Multi-omic analysis of the effects of low frequency ventilation during cardiopulmonary bypass surgery.

BACKGROUND: Heart surgery with cardio-pulmonary bypass (CPB) is associated with lung ischemia leading to injury and inflammation. It has been suggested this is a result of the lungs being kept deflated throughout the duration of CPB. Low frequency ventilation (LFV) during CPB has been proposed to reduce lung dysfunction. METHODS: We used a semi-biased multi-omic approach to analyse lung biopsies taken before and after CPB from 37 patients undergoing coronary artery bypass surgery randomised to both lungs left collapsed or using LFV for the duration of CPB. We also examined inflammatory and oxidative stress markers from blood samples from the same patients. RESULTS: 30 genes were induced when the lungs were left collapsed and 80 by LFV. Post-surgery 26 genes were significantly higher in the LFV vs. lungs left collapsed, including genes associated with inflammation (e.g. IL6 and IL8) and hypoxia/ischemia (e.g. HIF1A, IER3 and FOS). Relatively few changes in protein levels were detected, perhaps reflecting the early time point or the importance of post-translational modifications. However, pathway analysis of proteomic data indicated that LFV was associated with increased "cellular component morphogenesis" and a decrease in "blood circulation". Lipidomic analysis did not identify any lipids significantly altered by either intervention. DISCUSSION: Taken together these data indicate the keeping both lungs collapsed during CPB significantly induces lung damage, oxidative stress and inflammation. LFV during CPB increases these deleterious effects, potentially through prolonged surgery time, further decreasing blood flow to the lungs and enhancing hypoxia/ischemia.

Montelukast inhibits tumour necrosis factor-alpha-mediated interleukin-8 expression through inhibition of nuclear factor-kappaB p65-associated histone acetyltransferase activity.

BACKGROUND: Montelukast is a potent cysteinyl leukotriene-1 receptor antagonist possessing some anti-inflammatory effects although the molecular mechanism of these anti-inflammatory effects is unknown. In this study, we aimed to investigate the effect of montelukast on nuclear factor (NF)-kappaB-associated histone acetylation activity in phorbol myristate acetate (PMA)-differentiated U937 cells. METHODS: We examined the inhibitory effects of montelukast on TNF-alpha-induced IL-8 production in PMA-differentiated U-937 cells. U-937 cells were exposed to PMA (50 ng/mL) for 48 h to allow differentiation to macrophages. Macrophages were then exposed to TNF-alpha (10 ng/mL) in the presence or absence of montelukast (0.01-10 microm) for 24 h. After this time, the concentration of IL-8 in the culture supernatant was measured by sandwich-type ELISA kit. The effect of signalling pathways on TNF-alpha-induced IL-8 release was examined pharmacologically using selective NF-kappaB/IKK2 (AS602868, 3 microm), (PD98059, 10 microm) and p38 mitogen activated protein kinase (MAPK) (SB203580, 1 microm) inhibitors. NF-kappaB DNA binding activity was measured by a DNA-binding ELISA-based assay. NF-kappaB-p65-associated histone acetyltransferase (HAT) activity was measured by immunoprecipitation linked to commercial fluorescent HAT. RESULTS: TNF-alpha-induced IL-8 release was suppressed by an NF-kappaB inhibitor but not by MEK or p38 MAPK inhibitors. Montelukast induced a concentration-dependent inhibition of TNF-alpha-induced IL-8 release and mRNA expression that reached a plateau at 0.1 microm without affecting cell viability. Montelukast did not affect NF-kappaB p65 activation as measured by DNA binding but suppressed NF-kappaB p65-associated HAT activity. CONCLUSION: Montelukast inhibits TNF-alpha-stimulated IL-8 expression through changes in NF-kappaB p65-associated HAT activity. Drugs targeting these enzymes may enhance the anti-inflammatory actions of montelukast.

Effect of cigarette smoking on haem-oxygenase expression in alveolar macrophages.

We investigated the effect of chronic cigarette smoking on the expression of haem-oxygenase (HO)-1 and HO-2. Normal subjects and asymptomatic young current smokers with normal lung function tests underwent bronchoalveolar lavage for recovery of macrophages. Reverse transcription/polymerase chain reaction (RT-PCR) analysis showed no significant difference in HO-1 and HO-2 mRNA expression between the two groups. On the other hand, Western blot analysis showed a significant (P<0.05) reduction of HO-2 protein, but not of HO-1, in alveolar macrophages from smokers compared to normal. There was no significant differences by immunocytochemistry for HO-1 and HO-2 expression between the groups. We concluded that HO-2 expression is reduced in alveolar macrophages of smokers, possibly due to the oxidative stress of cigarette smoke. This may in turn lead to reduced protection against further oxidative insults.

Differential expression of IL-10 receptor by epithelial cells and alveolar macrophages.

BACKGROUND: Interleukin (IL)-10 is a pleiotropic cytokine with a broad spectrum of immunosuppressive and anti-inflammatory effects. IL-10 secretion from alveolar macrophages is defective in patients with asthma and lower concentrations of IL-10 are found in bronchoalveolar lavage (BAL) from asthmatic patients than in normal control subjects. Reduced IL-10 may result in exaggerated and more prolonged inflammatory responses in asthmatic airways. IL-10 acting through the IL-10 receptor (IL-10R) stimulates the transcription factors STAT1 and STAT3. METHODS: We investigated IL-10 and IL-10R expression in normal and asthmatic bronchial epithelium and BAL macrophages using reverse transcription-polymerase chain reaction, immunohistochemistry and Western blotting. The functional effect of IL-10 was examined using granulocyte-macrophage-colony stimulating factor, enzyme-linked immunosorbent assay and Western blotting for phosphorylated STAT1 and STAT3. RESULTS: IL-10 was not expressed in epithelial cells; furthermore these cells did not express the IL-10R and had no functional response to exogenous IL-10. Bronchial epithelial cells expressed variable levels of phosphorylated STAT1 and STAT3 with no change in expression between normal subjects and asthmatics. IL-10 protein and IL-10R expression was detected in alveolar macrophages from all subjects. CONCLUSION: Our study suggests that the bronchial epithelium is not a source of IL-10 and cannot respond to exogenous IL-10 because of a lack of IL-10R expression.

Expression of GATA family of transcription factors in T-cells, monocytes and bronchial biopsies.

GATA-binding proteins are a subfamily of zinc finger transcription factors with six members (GATA-1-6) that interact with the GATA deoxyribonucleic acid (DNA) sequence. This sequence is found in the regulatory regions of many genes including those encoding T-helper 2 (Th2)-like cytokines, receptors, adhesion molecules and enzymes, which may be important in the pathogenesis of bronchial asthma. The expression of GATA-3, 4 and -6 was investigated in peripheral blood T-lymphocytes and monocytes and bronchial biopsies from 11 normal subjects and 10 steroid-naive asthmatic patients. Using Western blot analysis, T-cells from asthmatic subjects expressed 5 times the level of GATA-3 compared to that in normals. Confocal microscopy indicated that GATA-3 expression was both nuclear and cytoplasmic. GATA DNA binding complex containing GATA-3 was elevated in Th2 cells as determined by electrophorectic mobility shift assay. In contrast, monocytes from normal and asthmatic subjects expressed GATA-4 and -6 in equal amounts, but no GATA-3 was found. Using immunohistochemistry in bronchial biopsies, epithelial cells expressed high levels of GATA-3, GATA-4 and GATA-6 proteins. Comparison of Western blots of bronchial biopsies showed no significant differences between normal and asthmatic subjects. In conclusion, the increased expression of GATA-3 in asthmatic T-cells may underlie augmented T-helper 2-like cytokines in this disease. However, the unaltered GATA-3 expression in epithelial cells suggests a distinct role for GATA-3 in these cells unrelated to T-helper 2-like cytokine release. Finally, no evidence was found for an increased expression of GATA-4 and GATA-6 in asthma.

p65-activated histone acetyltransferase activity is repressed by glucocorticoids: mifepristone fails to recruit HDAC2 to the p65-HAT complex.

Glucocorticoids acting through their specific receptor can either enhance or repress gene transcription. Dexamethasone represses interleukin-1beta-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor expression through a combination of direct inhibition of p65-associated histone acetyltransferase (HAT) activity and by recruiting histone deacetylase 2 (HDAC2) to the p65-HAT complex. Here we show that mifepristone, a glucocorticoid receptor partial agonist, has no ability to induce gene expression but represses interleukin-1beta-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor release by 50% maximally. Mifepristone was able to inhibit p65-associated HAT activity to the same extent as dexamethasone but failed to inhibit the natural promoter to an equal extent due to an inability to recruit HDAC2 to the p65-associated HAT complex. These data suggest that the maximal repressive actions of glucocorticoids require recruitment of HDAC2 to a p65-HAT complex. These data also suggest that pharmacological manipulation of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.

Probing the interaction of the cytotoxic bisdioxopiperazine ICRF-193 with the closed enzyme clamp of human topoisomerase IIalpha.

Topoisomerase II is an ATP-operated protein clamp that captures a DNA helix and transports it through another DNA duplex, allowing chromosome segregation at mitosis. A number of cytotoxic bisdioxopiperazines such as ICRF-193 target topoisomerase II by binding and trapping the closed enzyme clamp. To investigate this unusual mode of action, we have used yeast to select plasmid-borne human topoisomerase IIalpha alleles resistant to ICRF-193. Mutations in topoisomerase IIalpha of Leu-169 to Phe (L169F) (in the N-terminal ATPase domain) and Ala-648 to Pro (A648P) (in the core domain) were identified as conferring >50-fold and 5-fold resistance to ICRF-193 in vivo, respectively. The L169F mutation, located next to the Walker A box ATP-binding sequence, resulted in a mutant enzyme displaying ICRF-193-resistant topoisomerase and ATPase activities and whose closed clamp was refractory to ICRF-193-mediated trapping as an annulus on closed circular DNA. These data imply that the mutation interferes directly with ICRF-193 binding to the N-terminal ATPase gate. In contrast, the A648P enzyme displayed topoisomerase activities exhibiting wild-type sensitivity to ICRF-193. We suggest that the inefficient trapping of the A648P closed clamp results either from the observed increased ATP requirement, or more likely, from lowered salt stability, perhaps involving destabilization of ICRF-193 interactions with the B'-B' interface in the core domain. These results provide evidence for at least two different phenotypic classes of ICRF-193 resistance mutations and suggest that bisdioxopiperazine action involves the interplay of both the ATPase and core domains of topoisomerase IIalpha.

Nuclear distribution of human DNA topoisomerase IIbeta: a nuclear targeting signal resides in the 116-residue C-terminal tail.

We have analyzed the subcellular distribution of the beta isoform of human topoisomerase II using both isoform-specific antisera and an epitope-tagging approach. Previous immunocytochemical studies have yielded differing results with one reporting this isoform to be predominantly nucleolar. Later studies seem to refute this finding, as do our results with isoform-specific antisera reported here. Epitope tagging minimizes potential complications arising from the use of anti-topoisomerase II antisera that may recognize epitopes that are modified or masked in vivo and could lead to misleading results in immunocytochemical studies. A second strength of this approach is that it allowed a comparison with similarly tagged control proteins (derived from the nucleolar transcription factor UBF) that were known to localize unambiguously to the cytoplasmic, nucleoplasmic, or nucleolar compartments. We report that the C-terminal domain of topoisomerase IIbeta fused to a beta-galactosidase tag localizes to the nucleus (but not the nucleolar compartment) and that this is indistinguishable from the localization of native topoisomerase IIbeta detected by isoform-specific antisera. Further analysis revealed that the nuclear localization determinant lies within the 116-residue C-terminal tail of human topoisomerase IIbeta.

Cellular stress causes accumulation of the glucose transporter at the surface of cells independently of their insulin sensitivity.

The stimulation of glucose transport in response to various types of stress has been studied. There is no relationship between effects of stress-inducing agents on glucose transport and their effects on cellular protein synthesis. Although the effect of stress on glucose transport appears analogous to its stimulation by insulin, cells that are slightly insulin-sensitive in terms of glucose transport (BHK cells) show a similar degree of stimulation as highly insulin-sensitive cells (differentiated 3T3-L1 cells). External labeling of the transporter protein with a photoactivatable derivative of mannose, 2-N-4-(1-azi-2,2,2-trifluoroethyl) benzoyl-1, 3-bis-(D-mannos-4-yloxy)-propylamine, shows that most of the increased glucose transport activity correlates with an increase in the amount of the transporter on the cell surface. Cells subjected to K(+)-depletion, which inhibits endocytosis and results in an accumulation of receptors at the cell surface, show the same increase in glucose transport as cells exposed to stress; stressed cells show no further increase in glucose transport when subjected to K+ depletion. These results support the view (Widnell, C.C., Baldwin, S.A., Davies, A., Martin, S., Pasternak, C.A. 1990. FASEB J 4:1634-1637) that cellular stress increases glucose transport by promoting the accumulation of glucose transporter molecules at the cell surface.