-
[show abstract]
[hide abstract]
ABSTRACT: BACKGROUND: During aging, there is a decreased ability to maintain skeletal muscle mass and function (sarcopenia). Such changes in skeletal muscle are also co-morbidities of diseases including cancer, congestive heart failure and chronic obstructive pulmonary disease. The loss of muscle mass results in decreased strength and exercise tolerance and reduced ability to perform daily activities. Pharmacological agents addressing these pathologies could have significant clinical impact, but their identification requires understanding of mechanisms driving myotube formation (myogenesis) and atrophy and provision of relevant assays. The aim of this study was to develop robust in vitro methods to study human myogenesis. METHODS: Satellite cells were isolated by digestion of post-mortem skeletal muscle and selection using anti-CD56 MicroBeads. CD56(+) cell-derived myotubes were quantified by high content imaging of myosin heavy chains. TaqMan-polymerase chain reaction arrays were used to quantify expression of 41 selected genes during differentiation. The effects of activin receptor agonists and tumour necrosis factor alpha (TNFα) on myogenesis and gene expression were characterised. RESULTS: Large-scale isolation of CD56(+) cells enabled development of a quantitative myogenesis assay with maximal myotube formation 3 days after initiating differentiation. Gene expression analysis demonstrated expression of 19 genes changed substantially during myogenesis. TNFα and activin receptor agonists inhibited myogenesis and downregulated gene expression of muscle transcription factors, structural components and markers of oxidative phenotype, but only TNFα increased expression of pro-inflammatory markers. CONCLUSIONS: We have developed methods for large-scale isolation of satellite cells from muscle and quantitative assays for studying human myogenesis. These systems may prove useful as part of a screening cascade designed to identify therapeutic agents for improving muscle function.
Journal of cachexia, sarcopenia and muscle. 01/2013;
-
[show abstract]
[hide abstract]
ABSTRACT: Small molecule isoindoline and tetrahydroisoquinoline derivatives have been identified as selective agonists of human peroxisome proliferator-activated receptor δ (PPARδ. Compound 18 demonstrated efficacy in a biomarker for increased fatty acid oxidation, with upregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) in human primary myotubes.
Bioorganic & medicinal chemistry letters 01/2011; 21(1):492-6. · 2.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We describe the discovery of small molecule benzazepine derivatives as agonists of human peroxisome proliferator-activated receptor δ (PPARδ) that displayed excellent selectivity over the PPARα and PPARγ subtypes. Compound 8 displayed good PK in the rat and efficacy in upregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) mRNA in human primary myotubes, a biomarker for increased fatty acid oxidation.
Bioorganic & medicinal chemistry letters 10/2010; 21(1):531-6. · 2.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Alveolar macrophages have been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). In this setting they are routinely exposed to cigarette smoke and a range of pathogens including bacteria and viruses. The gene expression changes that result from these challenges may contribute to the initiation and progression of the disease. Understanding such changes is therefore of great interest and could aid the discovery of novel therapeutics. To study this, we stimulated monocyte-derived macrophages (MDM) from smokers and non-smokers with either cigarette smoke extract (CSE) or bacterially derived lipopolysaccharide (LPS) and profiled global transcriptional changes using Affymetrix arrays. LPS and CSE stimulation elicited markedly different transcriptome profiles with the former agent producing a larger number of significant changes. The CSE evoked changes showed some overlap with those observed when comparing habitual smokers with non-smokers, although the latter changes were generally of a more subtle nature. Detailed pathway analyses indicated that a number of genes involved in host defence were regulated following CSE stimulation and in MDM from smokers. In particular the interferon gamma (IFNgamma)-signalling pathway was significantly down-regulated following CSE stimulation, a finding that was confirmed by RT-PCR analysis. Furthermore, these changes were associated with suppressed release of the IFNgamma-induced chemokines, CXCL10 and CXCL9 from CSE treated MDM. In summary, our data provides evidence that smoking alters key mechanisms of host defence in macrophages. Such changes may explain the increased susceptibility of COPD patients to the lung infections that are associated with exacerbations of this disease.
Molecular Immunology 12/2009; 47(5):1058-65. · 2.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6-2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2YAC, (P2TAC or P2T in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y12 in keeping with current P2 receptor nomenclature.Three selective P2T receptor antagonists, with a range of affinities, inhibited ADP-induced aggregation of washed human or rat platelets, in a concentration-dependent manner, with a rank order of antagonist potency (pIC50, human: rat) of AR-C78511 (8.5 : 9.1)>AR-C69581 (6.2 : 6.0)>AR-C70300 (5.4 : 5.1). However, these compounds had no effect on ADP-induced platelet shape change.All three antagonists had no significant effect on the ADP-induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y1 receptor, when used at concentrations that inhibit platelet aggregation.These antagonists also blocked ADP-induced inhibition of adenylyl cyclase in rat platelets and C6-2B cells with identical rank orders of potency and overlapping concentration – response curves.RT – PCR and nucleotide sequence analyses revealed that the C6-2B cells express the P2Y12 mRNA.These data demonstrate that the P2YAC receptor in C6-2B cells is pharmacologically identical to the P2TAC receptor in rat platelets.British Journal of Pharmacology (2001) 133, 521–528; doi:10.1038/sj.bjp.0704114
British Journal of Pharmacology 01/2009; 133(4):521 - 528. · 4.41 Impact Factor
-
Brian Springthorpe,
Andrew Bailey,
Patrick Barton,
Timothy N Birkinshaw,
Roger V Bonnert,
Roger C Brown,
David Chapman,
John Dixon,
Simon D Guile,
Robert G Humphries, [......],
Michael P Mortimore,
Stuart W Paine,
Garry Pairaudeau,
Anil Patel,
Aaron J Rigby,
Robert J Riley,
Barry J Teobald, Wendy Tomlinson,
Peter J H Webborn,
Paul A Willis
[show abstract]
[hide abstract]
ABSTRACT: Starting from adenosine triphosphate (ATP), the identification of a novel series of P2Y(12) receptor antagonists and exploitation of their SAR is described. Modifications of the acidic side chain and the purine core and investigation of hydrophobic substituents led to a series of neutral molecules. The leading compound, 17 (AZD6140), is currently in a large phase III clinical trial for the treatment of acute coronary syndromes and prevention of thromboembolic clinical sequelae.
Bioorganic & Medicinal Chemistry Letters 12/2007; 17(21):6013-8. · 2.55 Impact Factor
