Nicholas S Kirkby

Imperial College London, Londinium, England, United Kingdom

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Publications (42)253.01 Total impact

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    ABSTRACT: Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system.
    Full-text · Article · Dec 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Aims: In vivo platelet function is a product of intrinsic platelet reactivity, modifiable by dual antiplatelet therapy (DAPT), and the extrinsic inhibitory endothelial mediators, nitric oxide (NO) and prostacyclin (PGI2 ), that are powerfully potentiated by P2Y12 receptor blockade. This implies that for individual patients endothelial mediator production is an important determinant of DAPT effectiveness. Here, we have investigated this idea using platelets taken from healthy volunteers treated with anti-platelet drugs. Methods: Three groups of male volunteers (n = 8) received either prasugrel (10 mg), aspirin (75 mg) or DAPT (prasugrel + aspirin) once daily for 7 days. Platelet reactivity in the presence of DEA/NONOate and PGI2 was studied before and following treatment. Results: Ex vivo, PGI2 and/or DEA/NONOate had little inhibitory effect on TRAP-6-induced platelet reactivity in control conditions. However, in the presence of DAPT, combination of DEA/NONOate + PGI2 reduced platelet aggregation (74 ± 3% to 19 ± 6%, p < 0.05). In vitro studies showed even partial (25%) P2Y12 receptor blockade produced a significant (67 ± 2% to 39 ± 10%, p < 0.05) inhibition when DEA/NONOate + PGI2 was present. Conclusions: We demonstrate that PGI2 and NO synergise with P2Y12 receptor antagonists to produce powerful platelet inhibition. Furthermore, even with submaximal P2Y12 blockade the presence of PGI2 and NO greatly enhances platelet inhibition. Our findings highlight the importance of endothelial mediator in vivo modulation of P2Y12 inhibition and introduces the concept of refining ex vivo platelet function testing by incorporating an assessment of endothelial function to better predict thrombotic outcomes and adjust therapy to prevent adverse outcomes in individual patients.
    No preview · Article · Nov 2015 · British Journal of Clinical Pharmacology
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    ABSTRACT: Reduced antiplatelet drug efficacy occurs in conditions of increased platelet turnover, associated with increased proportions of drug-free, that is, uninhibited, platelets. Here, we detail mechanisms by which drug-free platelets promote platelet aggregation in the face of standard antiplatelet therapy. To model standard antiplatelet therapy, platelets were treated in vitro with aspirin, the P2Y12 receptor blocker prasugrel active metabolite, and aspirin plus prasugrel active metabolite. Different proportions of uninhibited platelets were then introduced. Light transmission aggregometry analysis demonstrated clear positive associations between proportions of drug-free platelets and percentage platelet aggregation in response to a range of platelet agonists. Using differential platelet labeling coupled with advanced flow cytometry and confocal imaging, we found aggregates formed in mixtures of aspirin-inhibited platelets and drug-free platelets were characterized by intermingled platelet populations. This distribution is in accordance with the ability of drug-free platelets to generate thromboxane A2 and so drive secondary platelet activation. Conversely, aggregates formed in mixtures of prasugrel active metabolite-inhibited or aspirin plus prasugrel active metabolite-inhibited platelets together with drug-free platelets were characterized by distinct cores of drug-free platelets. This distribution is consistent with the ability of drug-free platelets to respond to the secondary activator ADP. These experiments are the first to image the interactions of inhibited and uninhibited platelets in the formation of platelet aggregates. They demonstrate that a general population of platelets can contain subpopulations that respond strikingly differently to overall stimulation of the population and so act as the seed for platelet aggregation. © 2015 The Authors.
    Full-text · Article · Aug 2015 · Arteriosclerosis Thrombosis and Vascular Biology
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    ABSTRACT: Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem we have developed a miniaturised whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor (PAR) 4 activating peptide. This loss was sensitive to well established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analysed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM) 1 identified a marked decrease in fibrinogen-dependent platelet monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation. Copyright © 2015 American Society of Hematology.
    Full-text · Article · Jul 2015 · Blood
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    ABSTRACT: Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.-Kirkby, N. S., Reed, D. M., Edin, M. L., Rauzi, F., Mataragka, S., Vojnovic, I., Bishop-Bailey, D., Milne, G. L., Longhurst, H., Zeldin, D. C., Mitchell, J. A., Warner, T. D. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation. © FASEB.
    No preview · Article · Jul 2015 · The FASEB Journal
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    ABSTRACT: Introduction Following ACS or PCI, P2Y12 receptor blockers such as clopidogrel or prasugrel are standardly prescribed alongside aspirin as dual anti-platelet therapy in a “one size fits all” approach, however recurrent thrombotic events occur. Despite the logical hypothesis that thrombotic risk should be associated with level of P2Y12 platelet inhibition, randomised control trials have repeatedly failed to show any clinical benefit of guiding anti-platelet therapy by platelet function tests (PFT). Whilst traditional PFT measure reactivity to ADP, they crucially ignore that P2Y12 receptor antagonists also produce powerful anti-thrombotic effects by potentiating the actions of the inhibitory endothelial mediators prostacyclin (PGI2) and nitric oxide (NO). Methods In vitro, IC50 curves produced from light transmission aggregation (LTA) traces for PGI2, NO and prasugrel active metabolite (PAM: 3 μM) were used to generate isobolograms. Ex vivo, healthy male volunteers (n = 8 each) received either prasugrel (10 mg) or aspirin (75 mg) for 7 days. Platelet-rich plasma (PRP) was obtained by centrifugation before and after treatment. Platelet responses to TRAP-6 amide (25 μM) or collagen (4 μg/ml) in the presence of PGI2 (1 nM) and/or the NO donor, DEA/NONOate (100 nM), or vehicle, were assessed by LTA, p-selectin binding by flow cytometry, and downstream cyclic nucleotide and VASP phosphorylation by immunoassay. Results Isobolographic analysis of in vitro studies showed P2Y12 blockade powerfully enhanced the synergy between NO and PGI2 (10 fold for TRAP-6). Ex-vivo platelet aggregation responses to TRAP-6 were unaffected by the addition of PGI2+NO prior to therapy. Aspirin monotherapy produced minimal inhibition of TRAP-6, though these became stronger in the presence of PGI2+NO (58%±8 to 28%±9; p < 0.05). Inhibitory effects were observed with prasugrel in the presence of PGI2 and NO individually (control, 63 ± 3%; PGI2, 43 ± 6%; NO, 50 ± 5%), and these became much greater with the combination (PGI2+NO, 7%±3). P-selectin expression was reduced from pre-treatment (vehicle, 32 ± 6%) to (PGI2+NO, 11 ± 4%) post aspirin, and from (vehicle, 25 ± 6%) to (PGI2+NO, 2 ± 4%) following prasugrel. cAMP levels increased with PGI2+NO in the aspirin group (0.97 ± 0.06 vs. 1.50 ± 0.19) compared to (1.41 ± 0.07 vs. 3.4 ± 0.58) in the prasugrel group. Conclusions PGI2 and NO synergise with P2Y12 blockade to cause powerful platelet inhibition suggesting that an individual’s endothelial function and production of NO and PGI2 are central determents of the anti-thrombotic protection from P2Y12 receptor blocker therapy. Whilst a therapeutic window of platelet reactivity is an attractive concept our research suggests that endothelial function testing alongside PFT would permit better risk stratification. Our research also suggests therapeutically that enhancing endothelial-derived mediators or optimising downstream cyclic nucleotide signalling could reduce both thrombotic and bleeding risk.
    No preview · Article · Jun 2015 · Heart (British Cardiac Society)
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    ABSTRACT: The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments. Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses. Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice. Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis. This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.
    Full-text · Article · May 2015 · Journal of Visualized Experiments
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    ABSTRACT: Endothelin (ET) receptor antagonism reduces neointimal lesion formation in animal models. This investigation addressed the hypothesis that the selective ETA receptor antagonist sitaxentan would be more effective than mixed ETA/B antagonism at inhibiting neointimal proliferation in a mouse model of intra-luminal injury. Antagonism of ETA receptors by sitaxentan (1-100nM) was assessed in femoral arteries isolated from adult, male C57Bl6 mice (25-35g) using isometric wire myography. Neointimal lesion development was induced by intraluminal injury in mice receiving sitaxentan (ETA antagonist; 15mg/kg/day), A192621 (ETB antagonist; 30 mg/kg/day), the combination of both antagonists, or vehicle (n=6-16). Treatment began one week before, and continued for 28 days after, surgery. Femoral arteries were then harvested for analysis of lesion size and composition. Sitaxentan produced a selective, concentration-dependent parallel rightward shift of ET-1-mediated contraction (pD2 ; 8.2±0.1 Control vs 7.2±0.1 100nM sitaxentan; P<0.001) in isolated femoral arteries. Sitaxentan reduced neointimal lesion size (23±5% vs 51±4%; P<0.05), whereas ETB (A192621; 61±7%) and combined ETA/B antagonism (51±7% P>0.05) did not. Macrophage and α-smooth muscle actin content were unaltered by ET antagonism but sitaxentan reduced the amount of collagen in lesions (14±2% vs 44±6%; p<0.01). These results suggest that ETA antagonism would be more effective than combined ETA / ETB antagonism at reducing neointimal lesion formation. This article is protected by copyright. All rights reserved.
    No preview · Article · Jan 2015 · British Journal of Pharmacology
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    ABSTRACT: -Cardiovascular side effects associated with cyclo-oxygenase-2 inhibitor drugs dominate clinical concern. Cyclo-oxygeanse-2 is expressed in the renal medulla where inhibition causes fluid retention and increased blood pressure. However, the mechanisms linking cyclo-oxygeanse-2 inhibition and cardiovascular events are unknown and no biomarkers have been identified. -Transcriptome analysis of wild-type and cyclo-oxygenase-2(-/-) mouse tissues revealed 1 gene altered in heart and aorta but >1000 genes in the renal medulla including those regulating the endogenous NO synthase inhibitors ADMA and L-NMMA; Cyclo-oxygeanse-2(-/-) mice had increased plasma levels of ADMA and L-NMMA and reduced endothelial NO responses. These genes and methylarginines were not similarly altered in mice lacking prostacyclin receptors (IP(-/-)). Wild-type mice or human volunteers taking cyclo-oxygeanse-2 inhibitors also showed increased plasma ADMA. Endothelial NO is cardio protective, reducing thrombosis and atherosclerosis. Consequently, increased ADMA is associated with cardiovascular disease. Thus, our study identifies ADMA as a biomarker and mechanistic bridge between renal cyclo-oxygenase-2 inhibition and systemic vascular dysfunction with non-steroidal anti-inflammatory drug usage. -We identify the endogenous eNOS inhibitor ADMA as a biomarker and mechanistic bridge between renal COX-2 inhibition and systemic vascular dysfunction.
    Full-text · Article · Dec 2014 · Circulation
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    ABSTRACT: Prostacyclin is a powerful cardioprotective hormone released by the endothelium of all blood vessels. Prostacyclin exists in equilibrium with other vasoactive hormones and a disturbance in the balance of these factors leads to cardiovascular disease including pulmonary arterial hypertension. Since it's discovery in the 1970s concerted efforts have been made to make the best therapeutic utility of prostacyclin, particularly in the treatment of pulmonary arterial hypertension. This has centred on working out the detailed pharmacology of prostacyclin and then synthesising new molecules based on its structure that are more stable or more easily tolerated. In addition, newer molecules have been developed that are not analogues of prostacyclin but that target the receptors that prostacyclin activates. Prostacyclin and related drugs have without doubt revolutionised the treatment and management of pulmonary arterial hypertension but are seriously limited by side effects within the systemic circulation. With the dawn of nanomedicine and targeted drug or stem cell delivery systems it will, in the very near future, be possible to make new formulations of prostacyclin that can evade the systemic circulation allowing for safe delivery to the pulmonary vessels. In this way, the full therapeutic potential of prostacyclin can be realised opening the possibility that pulmonary arterial hypertension will become, if not curable, a chronic manageable disease that is no longer fatal. This review discusses these and other issues relating to prostacyclin and its use in pulmonary arterial hypertension.
    Full-text · Article · Dec 2014
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    ABSTRACT: Endothelial cells form a highly specialised lining of all blood vessels where they provide an anti-thrombotic surface on the luminal side and protect the underlying vascular smooth muscle on the abluminal side. Specialised functions of endothelial cells include their unique ability to release vasoactive hormones and to morphologically adapt to complex shear stress. Stem cell derived-endothelial cells have a growing number of applications and will be critical in any organ regeneration programme. Generally endothelial cells are identified in stem cell studies by well-recognised markers such as CD31. However, the ability of stem cell-derived endothelial cells to release vasoactive hormones and align with shear stress has not been studied extensively. With this in mind, we have compared directly the ability of endothelial cells derived from a range of stem cell sources, including embryonic stem cells (hESC-EC) and adult progenitors in blood (blood out growth endothelial cells, BOEC) with those cultured from mature vessels, to release the vasoconstrictor peptide endothelin (ET)-1, the cardioprotective hormone prostacyclin, and to respond morphologically to conditions of complex shear stress. All endothelial cell types, except hESC-EC, released high and comparable levels of ET-1 and prostacyclin. Under static culture conditions all endothelial cell types, except for hESC-EC, had the typical cobblestone morphology whilst hESC-EC had an elongated phenotype. When cells were grown under shear stress endothelial cells from vessels (human aorta) or BOEC elongated and aligned in the direction of shear. By contrast hESC-EC did not align in the direction of shear stress. These observations show key differences in endothelial cells derived from embryonic stem cells versus those from blood progenitor cells, and that BOEC are more similar than hESC-EC to endothelial cells from vessels. This may be advantageous in some settings particularly where an in vitro test bed is required. However, for other applications, because of low ET-1 release hESC-EC may prove to be protected from vascular inflammation.
    No preview · Article · Nov 2014 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Cyxlo-oxygenase (COX)-2 inhibitors, including traditional nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with increased cardiovascular side effects, including myocardial infarction. We and others have shown that COX-1 and not COX-2 drives vascular prostacyclin in the healthy cardiovascular system, re-opening the question of how COX-2 might regulate cardiovascular health. In diseased, atherosclerotic vessels, the relative contribution of COX-2 to prostacyclin formation is not clear. Here we have used apoE-/-/COX-2-/- mice to show that, whilst COX-2 profoundly limits atherosclerosis, this protection is independent of local prostacyclin release. These data further illustrate the need to look for new explanations, targets and pathways to define the COX/NSAID/cardiovascular risk axis. Gene expression profiles in tissues from apoE-/-/COX-2-/- mice showed increased lymphocyte pathways that were validated by showing increased T-lymphocytes in plaques and elevated plasma Th1-type cytokines. In addition, we identified a novel target gene, rgl1, whose expression was strongly reduced by COX-2 deletion across all examined tissues. This study is the first to demonstrate that COX-2 protects vessels against atherosclerotic lesions independently of local vascular prostacyclin and uses systems biology approaches to identify new mechanisms relevant to development of next generation NSAIDs.
    Full-text · Article · Jun 2014 · PLoS ONE
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    ABSTRACT: Up to 1% of the general population have mild bleeding disorders, but these are often poorly characterized, particularly with regard to the roles of platelets. Here we have compared the usefulness of Optimul, a 96-well-plate-based assay of seven distinct pathways of platelet activation, to characterize inherited platelet defects in comparison to light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, TRAP-6, U46619, and ristocetin in samples from (i) healthy volunteers (n=50), (ii) healthy volunteers treated with antiplatelet agents in vitro (n=10), and (iii) patients with bleeding of unknown origin (n=65). The assays gave concordant results in 82% of cases (κ=0.62, p<0.0001). Normal platelet function results were particularly predictive (sensitivity 94%, negative predictive value 91%) whereas a positive result was not always substantiated by LTA (specificity 67%, positive predictive value 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which nonetheless presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This study is registered at www.isrctn.org, identifier: ISRCTN 77951167.
    Full-text · Article · Jan 2014 · Blood
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    ABSTRACT: Evidence is increasing of a link between interferon (IFN) and pulmonary arterial hypertension (PAH). Conditions with chronically elevated endogenous IFNs such as systemic sclerosis (SSc) are strongly associated with PAH. Furthermore, therapeutic use of type I IFN is associated with PAH. This was recognised at the 2013 World Symposium on Pulmonary Hypertension where the urgent need for research into this was highlighted. To explore the role of type I IFN in PAH. Cells were cultured using standard approaches. Cytokines were measured by ELISA. Gene and protein expression were measured using RT-PCR, Western blotting and immunohistochemistry. The role of type I IFN in PAH in vivo was determined using type I IFN receptor knock out (IFNAR1(-/-)) mice. Human lung cells responded to types I and II but not III IFN correlating with relevant receptor expression. Type I, II and III IFN levels were elevated in serum of SSc-PAH patients. Serum IP10 and ET-1 were raised and strongly correlated together. IP10 correlated positively with pulmonary haemodynamics and serum brain natriuretic peptide and negatively with 6-minute walk test and cardiac index. Endothelial cells grown out of the blood of PAH patients were more sensitive to the effects of type I IFN than cells from healthy donors. PAH lung demonstrated increased IFNAR1 protein levels. IFNAR1(-/-) mice were protected from the effects of hypoxia on the right heart, vascular remodelling and raised serum ET-1 levels. These data indicate that type I IFN, via an action of IFNAR1, mediates PAH.
    Full-text · Article · Dec 2013 · Circulation Research
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    ABSTRACT: Homocysteine is metabolized to methionine by the action of 5,10 methylenetetrahydrofolate reductase (MTHFR). Alternatively, by the transulfuration pathway, homocysteine is transformed to hydrogen sulphide (H2S), through multiple steps involving cystathionine β-synthase and cystathionine γ-lyase. Here we have evaluated the involvement of H2S in the thrombotic events associated with hyperhomocysteinemia. To this purpose we have used platelets harvested from healthy volunteers or patients newly diagnosed with hyperhomocysteinemia with a C677T polymorphism of the MTHFR gene (MTHFR++). NaHS (0.1-100 µM) or l-cysteine (0.1-100 µM) significantly increased platelet aggregation harvested from healthy volunteers induced by thrombin receptor activator peptide-6 amide (2 µM) in a concentration-dependent manner. This increase was significantly potentiated in platelets harvested from MTHFR++ carriers, and it was reversed by the inhibition of either cystathionine β-synthase or cystathionine γ-lyase. Similarly, in MTHFR++ carriers, the content of H2S was significantly higher in either platelets or plasma compared with healthy volunteers. Interestingly, thromboxane A2 production was markedly increased in response to both NaHS or l-cysteine in platelets of healthy volunteers. The inhibition of phospholipase A2, cyclooxygenase, or blockade of the thromboxane receptor markedly reduced the effects of H2S. Finally, phosphorylated-phospholipase A2 expression was significantly higher in MTHFR++ carriers compared with healthy volunteers. In conclusion, the H2S pathway is involved in the prothrombotic events occurring in hyperhomocysteinemic patients.
    Full-text · Article · Sep 2013 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Circulating platelets are constantly exposed to nitric oxide (NO) released from the vascular endothelium. This NO acts to reduce platelet reactivity, and in so doing blunts platelet aggregation and thrombus formation. For successful hemostasis, platelet activation and aggregation must occur at sites of vascular injury despite the constant presence of NO. As platelets aggregate, they release secondary mediators that drive further aggregation. Particularly significant among these secondary mediators is ADP, which, acting through platelet P2Y12 receptors, strongly amplifies aggregation. Platelet P2Y12 receptors are the targets of very widely used antithrombotic drugs such as clopidogrel, prasugrel, and ticagrelor. Here we show that blockade of platelet P2Y12 receptors dramatically enhances the antiplatelet potency of NO, causing a 1,000- to 100,000-fold increase in inhibitory activity against platelet aggregation and release reactions in response to activation of receptors for either thrombin or collagen. This powerful synergism is explained by blockade of a P2Y12 receptor-dependent, NO/cGMP-insensitive phosphatidylinositol 3-kinase pathway of platelet activation. These studies demonstrate that activation of the platelet ADP receptor, P2Y12, severely blunts the inhibitory effects of NO. The powerful antithrombotic effects of P2Y12 receptor blockers may, in part, be mediated by profound potentiation of the effects of endogenous NO.
    Full-text · Article · Sep 2013 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Cyclooxygenase 2 (COX)-2 is induced by bacterial and viral infections and has complex, poorly understood roles in anti-pathogen immunity. Here, we use a knock-in luciferase reporter model to image Cox2 expression across a range of tissues in mice following treatment with the either the prototypical bacterial pathogen-associated molecular pattern (PAMP), LPS, which activates Toll-like receptor (TLR)4, or with poly(I:C), a viral PAMP, which activates TLR3. LPS induced Cox2 expression in all tissues examined. In contrast, poly(I:C) elicited a milder response, limited to a subset of tissues. A panel of cytokines and interferons was measured in plasma of wild-type, Cox1(-/-) and Cox2(-/-) mice treated with LPS, poly(I:C), MALP2 (TLR2/6), Pam3CSK4 (TLR2/1), R-848 (TLR7/8) or CpG ODN (TLR9), to establish whether/how each COX isoform modulates specific PAMP/TLR responses. Only LPS induced notable loss of condition in mice (inactivity, hunching, piloerection). However, all TLR agonists produced cytokine responses, many of which were modulated in specific fashions by Cox1 or Cox2 gene deletion. Notably we observed opposing effects of Cox2 gene deletion on the responses to the bacterial PAMP, LPS, and the viral PAMP, poly(I:C), consistent with the differing abilities of the PAMPs to induce Cox2 expression. Cox2 gene deletion limited the plasma IL-1β and interferon-γ responses and hypothermia produced by LPS. In contrast, in response to poly(I:C), Cox2(-/-) mice exhibited enhanced plasma interferon (IFNα,β,γ,λ) and related cytokine responses (IP-10,IL-12). These observations suggest that a COX-2 selective inhibitor, given early in infection, may enhance and/or prolong endogenous interferon responses, and thereby increase anti-viral immunity.
    Full-text · Article · Jul 2013 · Biochemical and Biophysical Research Communications
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    ABSTRACT: There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.
    Full-text · Article · Jul 2013 · PLoS ONE
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    ABSTRACT: Inhibition of cyclooxygenase (COX)-2 increases cardiovascular deaths. Identifying a biomarker of COX-2 is desirable but difficult, since COX-1 and COX-2 ordinarily catalyze formation of an identical product, prostaglandin H2. When acetylated by aspirin, however, COX-2 (but not COX-1) can form 15(R)-HETE, which is metabolized to aspirin-triggered lipoxin (ATL), 15-epi-lipoxin A4. Here we have used COX-1- and COX-2-knockout mice to establish whether plasma ATL could be used as a biomarker of vascular COX-2 in vivo. Vascular COX-2 was low but increased by LPS (10 mg/kg; i.p). Aspirin (10 mg/kg; i.v.) inhibited COX-1, measured as blood thromboxane and COX-2, measured as lung PGE2. Aspirin also increased the levels of ATL in the lungs of LPS-treated wild-type C57Bl6 mice (vehicle: 25.5±9.3 ng/ml; 100 mg/kg: 112.0±7.4 ng/ml; P<0.05). Despite this, ATL was unchanged in plasma after LPS and aspirin. This was true in wild-type as well as COX-1(-/-) and COX-2(-/-) mice. Thus, in mice in which COX-2 has been induced by LPS treatment, aspirin triggers detectable 15-epi-lipoxin A4 in lung tissue, but not in plasma. This important study is the first to demonstrate that while ATL can be measured in tissue, plasma ATL is not a biomarker of vascular COX-2 expression.-Kirkby, N. S., Chan, M. V., Lundberg, M. H., Massey, K. A., Edmands, W. M. B., MacKenzie, L. S., Holmes, E., Nicolaou, A., Warner, T. D., Mitchell, J. A. Aspirin-triggered 15-epi-lipoxin A4 predicts cyclooxygenase-2 in the lungs of LPS-treated mice but not in the circulation: implications for a clinical test.
    Full-text · Article · Jun 2013 · The FASEB Journal
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    ABSTRACT: Acute renal failure (ARF) has high mortality and no effective treatment. Nitric oxide (NO) delivery represents a credible means of preventing the damaging effects of vasoconstriction, central to ARF, but design of drugs with the necessary renoselectivity is challenging. Here, we developed N-hydroxyguanidine NO donor drugs that were protected against spontaneous NO release by linkage to glutamyl adducts that could be cleaved by γ-glutamyl transpeptidase (γ-GT), found predominantly in renal tissue. Parent NO donor drug activity was optimized in advance of glutamyl adduct prodrug design. A lead compound that was a suitable substrate for γ-GT-mediated deprotection was identified. Metabolism of this prodrug to the active parent compound was confirmed in rat kidney homogenates, and the prodrug was shown to be an active vasodilator in rat isolated perfused kidneys (EC50 ∼50 μM). The data confirm that glutamate protection of N-hydroxyguanidines is an approach that might hold promise in ARF.
    No preview · Article · Jun 2013 · Journal of Medicinal Chemistry

Publication Stats

467 Citations
253.01 Total Impact Points

Institutions

  • 2010-2015
    • Imperial College London
      Londinium, England, United Kingdom
  • 2008-2015
    • The University of Edinburgh
      • Centre for Cardiovascular Science
      Edinburgh, Scotland, United Kingdom
  • 2014
    • The Heart Lung Center
      Londinium, England, United Kingdom
  • 2013
    • William Harvey Research Institute
      Londinium, England, United Kingdom
  • 2011-2012
    • Queen Mary, University of London
      • Barts and The London School of Medicine and Dentistry
      Londinium, England, United Kingdom