Donald R. Dunbar

Underestimated and under-recognized: the late consequences of acute coronary syndrome (GRACE UK–Belgian Study)

AIM To define the long-term outcome of patients presenting with acute coronary syndrome [ST-segment elevation myocardial infarction (STEMI), and non-STEMI and unstable angina acute coronary syndrome (ACS) without biomarker elevation] and to test the hypothesis that the GRACE (Global Registry of Acute Coronary Events) risk score predicts mortality and death/MI at 5 years. METHODS AND RESULTS In the GRACE long-term study, UK and Belgian centres prospectively recruited and followed ACS patients for a median of 5 years (1797 days). Primary outcome events: deaths, cardiovascular deaths (CVDs) and MIs. Secondary events: stroke and re-hospitalization for ACS. There were 736 deaths, 19.8% (482 CVDs, 13%) and 347 (9.3%) MIs (>24 h), 261 strokes (7.7%), and 452 (17%) subsequent revascularizations. Rehospitalization was common: average 1.6 per patient; 31.2% had >1 admission, 9.2% had 5+ admissions. These events were despite high rates of guideline indicated therapies. The GRACE score was highly predictive of all-cause death, CVD, and CVD/MI at 5 years (death: χ(2) likelihood ratio 632; Wald 709.9, P< 0.0001, C-statistic 0.77; for CVD C-statistic 0.75, P < 0.0001; CVD/MI C-statistic 0.70, P < 0.0001). Compared with the low-risk GRACE stratum (ESC Guideline criteria), those with intermediate [hazard ratio (HR) 2.14, 95% CI 1.63, 2.81] and those with high-risk (HR 6.36, 95% CI 4.95, 8.16) had two- and six-fold higher risk of later death (Cox proportional hazard). A landmark analysis after 6 months confirmed that the GRACE score predicted long-term death (χ(2) likelihood ratio 265.4; Wald 289.5, P < 0.0001). Although in-hospital rates of death and MI are higher following STEMI, the cumulative rates of death (and CVD) were not different, by class of ACS, over the duration of follow-up (Wilcoxon = 1.5597, df = 1, P = 0.21). At 5 years after STEMI 269/1403 (19%) died; after non-STEMI 262/1170 (22%) after unstable angina (UA) 149/850 (17%). Two-thirds (68%) of STEMI deaths occurred after initial hospital discharge, but this was 86% for non-STEMI and 97% for UA. CONCLUSION The GRACE risk score predicts early and 5 year death and CVD/MI. Five year morbidity and mortality are as high in patients following non-ST MI and UA as seen following STEMI. Their morbidity burden is high (MI, stroke, readmissions) and the substantial late mortality in non-STE ACS is under-recognized. The findings highlight the importance of pursuing novel approaches to diminish long-term risk.

IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels

The IUPHAR database (IUPHAR-DB) integrates peer-reviewed pharmacological, chemical, genetic, functional and anatomical information on the 354 nonsensory G protein-coupled receptors (GPCRs), 71 ligand-gated ion channel subunits and 141 voltage-gated-like ion channel subunits encoded by the human, rat and mouse genomes. These genes represent the targets of approximately one-third of currently approved drugs and are a major focus of drug discovery and development programs in the pharmaceutical industry. IUPHAR-DB provides a comprehensive description of the genes and their functions, with information on protein structure and interactions, ligands, expression patterns, signaling mechanisms, functional assays and biologically important receptor variants (e.g. single nucleotide polymorphisms and splice variants). In addition, the phenotypes resulting from altered gene expression (e.g. in genetically altered animals or in human genetic disorders) are described. The content of the database is peer reviewed by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR); the data are provided through manual curation of the primary literature by a network of over 60 subcommittees of NC-IUPHAR. Links to other bioinformatics resources, such as NCBI, Uniprot, HGNC and the rat and mouse genome databases are provided. IUPHAR-DB is freely available at http://www.iuphar-db.org.

Human Dendritic Cells Produce TGF-beta 1 under the Influence of Lung Carcinoma Cells and Prime the Differentiation of CD4(+)CD25(+)Foxp3(+) Regulatory T Cells

Dendritic cells (DCs) have a central role in the development of adaptive immune responses, including antitumor immunity. Factors present in the tumor milieu can alter the maturation of DCs and inhibit their capacity to activate T cells. Using gene expression analysis, we found that human DCs increased the expression of TGF-β1 transcripts following culture with human lung carcinoma cells (LCCs). These DCs produced increased amounts of TGF-β1 protein compared with DCs not exposed to tumor cells. LCCs also decreased the expression of CD86 and HLA-DR by immature DCs. Furthermore, LCCs decreased CD86 expression and the production of TNF-α and IL-12 p70 by mature DCs. Moreover, LCCs also converted mature DCs into cells producing TGF-β1. These TGF-β1-producing DCs were poor at eliciting the activation of naive CD4+ T cells and sustaining their proliferation and differentiation into Th1 (IFN-γ+) effectors. Instead, TGF-β1-producing DCs demonstrated an increased ability to generate CD4+CD25+Foxp3+ regulatory T cells that suppress the proliferation of T lymphocytes. These results identify a novel mechanism by which the function of human DCs is altered by tumor cells and contributes to the evasion of the immune response.

Cryptic loxP sites in mammalian genomes: Genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques

Cre is widely used for DNA tailoring and, in combination with recombineering techniques, to modify BAC/PAC sequences for generating transgenic animals. However, mammalian genomes contain recombinase recognition sites (cryptic loxP sites) that can promote illegitimate DNA recombination and damage when cells express the Cre recombinase gene. We have created a new bioinformatic tool, FuzznucComparator, which searches for cryptic loxP sites and we have applied it to the analysis of the whole mouse genome. We found that cryptic loxP sites occur frequently and are homogeneously distributed in the genome. Given the mammalian nature of BAC/PAC genomic inserts, we hypothesised that the presence of cryptic loxP sites may affect the ability to grow and modify BAC and PAC clones in E. coli expressing Cre recombinase. We have observed a defect in bacterial growth when some BACs and PACs were transformed into EL350, a DH10B-derived bacterial strain that expresses Cre recombinase under the control of an arabinose-inducible promoter. In this study, we have demonstrated that Cre recombinase expression is leaky in un-induced EL350 cells and that some BAC/PAC sequences contain cryptic loxP sites, which are active and mediate the introduction of single-strand nicks in BAC/PAC genomic inserts.

Novel Fat Depot–Specific Mechanisms Underlie Resistance to Visceral Obesity and Inflammation in 11β-Hydroxysteroid Dehydrogenase Type 1–Deficient Mice

OBJECTIVE The study objective was to determine the key early mechanisms underlying the beneficial redistribution, function, and inflammatory profile of adipose tissue in 11β-hydroxysteroid dehydrogenase type 1 knockout (11β-HSD1−/−) mice fed a high-fat (HF) diet. RESEARCH DESIGN AND METHODS By focusing on the earliest divergence in visceral adiposity, subcutaneous and visceral fat depots from 11β-HSD1−/− and C57Bl/6J control mice fed an HF diet for 4 weeks were used for comparative microarray analysis of gene expression, and differences were validated with real-time PCR. Key changes in metabolic signaling pathways were confirmed using Western blotting/immunoprecipitation, and fat cell size was compared with the respective chow-fed control groups. Altered adipose inflammatory cell content and function after 4 weeks (early) and 18 weeks (chronic) of HF feeding was investigated using fluorescence (and magnetic)-activated cell sorting analysis, immunohistochemistry, and in situ hybridization. RESULTS In subcutaneous fat, HF-fed 11β-HSD1−/− mice showed evidence of enhanced insulin and β-adrenergic signaling associated with accretion of smaller metabolically active adipocytes. In contrast, reduced 11β-HSD1−/− visceral fat accumulation was characterized by maintained AMP kinase activation, not insulin sensitization, and higher adipocyte interleukin-6 release. Intracellular glucocorticoid deficiency was unexpectedly associated with suppressed inflammatory signaling and lower adipocyte monocyte chemoattractant protein-1 secretion with strikingly reduced cytotoxic T-cell and macrophage infiltration, predominantly in visceral fat. CONCLUSIONS Our data define for the first time the novel and distinct depot-specific mechanisms driving healthier fat patterning and function as a result of reduced intra-adipose glucocorticoid levels.

A variant at chromosome 9p21 is associated with recurrent myocardial infarction and cardiac death after acute coronary syndrome: the GRACE Genetics Study

AIMS Recent genetic studies identified the rs1333049 variant on chromosome 9p21 as a major susceptibility locus for coronary artery disease and myocardial infarction (MI). Here, we evaluated whether this variant also contributes to recurrent MI or cardiac death following an acute coronary syndrome (ACS). METHODS AND RESULTS A total of 3247 patients with ACS enrolled in the Global Registry of Acute Coronary Events (GRACE) in three distinct populations (UK, Belgium and Poland) were prospectively followed for 6 months and genotyped for rs1333049, in addition to 3004 and 2467 healthy controls from the UK and Belgium. After having confirmed that the at-risk C allele of rs1333049 was associated with index ACS in the UK and Belgian populations, we found that the rs1333049 at-risk C allele was significantly and independently associated with recurrent MI [age- and gender-adjusted hazard ratio (HR) 1.48, CI = 1.00-2.19, P = 0.048; and multivariable-adjusted HR 1.47, CI = 0.99-2.18; P = 0.053] and with recurrent MI or cardiac death (age- and gender-adjusted HR 1.58, CI = 1.00-2.48; P = 0.045; and multivariable adjusted HR 1.49, CI = 1.03-1.98; P = 0.028) within 6 months after an index ACS. Inclusion of rs1333049 into the GRACE risk score significantly improved classification for recurrent MI or cardiac death (P = 0.040), as calculated by the integrated discrimination improvement method. CONCLUSION In this large observational study, the 9p21 variant was independently associated with adverse cardiac outcome after ACS.

Genome-wide mRNA expression profiling in vastus lateralis of COPD patients with low and normal fat free mass index and healthy controls

BackgroundChronic Obstructive Pulmonary Disease (COPD) has significant systemic effects beyond the lungs amongst which muscle wasting is a prominent contributor to exercise limitation and an independent predictor of morbidity and mortality. The molecular mechanisms leading to skeletal muscle dysfunction/wasting are not fully understood and are likely to be multi-factorial. The need to develop therapeutic strategies aimed at improving skeletal muscle dysfunction/wasting requires a better understanding of the molecular mechanisms responsible for these abnormalities. Microarrays are powerful tools that allow the investigation of the expression of thousands of genes, virtually the whole genome, simultaneously. We aim at identifying genes and molecular pathways involved in skeletal muscle wasting in COPD.MethodsWe assessed and compared the vastus lateralis transcriptome of COPD patients with low fat free mass index (FFMI) as a surrogate of muscle mass (COPDL) (FEV1 30 ± 3.6%pred, FFMI 15 ± 0.2 Kg.m−2) with patients with COPD and normal FFMI (COPDN) (FEV1 44 ± 5.8%pred, FFMI 19 ± 0.5 Kg.m−2) and a group of age and sex matched healthy controls (C) (FEV1 95 ± 3.9%pred, FFMI 20 ± 0.8 Kg.m−2) using Agilent Human Whole Genome 4x44K microarrays. The altered expression of several of these genes was confirmed by real time TaqMan PCR. Protein levels of P21 were assessed by immunoblotting.ResultsA subset of 42 genes was differentially expressed in COPDL in comparison to both COPDN and C (PFP < 0.05; −1.5 ≥ FC ≥ 1.5). The altered expression of several of these genes was confirmed by real time TaqMan PCR and correlated with different functional and structural muscle parameters. Five of these genes (CDKN1A, GADD45A, PMP22, BEX2, CGREF1, CYR61), were associated with cell cycle arrest and growth regulation and had been previously identified in studies relating muscle wasting and ageing. Protein levels of CDKN1A, a recognized marker of premature ageing/cell cycle arrest, were also found to be increased in COPDL.ConclusionsThis study provides evidence of differentially expressed genes in peripheral muscle in COPD patients corresponding to relevant biological processes associated with skeletal muscle wasting and provides potential targets for future therapeutic interventions to prevent loss of muscle function and mass in COPD.

Oncogenic Properties of Apoptotic Tumor Cells in Aggressive B Cell Lymphoma

Summary Background Cells undergoing apoptosis are known to modulate their tissue microenvironments. By acting on phagocytes, notably macrophages, apoptotic cells inhibit immunological and inflammatory responses and promote trophic signaling pathways. Paradoxically, because of their potential to cause death of tumor cells and thereby militate against malignant disease progression, both apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. We hypothesized that, in progression of malignant disease, constitutive loss of a fraction of the tumor cell population through apoptosis could yield tumor-promoting effects. Results Here, we demonstrate that apoptotic tumor cells promote coordinated tumor growth, angiogenesis, and accumulation of TAMs in aggressive B cell lymphomas. Through unbiased “in situ transcriptomics” analysis—gene expression profiling of laser-captured TAMs to establish their activation signature in situ—we show that these cells are activated to signal via multiple tumor-promoting reparatory, trophic, angiogenic, tissue remodeling, and anti-inflammatory pathways. Our results also suggest that apoptotic lymphoma cells help drive this signature. Furthermore, we demonstrate that, upon induction of apoptosis, lymphoma cells not only activate expression of the tumor-promoting matrix metalloproteinases MMP2 and MMP12 in macrophages but also express and process these MMPs directly. Finally, using a model of malignant melanoma, we show that the oncogenic potential of apoptotic tumor cells extends beyond lymphoma. Conclusions In addition to its profound tumor-suppressive role, apoptosis can potentiate cancer progression. These results have important implications for understanding the fundamental biology of cell death, its roles in malignant disease, and the broader consequences of apoptosis-inducing anti-cancer therapy.

Influence of 23 coronary artery disease variants on recurrent myocardial infarction or cardiac death: the GRACE Genetics Study

AIMS A pooled analysis of 14 genome-wide association studies revealed 23 susceptibility loci for coronary artery disease (CAD), thereby providing the most comprehensive genetic blueprint of CAD susceptibility. Here, we evaluated whether these 23 loci also predispose to recurrent myocardial infarction (MI) or cardiac death following an acute coronary syndrome (ACS). METHODS AND RESULTS A total of 2099 ACS patients enrolled in the Global Registry of Acute Coronary Events (GRACE) UK-Belgian study were prospectively followed for a median of 5 years (1668 days). C-allele carriers of the rs579459 variant, which is located upstream of the ABO gene and correlates with blood group A, were independently associated with recurrent MI [multivariable-adjusted hazard ratio (HR) 2.25, CI = 1.37-3.71; P = 0.001] and with recurrent MI or cardiac death [multivariable-adjusted (HR) 1.80, CI = 1.09-2.95; P = 0.021] within 5 years after an index ACS. The association of rs579459 was replicated in 1250 Polish patients with 6 months follow-up after an index ACS [multivariable-adjusted (HR) 2.70, CI = 1.26-5.82; P = 0.011 for recurrent MI]. Addition of rs579459 to a prediction model of 17 clinical risk factors improved risk classification for recurrent MI or cardiac death at 6 months as calculated by the integrated discrimination improvement method (P = 0.037), but not by C-statistics (P = 0.096). CONCLUSION In this observational study, rs579459 was independently associated with adverse cardiac outcome after ACS. A weak improvement in clinical risk prediction was also observed, suggesting that rs579459 should be further tested as a potentially relevant contributor to risk prediction models for adverse outcome following ACS.

A Stratified Transcriptomics Analysis of Polygenic Fat and Lean Mouse Adipose Tissues Identifies Novel Candidate Obesity Genes

Background Obesity and metabolic syndrome results from a complex interaction between genetic and environmental factors. In addition to brain-regulated processes, recent genome wide association studies have indicated that genes highly expressed in adipose tissue affect the distribution and function of fat and thus contribute to obesity. Using a stratified transcriptome gene enrichment approach we attempted to identify adipose tissue-specific obesity genes in the unique polygenic Fat (F) mouse strain generated by selective breeding over 60 generations for divergent adiposity from a comparator Lean (L) strain. Results To enrich for adipose tissue obesity genes a ‘snap-shot’ pooled-sample transcriptome comparison of key fat depots and non adipose tissues (muscle, liver, kidney) was performed. Known obesity quantitative trait loci (QTL) information for the model allowed us to further filter genes for increased likelihood of being causal or secondary for obesity. This successfully identified several genes previously linked to obesity (C1qr1, and Np3r) as positional QTL candidate genes elevated specifically in F line adipose tissue. A number of novel obesity candidate genes were also identified (Thbs1, Ppp1r3d, Tmepai, Trp53inp2, Ttc7b, Tuba1a, Fgf13, Fmr) that have inferred roles in fat cell function. Quantitative microarray analysis was then applied to the most phenotypically divergent adipose depot after exaggerating F and L strain differences with chronic high fat feeding which revealed a distinct gene expression profile of line, fat depot and diet-responsive inflammatory, angiogenic and metabolic pathways. Selected candidate genes Npr3 and Thbs1, as well as Gys2, a non-QTL gene that otherwise passed our enrichment criteria were characterised, revealing novel functional effects consistent with a contribution to obesity. Conclusions A focussed candidate gene enrichment strategy in the unique F and L model has identified novel adipose tissue-enriched genes contributing to obesity.