Imperial College London

DrNicholasKirkby

Faculty of MedicineNational Heart & Lung Institute

Senior Research Fellow
 
 
 
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Contact

 

+44 (0)20 7594 3075n.kirkby

 
 
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Location

 

364Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

130 results found

Vinokurova M, Lopes Pires M, Cypaite N, Shala F, Armstrong P, Ahmetaj-Shala B, Elghazouli Y, Nusing R, Liu B, Zhou Y, Hao C-M, Herschman H, Mitchell J, Kirkby Net al., 2024, Widening the prostacyclin paradigm: tissue fibroblasts are a critical site of production and anti-thrombotic protection, Arteriosclerosis, Thrombosis and Vascular Biology, Vol: 44, Pages: 271-286, ISSN: 1079-5642

BACKGROUND:Prostacyclin is a fundamental signaling pathway traditionally associated with the cardiovascular system and protection against thrombosis but which also has regulatory functions in fibrosis, proliferation, and immunity. Prevailing dogma states that prostacyclin is principally derived from vascular endothelium, although it is known that other cells can also synthesize it. However, the role of nonendothelial sources in prostacyclin production has not been systematically evaluated resulting in an underappreciation of their importance relative to better characterized endothelial sources.METHODS:To address this, we have used novel endothelial cell–specific and fibroblast-specific COX (cyclo-oxygenase) and prostacyclin synthase knockout mice and cells freshly isolated from mouse and human lung tissue. We have assessed prostacyclin release by immunoassay and thrombosis in vivo using an FeCl3-induced carotid artery injury model.RESULTS:We found that in arteries, endothelial cells are the main source of prostacyclin but that in the lung, and other tissues, prostacyclin production occurs largely independently of endothelial and vascular smooth muscle cells. Instead, in mouse and human lung, prostacyclin production was strongly associated with fibroblasts. By comparison, microvascular endothelial cells from the lung showed weak prostacyclin synthetic capacity compared with those isolated from large arteries. Prostacyclin derived from fibroblasts and other nonendothelial sources was seen to contribute to antithrombotic protection.CONCLUSIONS:These observations define a new paradigm in prostacyclin biology in which fibroblast/nonendothelial-derived prostacyclin works in parallel with endothelium-derived prostanoids to control thrombotic risk and potentially a broad range of other biology. Although generation of prostacyclin by fibroblasts has been shown previously, the scale and systemic activity was not tested and unappreciated. As such, this represents a basic

Journal article

Judina A, Niglas M, Leonov V, Kirkby NS, Diakonov I, Wright PT, Zhao L, Mitchell JA, Gorelik Jet al., 2023, Pulmonary Hypertension-Associated Right Ventricular Cardiomyocyte Remodelling Reduces Treprostinil Function., Cells, Vol: 12

(1) Pulmonary hypertension (PH)-associated right ventricular (RV) failure is linked to a reduction in pulmonary vasodilators. Treprostinil has shown effectiveness in PAH patients with cardiac decompensation, hinting at potential cardiac benefits. We investigated treprostinil's synergy with isoprenaline in RV and LV cardiomyocytes. We hypothesised that disease-related RV structural changes in cardiomyocytes would reduce contractile responses and cAMP/PKA signalling activity. (2) We induced PH in male Sprague Dawley rats using monocrotaline and isolated their ventricular cardiomyocytes. The effect of in vitro treprostinil and isoprenaline stimulation on contraction was assessed. FRET microscopy was used to study PKA activity associated with treprostinil stimulation in AKAR3-NES FRET-based biosensor-expressing cells. (3) RV cells exhibited maladaptive remodelling with hypertrophy, impaired contractility, and calcium transients compared to control and LV cardiomyocytes. Combining treprostinil and isoprenaline failed to enhance inotropy in PH RV cardiomyocytes. PH RV cardiomyocytes displayed an aberrant contractile behaviour, which the combination treatment could not rectify. Finally, we observed decreased PKA activity in treprostinil-treated PH RV cardiomyocytes. (4) PH-associated RV cardiomyocyte remodelling reduced treprostinil sensitivity, inotropic support, and impaired relaxation. Overall, this study highlights the complexity of RV dysfunction in advanced PH and suggests the need for alternative therapeutic strategies.

Journal article

Minghin Freitas Ferreira P, Vaja R, Lopes Pires M, Crescente M, Yu H, Nusing R, Liu B, Zhou Y, Yaqoob M, Zhang A, Rickman M, Longhurst H, Knowles R, Chan M, Warner T, Want E, Kirkby N, Mitchell Jet al., 2023, Renal function underpins the cyclooxygenase-2: ADMA axis in mouse and man, Kidney International Reports, Vol: 8, Pages: 1231-1238, ISSN: 2468-0249

IntroductionThrough the production of prostacyclin, cyclooxygenase (COX)-2 protects the cardiorenal system. Asymmetric dimethylarginine (ADMA), is a biomarker of cardiovascular and renal disease. Here we determined the relationship between COX-2/prostacyclin, ADMA, and renal function in mouse and human models.MethodsWe used plasma from COX-2 or prostacyclin synthase knockout mice and from a unique individual lacking COX-derived prostaglandins (PGs) because of a loss of function mutation in cytosolic phospholipase A2 (cPLA2), before and after receiving a cPLA2-replete transplanted donor kidney. ADMA, arginine, and citrulline were measured using UHPLC-MS/MS. ADMA and arginine were also measured by enzyme-linked immunosorbent assay (ELISA). Renal function was assessed by measuring cystatin C by immunoassay. ADMA and prostacyclin release from organotypic kidney slices were also measured by ELISA.ResultsLoss of COX-2 or prostacyclin synthase in mice increased plasma levels of ADMA, citrulline, arginine, and cystatin C. ADMA, citrulline, and arginine positively correlated with cystatin C. Plasma ADMA, citrulline, and cystatin C, but not arginine, were elevated in samples from the patient lacking COX/prostacyclin capacity compared to levels in healthy volunteers. Renal function, ADMA, and citrulline were returned toward normal range when the patient received a genetically normal kidney, capable of COX/prostacyclin activity; and cystatin C positively correlated with ADMA and citrulline. Levels of ADMA and prostacyclin in conditioned media of kidney slices were not altered in tissue from COX-2 knockout mice compared to wildtype controls.ConclusionIn human and mouse models, where renal function is compromised because of loss of COX-2/PGI2 signaling, ADMA levels are increased.

Journal article

Judina A, Endo A, Zhao L, Niglas M, Wright P, Kirkby N, Mitchell J, Gorelik Jet al., 2022, Activation of prostacyclin (PGI2) IP receptors by treprostinil in the right atrium is associated with stronger fibroblast PKA response, 24th World Congress of the International-Society-for-Heart-Research, Publisher: ELSEVIER SCI LTD, Pages: S156-S157, ISSN: 0022-2828

Conference paper

Pires ML, Vinokurova M, Shala F, Ahmetaj-Shala B, Armstrong PC, Herschman HR, Mitchell JA, Kirkby NSet al., 2022, Identifying Sources of Prostacyclin Outside the Vascular Wall and Their Contribution to Cardiovascular Protection, Scientific Sessions of the American-Heart-Association / Resuscitation Science Symposium, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322

Conference paper

Vaja R, Ferreira P, Collins P, Rickman M, Punjabi PP, Kirkby N, Mitchell Jet al., 2022, The Effects of Celecoxib on Endothelial Function in Healthy Volunteers; A Double Blind, Placebo Controlled Randomised Trial, Scientific Sessions of the American-Heart-Association / Resuscitation Science Symposium, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322

Conference paper

Pires ML, Ferreira P, Shala F, Cypaite N, Vinokurova M, Want E, Herschman H, Mitchell JA, Kirkby Net al., 2022, Using Renal-Specific Cyclo-Oxygenase-2-Deficient Mice to Understand the Pro-Thrombotic Effects of Non-Steroidal Anti-Inflammatory Drugs, Scientific Sessions of the American-Heart-Association / Resuscitation Science Symposium, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322

Conference paper

Ferreira PM, Kirkby N, Vaja R, Pires ML, Crescente M, Liu B, Yu H, Chan M, Nuesing R, Zhou Y, White W, Want E, Warner TD, Knowles R, Yaqoob M, Longhurst H, Zhang A, Rickman Met al., 2022, Renal Function Underpins Cox-2: Methylarginine Axis in Mouse and Man, Scientific Sessions of the American-Heart-Association / Resuscitation Science Symposium, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322

Conference paper

Vaja R, Ferreira PM, Want L, Ahmetaj-Shala B, Gashaw H, Kirkby NS, Mitchell JAet al., 2022, SWATH proteomic analysis from the standard care versus celecoxib outcome trial identifies renal biomarkers are associated with early cardiovascular events amongst NSAID users, Publisher: OXFORD UNIV PRESS, Pages: 3045-3045, ISSN: 0195-668X

Conference paper

Armstrong PC, Allan HE, Kirkby NS, Gutmann C, Joshi A, Crescente M, Mitchell JA, Mayr M, Warner TDet al., 2022, Temporal in vivo platelet labelling in mice reveals age-dependent receptor expression and conservation of specific mRNAs., Blood Advances, Vol: 6, Pages: 6028-6038, ISSN: 2473-9529

The proportion of young platelets, also known as newly formed or reticulated, within the overall platelet population has been clinically correlated with adverse cardiovascular outcomes. Our understanding of this is incomplete, however, because of limitations in the technical approaches available to study platelets of different ages. In this study we have developed and validated an in vivo 'temporal labelling' approach using injectable fluorescent anti-platelet antibodies to sub-divide platelets by age and assess differences in functional and molecular characteristics. With this approach we found that young platelets (<24h old) in comparison to older platelets respond to stimuli with greater calcium flux and degranulation, and contribute more to the formation of thrombi in vitro and in vivo. Sequential sampling confirmed this altered functionality to be independent of platelet size, with distribution of sizes of tracked platelets commensurate with the global platelet population throughout their 5-day lifespan in the circulation. The age associated decrease in thrombotic function was accompanied by significant decreases in the surface expression of GPVI and CD31 (PECAM-1) and an increase in CD9. Platelet mRNA content also decreased with age but at different rates for individual mRNAs indicating apparent conservation of those encoding granule proteins. Our pulse-chase type approach to define circulating platelet age has allowed timely re-examination of commonly held beliefs regarding size and reactivity of young platelets whilst providing novel insights into the temporal regulation of receptor and protein expression. Overall, future application of this validated tool will inform on age-based platelet heterogeneity in physiology and disease.

Journal article

MacIntyre IM, Turtle EJ, Farrah TE, Graham C, Dear JW, Webb DJ, McCallum M, Melville V, Fok H, McCrae JC, Sule A, Caparrotta TM, Kirkby NS, Mitchell JAet al., 2022, Regular Acetaminophen Use and Blood Pressure in People With Hypertension: The PATH-BP Trial, Circulation, Vol: 145, Pages: 416-423, ISSN: 0009-7322

<jats:sec> <jats:title>Background:</jats:title> <jats:p>Acetaminophen is widely used as first-line therapy for chronic pain because of its perceived safety and the assumption that, unlike nonsteroidal anti-inflammatory drugs, it has little or no effect on blood pressure (BP). Although observational studies suggest that acetaminophen may increase BP, clinical trials are lacking. We, therefore, studied the effects of regular acetaminophen dosing on BP in individuals with hypertension.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods:</jats:title> <jats:p>In this double-blind, placebo-controlled, crossover study, 110 individuals were randomized to receive 1 g acetaminophen 4× daily or matched placebo for 2 weeks followed by a 2-week washout period before crossing over to the alternate treatment. At the beginning and end of each treatment period, 24-hour ambulatory BPs were measured. The primary outcome was a comparison of the change in mean daytime systolic BP from baseline to end of treatment between the placebo and acetaminophen arms.</jats:p> </jats:sec> <jats:sec> <jats:title>Results:</jats:title> <jats:p> One-hundred three patients completed both arms of the study. Regular acetaminophen, compared with placebo, resulted in a significant increase in mean daytime systolic BP (132.8±10.5 to 136.5±10.1 mm Hg [acetaminophen] vs 133.9±10.3 to 132.5±9.9 mm Hg [placebo]; <jats:italic>P</jats:italic> &lt;0.0001) with a placebo-corrected increase of 4.7 mm Hg (95% CI, 2.9–6.6) and mean daytime diastolic BP (81.2±8.0 to 82.1±7.8 mm Hg [acetaminophen] vs 81.7±7.9 to 80.9±7.8 mm Hg [placebo]; <jats:italic>P

Journal article

Armstrong PC, Allan HE, Kirkby NS, Joshi A, Gutmann C, Crescente M, Mitchell JA, Mayr M, Warner TDet al., 2021, Temporal <i>in vivo</i> platelet labelling in mice reveals age-dependent receptor expression and conservation of specific mRNAs

<jats:title>Abstract</jats:title><jats:p>The proportion of young platelets, also known as newly formed or reticulated, within the overall platelet population has been clinically correlated with adverse cardiovascular outcomes. Our understanding of this is incomplete, however, because of limitations in the technical approaches available to study platelets of different ages. In this study we have developed and validated an <jats:italic>in vivo</jats:italic> ‘temporal labelling’ approach using injectable fluorescent anti-platelet antibodies to sub-divide platelets by age and assess differences in functional and molecular characteristics. With this approach we found that young platelets (&lt;24h old) in comparison to older platelets respond to stimuli with greater calcium flux and degranulation, and contribute more to the formation of thrombi <jats:italic>in vitro</jats:italic> and <jats:italic>in vivo</jats:italic>. Sequential sampling confirmed this altered functionality to be independent of platelet size with no size differences or changes relative to the global population seen at any age. The age associated decrease in thrombotic function was accompanied by significant decreases in the surface expression of GPVI and CD31 (PECAM-1) and an increase in CD9. Platelet mRNA content also decreased with age but at different rates for individual mRNAs indicating apparent conservation of those encoding granule proteins. Our pulse-chase type approach to define circulating platelet age has allowed timely re-examination of commonly held beliefs regarding size and reactivity of young platelets whilst providing novel insights into the temporal regulation of receptor and protein expression. Overall, future application of this validated tool will inform on age-based platelet heterogeneity in physiology and disease.</jats:p>

Working paper

Shih C-C, Chan M, Kirkby NS, Vojnovic I, Mitchell JA, Armstrong PC, Warner TDet al., 2021, Platelet inhibition by P2Y<sub>12</sub> antagonists is potentiated by adenosine signalling activators, BRITISH JOURNAL OF PHARMACOLOGY, Vol: 178, Pages: 4758-4771, ISSN: 0007-1188

Journal article

Al-Asfoor S, Mitchell JA, Mason P, Kirkby NS, Gashaw HHet al., 2021, DIFFERENTIAL EFFECTS OF OMEGA-3 FATTY ACIDS ON LIPOPOLYSACCHARIDE (LPS)-INDUCED MACROPHAGE ACTIVATION IN COMBINATION WITH COX INHIBITION, Publisher: ELSEVIER IRELAND LTD, Pages: E13-E14, ISSN: 0021-9150

Conference paper

Ahmetaj-Shala B, Marei I, Kawai R, Rothery S, Pericleous C, Mohamed N, Gashaw H, Bokea K, Samuel J, Vandenheste A, Shala F, Kirkby N, Mitchell Jet al., 2021, Activation and contraction of human ‘vascular’ smooth muscle cells grown from circulating blood progenitors, Frontiers in Cell and Developmental Biology, Vol: 9, Pages: 1-6, ISSN: 2296-634X

Blood outgrowth smooth muscle cells offer the means to study vascular cells without the requirement for surgery providing opportunities for drug discovery, tissue engineering and personalised medicine. However, little is known about these cells which has meant their therapeutic potential remains unexplored. Our objective was to investigate for the first time the ability of blood outgrowth smooth muscle cells and vessel derived smooth muscle cells to sense the thromboxane mimetic U46619 bymeasuring intracellular calcium elevation and contraction. U46619 (10 26 -6M) increased cytosolic calcium in blood outgrowth smooth muscle cells fibroblasts. Increased calcium signal peaked between 10-20 seconds after U46619 in both smoothmuscle cell types. Importantly, U46619 (10-9 to 10-6M) induced concentration-dependent contractions of both blood outgrowth smooth muscle cells and vascular smooth muscle cells but not in fibroblasts. In summary, we show that functional responses of blood outgrowth smooth muscle cells are in line with vascular smooth muscle cells providing critical evidence of their application in biomedical research.

Journal article

Mitchell J, Shala F, Lopes Pires M, Loy R, Ravendren A, Benson J, Urquhart P, Nicolaou A, Herschman H, Kirkby Net al., 2021, Endothelial cyclooxygenase-1 paradoxically drives local vasoconstriction and atherogenesis despite underpinning prostacyclin generation, Science Advances, Vol: 7, ISSN: 2375-2548

Endothelial cyclooxygenase-1–derived prostanoids, including prostacyclin, have clear cardioprotective roles associated with their anti-thrombotic potential but have also been suggested to have paradoxical pathological activities within arteries. To date it has not been possible to test the importance of this because no models have been available that separate vascular cyclooxygenase-1 products from those generated elsewhere. Here, we have used unique endothelial-specific cyclooxygenase-1 knockout mice to show that endothelial cyclooxygenase-1 produces both protective and pathological products. Functionally, however, the overall effect of these was to drive pathological responses in the context of both vasoconstriction in vitro and the development of atherosclerosis and vascular inflammation in vivo. These data provide the first demonstration of a pathological role for the vascular cyclooxygenase-1 pathway, highlighting its potential as a therapeutic target. They also emphasize that, across biology, the role of prostanoids is not always predictable due to unique balances of context, products, and receptors.

Journal article

Vaja R, Chan J, Ferreira P, Harky A, Rogers LJ, Gashaw HH, Kirkby NS, Mitchell JAet al., 2021, The COVID-19 ibuprofen controversy: A systematic review of NSAIDs in adult acute lower respiratory tract infections, BRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Vol: 87, Pages: 776-784, ISSN: 0306-5251

Journal article

Mohamed NA, Abou-Saleh H, Kameno Y, Marei I, de Nucci G, Ahmetaj-Shala B, Shala F, Kirkby NS, Jennings L, Al-Ansari DE, Davies RP, Lickiss PD, Mitchell JAet al., 2021, Studies on metal-organic framework (MOF) nanomedicine preparations of sildenafil for the future treatment of pulmonary arterial hypertension, Scientific Reports, Vol: 11, Pages: 1-8, ISSN: 2045-2322

Pulmonary arterial hypertension (PAH) is an incurable disease, although symptoms are treated with a range of dilator drugs. Despite their clinical benefits, these drugs are limited by systemic side-effects. It is, therefore, increasingly recognised that using controlled drug-release nanoformulation, with future modifications for targeted drug delivery, may overcome these limitations. This study presents the first evaluation of a promising nanoformulation (highly porous iron-based metal–organic framework (MOF); nanoMIL-89) as a carrier for the PAH-drug sildenafil, which we have previously shown to be relatively non-toxic in vitro and well-tolerated in vivo. In this study, nanoMIL-89 was prepared and charged with a payload of sildenafil (generating Sil@nanoMIL-89). Sildenafil release was measured by Enzyme-Linked Immunosorbent Assay (ELISA), and its effect on cell viability and dilator function in mouse aorta were assessed. Results showed that Sil@nanoMIL-89 released sildenafil over 6 h, followed by a more sustained release over 72 h. Sil@nanoMIL-89 showed no significant toxicity in human blood outgrowth endothelial cells for concentrations up to100µg/ml; however, it reduced the viability of the human pulmonary artery smooth muscle cells (HPASMCs) at concentrations > 3 µg/ml without inducing cellular cytotoxicity. Finally, Sil@nanoMIL-89 induced vasodilation of mouse aorta after a lag phase of 2–4 h. To our knowledge, this study represents the first demonstration of a novel nanoformulation displaying delayed drug release corresponding to vasodilator activity. Further pharmacological assessment of our nanoformulation, including in PAH models, is required and constitutes the subject of ongoing investigations.

Journal article

Mitchell JA, Kirkby NS, Ahmetaj-Shala B, Armstrong P, Crescente M, Ferreira P, Pires MEL, Vaja RK, Warner TDet al., 2021, Cyclooxygenases and the cardiovascular system, Pharmacology and Therapeutics, Vol: 217, Pages: 1-11, ISSN: 0163-7258

Cyclooxygenase (COX)-1 and COX-2 are centrally important enzymes within the cardiovascular system with a range of diverse, sometimes opposing, functions. Through the production of thromboxane, COX in platelets is a pro-thrombotic enzyme. By contrast, through the production of prostacyclin, COX in endothelial cells is antithrombotic and in the kidney regulates renal function and blood pressure. Drug inhibition of COX within the cardiovascular system is important for both therapeutic intervention with low dose aspirin and for the manifestation of side effects caused by nonsteroidal anti-inflammatory drugs. This review focuses on the role that COX enzymes and drugs that act on COX pathways have within the cardiovascular system and provides an in-depth resource covering COX biology and pharmacology. The review goes on to consider the role of COX in both discrete cardiovascular locations and in associated organs that contribute to cardiovascular health. We discuss the importance of, and strategies to manipulate, the thromboxane: prostacyclin balance. Finally within this review the authors discuss testable COX-2-hypotheses intended to stimulate debate and facilitate future research and therapeutic opportunities within the field.

Journal article

Ahmetaj-Shala B, Ricky V, Santosh A, Peter G, Nicholas K, Jane Met al., 2020, Cardiorenal tissues express SARS-CoV-2 entry genes and basigin (BSG/CD147) increases with age in endothelial cells, JACC: Basic to Translational Science, Vol: 5, Pages: 1111-1123, ISSN: 2452-302X

Objectives: To obtain mechanistic insight into COVID-19 within a cardiovascular setting.Background: Thrombosis and vascular dysfunction are part of the complex pathology seen in severe COVID-19 and advancing age is the most significant risk factor. Little is known about age and expression of pathways utilised by the COVID-19 virus, SARS-CoV-2, in cardiovascular tissues.Methods: We used publicly available databases (GTEx, GEO and Array Express) to investigate gene expression levels, in adult tissues, of the two putative SARS-CoV-2 receptors, ACE2 and BSG along with a selected range of genes thought to be involved in virus binding/processing. Our analysis included; vessels (aorta and coronary artery), heart (atrial appendage and left ventricle), kidney (cortex), whole blood, lung, colon and spleen along with endothelial cells, nasal and bronchial epithelium and peripheral blood mononuclear cells. Gene expression levels were then analysed for age associations.Results: We found: (i) cardiovascular tissues/endothelial cells express the required genes for SARS-CoV-2 infection, (ii) SARS-CoV-2 receptor pathways, ACE2/TMPRSS2 and BSG/PPIB(A) polarise to lung/epithelium and vessel/endothelium respectively, (iii) expression of host genes are relatively stable with age and (iv) notable exceptions are ACE2 which decreases with age in some tissues and BSG which increases with age in endothelial cells.Conclusion: Our data identifies a positive correlation of BSG with age in endothelial cells. Since BSG is utilised by other pathogens and is implicated in a range of cardiovascular disease, our observations may have relevance to our understanding of mechanisms associated with other pathogens and in the diseases associated with aging respectively.

Journal article

Kirkby N, Raouf J, Ahmetaj-Shala B, Liu B, Mazi S, Edin M, Geoffrey Chambers M, Korotkova M, Wang X, Wahli W, Zeldin D, Nusing R, Zhou Y, Jakobsson P-J, Mitchell Jet al., 2020, Mechanistic definition of the cardiovascular mPGES-1/COX-2/ADMA axis, Cardiovascular Research, Vol: 116, Pages: 1972-1980, ISSN: 0008-6363

Aims:Cardiovascular side effects caused by non-steroidal anti-inflammatory drugs (NSAIDs), which all inhibit cyclooxygenase (COX)-2, have prevented development of new drugs that target prostaglandins to treat inflammation and cancer. Microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors have efficacy in the NSAID arena but their cardiovascular safety is not known. Our previous work identified asymmetric dimethylarginine (ADMA), an inhibitor of eNOS, as a potential biomarker of cardiovascular toxicity associated with blockade of COX-2. Here we have used pharmacological tools and genetically modified mice to delineate mPGES-1 and COX-2 in the regulation of ADMA.Methods and Results:Inhibition of COX-2 but not mPGES-1 deletion resulted in increased plasma ADMA levels. mPGES-1 deletion but not COX-2 inhibition resulted in increased plasma prostacyclin levels. These differences were explained by distinct compartmentalisation of COX-2 and mPGES-1 in the kidney. Data from prostanoid synthase/receptor knockout mice showed that the COX-2/ADMA axis is controlled by prostacyclin receptors (IP and PPARβ/δ) and the inhibitory PGE2 receptor EP4, but not other PGE2 receptors.Conclusions:These data demonstrate that inhibition of mPGES-1 spares the renal COX-2/ADMA pathway and define mechanistically how COX-2 regulates ADMA.

Journal article

Armstrong PC, Ferreira PM, Chan MV, Lundberg Slingsby MH, Crescente M, Shih C-C, Kirkby NS, Hobbs AJ, Warner TDet al., 2020, Combination of cyclic nucleotide modulators with P2Y(12) receptor antagonists as anti-platelet therapy, Journal of Thrombosis and Haemostasis, Vol: 18, Pages: 1705-1713, ISSN: 1538-7836

BackgroundEndothelium‐derived prostacyclin and nitric oxide elevate platelet cyclic nucleotide levels and maintain quiescence. We previously demonstrated that a synergistic relationship exists between cyclic nucleotides and P2Y12 receptor inhibition. A number of clinically approved drug classes can modulate cyclic nucleotide tone in platelets including activators of NO‐sensitive guanylyl cyclase (GC) and phosphodiesterase (PDE) inhibitors. However, the doses required to inhibit platelets produce numerous side effects including headache.ObjectiveWe investigated using GC‐activators in combination with P2Y12 receptor antagonists as a way to selectively amplify the anti‐thrombotic effect of both drugs.MethodsIn vitro light transmission aggregation and platelet adhesion under flow were performed on washed platelets and platelet rich plasma. Aggregation in whole blood and a ferric chloride‐induced arterial thrombosis model were also performed.ResultsThe GC‐activator BAY‐70 potentiated the action of the P2Y12 receptor inhibitor prasugrel active metabolite in aggregation and adhesion studies and was associated with raised intra‐platelet cyclic nucleotide levels. Furthermore, mice administered sub‐maximal doses of the GC activator cinaciguat together with the PDE inhibitor dipyridamole and prasugrel, showed significant inhibition of ex vivo platelet aggregation and significantly reduced in vivo arterial thrombosis in response to injury without alteration in basal carotid artery blood flow.ConclusionsUsing in vitro, ex vivo, and in vivo functional studies, we show that low dose GC activators synergize with P2Y12 inhibition to produce powerful anti‐platelet effects without altering blood flow. Therefore, modulation of intra‐platelet cyclic nucleotide levels alongside P2Y12 inhibition can provide a strong, focused anti‐thrombotic regimen while minimizing vasodilator side effects.

Journal article

Crescente M, Armstrong P, Kirkby N, Edin M, Chan M, Lih F, Maffucci T, Allan H, Mein C, Gaston-Massuet C, Cottrell G, Zeldin D, Herschman H, Warner Tet al., 2020, Profiling the eicosanoid networks that underlie the anti- and pro-thrombotic effects of aspirin, The FASEB Journal, ISSN: 0892-6638

Aspirin preventsthrombosisby inhibiting platelet cyclooxygenase (COX)-1 activity and the production of thromboxane (Tx)A2, a pro-thrombotic eicosanoid. However, thenon-platelet actions ofaspirin limit its anti-thrombotic effects. Here we used platelet-COX-1-komice to define the platelet andnon-platelet eicosanoids affected by aspirin.Mass-spectrometry analysis demonstrated blood fromplatelet-COX-1-ko and global-COX-1-ko mice produced similar eicosanoid profiles in vitro: e.g. formation of TxA2,prostaglandin (PG) F2a, 11-HETE and 15-HETE was absent in both platelet-and global-COX-1-komice. Conversely, in vivo,platelet-COX-1-ko mice had a distinctly different profile from global-COX-1-koor aspirin-treated controlmice, notably significantly higher levels of PGI2metabolite. Ingenuity Pathway Analysis predicted that platelet-COX-1-ko mice would be protected from thrombosis,formingless prothrombotic TxA2 and PGE2. Conversely, aspirinor lack of systemic COX-1 activity decreased the synthesis of anti-aggregatory PGI2and PGD2 at non-platelet sites leading to predictedthrombosis increase. In vitroand in vivothrombosis studies proved these predictions. Overall, we have established the eicosanoid profileslinked to inhibition of COX-1 in platelets and in the remainder of the cardiovascular systemand linked them to anti-and pro-thrombotic effects of aspirin. These results explainwhy increasing aspirin dosage or aspirin addition to other drugs may lessenanti-thrombotic protection.

Journal article

Mitchell J, Zhao C, Gashaw H, Ferreira P, Kirkby Net al., 2020, Paracetamol and dipyrone share an identical pharmacological profile as inhibitors of COX activity in intact and homogenised cell preparations, Meeting of the British-Pharmacological-Society, Publisher: WILEY, Pages: 2620-2620, ISSN: 0007-1188

Conference paper

Kirkby N, Gashaw H, Perikleous A, Ferreira P, Ahmetaj-Shala B, Mitchell Jet al., 2020, Novel arginine formulations of celecoxib fully retain COX-2 inhibitory and anti-cancer activity and reverse L-NAME-induced endothelial dysfunction, Meeting of the British-Pharmacological-Society, Publisher: WILEY, Pages: 2491-2492, ISSN: 0007-1188

Conference paper

Ahmetaj-Shala B, Kawai R, Marei I, Nikolakopoulou Z, Shih C-C, Konain B, Reed DM, Mongey R, Kirkby NS, Mitchell JAet al., 2020, A bioassay system of autologous human endothelial, smooth muscle cells and leucocytes for use in drug discovery, phenotyping and tissue engineering, The FASEB Journal, Vol: 34, Pages: 1745-1754, ISSN: 0892-6638

Purpose: Blood vessels are comprised of endothelial and smooth muscle cells. Obtaining both types of cells from vessels of living donors is not possible without invasive surgery. To address this we have devised a strategy whereby human endothelial and smooth muscle cells derived from blood progenitors from the same donor could be cultured with autologous leucocytes to generate a same donor ‘vessel in a dish’ bioassay. Basic procedures: Autologous sets of blood outgrowth endothelial cells (BOECs), smooth muscle cells (BO-SMCs) and leucocytes were obtained from 4 donors. Cells were treated in mono and cumulative co-culture conditions. The endothelial specific mediator endothelin-1 along with interleukin (IL)-6, IL-8, tumour necrosis factor α, and interferon gamma-induced protein 10 were measured under control culture conditions and after stimulation with cytokines.Main findings: Co-cultures remained viable throughout. The profile of individual mediators released from cells was consistent with what we know of endothelial and smooth muscle cells cultured from blood vessels.Principle conclusions: For the first time, we report a proof of concept study where autologous blood outgrowth ‘vascular’ cells and leucocytes were studied alone and in co-culture. This novel bioassay has utility in vascular biology research, patient phenotyping, drug testing and tissue engineering.

Journal article

Mitchell J, Shala F, Elghazouli Y, Warner T, Gaston-Massuet C, Crescente M, Armstrong P, Herschman H, Kirkby Net al., 2019, Cell-Specific Gene Deletion Reveals the Antithrombotic Function of COX1 and Explains the Vascular COX1/Prostacyclin Paradox, Circulation Research, Vol: 125, Pages: 847-854, ISSN: 0009-7330

Rationale: Endothelial cells and platelets, which respectively produce anti-thrombotic prostacyclin and pro-thrombotic thromboxane A2, both express COX1. Consequently, there has been no way to delineate any anti-thrombotic role for COX1-derived prostacyclin from the pro-thrombotic effects of platelet COX1. By contrast an anti-thrombotic role for COX2, which is absent in platelets, is straightforward to demonstrate. This has resulted in an incomplete understanding of the relative importance of COX1 versus COX2 in prostacyclin production and anti-thrombotic protection in vivo.Objective: We sought to identify the role, if any, of COX1-derived prostacyclin in anti-thrombotic protection in vivo and compare this to the established protective role of COX2.Methods and Results: We developed vascular-specific COX1 knockout mice and studied them alongside endothelial-specific COX2 knockout mice. COX1 immunoreactivity and prostacyclin production were primarily associated with the endothelial layer of aortae; freshly isolated aortic endothelial cells released >10-fold more prostacyclin than smooth muscle cells. Moreover, aortic prostacyclin production, the ability of aortic rings to inhibit platelet aggregation and plasma prostacyclin levels were reduced when COX1 was knocked out in endothelial cells but not in smooth muscle cells. When thrombosis was measured in vivo after FeCl3 carotid artery injury, endothelial COX1 deletion accelerated thrombosis to a similar extent as prostacyclin receptor blockade. However, but this effect was lost when COX1 was deleted from both endothelial cells and platelets. Deletion of COX2 from endothelial cells also resulted in a pro-thrombotic phenotype that was independent of local vascular prostacyclin production.Conclusions:These data demonstrate for the first time that, in healthy animals, endothelial COX1 provides an essential anti-thrombotic tone, which is masked when COX1 activity is lost in both endothelial cells and platelets. These res

Journal article

Mohamed NA, Davies RP, Lickiss PD, Kameno Y, Marei I, Kirkby NS, Mitchell JA, Abou Saleh Het al., 2019, Mil-89 nanoformulation as a platform to improve pulmonary arterial hypertension treatment, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 3075-3075, ISSN: 0007-1188

Conference paper

Ahmetaj-Shala B, Kawai R, Marei I, Bhatti F, Gashaw H, Kirkby NS, Mitchell JAet al., 2019, A bioassay system of autologous human endothelial and smooth muscle cells for use in cardiovascular drug discovery and patient phenotyping, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 3040-3041, ISSN: 0007-1188

Conference paper

Mazi SI, Ahmetaj-Shala B, Warner TD, Mitchell JA, Kirkby NSet al., 2019, Omic profiling in healthy volunteers taking celecoxib reveals novel biomarkers regulated by cyclooxygenase-2, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 1628-1628, ISSN: 0306-5251

Conference paper

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