Untersuchungen zur Rolle von reaktiven Sauerstoffspezies bei der Schädigung der Haarzellen des Innenohres durch das Aminoglykosid-Antibiotikum Gentamicin und Zytostatikum Cisplatin

Um in vitro Einflussfaktoren der Innenohrschädigungen durch H2O2, Cisplatin und Gentamicin genauer erforschen zu können, wurde zunächst das in vitro-Modell des unfixierten neurosensorischen Epithels der Meerschweinchencochlea etabliert. Da die Vitalität der verschiedenen Zellen des Epithels in gepufferter Salzlösung (HBSS) mindestens 6 h erhalten blieb, wurden alle Untersuchungen innerhalb dieser Zeitspanne durchgeführt. Die empfindlichsten Zellen gegenüber allen eingesetzten Substanzen waren die äußeren Haarzellen, gefolgt von den inneren Haarzellen. Stützzellen (Hensen-Zellen und Deiters-Zellen) wurden nicht durch H2O2, Cisplatin oder Gentamicin geschädigt. Dieses Schädigungsmuster entspricht dem in vivo beobachteten Schädigungsverlauf nach Injektion von Cisplatin oder Gentamicin und auch nach traumatischen Lärm. Aus Versuchen mit H2O2 ging hervor, dass diese Sauerstoffspezies Haarzellen dosisunabhängig (200 µM oder 50 µM) sowohl kalzium- als auch eisenabhängig schädigt. Auch Antioxidantien wie Glutathion oder N-Acetylcystein spielen bei der Schädigung durch H2O2 (200 µM) eine wichtige Rolle. Die morphologischen Veränderungen der Haarzellen in Gegenwart der hohen H2O2-Konzentration deuteten auf einen nekrotischen, die in Gegenwart der niedrigeren Konzentration hingegen auf einen apoptotischen Zelltod hin. Das heißt, dass beide H2O2-induzierten Schädigungstypen sowohl durch Kalzium- als auch durch Eisenionen vermittelt werden. Auch die Schädigung der äußeren Haarzellen durch Cisplatin war teilweise eisen- und teilweise kalziumabhängig. Die Rolle des zellulären chelatisierbaren Eisenpools wurde mit dem Fluoreszenzindikator Phen Green SK durch Laser Scanning-Mikroskopie genauer charakterisiert. Es zeigte sich, dass die Eisenchelatoren 2,2´-Dipyridyl und Deferoxamin den Phen Green SK-detektierbaren chelatisierbaren Eisenpool der Zellen komplexieren konnten. Dabei war das lipophile 2,2´-Dipyridyl deutlich effektiver als das hydrophile Deferoxamin, was sich auch in den Befunden der Vitalitätstests widerspiegelte. Die mittels quantitativer Laser Scanning-Mikroskopie bestimmten Konzentrationen des cytosolischen chelatisierbaren Eisens variierten in den verschiedenen Zelltypen nur geringfügig (zwischen 1,3 ± 0,4 µM Eisen in den inneren Haarzellen und 3,7 ± 1,7 µM Eisen in den Hensen-Zellen) und können die unterschiedliche Empfindlichkeit der Zelltypen für H2O2, Cisplatin und auch Gentamicin nicht erklären. Die Zugabe von Cisplatin zu neurosensorischen Epithelien bewirkte keine Veränderung des Phen Green SK-detektierbaren chelatisierbaren Eisenpools aller vier Zelltypen. Durch eine Reduktion von Nitrotetrazoliumblauchlorid konnte jedoch, besonders in den Haarzellen, eine Erhöhung der Superoxidradikal-Anionen-Produktion in Gegenwart von Cisplatin gezeigt werden. Die cisplatininduzierte Schädigung der Haarzellen wird demnach durch eine erhöhte Produktion der Superoxidradikal-Anionen hervorgerufen, die dann teilweise eisen- und kalziumabhängig ohne detektierbare Veränderung des chelatisierbaren Eisenpools der Zellen verläuft. Die morphologischen Veränderungen der Zellen sprechen dafür, dass 50 µM Cisplatin eine Nekrose der Haarzellen auslöst. Gentamicin verursachte eine konzentrationsabhängige Schädigung der Haarzellen des neurosensorischen Epithels, die bei niedrigeren Konzentrationen (0,5 mM und 1mM) eine eisenunabhängige Kondensation des Chromatins der Haarzellen zeigte. Dieser Hinweis sowie die Beobachtung von Blebs oberhalb der Apikalplatten der Haarzellen deuten auf einen apoptotischen Zelltod hin. Ähnlich der Schädigung durch Cisplatin konnte auch die durch Gentamicin verursachte Haarzellschädigung durch Eisenchelatoren teilweise gehemmt werden, während Kalziumionen keinen Einfluss auf die Toxizität hatten. Trotz einer teilweise eisenabhängigen Schädigung der Haarzellen durch Gentamicin konnte weder eine Veränderung des chelatisierbaren Eisenpools der Zellen des neurosensorischen Epithels noch eine Erhöhung der Superoxidradikal-Anionen-Produktion detektiert werden. Bereits 1 Stunde vor Eintritt des Todes der äußeren Haarzellen kam es aber zu einem Verlust des mitochondrialen Membranpotenzials und Cyclosporin A, ein Inhibitor der mitochondrialen Permeabilitätspore, konnte vor dem durch Gentamicin verursachten Vitalitätsverlust der äußeren Haarzellen teilweise schützen. Zusammengefasst sind dies deutliche Hinweise dafür, dass Gentamicin eine Apoptose auslöst, bei der ein mitochondrialer Permeabilitätsübergang stattfindet. Trotz der zahlreichen parallelen Vorbefunde der Literatur beruht die Toxizität von Gentamicin auf einem anderen Mechanismus als die von Cisplatin. Gentamicin scheint in den äußeren Haarzellen des neurosensorischen Epithels der Meerschweinchencochlea ausschließlich eisenabhängig eine Apoptose auszulösen, in deren Verlauf ein mitochondrialer Permeabilitätsübergang stattfindet. Cisplatin hingegen löst bei vergleichbarer Toxizität in den äußeren Haarzellen Nekrose aus, die durch eine erhöhte Produktion von Superoxidradikal-Anionen verursacht wird und sowohl eisen- als auch kalziumabhängig ist. Teilweise eisen- und kalziumabhängig ist auch die Toxizität von H2O2, unabhängig davon, ob höhere Konzentrationen eine Nekrose oder niedrigere eine Apoptose verursachen. Die ermittelten Konzentrationen des chelatisierbaren Eisens der verschiedenen Zelltypen des neurosensorischen Epithels korrelierten nicht mit der Empfindlichkeit der Zellen gegenüber H2O2, Cisplatin und Gentamicin und lieferten somit keine Erklärung für die höhere Empfindlichkeit der Haarzellen

Hypoxic transcription gene profiles under the modulation of nitric oxide in nuclear run on-microarray and proteomics

Background: Microarray analysis still remains a powerful tool to identify new components of the transcriptosome and it has helped to increase the knowledge of targets triggered by stress conditions such as hypoxia and nitric oxide. However, analysis of transcriptional regulatory events remain elusive due to the contribution of altered mRNA stability to gene expression patterns, as well as changes in the half-life of mRNAs, which influence mRNA expression levels and their turn over rates. To circumvent these problems, we have focused on the analysis of newly transcribed (nascent) mRNAs by nuclear run on (NRO), followed by microarray analysis. Result: We identified 188 genes that were significantly regulated by hypoxia, 81 genes were affected by nitric oxide, and 292 genes were induced by the co-treatment of macrophages with both NO and hypoxia. Fourteen genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2, Son, Nnt, Lst1, Hps6 and Fxyd5) were common to hypoxia and/or nitric oxide treatments, but with different levels of expression. We observed that 166 transcripts were regulated only when cells were co-treated with hypoxia and NO but not with either treatment alone, pointing to the importance of a crosstalk between hypoxia and NO. In addition, both array and proteomics data supported a consistent repression of hypoxia regulated targets by NO. Conclusion: By eliminating the interference of steady state mRNA in gene expression profiling, we increased the sensitivity of mRNA analysis and identified previously unknown hypoxia-induced targets. Gene analysis profiling corroborated the interplay between NO- and hypoxia-induced signalling

Testing the 'zero-sum game' hypothesis: An examination of school health policy and practice and inequalities in educational outcomes

Background: There is recognition that health and education are intrinsically linked, through for example the World Health Organizations' Health Promoting Schools' (HPS) framework. Nevertheless, promoting health via schools is seen by some as a 'zero-sum game'; that is, schools have nothing to gain, and in fact may experience detriments to the core business of academic attainment as a result of focussing resources on health. Crucially, there is a paucity of evidence around the impacts of health and well-being policy and practice on attainment, with recent Cochrane reviews highlighting this gap. This study explored the 'zero-sum game' hypothesis among schools with varying levels of deprivation; that is, the role of health and wellbeing interventions in schools in reducing, or widening, socioeconomic inequality in educational attainment. Methods: Wales-wide, school-level survey data on health policies and practices, reflective of the HPS framework, were captured in 2016 using the School Environment Questionnaire (SEQ). SEQ data were linked with routinely collected data on academic attainment. Primary outcomes included attendance and attainment at Key Stages 3 and 4. Interaction terms were fitted to test whether there was an interaction between FSM,overall HPS activity, and outcomes. Linear regression models were constructed separately for high (>15% of pupils) and low (<15%) Free School Meal (FSM) schools, adjusting for confounders. Findings: The final analyses included 48 low and 49 high FSM secondary schools. Significant interactions were observed between FSM and overall HPS for KS3 attainment (b=0.28; 95% CI: 0.09, 0.47) and attendance(b=0.05; 95% CI: 0.02, 0.09), reflecting an association between health improvement activities and education outcomes among high, but not low FSM schools. There was no significant interaction for KS4 attainment (b=0.18; 95% CI: -0.22, 0.57).Interpretation: Our findings did not support the 'zero-sum game' hypothesis; in fact, among more deprived schools, there was a tendency for better attendance and attainment at Key Stage 3. Schools must equip students with the skills required for good physical, mental health and well-being in addition to academic and cognitive skills. The study included a large, nationally representative sample of secondary schools;however, the cross-sectional nature has implications for causality

Macrophage Polarization In The Tumor Microenvironment

Background: Tumor associated macrophages (TAMs) are known to support tumor progression and their accumulation is generally associated with poor prognosis. The shift from a tumor-attacking to a tumor-supportive macrophage phenotype is based on an educational program that, at least in part, is initiated by apoptotic tumor cells. Aims: We explored the macrophage phenotype shift during tumor progression by analyzing the macrophage NO-output system and examining potential NO targets. Methods: Biochemical and Molecular Biology-orientated cell culture experiments, in part using 3d-tumor spheroid models as well as animal experiments were used. Results: Apoptotic cells polarize macrophages towards a healing, tumor-supportive phenotype. Soluble mediators released from apoptotic cells, among them the lipid sphingosine-1-phosphate (S1P), cause expression of arginase 2 in macrophages, thereby lowering citrulline/NO formation but enhancing ornithine production. Mechanistically, this is achieved via the S1P2 receptor and the CRE (cAMP-response element) binding site in the arginase 2 promoter. Reduced NO-formation is also seen in ex vivo macrophages from a xenograft model allowing restricted vs. unrestricted tumor growth based on tumor-associated S1P-formation. The theoretical ability of NO to target hypoxia-inducible factor-1 (HIF-1) and jumonji histone demethylases (JHDMs) in cells of the tumor microenvironment will be discussed in light of the iNOS/arginase balance. Moreover, data on the importance of HIF-1 in macrophages for their interaction with tumor cells, polarization, and angiogenic potential will be presented. Conclusions: We hypothesize that apoptotic death of tumor cells and associated macrophage activation facilitates the progression of malignant disease. The macrophage polarization program affects the NO-output system and the capacity of macrophages to support or restrict tumor growth

SUMO signaling by hypoxic inactivation of SUMO-specific isopeptidases

Post-translational modification of proteins with ubiquitin-like SUMO modifiers is a tightly regulated and highly dynamic process. The SENP family of SUMO-specific isopeptidases comprises six cysteine proteases. They are instrumental in counterbalancing SUMO conjugation, but their regulation is not well understood. We demonstrate that in hypoxic cell extracts, the catalytic activity of SENP family members, in particular SENP1 and SENP3, is inhibited in a rapid and fully reversible process. Comparative mass spectrometry from normoxic and hypoxic cells defines a subset of hypoxia-induced SUMO1 targets, including SUMO ligases RanBP2 and PIAS2, glucose transporter 1, and transcriptional regulators. Among the most strongly induced targets, we identified the transcriptional co-repressor BHLHE40, which controls hypoxic gene expression programs. We provide evidence that SUMOylation of BHLHE40 is reversed by SENP1 and contributes to transcriptional repression of the metabolic master regulator gene PGC-1α. We propose a pathway that connects oxygen-controlled SENP activity to hypoxic reprogramming of metabolism

AMP-activated protein kinase suppresses arachidonate 15-lipoxygenase expression in interleukin 4-polarized human macrophages

Macrophages respond to the Th2 cytokine IL-4 with elevated expression of arachidonate 15-lipoxygenase (ALOX15). Although IL-4 signaling elicits anti-inflammatory responses, 15-lipoxygenase may either support or inhibit inflammatory processes in a context-dependent manner. AMP-activated protein kinase (AMPK) is a metabolic sensor/regulator that supports an anti-inflammatory macrophage phenotype. How AMPK activation is linked to IL-4-elicited gene signatures remains unexplored. Using primary human macrophages stimulated with IL-4, we observed elevated ALOX15 mRNA and protein expression, which was attenuated by AMPK activation. AMPK activators, e.g. phenformin and aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibited IL-4-evoked activation of STAT3 while leaving activation of STAT6 and induction of typical IL-4-responsive genes intact. In addition, phenformin prevented IL-4-induced association of STAT6 and Lys-9 acetylation of histone H3 at the ALOX15 promoter. Activating AMPK abolished cellular production of 15-lipoxygenase arachidonic acid metabolites in IL-4-stimulated macrophages, which was mimicked by ALOX15 knockdown. Finally, pretreatment of macrophages with IL-4 for 48 h increased the mRNA expression of the proinflammatory cytokines IL-6, IL-12, CXCL9, and CXCL10 induced by subsequent stimulation with lipopolysaccharide. This response was attenuated by inhibition of ALOX15 or activation of AMPK during incubation with IL-4. In conclusion, limiting ALOX15 expression by AMPK may promote an anti-inflammatory phenotype of IL-4-stimulated human macrophages

Myeloid knockout of HIF-1α does not markedly affect hemorrhage/resuscitation-induced inflammation and hepatic injury

Background: Hypoxia-inducible factor-1α (HIF-1α) and NF-κB play important roles in the inflammatory response after hemorrhagic shock and resuscitation (H/R). Here, the role of myeloid HIF-1α in liver hypoxia, injury, and inflammation after H/R with special regard to NF-κB activation was studied. Methods: Mice with a conditional HIF-1α knockout (KO) in myeloid cell-line and wild-type (WT) controls were hemorrhaged for 90 min ( mm Hg) and resuscitated. Controls underwent only surgical procedures. Results: After six hours, H/R enhanced the expression of HIF-1α-induced genes vascular endothelial growth factor (VEGF) and adrenomedullin (ADM). In KO mice, this was not observed. H/R-induced liver injury in HIF-1α KO was comparable to WT. Elevated plasma interleukin-6 (IL-6) levels after H/R were not reduced by HIF-1α KO. Local hepatic hypoxia was not significantly reduced in HIF-1α KO compared to controls after H/R. H/R-induced NF-κB phosphorylation in liver did not significantly differ between WT and KO. Conclusions: Here, deleting HIF-1α in myeloid cells and thereby in Kupffer cells was not protective after H/R. This data indicates that other factors, such as NF-κB, due to its upregulated phosphorylation in WT and KO mice, contrary to HIF-1α, are rather key modulators of inflammation after H/R in our model