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.

[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.

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.

Role of tumor necrosis factor alpha in hyperthermia-induced apoptosis of human leukemia cells.

We used the human myelomonoblastic leukemia cell line PLB-985 to study the effects of temperatures ranging from 37 degrees C to 43 degrees C for 1 h on the induction of apoptosis and cell cycle distribution in leukemia cells. The threshold temperature for the onset of apoptosis was 42 degrees C. Whereas hyperthermia exerted no effect on the expression of Bcl-2 and Bax, heat induced a >30-fold increase of tumor necrosis factor (TNF) alpha mRNA expression and a significant increase in TNF-alpha protein secretion. This endogenous production of TNF-alpha correlated directly with the temperature-induced apoptode effect. Blocking TNF-alpha expression via treatment with pyrrolidinedithiocarbamate or blocking TNF-alpha activity with neutralizing antibodies abrogated heat-provoked apoptosis. In addition, exposure of cell culture supernatant of heat-treated PLB-985 cells to untreated cells induced an apoptotic effect. These data indicate a TNF-a-mediated self eradication of the leukemia cells after heat exposure. Inducing apoptosis with wild-type TNF-alpha or p55 and p75 protein muteins demonstrated that this effect was mediated by the p55 receptor. Interestingly, the autocrine suicidal loop found in immature leukemia cells was lost after granulocytic differentiation with 0.5% N,N-dimethylformamide. These data should be of critical importance for the understanding of the biological impact of fever as well as for developing therapeutic approaches to malignant diseases

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.

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.

Thyroid hormones enhance hypoxia-induced erythropoietin production in vitro.

Effects of thyroid hormones on the production of erythropoietin (Epo) were investigated in isolated perfused rat kidneys and in the human hepatoma cell line, HepG2. Epo protein was measured by radioimmunoassay. L-triiodothyronine and L-thyroxine stimulated hypoxia-induced Epo formation both in the kidney and in HepG2 cells in a dose-dependent fashion. Quantitation of Epo mRNA by competitive polymerase chain reaction (PCR) showed that hypoxic HepG2 cells had three-fold higher Epo messenger RNA levels when treated with thyroid hormones for 3 hours. Measurements of oxygen consumption revealed that this effect was not due to an increase in the degree of hypoxia. Thus, apart from the known direct effect on erythroid precursors, thyroid hormones appear to stimulate erythropoiesis by a noncalorigenic increase in Epo production.

Erythropoietin induces Ca2+ mobilization and contraction in rat mesangial and aortic smooth muscle cultures.

Reportedly, recombinant human erythropoietin (rhEpo) can induce constriction of isolated resistance vessels. We have studied whether rhEpo affects cytosolic calcium concentration, [Ca2+]i, and contraction of cultured smooth-muscle cells grown from rat renal corpuscles and aortae. rhEpo at high dose (> or = 20 U/mL) induced a transient increase in [Ca2+]i as detected by fura-2 fluorescence analysis. The number of cells responding with an increase in [Ca2+]i was dose-dependent. No significant changes of [Ca2+]i occurred when lower doses of rhEpo (< 20 U/mL) were applied. The effect of Epo on contraction was studied by phase-contrast microscopy. The number of cells responding with contraction was dose-dependent, too (76% mesangial cells contracting at 200 U rhEpo per mL). The receptor mechanism of this unusual action of Epo still needs to be clarified.

Effects of pro- and antioxidative compounds on renal production of erythropoietin.

The most important stimulus for the enhanced synthesis of erythropoietin (Epo) is a lowered O2 tension in the tissue. However, the mechanism by which an impaired O2 supply is transduced into appropriate Epo production is still not fully understood. Recently, studies in human hepatoma cells (line HepG2) indicate that reactive O2 species are involved in the signal transduction from the cellular O2 sensor to the Epo gene. To clarify the role of reactive O2 species in the regulation of Epo synthesis in the kidney, the principal Epo-producing organ in vivo, we investigated the influence of potent pro- and antioxidants on Epo production in isolated perfused rat kidneys. Under normoxic conditions, the iron chelator desferrioxamine and the antioxidant vitamin A increased renal Epo production, mimicking hypoxic induction. In contrast, supplementation of the perfusion medium of hypoxically perfused kidneys with the prooxidant compounds H2O2 or pyrogallol caused a significant reduction of Epo synthesis. The inhibition of Epo formation by reactive O2 species could be completely antagonized by desferrioxamine and the hydroxyl radical-(OH*)-scavenger tetramethylthiourea. Vitamin A also antagonized the H2O2-dependent inhibition of hypoxically induced Epo synthesis. Interestingly, the addition of the antioxidant vitamin A to hypoxically perfused kidneys also induced Epo production significantly. Our data strongly support the idea that reactive O2 species, especially H2O2, are part of the signaling chain of the cellular O2-sensing mechanism regulating the renal synthesis of Epo.

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.

Effects of cobalt on haem proteins of erythropoietin-producing HepG2 cells in multicellular spheroid culture.

The hypoxia-induced increase of spectrophotometrically measured light absorption at 560 nm, considered as reduced cytochrome b, in HepG2 cells is diminished after exposure to cobalt chloride (50 or 100 microM) for 18-36 h. The redox state of cytochrome c and cytochrome aa3, however, remains stable, indicating a particular affinity of cytochrome b for cobalt. Erythropoietin production of HepG2 cells increases after application of cobalt chloride, whereas H2O2 production, as measured by the dihydrorhodamine technique, decreases. It is concluded that cobalt stimulates a signal cascade with cytochrome b as receptor and H2O2 as second messenger for regulating erythropoietin production.

The influence of phenobarbital on cytochromes and reactive oxygen species in erythropoietin producing HepG2 cells.

Light absorption photometry of HepG2 cells treated with phenobarbital for enhancing the content of cytochrome P-450 and the synthesis of erythropoietin revealed an influence on all cytochromes detectable in the wavelength range between 400 and 620 nm. No correlation was found between specific changes of cytochrome P-450 absorption and increased EPO synthesis as proposed earlier by Fandrey et al. (Life Sci. (1990) 47, 127-134). In the present study, however, the increased erythropoietin synthesis could be related to a decreased intracellular hydroxyl radical level described as crucial for the oxygen regulated gene expression (Kietzmann et al., Biochem. J. (1998) 335, 425-432; Porwol et al., Eur. J. Biochem. (1998) 256, 16-23).

Potentiation of angiotensin II-stimulated phosphoinositide hydrolysis, calcium mobilization and contraction of renal mesangial cells upon down-regulation of protein kinase C.

Long-term pretreatment of rat mesangial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) down-regulated protein kinase C activity and potentiated the angiotensin II-induced inositol trisphosphate (InsP3) formation. This increased response to angiotensin II occurred without a significant change in the receptor number or Kd value of angiotensin II binding to the cells. The biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect on angiotensin II-stimulated InsP3 generation. Long-term pretreatment with TPA also increased the angiotensin II-induced mobilization of Ca2+ and the subsequent contraction of mesangial cells.

Neopterin activates transcription factor nuclear factor-kappa B in vascular smooth muscle cells.

We have previously shown that the pteridine compound neopterin stimulates inducible nitric oxide synthase (iNOS) gene expression in vascular smooth muscle cells in vitro. The mechanisms whereby neopterin exhibits these effects remained unclear. The present study demonstrates that neopterin induces the translocation of the transcription factor nuclear factor-kappa B (NF-kappa B) to the nucleus. Pretreatment of cells with the antioxidant pyrrolidine dithiocarbamate completely suppressed the effects of neopterin on NF-kappa B activation, iNOS gene expression, and nitric oxide release. From these data we conclude that neopterin activates the translocation of NF-kappa B subunits to the nucleus by modulating the intracellular redox state. This is one possible explanation for the impact of neopterin on iNOS gene expression.

Tumor necrosis factor-alpha production by human hepatoma cell lines is resistant to drugs that are inhibitory to macrophages.

Little is known about the potential of immunomodulatory agents to lower tumor necrosis factor-alpha (TNF-alpha) synthesis in tissues of nonmonocytic origin. We studied effects of diverse drugs on the formation of immunoreactive TNF-alpha in the human hepatoma cell lines HepG2 and Hep3B, in which TNF-alpha production was induced by treatment (3 h incubation periods) with interleukin-1beta (IL-1beta, 300 pg/ml) or phorbol myristate acetate (PMA, 100 nmol/l). TNF-alpha production in IL-1beta-stimulated or PMA-stimulated hepatocyte cultures was not altered following the addition of dihydrocortisone (< or = 1 microg/ml), dibutyryl-cAMP (db-cAMP, < or = 100 micromol/l), adenosine (< or = 1 mmol/l), thalidomide (< or = 25 microg/ml), or cyclosporine (< or = 300 ng/ml). TNF-alpha production was inhibited by taurolidine (> or = 300 microg/ml), but this inhibition was associated with reduced cell viability. Pentoxifylline (1 mg/ml) did not influence PMA-induced TNF-alpha production, but it augmented IL-1beta-induced TNF-alpha production. Measurements of TNF-alpha mRNA by RT-PCR indicated that pentoxifylline exerted its effect posttranscriptionally. Additional studies with PMA-treated human whole blood cultures confirmed that pentoxifylline, db-cAMP, and adenosine reduced TNF-alpha production by leukocytes. These results provide first evidence to assume cell type-specific effects of immunomodulatory drugs on TFN-alpha synthesis, which may be relevant with respect to their clinical application.

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.

Effects of erythropoietin on endothelin-1 synthesis and the cellular calcium messenger system in vascular endothelial cells.

A rise in blood pressure is the main side effect of erythropoietin (EPO) treatment in patients with renal anemia. The mechanisms, however, by which EPO may cause hypertension are still unclear. We therefore investigated the effects of EPO on endothelin (ET) synthesis and cytosolic free calcium concentration ([Ca2+]i) in vascular endothelial cells. Porcine endothelial cells were isolated from thoracic aorta, pulmonary artery, and vena cava. Studies were performed with cells of the first subculture. ET concentrations were measured radioimmunologically. Changes in [Ca2+]i were determined with the fluorescent probe fura-2. Cytotoxicity was assessed by sodium 3'-[1-(phenyl-amino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)ben zene sulfonic acid hydrate (XTT) assay. ET synthesis was similar in cells of different vascular origins and was time-dependent, reaching approximately 2 pmol ET/mg protein within 12 h of incubation. EPO (12 to 200 U/mL) stimulated ET release time- and dose-dependently by up to 83.2% (P < .01) within 12 h in the absence of fetal calf serum and heparin. EPO induced an immediate significant rise in [Ca2+]i from 58 +/- 12 nmol/L to 495 +/- 85 nmol/L (P < .01) with a subsequent slow return to 257 +/- 3 nmol/L. During 2 h of incubation, the Ca-ionophore A 23187 (10(-8) mol/L) moderately but significantly stimulated endothelial ET synthesis. However, the Ca-channel blocker verapamil, the intracellular Ca-release blocker TMB-8, and nickel, an unspecific calcium channel blocker, had no consistent effects on [Ca2+]i or ET synthesis. The protein kinase C inhibitor H-7 stimulated basal [Ca2+]i and cellular ET synthesis. The tyrosine kinase inhibitor genistein suppressed the EPO-induced rise in [Ca2+]i and cellular ET synthesis. From these data we conclude that EPO may stimulate ET synthesis in vascular endothelial cells by activation of an EPO-receptor and via intracellular signalling mechanisms that comprise tyrosine kinase activation and a rise in [Ca2+]i. Therefore, the systemic hypertensive effects of EPO may be due at least in part to local stimulation of vascular endothelial ET synthesis via calcium mobilization.

Interleukin-1 and tumor necrosis factor-alpha inhibit erythropoietin production in vitro.

The effects of several immunomodulatory peptides (recombinant, human) on the in vitro production of erythropoietin (Epo) were studied in cultures of the human hepatoma cell line Hep G2. A dose-dependent decrease of up to 60% in Epo production was induced by interleukin-1 beta, interleukin-1 alpha, and tumor necrosis factor-alpha (in that order of potency). In contrast, moderately increased Epo levels resulted with interleukin-6 or interferon-gamma treatment at high concentrations. Concomitant measurements of the production of alpha-fetoprotein indicated that the observed effects were specific for Epo. Hence, we suspect a modulating role of the immune system in the in vivo control of Epo production and postulate that interleukin-1 and tumor necrosis factor-alpha are involved in some of the cases of lowered blood Epo levels in association with renal diseases, chronic inflammation, and malignancies.