Hypoxia-inducible factor 1 in dendritic cells is crucial for the activation of protective regulatory T cells in murine colitis.

Dendritic cells (DCs) serve as a bridge between innate and adaptive immunity and help to maintain intestinal homeostasis. Inflammatory bowel disease (IBD) is associated with dysregulation of the mucosal immune response. The concomitant hypoxic inflammation in IBD will activate the transcription factor hypoxia-inducible factor-1 (HIF-1) to also drive gene expression in DCs. Recent studies have described a protective role for epithelial HIF-1 in mouse models of IBD. We investigated the role of HIF-1 in DC function in a dextran sodium sulfate (DSS)-induced model of murine colitis. Wild-type and dendritic cell-specific HIF-1α knockout mice were treated with 3% DSS for 7 days. Knockout of HIF-1α in DCs led to a significantly larger loss of body weight in mice with DSS-induced colitis than in control mice. Knockout mice exhibited more severe intestinal inflammation with increased levels of proinflammatory cytokines and enhanced production of mucin. Induction of regulatory T cells (Tregs) was impaired, and the number of forkhead box P3 (Foxp3) Tregs was diminished by dendritic HIF-1α knockout. Our findings demonstrate that in DCs HIF-1α is necessary for the induction of sufficient numbers of Tregs to control intestinal inflammation.

[Erythropoiesis: Physiology, pathophysiology, and algorithm for classification of the type of anemia]. / Erythropoese : Physiologie, Pathophysiologie und Algorithmus zur Abklärung von Anämien.

Erythropoiesis is a continuous process that replaces 1% of all erythrocytes per day. To keep the erythrocyte count within stable limits about 3 million cells/s must be renewed. This enormous turnover requires folic acid and vitamin B12 for proper cell differentiation and iron for sufficient haemoglobin synthesis. In particular, iron metabolism underlies a precise regulation which may be disturbed by chronic bleeding, inflammatory disease or impaired dietary intake. If the loss of red blood cells due to physiological aging or bleeding is not balanced by sufficient erythropoiesis in the bone marrow, anaemia will develop. For the classification of various types of anaemia, a well-established algorithm has been proven useful. This algorithm addresses basic questions such as erythrocyte volume, the underlying mechanism, e.g. whether too many cells are destroyed or new cells are not sufficiently generated, and finally aims to define the main causes for the above identified disturbance of erythropoiesis.

Type I cell ROS kinetics under hypoxia in the intact mouse carotid body ex vivo: a FRET-based study.

Reactive oxygen species (ROS) mainly originating from NADPH oxidases have been shown to be involved in the carotid body (CB) oxygen-sensing cascade. For measuring ROS kinetics, type I cells of the mouse CB in an ex vivo preparation were transfected with the ROS sensor construct FRET-HSP33. After 2 days of tissue culture, type I cells expressed FRET-HSP33 as shown by immunohistochemistry. In one population of CBs, 5 min of hypoxia induced a significant and reversible decrease of type I cell ROS levels (n = 9 CBs; P < 0.015), which could be inhibited by 4-(2-aminoethyl)benzensulfonylfluorid (AEBSF), a highly specific inhibitor of the NADPH oxidase subunits p47(phox) and p67(phox). In another population of CBs, however, 5 min of hypoxia induced a significant and reversible increase of ROS levels in type I cells (n = 8 CBs; P < 0.05), which was slightly enhanced by administration of 3 mM AEBSF. These different ROS kinetics seemed to coincide with different mice breeding conditions. Type I cells of both populations showed a typical hypoxia-induced membrane potential (MP) depolarization, which could be inhibited by 3 mM AEBSF. ROS and MP closely followed the hypoxic decrease in CB tissue oxygen as measured with an O2-sensitive dye. We conclude that attenuated p47(phox) subunit activity of the NADPH oxidase under hypoxia is the physiological trigger for type I cell MP depolarization probably due to ROS decrease, whereas the observed ROS increase has no influence on type I cell MP kinetics under hypoxia.

Rocket fuel for the quantification of S-nitrosothiols. Highly specific reduction of S-nitrosothiols to thiols by methylhydrazine.

Reduction of S-nitrosothiols to the corresponding thiol function is the key step in analyzing S-nitrosocysteinyl residues in proteins. Though it has been shown to give low yields, ascorbate-dependent reduction is commonly performed in the frequently used biotin-switch technique. We demonstrate that the compound methylhydrazine can act as a specific and efficient reducing agent for S-nitrosothiols. The corresponding thiol function is exclusively generated from low molecular weight and proteinaceous S-nitrosothiols while methylhydrazine failed to reduce disulfides. It was possible to optimize the experimental conditions so that thiol autoxidation is excluded, and high reaction yields (>90%) are obtained for the thiol function. The biotin-switch technique performed with methylhydrazine-dependent reduction shows remarkably improved sensitivity compared to the ascorbate-dependent procedure.

The anemia of the newborn induces erythropoietin expression in the developing mouse retina.

The hematopoietic hormone erythropoietin (Epo), regularly produced by the kidneys and the liver, is also expressed in neuronal tissue, where it has been found to mediate paracrine neuroprotective effects. In most studies exploring the rescue effects of Epo, apoptosis was exogenously induced by different cell death stimuli. Herein, we set out to study the expression and function of Epo in physiologically occurring apoptosis in a model of retinal development. We made use of an organotypic retinal wholemount culture system that resembles the physiological in vivo situation with cell connections still retained. Epo mRNA expression in the retina, liver, and kidney showed a significant increase during early development, coinciding with the anemia of the newborn. In the retina of Epo-green fluorescent protein transgenic mice, Epo-expressing cells were identified and found to be distributed in the retinal ganglion cell layer. Treatment of retinal wholemount cultures with recombinant Epo resulted in a significant decrease of apoptotic ganglion cells as well as photoreceptor cells throughout retinal development. Moreover, transforming growth factor-beta-induced apoptosis was completely antagonized by Epo when both factors were simultaneously applied. Investigations on the signaling pathway revealed a decrease in Bax mRNA levels in Epo-treated retinal cells. We conclude that Epo exerts wide and prolonged neuroprotective activity in physiologically occurring apoptosis and thus contributes to proper retinal development.

Imaging of the hypoxia-inducible factor pathway: insights into oxygen sensing.

The transcription factor complex hypoxia-inducible factor (HIF)-1 controls the expression of most genes involved in adaptation to hypoxic conditions. HIF-1 is a heterodimer composed of oxygen-labile HIF-alpha and constitutively expressed HIF-beta subunits. The oxygen-dependent regulation of HIF-alpha is a multistep process that includes degradation under normoxia but stabilisation, translocation into the nucleus and activation under hypoxic conditions. The present paper summarises the contributions of optical methods to the understanding of oxygen-dependent regulation of the HIF-1 pathway. The tissue- and cell-specific distribution of HIF-alpha was visualised immunohistochemically and by immunofluorescence. Transcriptional activity of HIF-1 was monitored using green fluorescent protein as a reporter under control of hypoxia response elements in living cells, spheroids and tumour tissues in living mice. With cyan and yellow variants of green fluorescent protein fused to HIF subunits and regulatory proteins, subcellular distribution, migration and interaction were imaged in vivo by means of fluorescence recovery after photo-bleaching and fluorescence resonance energy transfer. Noninvasive imaging of these cellular and molecular processes by laser scanning microscopy complements ex vivo molecular biology assays and provides an additional spatial and temporal dimension to the understanding of the HIF-1 pathway.

Antagonism of lipopolysaccharide-induced blood pressure attenuation and vascular contractility.

OBJECTIVE: Aim was to assess whether lipopolysaccharide (LPS)-induced decrease of total peripheral resistance depends on Toll-like receptor (TLR)4 signaling and whether it is sensitive to NO-synthase or TLR4 antagonists. METHODS AND RESULTS: C3H/HeN mice (control), expressing a functional, and C3H/HeJ mice, expressing a nonfunctional TLR4, were compared. LPS (20 mg/kg) was injected i.p. 6 hours before hemodynamic measurements. L-NAME and SMT, inhibitors of NO production, and Eritoran, a TLR4 antagonist, were tested for their impact on vascular contractility. Aortic rings were incubated for 6 hours with or without LPS (1 microg/mL), or with LPS+Eritoran (2 microg/mL) and their phenylephrine-induced contractility was measured using a myograph. The expression of cytokines in aortic tissue was examined by real-time polymerase chain reaction. In control mice LPS induced a significant decrease of blood pressure and an increase of heart rate, whereas C3H/HeJ remained unaffected. LPS induced an increase of cytokine expression and a depression of vascular contractility only in control mice but not in C3H/HeJ. L-NAME and SMT increased contractility in all rings and restored LPS-dependent depression of contractility. Eritoran prevented LPS-induced loss of contractility. CONCLUSIONS: LPS upregulates cytokine expression via TLR4 and induces attenuation of smooth muscle contractility which can be effectively antagonized.

Organic cation transport in the rat kidney in vivo visualized by time-resolved two-photon microscopy.

Secretion of cationic drugs and endogenous metabolites is a major function of the kidney accomplished by tubular organic cation transport systems. A cationic styryl dye (ASP(+)) was developed as a fluorescent substrate for renal organic cation transporters. The dye was injected intravenously and continuously monitored in externalized rat kidneys by time-resolved two-photon laser scanning microscopy. To investigate changes in transport activity, cimetidine, a competitive inhibitor of organic cation transport was co-injected with ASP(+). Shortly after injection, fluorescence increased in peritubular capillaries. Simultaneously, fluorescence was transiently found at the basolateral membrane of the proximal and distal tubules at a higher intensity and shorter wavelength indicating membrane association of ASP(+). Subsequently, intracellular fluorescence increased steeply within 10 s. In the proximal tubules, intracellular fluorescence decreased by 50% within 5 min, while in the distal tubules the fluorescence decreased by only 5% within the same time frame. Intracellular uptake of ASP(+) into proximal tubules was significantly reduced by cimetidine. Our studies show that organic cation transport of the kidney can be visualized in vivo by two-photon laser scanning microscopy.

From critters to cancers: bridging comparative and clinical research on oxygen sensing, HIF signaling, and adaptations towards hypoxia.

The objective of this symposium at the First International Congress of Respiratory Biology (ICRB) was to enhance communication between comparative biologists and cancer researchers working on O(2) sensing via the HIF pathway. Representatives from both camps came together on August 13-16, 2006, in Bonn, Germany, to discuss molecular adaptations that occur after cells have been challenged by a reduced (hypoxia) or completely absent (anoxia) supply of oxygen. This brief "critters-to-cancer" survey discusses current projects and new directions aimed at improving understanding of hypoxic signaling and developing therapeutic interventions.

Nanostructure of specific chromatin regions and nuclear complexes.

Spatially modulated illumination (SMI) microscopy is a method of widefield fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale features in fluorescently labeled cells. Using this approach, structural changes in the context of gene activation and chromatin remodeling may be revealed. In this paper we present the application of SMI microscopy to size measurements of the 7q22 gene region, giving us a size estimate of 105+/-16 nm which corresponds to an average compaction ratio of 1:324. The results for the 7q22 domain are compared with the previously measured sizes of other fluorescently labeled gene regions, and to those obtained for transcription factories. The absence of a correlation between the measured and genomic sizes of the various gene regions indicate that a high variability in chromatin folding is present, with factors other than the sequence length contributing to the chromatin compaction. Measurements of the 7q22 region in different preparations and at different excitation wavelengths show a good agreement, thus demonstrating that the technique is robust when applied to biological samples.

ZAP-70 expression is a prognostic factor in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogenous disease with a highly variable clinical course. Recent studies have shown that expression of the protein tyrosine kinase ZAP-70 may serve as a prognostic marker in B-CLL. Employing a semiquantitative RT-PCR assay, we examined purified leukemia B cells of 39 CLL patients for the expression of ZAP-70 mRNA transcripts. Significant ZAP-70 mRNA levels exceeding those found in control samples with 5% T cells were detected in 36% of the CLL cases. Patients in the ZAP-70 positive cohort were characterized by an unfavorable clinical course with a significantly shorter progression-free survival as compared to the ZAP-70-negative patients (64%). These results were confirmed by flow-cytometric analysis of the ZAP-70 protein, and expanded to a larger patient cohort (n=67). A combined statistical analysis of 79 patients showed that the two patient subgroups also differed with regard to overall survival and a panel of known clinical prognostic factors including LDH, thymidine kinase serum levels and expression of the CD38 surface antigen by the leukemic cell clone. The level of ZAP-70 expression did not change over time in the majority of patients where sequential samples were available for analysis.

Cytosolic Ca(2+) levels and DNA synthesis of human umbilical vein endothelial cell cultures are unresponsive to thrombopoietin treatment.

The hormone thrombopoietin (TPO) induces proliferation of megakaryocytic progenitors and augments agonist-induced mobilization of Ca(2+) in platelets. Because the action of TPO is not restricted to the megakaryocytic lineage, we studied the occurrence of TPO receptor mRNA and protein, and effects of TPO on cytosolic Ca(2+) levels and DNA synthesis in human umbilical vein endothelial cell cultures (HUVECs). Polymerase chain reaction following reverse transcription (RT-PCR) of total mRNA revealed that TPO receptor (MPL) mRNA was expressed only at low level in our samples. TPO receptor protein was not detectable in HUVEC lysates investigated by immunoprecipitation and immunoblotting. In contrast to vascular endothelial growth factor (VEGF), TPO did neither alter fura2 fluorescence as a measure of cytosolic Ca(2+) levels nor increase 5-bromo-2'-deoxyuridine incorporation into DNA of HUVECs. In conclusion, our data demonstrate that HUVECs are neither structurally nor functionally responsive to TPO.

Accumulation of HIF-1alpha under the influence of nitric oxide.

The key player for adaptation to reduced oxygen availability is the transcription factor hypoxia-inducible factor 1 (HIF-1), composed of the redox-sensitive HIF-1alpha and the constitutively expressed HIF-1beta subunits. Under normoxic conditions, HIF-1alpha is rapidly degraded, whereas hypoxia, CoCl(2), or desferroxamine promote protein stabilization, thus evoking its transcriptional activity. Because HIF-1 is regulated by reactive oxygen species, investigation of the impact of reactive nitrogen species was intended. By using different nitric oxide (NO) donors, dose- and time-dependent HIF-1alpha accumulation in close correlation with the release of NO from chemically distinct NO donors was established. Intriguingly, small NO concentrations induced a faster but transient HIF-1alpha accumulation than higher doses of the same NO donor. In contrast, NO attenuated up-regulation of HIF-1alpha evoked by CoCl(2) in a concentration- and time-dependent manner, whereas the desferroxamine-elicited HIF-1alpha signal remained unaltered. To demonstrate an autocrine or paracrine signaling function of NO, we overexpressed the inducible NO synthase and used a coculture system of activated macrophages and tubular cells. Expression of the NO synthase induced HIF-1alpha accumulation, which underscored the role of NO as an intracellular activator for HIF-1. In addition, macrophage-derived NO triggered HIF-1alpha up-regulation in LLC-PK(1) target cells, which points to intercellular signaling properties of NO in achieving HIF-1 accumulation. Our results show that NO does not only modulate the HIF-1 response under hypoxic conditions, but it also functions as a HIF-1 inducer. We conclude that accumulation of HIF-1 occurs during hypoxia but also under inflammatory conditions that are characterized by sustained NO formation.

Chronic inborn erythrocytosis leads to cardiac dysfunction and premature death in mice overexpressing erythropoietin.

The most common cause of an increase of the hematocrit is secondary to elevated erythropoietin levels. Erythrocytosis is assumed to cause higher blood viscosity that could put the cardiovascular system at hemodynamic and rheological risks. Secondary erythrocytosis results from tissue hypoxia, and one can hardly define what cardiovascular consequences are caused by chronic erythrocytosis or hypoxia. Herein, a novel transgenic (tg) mouse line is characterized that is erythrocytotic because of chronic overexpression of the human erythropoietin gene. These mice grow up well, reaching a hematocrit of about 0.80 in adulthood. Blood volume of adult tg mice was markedly increased by 75%. Unexpectedly, blood pressure was not elevated and cardiac output was not decreased. Still, the adult tg mice showed features of cardiac dysfunction with increased heart weight. In vivo, high-frequency echocardiography revealed marked ventricular dilatation that was confirmed by histologic examination. Furthermore, by transmission electron microscopy, a prominent intracellular edema of the cardiomyocytes was seen. Exercise performance of the tg mice was dramatically reduced, unmasking the severity of their compromised cardiovascular function. In addition, life expectancy of the tg mice was significantly reduced to 7.4 months. Our findings suggest that severe erythrocytosis per se results in cardiac dysfunction and markedly reduced life span.

Hepatic thrombopoietin mRNA is increased in acute inflammation.

The plasma concentration of thrombopoietin (TPO) in general is inversely related to the mass of platelets and megakaryocytes. However, reactive thrombocytosis of inflammatory disease is accompanied by elevated TPO levels. To investigate whether the rate of TPO mRNA expression is altered during acute inflammation, rats were injected with bacterial lipopolysaccharide (LPS). After 6 h, total RNA from liver and kidney was reverse transcribed and analyzed by competitive PCR for TPO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). LPS-treated rats showed a significant increase in hepatic TPO mRNA concentration. The ratio of TPO to GAPDH mRNA was 3.5 +/- 0.6% in the livers of control rats and 8.3 +/- 2.0% in the livers of LPS-treated rats (mean +/- SD). Thus, reactive thrombocytosis of inflammatory disease might result from an increase in hepatic TPO production. Since platelets are involved in the immune reaction, reactive thrombocytosis may be a mechanism of host defense.

Nitric oxide affects the production of reactive oxygen species in hepatoma cells: implications for the process of oxygen sensing.

Treatment of human hepatoma cells (HepG2) with NO-donors for 24 h inhibited hypoxia-induced erythropoietin (EPO) gene activation. NO was found to increase the production of reactive oxygen species (ROS), the putative signaling molecules between a cellular O2-sensor and hypoxia inducible factor 1 (HIF-1). HIF-1 is the prime regulator of O2-dependent genes such as EPO. NO-treatment for more than 20 h reduced HIF-1-driven reporter gene activity. In contrast, immediately after the addition of NO, ROS levels in HepG2 cells decreased below control values for as long as 4 h. Corresponding to these lowered ROS-levels, HIF-1 reporter gene activity and EPO gene expression transiently increased but were reduced when ROS levels rose thereafter. Our findings of a bimodal effect of NO on ROS production shed new light on the involvement of ROS in the mechanism of O2-sensing and may explain earlier conflicting data about the effect of NO on O2-dependent gene expression.

VEGF production, cell proliferation and apoptosis of human IGR 1 melanoma cells under nIFN-alpha/beta and rIFN-gamma treatment.

The effect of natural and recombinant interferons (nIFN, rIFN) on cell growth, apoptosis and the production of vascular endothelial growth factor (VEGF) was investigated in the human melanoma cell line IGR 1. We determined cell proliferation, cell vitality, DNA synthesis, apoptosis, intracellular oxygen radicals (ROS) and VEGF-mRNA as well as VEGF-protein levels. rIFN-gamma significantly inhibited growth by decreasing DNA synthesis and increasing apoptosis. Less pronounced was the growth inhibitory effect of nIFN-beta because an increased rate of apoptosis was outweighed by enhanced DNA synthesis. nIFN-alpha only had minor effects on cell growth parameters. Under long-term incubation (144 h) nIFN-beta decreased, but rIFN-gamma increased production of the angiogen VEGF. Our data underscore the multiple effects of IFNs on melanoma cells and may contribute to the understanding of ambivalent results of melanoma therapy by IFNs. Particularly, the increased VEGF production under long-term treatment with serum IFN levels between 100 and 1,200 IU/ml should be kept in mind.