Pankaj K. Bhavsar

Relative corticosteroid insensitivity of alveolar macrophages in severe asthma compared with non-severe asthma

Background: About 5–10% of patients with asthma suffer from poorly controlled disease despite corticosteroid (CS) treatment, which may indicate the presence of CS insensitivity. A study was undertaken to determine whether relative CS insensitivity is present in alveolar macrophages from patients with severe asthma and its association with p38 mitogen-activated protein kinase (MAPK) activation and MAPK phosphatase-1 (MKP-1). Methods: Fibreoptic bronchoscopy and bronchoalveolar lavage (BAL) were performed in 20 patients with severe asthma and 19 with non-severe asthma and, for comparison, in 14 normal volunteers. Alveolar macrophages were exposed to lipopolysaccharide (LPS, 10 μg/ml) and dexamethasone (10−8 and 10−6 M). Supernatants were assayed for cytokines using an ELISA-based method. p38 MAPK activity and MKP-1 messenger RNA expression were assayed in cell extracts. Results: The inhibition of LPS-induced interleukin (IL)1β, IL6, IL8, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α release by dexamethasone (10−6 M) was significantly less in macrophages from patients with severe asthma than in macrophages from patients with non-severe asthma. There was increased p38 MAPK activation in macrophages from patients with severe asthma. MKP-1 expression induced by dexamethasone and LPS, expressed as a ratio of LPS-induced expression, was reduced in severe asthma. Conclusion: Alveolar macrophages from patients with severe asthma demonstrate CS insensitivity associated with increased p38 MAPK activation that may result from impaired inducibility of MKP-1.

Effect of p38 MAPK inhibition on corticosteroid suppression of cytokine release in severe asthma

Patients with severe asthma respond less well to corticosteroids than those with non-severe asthma. Increased p38 mitogen-activated protein kinase (MAPK) activation in alveolar macrophages (AMs) from severe asthma patients has been associated with a reduced inhibition of cytokine release by dexamethasone. We determined whether p38 MAPK inhibitors would modulate corticosteroid suppression of cytokine release from AMs and peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from venous blood and AMs by bronchoalveolar lavage in severe and non-severe asthma patients. PBMCs and AMs were exposed to lipopolysaccharide (LPS) with and without the p38 MAPK inhibitor, SD282, or dexamethasone. We determined the concentration-dependent effects of another p38 MAPK inhibitor, GW-A, on dexamethasone-induced inhibition of interleukin (IL)-8 release from PBMCs. Cytokines were assayed using an ELISA-based method. SD282 (10−7u2005M), with dexamethasone (10−6u2005M), caused a greater inhibition of release of IL-1β, IL-6, macrophage inflammatory protein-1α and IL-10, than with dexamethasone alone in AMs from severe and non-severe asthma. At 10−9 and 10−10u2005M, GW-A, that had no direct effects, increased the inhibitory activity of dexamethasone (10−8 and 10−6u2005M) on LPS-induced IL-8 release in PBMCs from severe asthma. Corticosteroid insensitivity in severe asthma patients may be improved by inhibitors of p38 MAPK.

Glucocorticoid suppression of CX3CL1 (fractalkine) by reduced gene promoter recruitment of NF-κB

Glucocorticoids are an important antiinflammatory treatment of many inflammatory diseases including asthma. However, the mechanisms by which they mediate their suppressive effects are not fully understood. Respiratory epithelial cells are a source of CX3CL1 (fractalkine), which mediates cell adhesion and acts as a chemoattractant for monocytes, T cells, and mast cells. We show, in lung A549 epithelial cells, that the tumor necrosis factor‐α (TNF‐α) and IFNγ synergistically induced protein release and mRNA expression of CX3CL1 is inhibited by dexamethasone, without interfering with cytokine‐induced nuclear translocation of NF‐κB, and by an inhibitor of IκB kinase 2, AS602868. DNA binding assays confirmed the ability of NF‐κB to bind to the proximal CX3CL1 promoter. Chromatin immunoprecipitation assays showed a 5‐fold increase in the recruitment of NF‐κB to the CX3CL1 gene promoter in response to IFNγ/TNF‐α; this too was reversed by dexamethasone. In contrast, dexameth‐asone did not displace NF‐κB from the granulocyte‐macrophage colony‐stimulating factor gene promoter. We conclude that CX3CL1 expression is regulated through the NF‐κB pathway and that dexamethasone inhibits CX3CL1 expression through a glucocorticoid receptor‐dependent (RU486 sensitive) mechanism. This study also provides support for the action of glucocorticoids mediating their suppressive effects on expression by interfering with the binding of transcriptional activators at native gene promoters.— Bhavsar P. K., Sukkar, M. B., Khorasani, N., Lee, K.‐Y., Chung K. F. Glucocorticoid suppression of CX3CL1 (frac‐talkine) by reduced gene promoter recruitment of NF‐kB. FASEB J. 22, 1807–1816 (2008)

Identification of novel, cardiac-restricted transcription factors binding to a CACC-box within the human cardiac troponin I promoter

OBJECTIVESnThe expression of the human cardiac troponin I (hTnIc) gene is developmentally regulated and tissue-specific. In analysing the putative binding elements within the proximal promoter, a CACC-box sequence overlapping a consensus Sp1 element has been identified. The aim of this study was to characterise the factors binding to this element and to determine their importance in the transcriptional activity of the promoter.nnnMETHODSnA combination of supershift and competition electrophoretic mobility shift assays (EMSA) were used to identify the binding of factors to the overlapping CACC-box/Sp1 consensus element. The functional importance of this element was tested by transient transfection into primary neonatal rat cardiac myocytes in culture.nnnRESULTSnAt least four factors were able to interact with this region including the zinc finger proteins Sp1, Sp3 and two potentially novel factors. Whereas both Sp1 and Sp3 bound to the consensus Sp1 element, and to a lesser extent the CACC-box, two of the complexes required the intact CACC-box for binding. Site-directed mutagenesis of this region showed that the CACC-box is essential for hTnIc promoter-reporter activity. Further characterisation using EMSA indicated that the factors binding the hTnIc CACC-box are unlikely to be zinc finger proteins as they are insensitive to the addition of divalent cation chelating agents. They were also unable to bind to other known CACC-box elements. These factors are present in both human and rat cardiac muscle but absent from a number of cell lines including several derived from skeletal muscle.nnnCONCLUSIONnThe human cardiac troponin I gene promoter requires an upstream CACC-box element for full activity. This element binds at least two complexes which represent novel, tissue-restricted DNA-binding activity present in the heart which we have named HCB1 and HCB2 for heart CACC-box binding factors.

Clenbuterol Induces Cardiac Myocyte Hypertrophy via Paracrine Signalling and Fibroblast-derived IGF-1

The β2-selective adrenoreceptor agonist clenbuterol promotes both skeletal and cardiac muscle hypertrophy and is undergoing clinical trials in the treatment of muscle wasting and heart failure. We have previously demonstrated that clenbuterol induces a mild physiological ventricular hypertrophy in vivo with normal contractile function and without induction of α-skeletal muscle actin (αSkA), a marker of pathological hypertrophy. The mechanisms of this response remain poorly defined. In this study, we examine the direct action of clenbuterol on cardiocyte cultures in vitro. Clenbuterol treatment resulted in increased cell size of cardiac myocytes with increased protein accumulation and myofibrillar organisation characteristic of hypertrophic growth. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed elevated mRNA expression of ANP and brain natriuretic peptide (BNP) but without change in αSkA, consistent with physiological hypertrophic growth. Clenbuterol-treated cultures also showed elevated insulin-like growth factor I (IGF-1) mRNA and activation of the protein kinase Akt. Addition of either IGF-1 receptor-blocking antibodies or LY294002 in order to inhibit phosphatidylinositol 3-kinase, a downstream effector of the IGF-1 receptor, inhibited the hypertrophic response indicating that IGF-1 signalling is required. IGF-1 expression localised primarily to the minor population of cardiac fibroblasts present in the cardiocyte cultures. Together these data show that clenbuterol acts to induce mild cardiac hypertrophy in cardiac myocytes via paracrine signalling involving fibroblast-derived IGF-1.

Human Troponin Genes: Transcriptional Regulation and Chromosomal Organization

The troponin complex forms the calcium-sensitive molecular switch which regulates striated muscle contraction in response to alterations in intracellular calcium concentration. It is located on the thin filament of the sarcomere and is composed of three subunits: troponin C, the calcium binding subunit; troponin T, which is involved in the attachment of the complex to tropomyosin; and troponin I, the inhibitory subunit. Multiple isoforms of each of these subunits have been identified (see table 1) which are expressed with distinct tissue-specificity and developmental regulation [1,2]. In the case of troponin I, three isoforms have been identified in vertebrate striated muscle and in the adult these are expressed in cardiac muscle, slow skeletal muscle and fast skeletal muscle respectively. In the adult heart, cardiac troponin I is the only troponin I isoform detected in the bulk of the myocardium. However, during development the predominant isoform expressed is slow skeletal troponin I [2]. We have previously documented aspects of troponin expression in the human heart [3–6] and demonstrated a developmental switch in troponin I expression during human development. Analysis of mRNA and protein levels suggests that the increase in expression of the cardiac troponin I gene seen in late fetal stages in man is due to an increase in transcription. Here, we present data on the basic machinery required for the expression of the human cardiac troponin I gene. In other studies we have analyzed the organization of the troponin gene families and revealed that the six human genes encoding the different troponin I and T isoforms are organized as paralogous pairs located at three different chromosomal sites. Analysis of the pair comprized of the cardiac troponin I and slow skeletal troponin T genes reveals that they are organized head to tail and lie within 3 kb of each other. Close physical linkage raises questions concerning the evolution of these two troponin gene families, for their regulation and for the analysis of mutations suspected to result in cardiomyopathy.

S64 Clinical and transcriptomic profiles of severe asthmatics with high or low expression of the glucocorticoid receptor and importin-7

Introduction and Objectives The majority of asthmatics can be well-controlled using inhaled corticosteroids (ICS) and long-acting β2-agonists (LABAs). However, approximately 5% have a severe, refractory form of the disease and are often difficult to treat. Corticosteroid (CS) Insensitivity is the defining feature of these asthmatics known as “severe” asthmatics. The molecular mechanisms underlying CS insensitivity are not fully known. Here, we hypothesise that reduction in the expression of the Glucocorticoid receptor (GR) and Importin 7 are related to CS insensitivity. This aims project were to compare the gene expression of GR and Importin 7 between severe asthmatics and mild/moderate asthmatics or healthy controls of the U-BIOPRED cohort. We then investigated whether changes in their expression, correlated with changes in clinical features and expression of other genes. Methods The U-BIOPRED database contains data on mRNA expression, lung function, medication usage, blood, urine and sputum samples for their subjects (n=611). Using an unbiased approach to analyse the data we will initially used Gene Set Variation Analysis (GSVA) to look for differences in expression of GR and Importin 7 between the severe asthma, mild/moderate asthma and healthy volunteer cohorts. We then characterised the asthmatics into subjects with high (top 25%, compared to healthy controls) or low (bottom 25%) expression of GR or Importin 7 and then compared clinical characteristics and gene expression profiles between the high and low expressing GR or Importin-7 groups. Results Severe and non-severe asthmatics had reduced GR expression in endobronchial biopsy and brushings samples compared to healthy controls. There were no significant differences in lung function, blood analytes or exacerbation rates between high or low GR expression groups. Severe non-smoking asthmatics had reduced Importin 7 expression in sputum compared to mild/moderate asthmatics and healthy controls. Low Importin 7 group had lower means in FEV1%, FEV1/FVC, higher means in blood and sputum neutrophils%, IL-6 and hCRP. Conclusions Reduced Importin-7 expression in sputum samples of asthmatics correlated with reduced lung function scores, increased neutrophilic inflammation and more oral CS use.

S90 The effect of long acting beta-agonists on glucocorticoid receptor and importin-7 nuclear translocation in airway smooth muscle cells

Introduction and Objectives 5%–10% of asthmatic patients display relative insensitivity to the therapeutic benefits corticosteroids and are termed severe asthmatics. This has been attributed, in severe asthma (SA) airway smooth muscle cells (ASMCs), to defective corticosteroid-induced nuclear translocation of the glucocorticoid receptor (GR). Importins, including importin-7, mediate GR nuclear translocation. Studies report increased importin-7-GR interaction in response to corticosteroids in ASMCs. Long-acting beta agonists (LABAs) augment corticosteroid-induced GR nuclear translocation in COPD macrophages. The primary aim of this project was to determine whether the LABA salmeterol could reverse impaired corticosteroid (dexamethasone)-induced GR nuclear translocation in severe asthmatic ASMCs through an importin-7-dependent mechanism. Methods ASMCs derived from bronchial biopsies from patients with non-severe asthma (NSA) (n=4) and SA (n=4) were cultured and treated with dexamethasone (10–8 M) in the absence or presence of salmeterol (10–10 M or 10–8 M). 30u2009min post-stimulation, nuclear and cytoplasmic protein fractions underwent western blot analysis to determine levels of importin-7 and GR. RNA extraction was conducted at 4u2009hours post-stimulation followed by RT-qPCR to investigate the levels of the GR-activated genes, mitogen activated protein kinase phosphatase-1 (MKP-1) and glucocorticoid leucine zipper mRNA levels (GILZ). Results The Results show a reduced dexamethasone-induced GR nuclear translocation in SA when compared to NSA ASMCs, which was increased with co-treatment with dexamethasone and salmeterol. Co-treatment with dexamethasone and salmeterol also increased GR-activated gene expression leading to an increase in the expression of MKP-1 in SA ASMCs compared to dexamethasone alone. Nuclear importin-7 levels in NSA and SA ASMCs were not increased by co-treatment compared to dexamethasone alone. Conclusion We have demonstrated impaired dexamethasone-induced GR nuclear translocation in SA ASMCs, which can be reversed by the addition of the LABA salmeterol in combination with dexamethasone. No differences were observed in importin-7 nuclear localisation after co-treatment compared to dexamethasone alone in SA ASMCs. This could suggest that importin-7 is either rapidly recycled back into the cytoplasm after entry into the nucleus, or that the effect of LABAs are not mediated through Importin-7 in severe asthmatic patients. Further real-time experiments would elucidate the role of importin-7 in LABA mediated GR nuclear translocation.

S12 Plasma syndecan-1 level as a predictive marker of vasoplegia associated with surgery requiring cardiopulmonary bypass and possible involvement of oxidative stress

Background Vasoplegic syndrome (severe refractory hypotension) is associated with oxidative stress leading to endothelial dysfunction and complicates 10 to 40% of surgery requiring cardiopulmonary bypass (CPB). Whilst operative mortality is low, recovery is often prolonged in patients developing vasoplegia. There are, as yet, no validated biomarkers for vasoplegia that could be used to identify ‘at risk’ patients. We hypothesised that plasma levels of the endothelial surface layer (glycocalyx) protein, syndecan-1, shed during CPB, will be higher in patients who develop vasoplegia and that leukocyte responses to oxidative stress will be altered. Methods Patients (n = 48) undergoing cardiac surgery requiring CPB were, prospectively, enrolled; blood collected and indices of outcome recorded. A surrogate index of vasoplegia was adopted: requirement for infusion of vasoconstrictor agents for longer than 48h. An enzyme-linked immunosorbent assay was used to measure plasma levels of syndecan-1 at four time-points: after induction of anaesthesia but before CPB (T1); within 30 min of CPB ending (T2); 2h (T3) and 24h (T4) post-CPB. Real time qPCR was used to determine, in patient leukocytes (n = 20), relative expression (to house-keeping gene18S) of mRNA for markers of oxidative stress; NQO1 and SOD2, cytoplasmic and mitochondrial enzymes, respectively; and for comparison, TNFα. Results Syndecan-1 levels at T2 were significantly higher in vasoplegic patients (110.7 ng/mL, IQR 65.46–155.2) than non-vasoplegic patients (53.8 ng/mL, IQR 40.67–102.2; p < 0.001). ROC curve analysis showed syndecan-1 had significant (p = 0.009) predictive power for onset of vasoplegia, with an area under the curve of 0.766 (95% CI: 0.6019–0.9301); and a cut-off of 63.33 ng/mL (83.33% sensitivity, 69.23% specificity). Syndecan-1 levels were higher in patients whose intensive care unit length of stay (LOS) and hospital LOS were above corresponding medians for the cohort (p = 0.0061 and p = 0.0148, respectively). NQO1 relative expression was significantly higher (p = 0.022) in vasoplegic patients (3.779 ± 1.036) than non-vasoplegic patients (1.3 ± 0.302); whereas, neither SOD2 nor TNFα expression were significantly altered. Conclusion Plasma syndecan-1 measured immediately post-CPB had good predictive power for patients at risk of vasoplegia. Greater relative expression of leukocyte NQO1 in vasoplegic patients indicates activation of antioxidant defence mechanisms in response to oxidative stress, which could contribute to syndecan-1 shedding.