Publications by authors named "Simon C Pitchford"

32 Publications

Platelets Independently Recruit into Asthmatic Lungs and Models of Allergic Inflammation via CCR3.

Am J Respir Cell Mol Biol 2021 Feb 8. Epub 2021 Feb 8.

King's College London, Pharmaceutical Sciences, London, United Kingdom of Great Britain and Northern Ireland;

Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, where leukocyte infiltration, airway remodelling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. To assess the mechanism of platelet recruitment to extravascular compartments of lungs from asthmatic patients, and following allergen challenge of house dust mite (HDM, containing the DerP1 allergen) sensitized mice; additionally we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice visualised platelets tagged with an anti-mouse CD49b-PE antibody. Platelet-endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsies taken from subjects with steroid naïve mild asthma. Platelets were significantly raised in lung parenchyma from patients with fatal asthma compared with post mortem control lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, adhesion; and recruitment of platelets into airway walls compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular non-aggregated platelets occurs in lungs of asthmatic patients. In allergic mice, platelet recruitment occurs via recognised vascular adhesive and migratory events, independently of leukocytes via a CCR3 dependent mechanism.
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http://dx.doi.org/10.1165/rcmb.2020-0425OCDOI Listing
February 2021

Red Blood Cells Elicit Platelet-Dependent Neutrophil Recruitment into Lung Airspaces.

Shock 2020 Dec 9. Epub 2020 Dec 9.

Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, King's College London, London, UK.

Haemolysis that occurs in intravascular haemolytic disorders, such as sickle cell disease and malaria, is associated with inflammation and platelet activation. Alveolar haemorrhage, for example following primary blast lung injury (PBLI) or acute respiratory distress syndrome (ARDS), results in the escape of erythrocytes (RBCs) into alveolar spaces, where they subsequently lyse and release their intracellular contents. However, the inflammatory effects of RBCs in the airways are not fully understood. We hypothesized that RBCs in the airway induce an inflammatory response, associated with platelet activation. By instilling whole RBCs or lysed RBCs into the airways of mice, we have demonstrated that whole RBCs elicit macrophage accumulation in the lung. However, lysed RBCs induce significant inflammatory cell recruitment, particularly neutrophils and this was associated with a 50% increase in circulating platelet neutrophil complexes (PNCs). Platelet depletion prior to lysed RBC exposure in the lung resulted in reduced neutrophil recruitment, suggesting that the presence of intracellular RBC components in the airways can elicit inflammation that is platelet dependent. To identify specific platelet dependent signalling pathways involved in neutrophil recruitment, anti-P-selectin ligand and anti-PSGL1 blocking antibodies were tested, however neither affected neutrophil recruitment. These findings implicate an involvement for other, as yet unidentified platelet-dependent signalling and adhesion mechanisms. Further understanding of how platelets contribute to lung inflammation induced by the presence of RBCs could offer novel therapeutic approaches to attenuate inflammation that occurs in conditions associated with alveolar haemorrhage.
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http://dx.doi.org/10.1097/SHK.0000000000001705DOI Listing
December 2020

A pleurocidin analogue with greater conformational flexibility, enhanced antimicrobial potency and in vivo therapeutic efficacy.

Commun Biol 2020 Nov 27;3(1):697. Epub 2020 Nov 27.

Institute of Pharmaceutical Science, School of Cancer & Pharmaceutical Science, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK.

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.
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http://dx.doi.org/10.1038/s42003-020-01420-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699649PMC
November 2020

Animal models of mechanisms of SARS-CoV-2 infection and COVID-19 pathology.

Br J Pharmacol 2020 11 19;177(21):4851-4865. Epub 2020 Jul 19.

Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, School of Cancer and Pharmaceutical Sciences, King's College London, London, UK.

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 infections has led to a substantial unmet need for treatments, many of which will require testing in appropriate animal models of this disease. Vaccine trials are already underway, but there remains an urgent need to find other therapeutic approaches to either target SARS-CoV-2 or the complications arising from viral infection, particularly the dysregulated immune response and systemic complications which have been associated with progression to severe COVID-19. At the time of writing, in vivo studies of SARS-CoV-2 infection have been described using macaques, cats, ferrets, hamsters, and transgenic mice expressing human angiotensin I converting enzyme 2 (ACE2). These infection models have already been useful for studies of transmission and immunity, but to date only partly model the mechanisms involved in human severe COVID-19. There is therefore an urgent need for development of animal models for improved evaluation of efficacy of drugs identified as having potential in the treatment of severe COVID-19. These models need to reproduce the key mechanisms of COVID-19 severe acute respiratory distress syndrome and the immunopathology and systemic sequelae associated with this disease. Here, we review the current models of SARS-CoV-2 infection and COVID-19-related disease mechanisms and suggest ways in which animal models can be adapted to increase their usefulness in research into COVID-19 pathogenesis and for assessing potential treatments. LINKED ARTICLES: This article is part of a themed issue on The Pharmacology of COVID-19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc.
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http://dx.doi.org/10.1111/bph.15143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283621PMC
November 2020

Editorial overview: Emerging anti-inflammatory approaches for the treatment of respiratory diseases.

Curr Opin Pharmacol 2019 06 2;46:iii-v. Epub 2019 Jul 2.

Institute of Pharmaceutical Science, School of Cancer and Pharmaceutical Sciences, King's College London, London, SE1 9NH, UK. Electronic address:

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http://dx.doi.org/10.1016/j.coph.2019.06.004DOI Listing
June 2019

LPS-induced Lung Platelet Recruitment Occurs Independently from Neutrophils, PSGL-1, and P-Selectin.

Am J Respir Cell Mol Biol 2019 08;61(2):232-243

1Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science and.

Platelets are recruited to inflammatory foci and contribute to host defense and inflammatory responses. Compared with platelet recruitment in hemostasis and thrombosis, the mechanisms of platelet recruitment in inflammation and host defense are poorly understood. Neutrophil recruitment to lung airspaces after inhalation of bacterial LPS requires platelets and PSGL-1 in mice. Given this association between platelets and neutrophils, we investigated whether recruitment of platelets to lungs of mice after LPS inhalation was dependent on PSGL-1, P-selectin, or interaction with neutrophils. BALB/c mice were administered intranasal LPS (O55:B5, 5 mg/kg) and, 48 hours later, lungs were collected and platelets and neutrophils quantified in tissue sections by immunohistochemistry. The effects of functional blocking antibody treatments targeting the platelet-neutrophil adhesion molecules, P-selectin or PSGL-1, or treatment with a neutrophil-depleting antibody targeting Ly6G, were tested on the extent of LPS-induced lung platelet recruitment. Separately in Pf4-Cre × mTmG mice, two-photon intravital microscopy was used to image platelet adhesion in live lungs. Inhalation of LPS caused both platelet and neutrophil recruitment to the lung vasculature. However, decreasing lung neutrophil recruitment by blocking PSGL-1, P-selectin, or depleting blood neutrophils had no effect on lung platelet recruitment. Lung intravital imaging revealed increased adhesion of platelets in the lung microvasculature which was not associated with thrombus formation. In conclusion, platelet recruitment to lungs in response to LPS occurs through mechanisms distinct from those mediating neutrophil recruitment, or the occurrence of pulmonary emboli.
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http://dx.doi.org/10.1165/rcmb.2018-0182OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6670039PMC
August 2019

A dichotomy in platelet activation: Evidence of different functional platelet responses to inflammatory versus haemostatic stimuli.

Thromb Res 2018 12 25;172:110-118. Epub 2018 Oct 25.

Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, Room 5.06 Franklin-Wilkins Building, Waterloo Campus, King's College London, London SE1 9NH, UK.

Introduction: Platelets participate in inflammatory disorders through a variety of different functional responses, including chemotaxis, platelet-leukocyte complex formation and facilitation of leukocyte recruitment that are thought to be distinct from platelet aggregation. This may account for why classical anti-platelet drugs have failed to ameliorate inflammatory disorders where platelets are known to participate, suggesting that distinct pathways may control inflammatory and haemostatic functions of platelets. In the present study, we have therefore investigated the effect of different stimuli on several different functions of platelets preferentially involved either in haemostasis or in inflammation.

Materials And Methods: Human platelets were stimulated with either inflammatory (fMLP, histamine, IL-1β, LPS, MDC/CCL22, SDF-1α/CXCL12 and 5-HT) or haemostatic (ADP, collagen, convulxin, epinephrine, TRAP-6 and U46619) stimuli. Aggregation, platelet-leukocyte complex formation, platelet migration and platelet protein phosphorylation were assessed.

Results: Haemostatic stimuli induced platelet aggregation, whilst inflammatory agonists induced platelet migration. The haemostatic stimuli, with the exception of epinephrine, and some of the inflammatory stimuli induced platelet-leukocyte complex formation, even if to a different extent. Furthermore, inflammatory stimuli induced a shorter lasting profile of platelet protein phosphorylation compared with haemostatic stimuli.

Conclusions: Stimulation of platelets with inflammatory stimuli triggers the activation of non haemostatic functions different from those induced by haemostatic stimuli, supporting the existence of alternative platelet responses depending on the stimulus (haemostatic or inflammatory). A deeper understanding of the biochemical pathways behind these functional differences may lead to the development of novel therapeutic options targeting the inflammatory actions of platelets, without affecting their critical role in haemostasis.
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http://dx.doi.org/10.1016/j.thromres.2018.10.019DOI Listing
December 2018

Sensory nerves mediate spontaneous behaviors in addition to inflammation in a murine model of psoriasis.

FASEB J 2019 02 11;33(2):1578-1594. Epub 2018 Sep 11.

British Heart Foundation (BHF) Cardiovascular Centre of Research Excellence, Vascular Biology and Inflammation Section, King's College London, London, United Kingdom.

Psoriasis is characterized by keratinocyte hyperproliferation, erythema, as well as a form of pruritus, involving cutaneous discomfort. There is evidence from both clinical and murine models of psoriasis that chemical or surgical depletion of small-diameter sensory nerves/nociceptors benefits the condition, but the mechanisms are unclear. Hence, we aimed to understand the involvement of sensory nerve mediators with a murine model of psoriasis and associated spontaneous behaviors, indicative of cutaneous discomfort. We have established an Aldara model of psoriasis in mice and chemically depleted the small-diameter nociceptors in a selective manner. The spontaneous behaviors, in addition to the erythema and skin pathology, were markedly improved. Attenuated inflammation was associated with reduced dermal macrophage influx and production of reactive oxygen/nitrogen species (peroxynitrite and protein nitrosylation). Subsequently, this directly influenced observed behavioral responses. However, the blockade of common sensory neurogenic mechanisms for transient receptor potential (TRP)V1, TRPA1, and neuropeptides (substance P and calcitonin gene-related peptide) using genetic and pharmacological approaches inhibited the behaviors but not the inflammation. Thus, a critical role of the established sensory TRP-neuropeptide pathway in influencing cutaneous discomfort is revealed, indicating the therapeutic potential of agents that block that pathway. The ongoing inflammation is mediated by a distinct sensory pathway involving macrophage activation.-Kodji, X., Arkless, K. L., Kee, Z., Cleary, S. J., Aubdool, A. A., Evans, E., Caton, P., Pitchford, S. C., Brain, S. D. Sensory nerves mediate spontaneous behaviors in addition to inflammation in a murine model of psoriasis.
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http://dx.doi.org/10.1096/fj.201800395RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338626PMC
February 2019

Diverse signalling of the platelet P2Y receptor leads to a dichotomy in platelet function.

Eur J Pharmacol 2018 May 11;827:58-70. Epub 2018 Mar 11.

Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, King's College London, London SE1 9NH, United Kingdom. Electronic address:

Platelet P2Y receptor signalling via RhoGTPases is necessary for platelet-dependent leukocyte recruitment, where no platelet aggregation is observed. We investigated signalling cascades involved in distinct P2Y-dependent platelet activities in vitro, using specific inhibitors for phospholipase C (PLC) (U73122, to inhibit the canonical pathway), and RhoGTPases: Rac1 (NSC23766) and RhoA (ROCK inhibitor GSK429286). Human platelet rich plasma (for platelet aggregation) or isolated washed platelets (for chemotaxis assays) was treated with U73122, GSK429286 or NSC23766 prior to stimulation with adenosine diphosphate (ADP) or the P2Y specific agonist MRS2365. Aggregation, chemotaxis (towards f-MLP), or platelet-induced human neutrophil chemotaxis (PINC) towards macrophage derived chemokine (MDC) was assessed. Molecular docking of ADP and MRS2365 to P2Y was analysed using AutoDock Smina followed by GOLD molecular docking in the Accelrys Discovery Studio software. Inhibition of PLC, but not Rac1 or RhoA, suppressed platelet aggregation induced by ADP and MRS2365. In contrast, platelet chemotaxis and PINC, were significantly attenuated by inhibition of platelet Rac1 or RhoA, but not PLC. MRS2365, compared to ADP had a less pronounced effect on P2Y-induced aggregation, but a similar efficacy to stimulate platelet chemotaxis and PINC, which might be explained by differences in molecular interaction of ADP compared to MRS2365 with the P2Y receptor. Platelet P2Y receptor activation during inflammation signals through alternate pathways involving Rho GTPases in contrast to canonical P2Y receptor induced PLC signalling. This might be explained by selective molecular interactions of ligands within the orthosteric site of the P2Y receptor.
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http://dx.doi.org/10.1016/j.ejphar.2018.03.014DOI Listing
May 2018

Platelets Play a Central Role in Sensitization to Allergen.

Am J Respir Cell Mol Biol 2018 07;59(1):96-103

Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, King's College London, London, United Kingdom.

Platelet activation occurs in patients with allergic inflammation, and platelets can be activated directly by allergen via an IgE-dependent process. Platelets have been shown to activate APCs such as CD11c dendritic cells in vitro. Although CD11c dendritic cells are a requisite for allergen sensitization, the role of platelets in this process is unknown. In this study, we investigated whether platelets were necessary for allergen sensitization. Balb/c mice sensitized to ovalbumin were exposed to subsequent aerosolized allergen (ovalbumin challenge). We analyzed lung CD11c cell activation, colocalization with platelets, and some other indices of inflammation. The role of platelets at the time of allergen sensitization was assessed through platelet depletion experiments restricted to the period of sensitization. Platelets colocalized with airway CD11c cells, and this association increased after allergen sensitization as well as after subsequent allergen exposure. Temporary platelet depletion (>95%) at the time of allergen sensitization led to a suppression of IgE and IL-4 synthesis and to a decrease in the pulmonary recruitment of eosinophils, macrophages, and lymphocytes after subsequent allergen exposure. Furthermore, in mice previously depleted of platelets at the time of sensitization, the recovered platelet population was shown to have reduced expression of FcεRI. Pulmonary CD11c cell recruitment was suppressed in these mice after allergen challenge, suggesting that the migration of CD11c cells in vivo may be dependent on direct platelet recognition of allergen. We conclude that platelets are necessary for efficient host sensitization to allergen. This propagates the subsequent inflammatory response during secondary allergen exposure and increases platelet association with airway CD11c cells.
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http://dx.doi.org/10.1165/rcmb.2017-0401OCDOI Listing
July 2018

Platelet Depletion Impairs Host Defense to Pulmonary Infection with Pseudomonas aeruginosa in Mice.

Am J Respir Cell Mol Biol 2018 03;58(3):331-340

1 Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science and.

Platelets have been implicated in pulmonary inflammatory cell recruitment after exposure to allergic and nonallergic stimuli, but little is known about the role of platelets in response to pulmonary infection with Pseudomonas aeruginosa. In this study, we have investigated the impact of the experimental depletion of circulating platelets on a range of inflammatory and bacterial parameters, and their subsequent impact on mortality in a murine model of pulmonary infection with P. aeruginosa. P. aeruginosa infection in mice induced a mild, but significant, state of peripheral thrombocytopenia in addition to pulmonary platelet accumulation. Increased platelet activation was detected in infected mice through increased levels of the platelet-derived mediators, platelet factor-4 and β-thromboglobulin, in BAL fluid and blood plasma. In mice depleted of circulating platelets, pulmonary neutrophil recruitment was significantly reduced 24 hours after infection, whereas the incidence of systemic dissemination of bacteria was significantly increased compared with non-platelet-depleted control mice. Furthermore, mortality rates were increased in bacterial-infected mice depleted of circulating platelets. This work demonstrates a role for platelets in the host response toward a gram-negative bacterial respiratory infection.
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http://dx.doi.org/10.1165/rcmb.2017-0083OCDOI Listing
March 2018

Platelet-Eosinophil Interactions As a Potential Therapeutic Target in Allergic Inflammation and Asthma.

Front Med (Lausanne) 2017 8;4:129. Epub 2017 Aug 8.

Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, King's College London, London, United Kingdom.

The importance of platelet activation during hemostasis is well understood. An understanding of these mechanisms has led to the use of several classes of anti-platelet drugs to inhibit aggregation for the prevention of thrombi during cardiovascular disease. It is now also recognized that platelets can function very differently during inflammation, as part of their role in the innate immune response against pathogens. This dichotomy in platelet function occurs through distinct physiological processes and alternative signaling pathways compared to that of hemostasis (leading to platelet aggregation) and is manifested as increased rheological interactions with leukocytes, the ability to undergo chemotaxis, communication with antigen-presenting cells, and direct anti-pathogen responses. Mounting evidence suggests platelets are also critical in the pathogenesis of allergic diseases such as asthma, where they have been associated with antigen presentation, bronchoconstriction, bronchial hyperresponsiveness, airway inflammation, and airway remodeling in both clinical and experimental studies. In particular, platelets have been reported bound to eosinophils in the blood of patients with asthma and the incidence of these events increases after both spontaneous asthma attacks in a biphasic manner, or after allergen challenge in the clinic. Platelet depletion in animal models of allergic airway inflammation causes a profound reduction in eosinophil recruitment to the lung, suggesting that the association of platelets with eosinophils is indeed an important event during eosinophil activation. Furthermore, in cases of severe asthma, and in animal models of allergic airways inflammation, platelet-eosinophil complexes move into the lung through a platelet P-selectin-mediated, eosinophil β1-integrin activation-dependent process, while platelets increase adherence of eosinophils to the vascular endothelium , demonstrating a clear interaction between these cell types in allergic inflammatory diseases. This review will explore non-thrombotic platelet activation in the context of allergy and the association of platelets with eosinophils, to reveal how these phenomena may lead to the discovery of novel therapeutic targets.
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http://dx.doi.org/10.3389/fmed.2017.00129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550710PMC
August 2017

Base-modified UDP-sugars reduce cell surface levels of P-selectin glycoprotein 1 (PSGL-1) on IL-1β-stimulated human monocytes.

Glycobiology 2016 10 27;26(10):1059-1071. Epub 2016 May 27.

Department of Chemistry, Faculty of Natural & Mathematical Sciences, King's College London, Britannia House, 7 Trinity Street, London, SE1 1DB, UK

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a cell-surface glycoprotein that is expressed, either constitutively or inducibly, on all myeloid and lymphoid cell lineages. PSGL-1 is implicated in cell-cell interactions between platelets, leukocytes and endothelial cells, and a key mediator of inflammatory cell recruitment and transmigration into tissues. Here, we have investigated the effects of the β-1,4-galactosyltransferase inhibitor 5-(5-formylthien-2-yl) UDP-Gal (5-FT UDP-Gal, compound 1: ) and two close derivatives on the cell surface levels of PSGL-1 on human peripheral blood mononuclear cells (hPBMCs). PSGL-1 levels were studied both under basal conditions, and upon stimulation of hPBMCs with interleukin-1β (IL-1β). Between 1 and 24 hours after IL-1β stimulation, we observed initial PSGL-1 shedding, followed by an increase in PSGL-1 levels on the cell surface, with a maximal window between IL-1β-induced and basal levels after 72 h. All three inhibitors reduce PSGL-1 levels on IL-1β-stimulated cells in a concentration-dependent manner, but show no such effect in resting cells. Compound 1: also affects the cell surface levels of adhesion molecule CD11b in IL-1β-stimulated hPBMCs, but not of glycoproteins CD14 and CCR2. This activity profile may be linked to the inhibition of global Sialyl Lewis presentation on hPBMCs by compound 1: , which we have also observed. Although this mechanistic explanation remains hypothetical at present, our results show, for the first time, that small molecules can discriminate between IL-1β-induced and basal levels of cell surface PSGL-1. These findings open new avenues for intervention with PSGL-1 presentation on the cell surface of primed hPBMCs and may have implications for anti-inflammatory drug development.
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http://dx.doi.org/10.1093/glycob/cww053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072147PMC
October 2016

Uncharged nucleoside inhibitors of β-1,4-galactosyltransferase with activity in cells.

Chem Commun (Camb) 2016 Mar 16;52(20):3955-8. Epub 2016 Feb 16.

Department of Chemistry, King's College London, Faculty of Natural & Mathematical Sciences, Britannia House, 7 Trinity Street, London, SE1 1DB, UK.

We report 5-substituted uridine derivatives as novel, uncharged inhibitors of β-1,4-galactosyltransferase and chemical tools for cellular applications. The new inhibitors reduce P-selectin glycoprotein 1 (PSGL-1) expression in human monocytes. Our results also provide novel insights into a unique mode of glycosyltransferase inhibition.
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http://dx.doi.org/10.1039/c5cc09289bDOI Listing
March 2016

The stop clock of platelet activation.

Blood 2015 Dec;126(24):2538-9

KING'S COLLEGE LONDON.

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http://dx.doi.org/10.1182/blood-2015-10-672634DOI Listing
December 2015

P-Rex and Vav Rac-GEFs in platelets control leukocyte recruitment to sites of inflammation.

Blood 2015 Feb 23;125(7):1146-58. Epub 2014 Dec 23.

Signalling Programme, Babraham Institute, Cambridge, United Kingdom; and.

The small GTPase Rac is required for neutrophil recruitment during inflammation, but its guanine-nucleotide exchange factor (GEF) activators seem dispensable for this process, which led us to investigate the possibility of cooperation between Rac-GEF families. Thioglycollate-induced neutrophil recruitment into the peritoneum was more severely impaired in P-Rex1(-/-) Vav1(-/-) (P1V1) or P-Rex1(-/-) Vav3(-/-) (P1V3) mice than in P-Rex null or Vav null mice, suggesting cooperation between P-Rex and Vav Rac-GEFs in this process. Neutrophil transmigration and airway infiltration were all but lost in P1V1 and P1V3 mice during lipopolysaccharide (LPS)-induced pulmonary inflammation, with altered intercellular adhesion molecule 1-dependent slow neutrophil rolling and strongly reduced L- and E-selectin-dependent adhesion in airway postcapillary venules. Analysis of adhesion molecule expression, neutrophil adhesion, spreading, and migration suggested that these defects were only partially neutrophil-intrinsic and were not obviously involving vascular endothelial cells. Instead, P1V1 and P1V3 platelets recapitulated the impairment of LPS-induced intravascular neutrophil adhesion and recruitment, showing P-Rex and Vav expression in platelets to be crucial. Similarly, during ovalbumin-induced allergic inflammation, pulmonary recruitment of P1V1 and P1V3 eosinophils, monocytes, and lymphocytes was compromised in a platelet-dependent manner, and airway inflammation was essentially abolished, resulting in improved airway responsiveness. Therefore, platelet P-Rex and Vav family Rac-GEFs play important proinflammatory roles in leukocyte recruitment.
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http://dx.doi.org/10.1182/blood-2014-07-591040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4326774PMC
February 2015

RhoA signaling through platelet P2Y₁ receptor controls leukocyte recruitment in allergic mice.

J Allergy Clin Immunol 2015 Feb 31;135(2):528-38. Epub 2014 Oct 31.

Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, King's College London, London, United Kingdom. Electronic address:

Background: Clinical studies reveal platelet activation in patients with asthma, allergic rhinitis, and eczema. This is distinct from platelet aggregation, which is critical for the maintenance of hemostasis and in which a role for platelet purinergic receptors is well documented. However, purines are also essential for inflammatory cell trafficking in animal models of allergic lung inflammation, which are known to be platelet dependent, yet the role of purines in the platelet activation accompanying inflammation is unknown.

Objectives: We investigated whether the involvement of purine activation of platelets during allergic inflammation is distinct from purine involvement in platelet aggregation.

Methods: BALB/c mice were sensitized to ovalbumin and subsequent airway ovalbumin challenge. Bronchoalveolar lavage fluid was analyzed for inflammatory cells, and blood samples were assessed for platelet activation. The role of platelet purinergic receptors and associated signaling mechanisms (RhoA) were assessed.

Results: P2Y₁, but not P2Y₁₂ or P2X₁, antagonism inhibited pulmonary leukocyte recruitment. The formation of platelet-leukocyte complexes in vivo and platelet/P-selectin-dependent polymorphonuclear cell migration in vitro were exclusively platelet P2Y₁ receptor dependent. Furthermore, platelet P2Y₁ activation resulted in RhoA activity in vivo after allergen challenge, and RhoA signaling in platelets through P2Y₁ stimulation was required for platelet-dependent leukocyte chemotaxis in vitro. Leukocyte recruitment in thrombocytopenic mice remained suppressed after reinfusion of platelets pretreated with a P2Y₁ antagonist or a Rho-associated kinase 1 inhibitor, confirming the crucial role of platelet P2Y₁ receptor and subsequent activation of RhoA.

Conclusion: RhoA signaling downstream of platelet P2Y₁, but not P2Y₁₂, represents a clear dichotomy in platelet activation during allergic inflammation versus hemostasis.
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http://dx.doi.org/10.1016/j.jaci.2014.09.032DOI Listing
February 2015

Combinatorial stem cell mobilization in animal models.

Methods Mol Biol 2012 ;904:139-54

Leukocyte Biology Section, Faculty of Medicine National Heart and Lung Institute, Imperial College London, London, UK.

It has long been recognized that single therapies, such as G-CSF, have a limited capacity to mobilize hematopoietic progenitor cells from the bone marrow. As a consequence in ∼20% of patients insufficient numbers of HPCs are mobilized to perform a bone marrow transplant. Recent studies have shown synergistic mobilization of HPCs when G-CSF pretreatment is combined with acute administration of a CXCR4 antagonist suggesting that combinatorial therapies may have therapeutic potential. In addition to HPCs, endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) reside in the bone marrow. These progenitor cells contribute to tissue regeneration and there is currently much interest in identifying the factors and mechanisms that regulate their mobilization. We describe a methodology for an in situ perfusion system of the mouse hind limb that permits direct quantification of stem and progenitor cell egress from the bone marrow. Progenitor cells are quantified by colony forming assays and immunohistochemistry. A strength of the methodology described is the ability to simultaneously quantify the mobilization of HPCs, EPCs and MSCs. Using this system we have shown that it is possible to achieve differential mobilization of these stem cell subsets using discrete combination therapies. Identification of such novel pharmacological regimens that stimulate the selective mobilization of EPCs and MSCs might be exploited in the future for tissue regeneration.
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http://dx.doi.org/10.1007/978-1-61779-943-3_12DOI Listing
December 2012

VEGFR1 stimulates a CXCR4-dependent translocation of megakaryocytes to the vascular niche, enhancing platelet production in mice.

Blood 2012 Oct 31;120(14):2787-95. Epub 2012 May 31.

Leukocyte Biology Section, National Heart and Lung Institute, Imperial College, London, UK.

It has previously been reported that VEGF-A stimulates megakaryocyte (MK) maturation in vitro. Here we show that treatment of mice with the isoform VEGF-A(165) resulted in a significant increase in circulating numbers of platelets. Using specific VEGFR1 and VEGFR2 blocking mAbs and selective VEGFR1 and 2 agonists, PlGF-2 and VEGF-E, respectively, we show directly that stimulation of VEGFR1, but not VEGFR2, increases circulating platelet numbers in vivo. Using flow cytometric analysis of harvested MKs, we show that while PlGF does not change the absolute numbers of MKs present in the bone marrow and the spleen, it increases both their maturation and cell-surface expression of CXCR4 in the bone marrow. Histology of the bone marrow revealed a redistribution of MKs from the endosteal to the vascular niche in response to both VEGF-A(165) and PlGF-2 treatment in vivo. Antagonism of CXCR4 suppressed both the VEGFR1-stimulated redistribution of megakyocytes within the bone marrow compartment and the VEGF-A(165)-induced thrombocytosis. In conclusion, we define a novel proinflammatory VEGFR1-mediated pathway that stimulates the maturation and up-regulation of CXCR4 on megakaryocytes, leading to their redistribution within the bone marrow environment, thereby enhancing platelet production in vivo.
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http://dx.doi.org/10.1182/blood-2011-09-378174DOI Listing
October 2012

Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration.

J Appl Physiol (1985) 2010 Sep 17;109(3):758-67. Epub 2010 Jun 17.

Sackler Institute of Pulmonary Pharmacology and Therapeutics, Division of Pharmaceutical Sciences, School of Biomedical and Health Sciences, King’s College, London, UK.

Previous studies in our laboratory have shown that platelets are essential for the migration of eosinophils into the lungs of allergic mice, and that this is dependent on the functional expression of platelet P-selectin. We sought to investigate whether the same is true for nonallergic, acute inflammatory stimuli administered to distinct anatomic compartments. Neutrophil trafficking was induced in two models, namely zymosan-induced peritonitis and LPS-induced lung inflammation, and the platelet dependence of these responses investigated utilizing mice rendered thrombocytopenic. The relative contribution of selectins was also investigated. The results presented herein clearly show that platelet depletion (>90%) significantly inhibits neutrophil recruitment in both models. In addition, we show that P-selectin glycoprotein ligand-1, but not P-selectin, is essential for neutrophil recruitment in mice in vivo, thus suggesting the existence of different regulatory mechanisms for the recruitment of leukocyte subsets in response to allergic and nonallergic stimuli. Further studies in human blood demonstrate that low-dose prothrombotic and pro-inflammatory stimuli (CCL17 or CCL22) synergize to induce platelet and neutrophil activation, as well as the formation of platelet-neutrophil conjugates. We conclude that adhesion between platelets and neutrophils in vivo is an important event in acute inflammatory responses. Targeting this interaction may be a successful strategy for inflammatory conditions where current therapy fails to provide adequate treatment.
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http://dx.doi.org/10.1152/japplphysiol.01086.2009DOI Listing
September 2010

Troubleshooting: Quantification of mobilization of progenitor cell subsets from bone marrow in vivo.

J Pharmacol Toxicol Methods 2010 Mar-Apr;61(2):113-21. Epub 2010 Feb 6.

Sackler Institute of Pulmonary Pharmacology, Division of Pharmaceutical Sciences, King's College London, SE1 9NH, UK.

Introduction: The molecular mechanisms that control the mobilization of specific stem cell subsets from the bone marrow are currently being intensely investigated. It is anticipated that boosting the mobilization of these stem cells via pharmacological intervention will not only produce more effective strategies for bone marrow transplant patients, but also provide novel therapeutic approaches for tissue regeneration.

Methods: Measurement of stem cell mobilization by sampling peripheral blood is problematic because it is technically difficult to accurately determine absolute numbers of rare progenitor cells by blood sampling. Furthermore a rise in progenitors may be caused by release of stem cells from tissues other than the bone marrow (e.g. spleen and adipose), or indeed an inhibition of stem cell homing back to the bone marrow or other tissues. Finally it is not possible to distinguish whether the pharmacological agent is acting directly at the level of the bone marrow or mobilizing progenitors by a distinct indirect mechanism. To resolve these problems, we have developed a technique that allows perfusion of the vasculature of the hind limb bone marrow in situ in mice. In this system, the femoral artery and vein are cannulated in situ such that the femur and tibia bone marrow are perfused in isolation under anaesthesia. As such, pharmacological agents can be administered directly into the bone marrow vasculature. Mobilized cells are then collected via the femoral vein and colony assays performed in defined growth media to allow identification of haematopoietic, endothelial, and mesenchymal progenitor cells. We have used this system to determine the ability of a CXCR4 antagonist to mobilize these distinct types of progenitor cells from the bone marrow of mice pre-conditioned with either G-CSF or VEGF.

Results And Conclusion: This isolated hind limb perfusion system has allowed comparisons to be made between cytokines (G-CSF and VEGF) that act chronically, either alone or in combination with agents that act acutely on the bone marrow (CXCR4 antagonist) on their ability to directly mobilize specific populations of stem cells. Data obtained therefore gives a more accurate understanding of the efficacy of different mobilizing strategies compared to peripheral blood analysis.
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http://dx.doi.org/10.1016/j.vascn.2010.01.013DOI Listing
June 2010

Deficiency of bone marrow beta3-integrin enhances non-functional neovascularization.

J Pathol 2010 Mar;220(4):435-45

Histopathology Unit, Cancer Research UK London Research Institute, UK.

beta3-Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in beta3-integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on beta3-null endothelial cells. Here we transplanted beta3-null bone marrow (BM) into wild-type (WT) mice to dissect the role of BM beta3-integrin deficiency in pathological angiogenesis. Mice transplanted with beta3-null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow beta3-integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM beta3-integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with beta3-null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with beta3-null bone marrow a significant proportion of tumour blood vessels are non-functional when compared with tumour blood vessels in WT-transplanted controls. Furthermore, beta3-null-transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT-transplanted animals. BM beta3-integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF-induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with beta3-null bone marrow when compared with WT-transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in beta3-null-transplanted mice. In conclusion, although BM beta3-integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM beta3-integrin in VEGF-induced mobilization of bone marrow-derived cells to the peripheral circulation and for the functionality of those vessels in which BM-derived cells become incorporated.
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http://dx.doi.org/10.1002/path.2660DOI Listing
March 2010

Asthma: what's the bleeding point?

Thorax 2009 Dec;64(12):1014-5

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http://dx.doi.org/10.1136/thx.2009.120279DOI Listing
December 2009

CXCR2 mediates the recruitment of endothelial progenitor cells during allergic airways remodeling.

Stem Cells 2009 Dec;27(12):3074-81

Leukocyte Biology Section, National Heart and Lung Institute, Imperial College London, United Kingdom.

Airway remodeling is a central feature of asthma and includes the formation of new peribronchial blood vessels, which is termed angiogenesis. In a number of disease models, bone marrow-derived endothelial progenitor cells (EPCs) have been shown to contribute to the angiogenic response. In this study we set out to determine whether EPCs were recruited into the lungs in a model of allergic airways disease and to identify the factors regulating EPC trafficking in this model. We observed a significant increase in the number of peribronchial blood vessels at day 24, during the acute inflammatory phase of the model. This angiogenic response was associated with an increase in the quantity of EPCs recoverable from the lung. These EPCs formed colonies after 21 days in culture and were shown to express CD31, von Willebrand factor, and vascular endothelial growth factor (VEGF) receptor 2, but were negative for CD45 and CD14. The influx in EPCs was associated with a significant increase in the proangiogenic factors VEGF-A and the CXCR2 ligands, CXCL1 and CXCL2. However, we show directly that, while the CXCL1 and CXCL2 chemokines can recruit EPCs into the lungs of allergen-sensitized mice, VEGF-A was ineffective in this respect. Further, the blockade of CXCR2 significantly reduced EPC numbers in the lungs after allergen exposure and led to a decrease in the numbers of peribronchial blood vessels after allergen challenge with no effect on inflammation. The data presented here provide in vivo evidence that CXCR2 is critical for both EPC recruitment and the angiogenic response in this model of allergic inflammation of the airways.
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http://dx.doi.org/10.1002/stem.222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3385349PMC
December 2009

Differential mobilization of subsets of progenitor cells from the bone marrow.

Cell Stem Cell 2009 Jan;4(1):62-72

Leukocyte Biology Section, National Heart and Lung Institute, Imperial College, London SW7 2AZ, UK.

G-CSF stimulates mobilization of hematopoietic progenitor cells (HPCs) from bone marrow by disrupting the CXCR4/SDF-1alpha retention axis. We show here that distinct factors and mechanisms regulate the mobilization of endothelial (EPCs) and stromal progenitor cells (SPCs). Pretreatment of mice with VEGF did not disrupt the CXCR4/SDF-1alpha chemokine axis but stimulated entry of HPCs into the cell cycle via VEGFR1, reducing their migratory capacity in vitro and suppressing their mobilization in vivo. In contrast, VEGF pretreatment enhanced EPC mobilization via VEGFR2 in response to CXCR4 antagonism. Furthermore, SPC mobilization was detected when the CXCR4 antagonist was administered to mice pretreated with VEGF, but not G-CSF. Thus, differential mobilization of progenitor cell subsets is dependent upon the cytokine milieu that regulates cell retention and proliferation. These findings may inform studies investigating mechanisms that regulate progenitor cell recruitment in disease and can be exploited to provide efficacious stem cell therapy for tissue regeneration.
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http://dx.doi.org/10.1016/j.stem.2008.10.017DOI Listing
January 2009

Real-time measurement of non-lethal platelet thromboembolic responses in the anaesthetized mouse.

Thromb Haemost 2008 Feb;99(2):435-40

Sir Alexander Fleming Building, National Heart and Lung Institute, Imperial College London, Exhibition Road, London, UK.

Identifying and evaluating new therapeutic targets in platelets requires advanced animal models in which platelet responses can be measured directly and in situ. This is important because platelet function is strongly influenced by external factors such as those originating from the vascular endothelium. Our objectives were to record graded, non-lethal thromboembolic platelet responses to platelet agonists in situ in the mouse and to demonstrate an inhibitory effect of aspirin in our model. Radiolabelled platelets were infused into anaesthetized mice and responses to ADP, collagen and thrombin measured as changes in platelet associated counts in miniaturized detection probes placed over the thoracic region. All agonists induced dose-dependent changes in platelet counts due to accumulation of thrombi in the pulmonary vasculature. We confirmed a specific platelet effect by comparing platelet and erythrocyte responses and showing platelet aggregates in the lung vasculature histologically. Simultaneous injection of collagen and adrenaline induced increased and protracted synergistic platelet responses compared with individual injection of these agents and aspirin inhibited collagen-induced responses. We confirmed the clinical relevance of our model by showing that platelet thromboembolism in the mouse, like pulmonary embolism in humans, impaired cardiovascular performance. We present a refined method for measuring platelet agonist dose-responses and thromboembolism in real-time without inducing mortality in the mouse. Our technique will be useful in investigating the molecular determinants of physiological and pathophysiological platelet function in an in-vivo context and will enable investigations of both platelet and non-platelets mediators of thrombus formation.
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http://dx.doi.org/10.1160/TH07-07-0479DOI Listing
February 2008

Allergen induces the migration of platelets to lung tissue in allergic asthma.

Am J Respir Crit Care Med 2008 Mar 20;177(6):604-12. Epub 2007 Dec 20.

Department of Internal Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Via E. dal Pozzo, I-06126 Perugia, Italy.

Rationale: Platelets are essential for pulmonary leukocyte recruitment, airway hyperresponsiveness, and bronchial remodeling in animals with allergic inflammation and can be found in bronchoalveolar lavage of sensitized animals. No studies, however, have explored the direct migration of platelets to lungs.

Objectives: To assess whether platelets migrate into lung parenchyma in response to inhaled allergen in ovalbumin-sensitized mice; to assess the role of the FcepsilonRI receptor in this phenomenon; and to evaluate whether platelets from patients with asthma, or from sensitized mice, undergo chemotaxis in vitro in response to relevant antigens.

Methods: Ovalbumin-sensitized wild-type (WT) mice, or FcRgamma(-/-) mice lacking the FcepsilonRIgamma, were challenged with aerosolized allergen and lungs analyzed by platelet-specific immunohistochemistry. In some experiments, mice were depleted of platelets and cross-transfused with either WT or FcRgamma(-/-) platelets to assess the role of platelet FcRgamma(-/-). Chemotaxis of platelets from patients with asthma or from sensitized mice was studied in vitro.

Measurements And Main Results: Histology of lungs revealed isolated platelets, migrating out of vessels and localizing underneath the airways after allergen challenge in WT but not in FcRgamma(-/-) mice. Platelets from patients with asthma and from sensitized WT mice, but not from sensitized FcRgamma(-/-) mice, migrated in vitro toward the relevant allergen or an anti-IgE. Platelets from normal mice were found to express FcepsilonRIgamma and platelet-bound IgEs were increased in sensitized mice.

Conclusions: Platelets migrate extravascularly in response to a sensitizing allergen via a mechanism dependent on the interaction among allergen, allergen-specific IgE, and the FcepsilonRI, and this may allow them to participate directly in allergic tissue inflammation.
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http://dx.doi.org/10.1164/rccm.200702-214OCDOI Listing
March 2008

The coordinated action of G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute inflammation.

Blood 2008 Jan 10;111(1):42-9. Epub 2007 Oct 10.

Leukocyte Biology Section, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London, United Kingdom.

In this study, we have identified a unique combinatorial effect of the chemokines KC/MIP-2 and the cytokine granulocyte colony-stimulating factor (G-CSF) with respect to the rapid mobilization of neutrophils from the bone marrow in a model of acute peritonitis. At 2 hours following an intraperitoneal injection of thioglycollate, there was a 4.5-fold increase in blood neutrophil numbers, which was inhibited 84% and 72% by prior administration of blocking mAbs against either the chemokines KC/MIP-2 or G-CSF, respectively. An intraperitoneal injection of G-CSF acted remotely to stimulate neutrophil mobilization, but did not elicit recruitment into the peritoneum. Further, in vitro G-CSF was neither chemotactic nor chemokinetic for murine neutrophils, and had no priming effect on chemotaxis stimulated by chemokines. Here, we show that, in vitro and in vivo, G-CSF induces neutrophil mobilization by disrupting their SDF-1alpha-mediated retention in the bone marrow. Using an in situ perfusion system of the mouse femoral bone marrow to directly assess mobilization, KC and G-CSF mobilized 6.8 x 10(6) and 5.4 x 10(6) neutrophils, respectively, while the infusion of KC and G-CSF together mobilized 19.5 x 10(6) neutrophils, indicating that these factors act cooperatively with respect to neutrophil mobilization.
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http://dx.doi.org/10.1182/blood-2007-07-099648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575836PMC
January 2008

Nitroaspirin plus clopidogrel versus aspirin plus clopidogrel against platelet thromboembolism and intimal thickening in mice.

Thromb Haemost 2005 Mar;93(3):535-43

Department of Internal Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy.

Clopidogrel plus aspirin is the treatment of choice for patients undergoing percutaneous, coronary interventions with stenting, but it does not prevent restenosis. NCX-4016, a nitric oxide-releasing aspirin (nitroaspirin), exerts a wider range of antiplatelet actions compared to aspirin, superior antithrombotic activity and reduces restenosis after arterial injury in animals. The aim of the present study was to compare the combination of nitroaspirin plus clopidogrel with aspirin plus clopidogrel in a model of platelet pulmonary thromboembolism, bleeding and intimal thickening in mice. Drugs were administered orally for 5 days; the antithrombotic effects were evaluated against collagen plus epinephrine-induced pulmonary thromboembolism, the haemorrhagic effects by tail transection bleeding time and the effects on neointima proliferation by histomorphology of photochemically injured femoral arteries. Lung platelet emboli were reduced significantly and more effectively by nitroaspirin plus clopidogrel (-56%, p<0.05 vs control) than by aspirin plus clopidogrel (-26%, p<0.05 vs control). Ex vivo platelet aggregation was inhibited maximally by nitroaspirin plus clopidogrel. Aspirin plus clopidogrel strikingly prolonged the bleeding time while nitroaspirin plus clopidogrel induced a lesser prolongation. Nitroaspirin plus clopidogrel significantly reduced intimal thickening of the femoral artery while aspirin plus clopidogrel was ineffective. Nitroaspirin plus clopidogrel is more effective and less prohaemorrhagic than aspirin plus clopidogrel in mice; provided these data are confirmed in other animal models, nitroaspirin plus clopidogrel may represent a new regimen to be tested in patients undergoing coronary revascularization procedures.
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http://dx.doi.org/10.1160/TH04-07-0464DOI Listing
March 2005

Platelet P-selectin is required for pulmonary eosinophil and lymphocyte recruitment in a murine model of allergic inflammation.

Blood 2005 Mar 4;105(5):2074-81. Epub 2004 Nov 4.

Department of Internal Medicine, Division of Internal and Cardiovascular Medicine, University of Perugia, Via E dal Pozzo, 06126 Italy.

Platelets are necessary for lung leukocyte recruitment in a murine model of allergic inflammation, and platelet-leukocyte aggregates are formed in circulating blood of patients with asthma after allergen exposure. However, it is unknown how platelets induce pulmonary leukocyte recruitment in asthma. Here, we have investigated the importance of platelet adhesion molecule expression on pulmonary eosinophil and lymphocyte recruitment and on leukocyte CD11b and very late antigen (VLA)-4 expression in mice. Pulmonary leukocyte recruitment in platelet-depleted mice (sensitized and exposed to ovalbumin) transfused with fixed, unstimulated platelets (FUSPs) was abolished, whereas transfusion with platelets stimulated and fixed (FSPs), expressing P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1), restored eosinophil and lymphocyte recruitment. Transfusion with platelets from P-selectin-deficient mice, or with FSPs stimulated in the presence of a blocking anti-P-selectin antibody, were unable to restore pulmonary leukocyte recruitment. Flow cytometric analysis revealed increased expression of CD11b and VLA-4 on leukocytes attached to platelets after allergen exposure, and CD11b expression on leukocytes was suppressed in thrombocytopenic mice but was restored with the transfusion of FSPs, but not FUSPs, a phenomenon concurrent with the formation of platelet-leukocyte complexes. P-selectin expression on the surfaces of platelets is a major requirement for pulmonary eosinophil and lymphocyte recruitment, allowing circulating platelets to bind to and stimulate leukocytes for endothelial attachment.
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http://dx.doi.org/10.1182/blood-2004-06-2282DOI Listing
March 2005