Enhanced pulmonary expression of the TrkB neurotrophin receptor in hypoxic rats is associated with increased acetylcholine-induced airway contractility

AIM We have recently reported that hypoxia stimulates transcription of the TrkB neurotrophin receptor in cultured cells via stabilization of hypoxia-inducible factor-1alpha. Here we investigated whether the expression of TrkB and other neurotrophin receptors is oxygen-sensitive also in vivo, and explored the functional consequences of an oxygen-regulated TrkB expression. METHODS Rats were exposed either to 21% O(2) or 8% O(2) for 6 h and TrkB was analysed by reverse transcription real-time PCR, in situ mRNA hybridization, and immunological techniques. The importance of the brain-derived neurotrophic factor (BDNF)-TrkB pathway in the control of mechanical airway function was assessed on isolated tracheal segments from normoxic and hypoxic rats. RESULTS TrkB transcripts were increased approx. 15-fold in the lungs of hypoxic rats, and the respiratory epithelium was identified as the site of enhanced TrkB expression in hypoxia. The TrkB ligand, BDNF, significantly increased the contractile response to acetylcholine (ACh) of isolated tracheal segments from hypoxic but not from normoxic rats. This effect of BDNF was prevented by pre-incubation of the tissue specimens with the tyrosine kinase inhibitor K252a and by mechanical removal of the TrkB containing airway epithelium. Likewise, the nitric oxide (NO) synthase inhibitor l-NAME abrogated the influence of BDNF on ACh-induced contractions of isolated tracheal segments from hypoxic rats. CONCLUSION These results demonstrate that systemic hypoxia stimulates expression of the TrkB neurotrophin receptor in the airway epithelium. Furthermore, activation of TrkB signalling by BDNF in hypoxia enhances mechanical airway contractility to ACh through a mechanism that requires NO

Change point detection for clustered expression data

Background: To detect changes in biological processes, samples are often studied at several time points. We examined expression data measured at different developmental stages, or more broadly, historical data. Hence, the main assumption of our proposed methodology was the independence between the examined samples over time. In addition, however, the examinations were clustered at each time point by measuring littermates from relatively few mother mice at each developmental stage. As each examination was lethal, we had an independent data structure over the entire history, but a dependent data structure at a particular time point. Over the course of these historical data, we wanted to identify abrupt changes in the parameter of interest - change points. Results: In this study, we demonstrated the application of generalized hypothesis testing using a linear mixed effects model as a possible method to detect change points. The coefficients from the linear mixed model were used in multiple contrast tests and the effect estimates were visualized with their respective simultaneous confidence intervals. The latter were used to determine the change point(s). In small simulation studies, we modelled different courses with abrupt changes and compared the influence of different contrast matrices. We found two contrasts, both capable of answering different research questions in change point detection: The Sequen contrast to detect individual change points and the McDermott contrast to find change points due to overall progression. We provide the R code for direct use with provided examples. The applicability of those tests for real experimental data was shown with in-vivo data from a preclinical study. Conclusion: Simultaneous confidence intervals estimated by multiple contrast tests using the model fit from a linear mixed model were capable to determine change points in clustered expression data. The confidence intervals directly delivered interpretable effect estimates representing the strength of the potential change point. Hence, scientists can define biologically relevant threshold of effect strength depending on their research question. We found two rarely used contrasts best fitted for detection of a possible change point: the Sequen and McDermott contrasts

The Erythropoietin Promoter Variant rs1617640 Is Not Associated with Severe Retinopathy of Prematurity, Independent of Treatment with Erythropoietin

In this case-control study, the erythropoietin (EPO) promoter variant s1617640, linked to high intravitreal EPO concentrations and increased risk of diabetic retinopathy, was not associated with severe retinopathy of prematurity. This finding was observed both in infants with and without recombinant EPO administration

Validation of a New PCR-Based Screening Method for Prevention of Serratia marcescens Outbreaks in the Neonatal Intensive Care Unit

Background: Serratia marcescens may cause severe nosocomial infections, mostly in very low birth weight infants. Since S. marcescens exhibits by far the highest adjusted incidence rate for horizontal transmission, it can cause complex outbreak situations in neonatal intensive care units. Objective: The aim of this study was to establish a fast and highly sensitive colonization screening for prompt cohorting and barrier nursing strategies. Methods: A probe-based duplex PCR assay targeting the 16S rRNA gene of S. marcescens was developed and validated by using 36 reference strains, 14 S. marcescens outbreak- and nonoutbreak isolates, defined by epidemiological linkage and molecular typing, and applied in 1,347 clinical specimens from 505 patients. Results and Conclusions: The novel PCR assay proved to be highly specific and had an in vitro sensitivity of 100 gene copies per reaction (∼15 bacteria). It showed a similar (in laryngeal/tracheal specimens) or even higher (in rectal/stoma swabs) in vivo sensitivity in comparison to routine microbial culture and was much quicker (<24 h vs. 2 days). By combining different oligonucleotide primers, there was robust detection of genetic variants of S. marcescens strains. PCR inhibition was low (1.6%) and observed with rectal swabs only. Cohort analysis illustrated applicability of the PCR assay as a quick tool to prevent outbreak scenarios by allowing rapid decisions on cohorting and barrier nursing. In summary, this novel molecular screening for colonization by S. marcescens is specific, highly sensitive, and substantially accelerates detection

The circadian clock regulates rhythmic erythropoietin expression in the murine kidney

Generation of circadian rhythms is cell-autonomous and relies on a transcription/translation feedback loop controlled by a family of circadian clock transcription factor activators including CLOCK, BMAL1 and repressors such as CRY1 and CRY2. The aim of the present study was to examine both the molecular mechanism and the hemopoietic implication of circadian erythropoietin expression. Mutant mice with homozygous deletion of the core circadian clock genes cryptochromes 1 and 2 (Cry-null) were used to elucidate circadian erythropoietin regulation. Wild-type control mice exhibited a significant difference in kidney erythropoietin mRNA expression between circadian times 06 and 18. In parallel, a significantly higher number of erythropoietin-producing cells in the kidney (by RNAscope®) and significantly higher levels of circulating erythropoietin protein (by ELISA) were detected at circadian time 18. Such changes were abolished in Cry-null mice and were independent from oxygen tension, oxygen saturation, or expression of hypoxia-inducible factor 2 alpha, indicating that circadian erythropoietin expression is transcriptionally regulated by CRY1 and CRY2. Reporter gene assays showed that the CLOCK/BMAL1 heterodimer activated an E-box element in the 5' erythropoietin promoter. RNAscope® in situ hybridization confirmed the presence of Bmal1 in erythropoietin-producing cells of the kidney. In Cry-null mice, a significantly reduced number of reticulocytes was found while erythrocyte numbers and hematocrit were unchanged. Thus, circadian erythropoietin regulation in the normoxic adult murine kidney is transcriptionally controlled by master circadian activators CLOCK/BMAL1, and repressors CRY1/CRY2. These findings may have implications for kidney physiology and disease, laboratory diagnostics, and anemia therapy

Pooling, room temperature, and extended storage time increase the release of adult‐specific biologic response modifiers in platelet concentrates: a hidden transfusion risk for neonates?

BACKGROUND: Adult donor platelets (PLTs) are frequently transfused to prevent or stop bleeding in neonates with thrombocytopenia. There is evidence for PLT transfusion-related morbidity and mortality, leading to the hypothesis on immunomodulatory effects of transfusing adult PLTs into neonates. Candidate factors are biologic response modifiers (BRMs) that are expressed at higher rates in adult than in neonatal PLTs. This study investigated whether storage conditions or preparation methods impact on the release of those differentially expressed BRMs. STUDY DESIGN AND METHODS: Pooled PLT concentrates (PCs) and apheresis PCs (APCs) were stored under agitation for up to 7 days at room temperature (RT) or at 2 to 8 degrees C. The BRMs CCL5/RANTES, TGF beta 1, TSP1, and DKK1 were measured in PCs' supernatant, lysate, and corresponding plasma. PLT function was assessed by light transmission aggregometry. RESULTS: Concerning the preparation method, higher concentrations of DKK1 were found in pooled PCs compared to APCs. In supernatants, the concentrations of CCL5, TGF beta 1, TSP1, and DKK1 significantly increased, both over standard (≤ 4 days) and over extended storage times (7 days). Each of the four BRMs showed an up to twofold increase in concentration after storage at RT compared to cold storage (CS). There was no difference in the aggregation capacity. CONCLUSION: This analysis shows that the release of adult-specific BRMs during storage is lowest in short- and CS APCs. Our study points to strategies for reducing the exposure of sick neonates to BRMs that can be specifically associated to PLT transfusion-related morbidity

Mechanisms and (patho)physiological consequences of an oxygen-dependent regulation of the TrkB neurotrophin receptor gene

Hypoxische TrkB-Expression in vitro: Neurotrophine und ihre Rezeptoren spielen eine Schlüsselrolle in der Entwicklung und Funktion des Nervensystems. Hohe Expressionsniveaus des Neurotrophinrezeptors TrkB und seiner Liganden sind in Neuroblastomen mit einer ungünstigen Prognose assoziiert. In der vorliegenden Arbeit wird dargestellt, dass das für die Rezeptortyrosinkinase TrkB kodierende Gen NTRK2 in verschiedenen Zelllinien sauerstoffabhängig reguliert ist und durch den Hypoxie-induzierbaren Faktor-1 (HIF-1) stimuliert wird. Die mRNA und –Proteinniveaus von TrkB waren in der Neuroblastom-Zelllinie Kelly unter Hypoxie (1 % O2) vs. Normoxie (21 % O2) ca. 28-fach erhöht. Ein Luziferase-Reporterkonstrukt, das ca. 2,1 kbp des humanen TrkB-Promotors enthielt, wurde unter Hypoxie und nach Stimulation mit dem Hypoxiemimetikum 2,2’-Dipyridyl (DP, 100 µM) bei 21 % O2 ca. 6-fach stärker abgelesen. Die Luziferaseaktivität in der Anwesenheit von DP war durch RNA-Knockdown mit siRNAs gegen HIF-1, nicht aber gegen HIF-2, signifikant reduziert. Entsprechend konnte Hypoxie den TrkB-Promotor in embryonalen Mausfibroblasten, denen HIF-1 fehlte, nicht stimulieren. Die hypoxieresponsive TrkB- Promotorregion konnte auf drei Bindungsstellen für HIF-1 eingegrenzt werden, die sich im Bereich zwischen 923 und 879 bp relativ zur Transkriptionsstartstelle befanden. Die Migration von kultivierten Neuroblastomzellen war durch Inkubation bei 1 vs. 21 % O2 ungefähr verdoppelt. Dieser Hypoxieeffekt wurde durch Weglassen des TrkB-Liganden Brain-derived Neurotrophic Factor (BDNF) und den Tyrosinkinase-Inhibitor K252a verhindert. Zusammenfassend könnte die durch Hypoxie verstärkte TrkB-Expression einen kritischen Schalter für die bereits beschriebene Dedifferenzierung von Neuroblastomzellen unter hypoxischen Bedingungen darstellen und Teil der genetischen Adaptation im Rahmen der Tumorprogression sein. Hypoxische TrkB- Expression in vivo: Zur Untersuchung, ob die Expression von TrkB oder anderer Neurotrophinrezeptoren auch in vivo sauerstoffsensitiv ist, wurden Ratten für 6 h entweder bei Normoxie (21 % O2) oder normobarer Hypoxie (8 % O2) exponiert und die Genexpressionsniveaus bestimmt. Das markanteste Ergebnis war eine fast 15-fache Steigerung der TrkB-mRNA in den Lungen hypoxischer Ratten, aber in keinem anderen der untersuchten Organe. Mittels in situ mRNA-Hybridisierung und Immunhistochemie wurden die Basalzellen des Bronchialepithels und die alveolären Epithelzellen als Ort der verstärkten TrkB-Expression unter Hypoxie identifiziert. Der TrkB-Ligand BDNF erhöhte die Acetylcholin(ACh)-induzierte Kontraktionsantwort isolierter Trachealsegmente hypoxischer, nicht aber normoxischer Ratten signifikant. Dieser BDNF-Effekt wurde durch Präinkubation der Gewebeproben mit dem Tyrosinkinase-Inhibitor K252a und durch mechanische Entfernung des TrkB-exprimierenden Atemwegsepithels aufgehoben, während der Stickoxidsynthase(NOS)-Inhibitor L-Nitroargininmethylester (L NAME, 0,1 mM) den Einfluss von BDNF auf die ACh-induzierte Kontraktion hypoxischer Trachealsegmente verhinderte. Diese Ergebnisse zeigen, dass systemische Hypoxie die Expression des Neurotrophinrezeptors TrkB im Atemwegsepithel stimuliert. Zusätzlich verstärkt die Aktivierung der TrkB-Signalwege durch BDNF unter Hypoxie die ACh-induzierte Atemwegskontraktilität durch einen Mechanismus, der NO erfordert, da er unter Inhibition der NO-Synthasen mit L NAME nicht reproduziert werden konnte.Hypoxic TrkB expression in vitro: Neurotrophins and their receptors play a critical role in development and function of the nervous system. High expression levels of the TrkB neurotrophin receptor and its ligands in neuroblastoma are associated with a poor prognosis. The present study shows that the TrkB receptor tyrosine kinase-encoding gene NTRK2 is regulated oxygen-dependendly in different cell lines and that it is stimulated by the Hypoxia inducible Factor-1 (HIF-1). In the neuroblastoma cell line Kelly, mRNA and protein levels of TrkB were enhanced approx. 28 fold in hypoxia (1 % O2) vs. normoxia (21 % O2). A luciferase reporter construct that contained approx. 2,1 kbp of the human TrkB promoter was induced approx. 6 fold in hypoxia and after stimulation with the hypoxia mimetic 2,2’-Dipyridyl (DP, 100 µM) at 21 % O2. Luciferase activity in the presence of DP was significantly reduced by RNA knockdown with siRNAs against HIF-1 but not against HIF-2. Likewise, hypoxia was not able to stimulate the TrkB promoter in embryonic mouse fibroblasts which lacked HIF-1. The hypoxia-responsive TrkB promoter region could be confined to three binding sites for HIF-1 which were located to a region between 923 and 879 bp relative to the transcription start site. Migration of cultivated neuroblastoma cells was doubled after incubation at 1 vs. 21 % O2. This hypoxia effect was inhibited by omission of the TrkB ligand Brain-derived Neurotrophic Factor (BDNF) and the tyrosine kinase inhibitor K252a. In summary, hypoxia-induced TrkB expression could represent a critical switch for the formerly described dedifferentiation of neuroblastoma cells under hypoxic conditions and be part of the genetic adaptation during tumor progression. Hypoxic TrkB expression in vivo: To investigate whether expression of TrkB or other neurotrophin receptors was oxygen-sensitive in vivo, rats were exposed for 6 h at normoxia (21 % O2) or normobaric hypoxia (8 % O2), respectively, and the gene expression levels were determined. The most striking result was an approx. 15-fold induction of the TrkB mRNA in the lungs of hypoxic rats but in no other organ studied. By the means of in situ mRNA hybridisation and protein immunostaining basal cells of the bronchial epithelium and alveolar epithelial cells were identified as site of the enhanced TrkB expression in hypoxia. The TrkB ligand BDNF enhanced Acetylcholine(ACh)-induced contractional responses of isolated tracheal segments of hypoxic, but not normoxic rats, significantly. This BDNF effect was abolished by preincubation of the tissue samples with the tyrosine kinase inhibitor K252a and ba mechanic removal of the TrkB-expressing airway epithelium while the nitric oxide synthase (NOS) inhibitor L-Nitro-Arginine Methyl Ester (L NAME, 0,1 mM) inhibited the BDNF effect on the ACh-induced contraction of hypoxic tracheal segments. These results show that systemic hypoxia stimulates expression of the neurotrophin receptor TrkB in the airway epithelium. Additionally, activation of TrkB signalling by BDNF in hypoxia enhances the ACh-induced airway contractility by a mechanism that requires NO as it could not be reproduced when NO synthases were inhibited with L NAME

Common genetic variants at the 11q13.3 renal cancer susceptibility locus influence binding of HIF to an enhancer of cyclin D1 expression

Although genome-wide association studies (GWAS) have identified the existence of numerous population-based cancer susceptibility loci, mechanistic insights remain limited, particularly for intergenic polymorphisms. Here, we show that polymorphism at a remote intergenic region on chromosome 11q13.3, recently identified as a susceptibility locus for renal cell carcinoma, modulates the binding and function of hypoxia-inducible factor (HIF) at a previously unrecognized transcriptional enhancer of CCND1 (encoding cyclin D1) that is specific for renal cancers characterized by inactivation of the von Hippelg-Lindau tumor suppressor (pVHL). The protective haplotype impairs binding of HIF-2, resulting in an allelic imbalance in cyclin D1 expression, thus affecting a link between hypoxia pathways and cell cycle control. © 2012 Nature America, Inc. All rights reserved

Adaptation of the Oxygen Sensing System during Lung Development

During gestation, the most drastic change in oxygen supply occurs with the onset of ventilation after birth. As the too early exposure of premature infants to high arterial oxygen pressure leads to characteristic diseases, we studied the adaptation of the oxygen sensing system and its targets, the hypoxia-inducible factor- (HIF-) regulated genes (HRGs) in the developing lung. We draw a detailed picture of the oxygen sensing system by integrating information from qPCR, immunoblotting, in situ hybridization, and single-cell RNA sequencing data in ex vivo and in vivo models. HIF1α protein was completely destabilized with the onset of pulmonary ventilation, but did not coincide with expression changes in bona fide HRGs. We observed a modified composition of the HIF-PHD system from intrauterine to neonatal phases: Phd3 was significantly decreased, while Hif2a showed a strong increase and the Hif3a isoform Ipas exclusively peaked at P0. Colocalization studies point to the Hif1a-Phd1 axis as the main regulator of the HIF-PHD system in mouse lung development, complemented by the Hif3a-Phd3 axis during gestation. Hif3a isoform expression showed a stepwise adaptation during the periods of saccular and alveolar differentiation. With a strong hypoxic stimulus, lung ex vivo organ cultures displayed a functioning HIF system at every developmental stage. Approaches with systemic hypoxia or roxadustat treatment revealed only a limited in vivo response of HRGs. Understanding the interplay of the oxygen sensing system components during the transition from saccular to alveolar phases of lung development might help to counteract prematurity-associated diseases like bronchopulmonary dysplasia