97 results found
Huang Y, Gu B, Salles II, et al., 2021, Fibrinogen-mimicking, multi-arm nanovesicles for human thrombus-specific delivery of tissue plasminogen activator and targeted thrombolytic therapy, Science Advances, Vol: 7, ISSN: 2375-2548
Clinical use of tissue plasminogen activator (tPA) in thrombolytic therapy is limited by its short circulation time and hemorrhagic side effects. Inspired by fibrinogen binding to activated platelets, we report a fibrinogen-mimicking, multi-arm nanovesicle for thrombus-specific tPA delivery and targeted thrombolysis. This novel system is based on the lipid nanovesicle coated with polyethylene glycol (PEG) terminally conjugated with a cyclic RGD (cRGD) peptide. Our experiments with human blood demonstrated its highly selective binding to activated platelets and efficient tPA release at a thrombus site under both static and physiological flow conditions. Its clot dissolution time in a microfluidic system was comparable to that of free tPA. Furthermore, we report a purpose-built computational model capable of simulating targeted thrombolysis of the tPA-loaded nanovesicle and with potential in predicting the dynamics of thrombolysis in physiologically realistic scenarios. This combined experimental and computational work presents a promising platform for development of thrombolytic nanomedicines.
Khawaja AA, Taylor KA, Lovell AO, et al., 2020, HIV antivirals affect endothelial activation and endothelial-platelet crosstalk., Circulation Research, Vol: 127, Pages: 1365-1380, ISSN: 0009-7330
Rationale: People living with human immunodeficiency virus (PLHIV) on effective antiretroviral therapy are at increased risk of cardiovascular complications, possibly due to off-target drug effects. Some studies have associated antiretroviral therapy with increased risk of myocardial infarction and endothelial dysfunction, but a link between endothelial function and antiretrovirals has not been established. Objective: To determine the effects of antiretrovirals in common clinical use upon in vitro endothelial function in order to better understand cardiovascular risk in PLHIV. Methods and Results: Human umbilical cord vein endothelial cells (HUVEC) or human coronary artery endothelial cells (HCAEC) were pre-treated with the antiretrovirals abacavir sulphate (ABC), tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF). Expression of adhesion molecules, ectonucleotidases (CD39 and CD73), tissue factor (TF), endothelial-derived microparticle (EMP) numbers and phenotype, and platelet activation were evaluated by flow cytometry. TF and ectonucleotidase activities were measured using colourimetric plate-based assays. ABC-treated endothelial cells had higher levels of ICAM-1 and TF expression following TNF-α stimulation. In contrast, TDF and TAF treatment gave rise to greater populations of CD39+CD73+ cells. These cell surface differences were also observed within EMP repertoires. ABC-treated cells and EMP had greater TF activity, whilst TDF- and TAF-treated cells and EMP displayed higher ectonucleotidase activity. Finally, EMP isolated from ABC-treated cells enhanced collagen-evoked platelet integrin activation and α-granule release. Conclusions: We report differential effects of antiretrovirals used in the treatment of HIV upon endothelial function. ABC treatment led to an inflammatory, pro-thrombotic endothelial phenotype that promoted platelet activation. In contrast, TDF and TAF conferred potentially cardioprotective properties associated with
Khawaja AA, Maughan RT, Paschalaki KE, et al., 2020, Modelling endothelial function in vitro and via blood sampling to evaluate cardiovascular risk in people living with HIV, Publisher: WILEY, Pages: 39-39, ISSN: 1464-2662
Khawaja AA, Taylor KA, Lovell AO, et al., 2020, Differential effects of HIV antiretrovirals on human coronary artery endothelial cells, Publisher: WILEY, Pages: 7-7, ISSN: 1464-2662
Taylor K, Khawaja A, Lovell A, et al., 2020, Analysis of platelet and endothelial function after daily doravirine in HIV-negative volunteers, Publisher: WILEY, Pages: 51-51, ISSN: 1464-2662
du Sert NP, Hurst V, Ahluwalia A, et al., 2020, The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research, EXPERIMENTAL PHYSIOLOGY, Vol: 105, Pages: 1459-1466, ISSN: 0958-0670
Percie du Sert N, Hurst V, Ahluwalia A, et al., 2020, The ARRIVE guidelines 2.0: updated guidelines for reporting animal research, JOURNAL OF PHYSIOLOGY-LONDON, Vol: 598, Pages: 3793-3801, ISSN: 0022-3751
du Sert NP, Ahluwalia A, Alam S, et al., 2020, Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0, PLoS Biology, Vol: 18, Pages: 1-65, ISSN: 1544-9173
Improving the reproducibility of biomedical research is a major challenge. Transparent and accurate reporting is vital to this process; it allows readers to assess the reliability of the findings and repeat or build upon the work of other researchers. The ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments) were developed in 2010 to help authors and journals identify the minimum information necessary to report in publications describing in vivo experiments. Despite widespread endorsement by the scientific community, the impact of ARRIVE on the transparency of reporting in animal research publications has been limited. We have revised the ARRIVE guidelines to update them and facilitate their use in practice. The revised guidelines are published alongside this paper. This explanation and elaboration document was developed as part of the revision. It provides further information about each of the 21 items in ARRIVE 2.0, including the rationale and supporting evidence for their inclusion in the guidelines, elaboration of details to report, and examples of good reporting from the published literature. This document also covers advice and best practice in the design and conduct of animal studies to support researchers in improving standards from the start of the experimental design process through to publication.
du Sert NP, Hurst V, Ahluwalia A, et al., 2020, The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research*, JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM, Vol: 40, Pages: 1769-1777, ISSN: 0271-678X
du Sert NP, Hurst V, Ahluwalia A, et al., 2020, The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research, British Journal of Pharmacology, Vol: 177, Pages: 3617-3624, ISSN: 0007-1188
Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the “ARRIVE Essential 10,” which constitutes the minimum requirement, and the “Recommended Set,” which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration (E&E) document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.
Percie du Sert N, Hurst V, Ahluwalia A, et al., 2020, The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research, BMC Veterinary Research, Vol: 16, Pages: 1-7, ISSN: 1746-6148
Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the “ARRIVE Essential 10,” which constitutes the minimum requirement, and the “Recommended Set,” which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.
Khawaja AA, Maughan RT, Paschalaki KE, et al., 2020, Modelling endothelial function in vitro and via blood sampling to assess cardiovascular risk in people living with HIV, Publisher: JOHN WILEY & SONS LTD, Pages: 105-105
du Sert NP, Hurst V, Ahluwalia A, et al., 2020, The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research, PLOS BIOLOGY, Vol: 18, ISSN: 1544-9173
Khawaja A, Taylor K, Lovell A, et al., 2020, Differential effects of HIV antiretroviral drugs upon pro-inflammatory and cardioprotective properties of vascular endothelial cells, Meeting of the British-Pharmacological-Society, Publisher: WILEY, Pages: 2578-2579, ISSN: 0007-1188
Cleary SJ, Rauzi F, Smyth E, et al., 2020, Radiolabelling and immunohistochemistry reveal platelet recruitment into lungs and platelet migration into airspaces following LPS inhalation in mice, Journal of Pharmacological and Toxicological Methods, Vol: 102, Pages: 106660-106660, ISSN: 1056-8719
INTRODUCTION: Platelets are under investigation for their role in host defence and inflammatory lung diseases and have been demonstrated to be recruited to the lung. However, the mechanisms and consequences of platelet recruitment into lungs are poorly understood. We have utilised a murine model to investigate the mechanisms of platelet involvement in lung inflammation induced by intranasal administration of LPS. OBJECTIVES: Our aim was to characterise lung platelet recruitment following LPS inhalation in mice using immunohistochemistry, and non-invasive and invasive radiolabelled platelet tracking techniques. RESULTS: Intranasal administration of LPS caused an increase in lung platelet staining in lung tissue and elicited the recruitment of radiolabelled platelets into the lung. Prior to these responses in the lung, we observed an earlier decrease in blood platelet counts, temporally associated with platelet recruitment to the liver and spleen. Non-invasive measurements of thoracic radioactivity reflected changes in blood counts rather than extravascular lung platelet recruitment. However, both in situ counting of radiolabelled platelets and immunostaining for platelet surface markers showed LPS-induced increases in extravascular platelets into lung airspaces suggesting that some of the platelets recruited to the lung enter air spaces. CONCLUSIONS: Intranasal administration of LPS activates the innate immune response which includes a fall in peripheral blood platelet counts with subsequent platelet recruitment to the lung, spleen and liver, measured by immunohistochemistry and radiolabelling techniques.
Khawaja AA, Taylor KA, Lovell AO, et al., 2019, Distinct pro-inflammatory and cardio-protective effects of antiretroviral drugs in vascular endothelial cells, Publisher: WILEY, Pages: 230-230, ISSN: 1464-2662
Lovell AO, Taylor KA, Winston A, et al., 2019, Investigation of the impact of antiretroviral therapy upon platelet activation to determine HIV-associated cardiovascular risk, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 3047-3047, ISSN: 0007-1188
Khawaja A, Taylor K, Nelson M, et al., 2019, Abacavir sulphate and tenofovir disoproxil fumarate/alafenamide differentially regulate endothelial dysfunction, Publisher: WILEY, Pages: 24-24, ISSN: 1464-2662
Taylor KA, Smyth E, Rauzi F, et al., 2019, Pharmacological impact of antiretroviral therapy on platelet function to investigate HIV-associated cardiovascular risk, British Journal of Pharmacology, Vol: 176, Pages: 879-889, ISSN: 0007-1188
Background and purposeSome clinical studies have reported increased myocardial infarction in people living with HIV taking the antiretroviral abacavir sulphate (ABC). Given that clinical studies contain confounding variables (e.g. HIV status), we investigated the pharmacological impact of antiretrovirals on platelet function in HIV‐negative volunteers in order to identify mechanisms of increased cardiovascular risk.Experimental approachPlatelets were isolated from healthy volunteers and HIV‐negative subjects enrolled on a Phase I clinical trial and platelet function evaluated using aggregometry and flow cytometry. In vivo platelet thromboembolism was monitored in anaesthetised mice.Key resultsHuman platelet aggregation was unaffected by all antiretrovirals tested but ABC treatment led uniquely to increased platelet granule release. ABC also interrupted nitric oxide (NO)‐mediated inhibition of platelet aggregation and increased in vivo aggregation in mice. An alternative antiretroviral, tenofovir, did not affect platelet function. Furthermore, aggregation and activation of platelets isolated from twenty subjects taking clinically‐relevant doses of tenofovir were comparable to baseline samples.Conclusions and implicationsABC can enhance platelet activation, independently of HIV status suggesting a potential pharmacological effect that is absent with tenofovir. Mechanistically, we propose that ABC enhances platelet degranulation and interrupts NO‐mediated platelet inhibition. The interaction of ABC with NO signalling is supported by data demonstrating ABC‐mediated enhancement of aggregation in vivo and in vitro responses that persist in the presence of NO. Although an association between ABC and platelet activation has not been confirmed in patients, these findings provide evidence of a mechanistic link between platelet activation and antiretroviral therapy.
Khawaja AA, Taylor KA, Nelson M, et al., 2018, Differential Effects of Antiretroviral Drugs Upon Endothelial Activation and Microparticle Repertoire, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Gillespie S, Holloway PM, Becker F, et al., 2018, The isothiocyanate sulforaphane modulates platelet function and protects against cerebral thrombotic dysfunction, British Journal of Pharmacology, Vol: 175, Pages: 3333-3346, ISSN: 1476-5381
Background and purposePlatelet activation provides a critical link between inflammation and thrombosis. Sulforaphane (SFN), a naturally occurring isothiocyanate, has been shown to display both anti‐inflammatory and anti‐thrombotic actions in the systemic microvasculature. As inflammation promotes thrombosis and vice versa, this study investigates whether SFN is able to reduce inflammatory potentiation of thrombotic events, suppress platelet activation and thrombus formation in the cerebral microvasculature.Experimental approachThrombosis was induced in the murine brain using the light/dye‐injury model, in conjunction with lipopolysaccharide (LPS) treatment, with and without SFN treatment. In vitro and in vivo platelet assays (aggregation, flow and other functional tests) were also employed, using both human and murine platelets.Key ResultsSFN was found to reduce LPS mediated enhancement of thrombus formation in the cerebral microcirculation. In tail bleed experiments LPS treatment prolonged bleeding time, and SFN treatment was found to protect against this LPS‐induced derangement of platelet function. SFN inhibited collagen‐mediated platelet aggregation in vitro and in vivo and the associated adhesion and impaired calcium signalling. Furthermore, Glycoprotein VI (GPVI) is involved in the protective effects observed with SFN treatment.Conclusions and ImplicationsData presented here provides evidence for the use of SFN in preventing stroke in selected high‐risk patient cohorts.
du Sert NP, Hurst V, Ahluwalia A, et al., 2018, Revision of the ARRIVE guidelines: rationale and scope., BMJ Open Sci, Vol: 2
In 2010, the NC3Rs published the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines to improve the reporting of animal research. Despite considerable levels of support from the scientific community, the impact on the quality of reporting in animal research publications has been limited. This position paper highlights the strategy of an expert working group established to revise the guidelines and facilitate their uptake. The group's initial work will focus on three main areas: prioritisation of the ARRIVE items into a tiered system, development of an explanation and elaboration document, and revision of specific items.
Taylor KA, Emerson M, 2018, Refinement of a mouse cardiovascular model: development, application and dissemination, F1000Research, Vol: 7, ISSN: 2046-1402
European and UK legislation requires all animal procedures to be conductedwith consideration to reduction, refinement and replacement. In this review,3Rs developments are discussed in the field of platelet biology andthromboembolism. Platelet research requires the use of animal models, andmice are widely used in the field. When working , conventional lightin vitrotransmission techniques have been scaled down allowing reduction in animalnumbers. , vascular injury models are widely used and work is ongoing toIn vivodevelop approaches that use fewer animals. Thromboembolic mortalityex vivomodels, which inflict considerable pain and suffering, have also been usedwidely. A published and characterised refinement of this mortality model allowsreal-time monitoring of radiolabelled platelets under general anaesthesia andreduces both the severity level and the numbers of mice used in a typicalexperiment. This technique is more sensitive than the mortality approach andhas opened up new avenues of research, which would not have been feasibleby using death as an end-point. To drive uptake of real-time monitoring, a moresimplistic approach has been developed involving micro-sampling and cellcounting. Thromboembolic mortality models should therefore be consideredobsolete due to the emergence of 3Rs models with improved scientificoutcomes and that can be implemented relatively easily.
Rauzi F, Smyth E, Emerson M, 2017, Refinement of mouse protocols for the study of platelet thromboembolic responses in vivo, Thrombosis and Haemostasis, Vol: 117, Pages: 2283-2290, ISSN: 0340-6245
Mouse models of thromboembolism are frequently used to investigate platelet function in vivo and, according to European Union (EU) legislation, must be conducted in the context of replacement, refinement and reduction. We have previously developed a refined real-time mouse model of thromboembolism as an alternative to models of thromboembolic mortality which inflict considerable pain and suffering. Real-time monitoring involves infusion of radiolabelled platelets into the circulation of anaesthetized mice, and platelet aggregation is measured as increases in platelet-associated counts in the pulmonary vasculature following injection of platelet agonists. This gives a definitive data set on the tissue localization and extent of platelet activation. We developed an additional, more simplistic alternative to mortality models based on blood microsampling which entails the measurement of circulating platelet counts following agonist stimulation. Blood microsamples were collected from the tail vein of anaesthetized mice at three different time points leading to a reduction in animal numbers. Platelet counts significantly dropped 1 minute after stimulation with collagen or thrombin and were restored over 10 minutes. These results correlate with those obtained via real-time monitoring and were confirmed by immunohistochemistry. Pre-treatment of mice with aspirin significantly inhibited the decrease in platelet counts following collagen. These data suggest that blood microsampling may be implemented as a simplistic refined alternative to mortality models of thromboembolism when specialized monitoring equipment, or use of radioactive isotopes for real-time monitoring, which remains the ‘gold standard’, is not feasible. Microsampling refines and reduces animal procedures in compliance with current EU legislation
Smyth E, Solomon A, Birrell MA, et al., 2017, Effects of diesel exhaust particles on coagulation., British Journal of Pharmacology, Vol: 174, Pages: 4200-4200, ISSN: 1476-5381
Smyth E, Solomon A, Birrell MA, et al., 2017, Influence of inflammation and nitric oxide upon platelet aggregation following deposition of diesel exhaust particles in the airways., British Journal of Pharmacology, Vol: 174, Pages: 2130-2139, ISSN: 0007-1188
Background and Purpose: Exposure to nanoparticulate pollution has been implicated in platelet-driven thrombotic events such as myocardial infarction. Inflammation and impairment of NO bioavailability have been proposed as potential causative mechanisms. It is unclear, however, whether airways exposure to combustion-derived nanoparticles such as diesel exhaust particles (DEP) or carbon black (CB) can augment platelet aggregation in vivo and the underlying mechanisms remain undefined. We aimed to investigate the effects of acute lung exposure to DEP and CB on platelet activation and the associated role of inflammation and endothelial-derived NO.Experimental Approach: DEP and CB were intratracheally instilled into wild-type (WT) and eNOS−/− mice and platelet aggregation was assessed in vivo using an established model of radio-labelled platelet thromboembolism. The underlying mechanisms were investigated by measuring inflammatory markers, NO metabolites and light transmission aggregometry.Key Results: Platelet aggregation in vivo was significantly enhanced in WT and eNOS−/− mice following acute airways exposure to DEP but not CB. CB exposure, but not DEP, was associated with significant increases in pulmonary neutrophils and IL-6 levels in the bronchoalveolar lavage fluid and plasma of WT mice. Neither DEP nor CB affected plasma nitrate/nitrite concentration and DEP-induced human platelet aggregation was inhibited by an NO donor.Conclusions and Implications: Pulmonary exposure to DEP and subsequent platelet activation may contribute to the reports of increased cardiovascular risk, associated with exposure to airborne pollution, independent of its effects on inflammation or NO bioavailability.
Smyth EJ, Nelson M, Emerson M, 2015, Comparative impacts of antiretroviral HIV therapies on platelet function. Abstracts of the XXV Congress of the International Society on Thrombosis and Haemostasis., Journal of Thrombosis and Haemostasis, ISSN: 1538-7933
Emerson M, Ahmad O, Rauzi F, et al., 2015, Physiological regulation of platelet aggregation by the gasotransmitter hydrogen sulfide, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 13, Pages: 406-406, ISSN: 1538-7933
Smyth E, Nelson M, Emerson M, 2015, Comparative impacts of antiretroviral HIV therapies on platelet function, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 13, Pages: 651-652, ISSN: 1538-7933
Emerson M, Ilkan Z, Mustafa F, et al., 2015, Inhibitory regulation of platelets by hydrogen sulphide, 3rd European Conference on the Biology of Hydrogen Sulfide (H2S), Publisher: ACADEMIC PRESS INC ELSEVIER SCIENCE, Pages: S14-S15, ISSN: 1089-8603
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