive transplantation. The conserved role for PGE2 in the regulation of vertebrate HSC homeostasis indicates that modulation of the prostaglandin pathway may facilitate expansion of HSC number for therapeutic purposes. more...

SummaryRecently, we cloned from platelet mRNA a novel cyclooxygenase (COX)-2 splice variant, designated COX-2a, which is characterized by a partial deletion of exon 5. Preliminary studies of mRNA distribution of COX-2 isoforms in platelets from coronary artery bypass grafting (CABG) patients showed a variable increase in COX-2a mRNA expression after cardiac surgery. Thus, we assessed whether this variant may play a functional role in these patients. We report a marked (about 20346-fold) increase in the expression of COX-2a mRNA after CABG. Evidence is presented that ribosomal frame-shifting may correct the coding sequence resulting in the expression of a full-length COX-2a pro-tein. In addition, a reading frame-corrected COX-2a mutant (COX-2aΔG) was generated by site-directed mutagenesis and expressed in COS-7 cells using an adenoviral expression system. However, COX-2a protein was not active in terms of prostaglandin formation. Thus, alternative mRNA splicing might represent an intriguing posttranscriptional mechanism to oppose a transcriptional activation of the COX-2 gene. Evolutionary, this mechanism may prevent COX-2-dependent thromboxane synthesis in the platelet, which would potentiate the likelihood of thrombosis; pharmacologically, this mechanism would prevent an aspirin-insensitive pathway of thromboxane formation. more...

The absence of Bmal1, a core clock gene, results in a loss of circadian rhythms, an acceleration of aging, and a shortened life span in mice. To address the importance of circadian rhythms in the aging process, we generated conditional Bmal1 knockout mice that lacked the BMAL1 protein during adult life and found that wild-type circadian variations in wheel-running activity, heart rate, and blood pressure were abolished. Ocular abnormalities and brain astrogliosis were conserved irrespective of the timing of Bmal1 deletion. However, life span, fertility, body weight, blood glucose levels, and age-dependent arthropathy, which are altered in standard Bmal1 knockout mice, remained unaltered, whereas atherosclerosis and hair growth improved, in the conditional adult-life Bmal1 knockout mice, despite abolition of clock function. Hepatic RNA-Seq revealed that expression of oscillatory genes was dampened in the adult-life Bmal1 knockout mice, whereas overall gene expression was largely unchanged. Thus, many phenotypes in conventional Bmal1 knockout mice, hitherto attributed to disruption of circadian rhythms, reflect the loss of properties of BMAL1 that are independent of its role in the clock. These findings prompt reevaluation of the systemic consequences of disruption of the molecular clock. more...

Randomized controlled trials of cyclooxygenase (COX)-2 selective NSAIDs revealed that these drugs confer a cardiovascular hazard, resulting mainly from suppression of COX-2 derived prostacyclin (PGI2). Disruption of rodent COX-2-derived PGI2 in vascular cells and cardiomyocytes has recapitulated all elements of the cardiovascular hazard attributable to NSAIDs in humans. To investigate further the impact of COX-2 inhibition in the vasculature, we examined the effects of therapeutic concentrations of two selective COX-2 inhibitors, celecoxib and rofecoxib, on thrombosis and vascular injury. Furthermore, we used RNA sequencing (RNA-Seq) to assess whether celecoxib and rofecoxib might elicit distinct genomic effects in mouse aorta. Celecoxib (100 mg/Kg) and rofecoxib (50 mg/Kg) caused a selective and equipotent COX-2 inhibition as indicated by measuring thromboxane (TXB-M, an in vivo index of COX-1 activity) and prostacyclin (PGI-M, an in vivo index of COX-2 activity) urinary metabolites by mass spectrometry. Both celecoxib and rofecoxib augmented platelet deposition and prolonged platelet disaggregation after laser-induced injury in the cremaster arterioles. A similar effect was observed in mice lacking PGI2 receptor IP. There was no additional pro-thrombotic effect of the drugs on an IPKO background, consistent with suppression of COX-2 dependent PGI2 mediating the predisposition to thrombosis induced by celecoxib and rofecoxib. Celecoxib, but not rofecoxib, caused a reduction of neointimal hyperplasia and the intima/media ratio of injured femoral artery at 3 weeks after wire injury. RNA-seq analysis revealed 903 transcripts differentially expressed between celecoxib and control group and 3,127 transcripts between rofecoxib and control group (at a false discovery rate of 10%) in mouse aorta. Thus, both celecoxib and rofecoxib cause a similar predisposition to thrombosis in mice. Celecoxib, but not rofecoxib, reduces neointimal hyperplasia after vascular injury and COX-2 independent genetic actions might modulate this effect. The predisposition to thrombosis and attenuation of the response to vascular injury by celecoxib recapitulates what has been found in humans. more...

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 more...

Selective inhibitors of cyclooxygenase (COX)-2, the coxibs, were developed to inhibit inflammatory prostaglandins derived from COX-2, while sparing gastroprotective prostaglandins primarily formed by COX-1. However, COX-2-derived prostaglandins mediate not only pain and inflammation but also affect vascular function, the regulation of hemostasis/ thrombosis, and blood pressure control. All coxibs depress COX-2-dependent prostacyclin (PGI2) biosynthesis without effective suppression of platelet COX-1-derived thromboxane (Tx) A2, unlike aspirin or traditional nonsteroidal anti-inflammatory drugs, which inhibit both COX-1 and COX-2. The actions of PGI2 oppose mediators, which stimulate platelets, elevate blood pressure, and accelerate atherogenesis, including TxA2. Indeed, structurally distinct inhibitors of COX-2 have increased the likelihood of hypertension, myocardial infarction and stroke in controlled clinical trials. The detection of these events in patients is related to the duration of exposure and to their baseline risk of cardiovascular disease. Thus, coxibs should be withheld from patients with preexisting cardiovascular risk factors, and exposed patients at low cardiovascular baseline risk should be monitored for changes in their risk factor profile, such as increases in arterial blood pressure. more...

Study of the cyclooxygenases (COXs) has been limited by the role of COX-2 in murine reproduction and renal organogenesis. We sought to characterize COX expression and function in zebrafish (z). Full-length cDNAs of zCOX-1 and zCOX-2 were cloned and assigned to conserved regions of chromosomes 5 and 2, respectively. The deduced proteins are 67% homologous with their human orthologs. Prostaglandin (PG) E 2 is the predominant zCOX product detected by mass spectrometry. Pharmacological inhibitors demonstrate selectivity when directed against heterologously expressed zCOX isoforms. Zebrafish thrombocyte aggregation ex vivo and hemostasis in vivo are sensitive to inhibition of zCOX-1, but not zCOX-2. Both zCOXs were widely expressed during development, and knockdown of zCOX-1 causes growth arrest during early embryogenesis. zCOX-1 is widely evident in the embryonic vasculature, whereas zCOX-2 exhibits a more restricted pattern of expression. Both zCOX isoforms are genetically and functionally homologous to their mammalian orthologs. The zebrafish affords a tractable model system for the study of COX biology and development. more...

Drs. Cerit and Duygu raise an interesting question of whether diabetic gastroparesis may play a role in apparent “aspirin resistance” in diabetic patients [(1)][1]. It is possible that in some patients, diabetic gastroparesis impairs absorption of a number of enteric-coated (EC) drugs, including more...

Apoptosis of vascular smooth muscle cells is critically involved in progression of atherosclerosis and may prevent intimal hyperplasia in restenosis and vascular remodeling. Nitric oxide (NO) is known to induce apoptosis, but the signaling pathways still remain unclear. We investigated p53 accumulation, protein kinase C (PKC) activation and nuclear transcription factor (NF-kappaB) binding activity as possible signaling mechanisms of NO-induced apoptosis. Apoptosis was induced dose-dependently with the NO-donors sodiumnitroprusside (SNP: 232+/-48%) and SIN-1 (241+/-90% of actinomycin D induced apoptosis; means +/- SEM, *por =0.05 vs. control) in HSMC. Inhibition of PKC significantly attenuated NO-induced apoptosis. Staurosporine reduced SIN-1/SNP-mediated DNA fragmentation by 55.3+/-13.8% and 38.3+/-13.9% respectively. Comparable results were obtained for calphostin C. However, NO-mediated induction of apoptosis was not preceded by p53 accumulation. SNP decreased NF-kappaB binding activity in HSMC. These results suggest that induction of apoptosis by exogenous NO in HSMC is not dependent on p53 accumulation but involves protein kinase C signaling and regulation of NF-kappaB binding activity. This opens a new therapeutical approach in preventing restenosis after angioplasty. more...

Smooth muscle cell (SMC) proliferation can result in luminal reduction of a vessel following balloon angioplasty. This study was designed (i) to determine if local administration of trapidil (triazolopyrimidine) into a vessel wall reduces neointima formation, and (ii) to explore the mechanism involved in the subsequent reduction in cell proliferation. Following balloon angioplasty in 40 anaesthetized New Zealand White rabbits, trapidil (50–200 mg) or its vehicle (saline) was injected into the dilated vessel wall of the right femoral artery. Experimental groups and time of investigation: (I) vehicle (2 weeks, n=3), (II) trapidil-100 mg (2 weeks, n=3), (III) vehicle (3 weeks, n=8), (IV) trapidil-50 mg (3 weeks, n=5); (V) trapidil-100 mg (3 weeks, n=9) or (V) trapidil-200 mg (3 weeks, n=7). After 2 weeks, there was a significant reduction of intimal hyperplasia (expressed as intima to media area ratio) in the trapidil group compared with vehicle (0.44±0.04 vs 0.93±0.04, *P more...

This study compares the antimitogenic effects of iloprost and prostaglandin E 1 on platelet-derived growth factor-BB stimulated DNA synthesis ([ 3 H]thymidine incorporation) in bovine coronary artery smooth muscle cells. When added 20–24 h after stimulation with platelet-derived growth factor-BB (20 ng/ml), both iloprost and prostaglandin E 1 , concentration-dependently (IC 50 3–5 nM) inhibited DNA synthesis. However, when added together with the growth factor (0–24 h), the inhibition of DNA synthesis by iloprost was markedly attenuated, indicating tolerance development. In contrast, no tolerance to antimitogenic effects of prostaglandin E 1 or forskolin were observed. When added to iloprost-tolerant cells, both prostaglandin E 1 and forskolin, still inhibited DNA synthesis. There was no evidence for transcriptional down-regulation of prostacyclin receptor gene by iloprost. The data demonstrate a tolerance development to antimitogenic actions of prostacyclin but not of prostaglandin E 1 and suggest that the receptors, mediating the antiproliferative actions of these prostaglandins, may be different. more...

The cardiovascular safety of nonsteroidal antiinflammatory drugs (NSAIDs) may be influenced by interactions with antiplatelet doses of aspirin. We sought to quantitate precisely the propensity of commonly consumed NSAIDs—ibuprofen, naproxen, and celecoxib—to cause a drug-drug interaction with aspirin in vivo by measuring the target engagement of aspirin directly by MS. We developed a novel assay of cyclooxygenase-1 (COX-1) acetylation in platelets isolated from volunteers who were administered aspirin and used conventional and microfluidic assays to evaluate platelet function. Although ibuprofen, naproxen, and celecoxib all had the potential to compete with the access of aspirin to the substrate binding channel of COX-1 in vitro, exposure of volunteers to a single therapeutic dose of each NSAID followed by 325 mg aspirin revealed a potent drug-drug interaction between ibuprofen and aspirin and between naproxen and aspirin but not between celecoxib and aspirin. The imprecision of estimates of aspirin consumption and the differential impact on the ability of aspirin to inactivate platelet COX-1 will confound head-to-head comparisons of distinct NSAIDs in ongoing clinical studies designed to measure their cardiovascular risk. more...