Heimo Mairbäurl

Role of alveolar epithelial sodium transport in high altitude pulmonary edema (HAPE)

Alveolar edema results from an imbalance between fluid filtration into the alveolar space and removal by reabsorption. Hypoxia increases filtration by raising pulmonary capillary pressure and increasing endothelial and epithelial permeability allowing fluid and blood cells to access the alveoli. Active Na-reabsorption drives the fluid reabsorption from the alveolar space, but hypoxia inhibits reabsorption by inhibition of epithelial Na-channels (ENaC) and Na/K-ATPase. A (genetically determined) low activity of alveolar reabsorption in normoxia and further inhibition by hypoxia might cause HAPE-susceptibility, since at some point the depressed reabsorption may not keep pace with increased filtration. Na-reabsorption might even prove totally inefficient in the presence of large leaks of the alveolar barrier. Alveolar Na-reabsorption has not been measured in HAPE. Nasal epithelial Na-transport has been used as surrogate marker based on similarities in subunit expression of ENaC in nasal, airway, and alveolar epithelium. At high altitude cold, dryness, and nasal infections affect the nasal potential making any extrapolation to processes at the alveolar epithelium unreliable. The variability in nasal Na- and Cl-transport reduces the usefulness of nasal potentials to diagnose HAPE-susceptibility.

Factor inhibiting HIF-1 (FIH-1) modulates protein interactions of apoptosis-stimulating p53 binding protein 2 (ASPP2).

Summary The asparaginyl hydroxylase factor inhibiting HIF-1 (FIH-1) is an important suppressor of hypoxia-inducible factor (HIF) activity. In addition to HIF-&agr;, FIH-1 was previously shown to hydroxylate other substrates within a highly conserved protein interaction domain, termed the ankyrin repeat domain (ARD). However, to date, the biological role of FIH-1-dependent ARD hydroxylation could not be clarified for any ARD-containing substrate. The apoptosis-stimulating p53-binding protein (ASPP) family members were initially identified as highly conserved regulators of the tumour suppressor p53. In addition, ASPP2 was shown to be important for the regulation of cell polarity through interaction with partitioning defective 3 homolog (Par-3). Using mass spectrometry we identified ASPP2 as a new substrate of FIH-1 but inhibitory ASPP (iASPP) was not hydroxylated. We demonstrated that ASPP2 asparagine 986 (N986) is a single hydroxylation site located within the ARD. ASPP2 protein levels and stability were not affected by depletion or inhibition of FIH-1. However, FIH-1 depletion did lead to impaired binding of Par-3 to ASPP2 while the interaction between ASPP2 and p53, apoptosis and proliferation of the cancer cells were not affected. Depletion of FIH-1 and incubation with the hydroxylase inhibitor dimethyloxalylglycine (DMOG) resulted in relocation of ASPP2 from cell–cell contacts to the cytosol. Our data thus demonstrate that protein interactions of ARD-containing substrates can be modified by FIH-1-dependent hydroxylation. The large cellular pool of ARD-containing proteins suggests that FIH-1 can affect a broad range of cellular functions and signalling pathways under certain conditions, for example, in response to severe hypoxia.

Downregulation of the TGFβ Pseudoreceptor BAMBI in Non-Small Cell Lung Cancer Enhances TGFβ Signaling and Invasion.

Non-small cell lung cancer (NSCLC) is characterized by early metastasis and has the highest mortality rate among all solid tumors, with the majority of patients diagnosed at an advanced stage where curative therapeutic options are lacking. In this study, we identify a targetable mechanism involving TGFβ elevation that orchestrates tumor progression in this disease. Substantial activation of this pathway was detected in human lung cancer tissues with concomitant downregulation of BAMBI, a negative regulator of the TGFβ signaling pathway. Alterations of epithelial-to-mesenchymal transition (EMT) marker expression were observed in lung cancer samples compared with tumor-free tissues. Distinct alterations in the DNA methylation of the gene regions encoding TGFβ pathway components were detected in NSCLC samples compared with tumor-free lung tissues. In particular, epigenetic silencing of BAMBI was identified as a hallmark of NSCLC. Reconstitution of BAMBI expression in NSCLC cells resulted in a marked reduction of TGFβ-induced EMT, migration, and invasion in vitro, along with reduced tumor burden and tumor growth in vivo In conclusion, our results demonstrate how BAMBI downregulation drives the invasiveness of NSCLC, highlighting TGFβ signaling as a candidate therapeutic target in this setting. Cancer Res; 76(13); 3785-801. ©2016 AACR.

Beta2-adrenergic stimulation blunts inhibition of epithelial ion transport by hypoxia of rat alveolar epithelial cells.

Hypoxia impairs alveolar fluid clearance by inhibition of Na+ reabsorption, and also impairs β 2 adrenergic signaling in alveolar epithelium. Since both are major rescue mechanisms preventing pulmonary edema, we studied whether acute and prolonged treatment with terbutaline would prevent hypoxic inhibition of ion transport. Short circuit currents (ISC) were measured on normoxic and hypoxic (1.5% O2; 24h) primary rat alveolar type II (ATII) cells in absence and presence of terbutaline (1 to 100 μM, 24h). Control and pre-treated cells were stimulated acutely with terbutaline. Transepithelial transport was measured as short circuit current (ISC) in Ussing chambers. Terbutaline induced a rapid decrease ISC (-20%) followed by a slow raise. The transient change in ISC was not inhibited by amiloride but was prevented after Cl- depletion indicating a Cl- current. The slow increase after this transient was amiloride-sensitive indicating a Na+ current. Total ISC, its amiloride-sensitive component, and the transient decrease upon terbutaline stimulation were decreased by hypoxia. 24h treatment with terbutaline stimulated these currents in normoxia and hypoxia, although stimulation was less in the latter. 24h treatment with terbutaline increased the capacity of Na+/K+-ATPase and ENaC as measured after permeabilization with amphotericin. These changes were not paralleled by altered mRNA expression. Acutely applied terbutaline induced a 4-fold increase in cAMP formation in normoxia; terbutaline-induced cAMP-formation was impaired by hypoxia (-20%). Pre-treatment with terbutaline for 24h decreased terbutaline-induced cAMP formation by 85%. Despite this desensitization, addition of terbutaline to terbutaline pre-treated cells caused a larger increase in Cl- and Na+ transport both in normoxia and hypoxia than in non pre-treated cells. These results indicate that β 2 adrenergic stimulation increased Na+- and Cl- transport in ATII cells even in hypoxia thus restoring normal reabsorption.

Increased hepcidin levels in high-altitude pulmonary edema.

Low iron availability enhances hypoxic pulmonary vasoconstriction (HPV). Considering that reduced serum iron is caused by increased erythropoiesis, insufficient reabsorption, or elevated hepcidin levels, one might speculate that exaggerated HPV in high-altitude pulmonary edema (HAPE) is related to low serum iron. To test this notion we measured serum iron and hepcidin in blood samples obtained in previously published studies at low altitude and during 2 days at 4,559 m (HA1, HA2) from controls, individuals with HAPE, and HAPE-susceptible individuals where prophylactic dexamethasone and tadalafil prevented HAPE. As reported, at 4,559 m pulmonary arterial pressure was increased in healthy volunteers but reached higher levels in HAPE. Serum iron levels were reduced in all groups at HA2. Hepcidin levels were reduced in all groups at HA1 and HA2 except in HAPE, where hepcidin was decreased at HA1 but unexpectedly high at HA2. Elevated hepcidin in HAPE correlated with increased IL-6 at HA2, suggesting that an inflammatory response related to HAPE contributes to increased hepcidin. Likewise, platelet-derived growth factor, a regulator of hepcidin, was increased at HA1 and HA2 in controls but not in HAPE, suggesting that hypoxia-controlled factors that regulate serum iron are inappropriately expressed in HAPE. In summary, we found that HAPE is associated with inappropriate expression of hepcidin without inducing expected changes in serum iron within 2 days at HA, likely due to too short time. Although hepcidin expression is uncoupled from serum iron availability and hypoxia in individuals developing HAPE, our findings indicate that serum iron is not related with exaggerated HPV.

Inhaled budesonide does not prevent acute mountain sickness after rapid ascent to 4559 m

Recent studies showed that inhaled budesonide (200u2005µg twice per day) reduced the incidence of acute mountain sickness (AMS) after passive ascent to 3700 and 3900u2005m [1, 2]. These findings raised the possibility that mediators released from the hypoxic lung transmit signals to the brain which contribute to the cerebral processes leading to AMS [3]. Because neither of these studies reflect alpine-style climbing, the present study was performed to test whether inhalation of budesonide at two different doses (200 and 800u2005µg twice per day) prior to active and rapid ascent (<20u2005h) to 4559u2005m prevents AMS in this high-risk setting. Prophylactic inhalation of budesonide does not prevent acute mountain sickness after rapid ascent to high-altitude http://ow.ly/Bc9p30dOz46

Intravenous S-ketamine does not inhibit alveolar fluid clearance in a septic rat model.

We previously demonstrated that intratracheally administered S-ketamine inhibits alveolar fluid clearance (AFC), whereas an intravenous (IV) bolus injection had no effect. The aim of the present study was to characterize whether continuous IV infusion of S-ketamine, yielding clinically relevant plasma concentrations, inhibits AFC and whether its effect is enhanced in acute lung injury (ALI) which might favor the appearance of IV S-ketamine at the alveolar surface. AFC was measured in fluid-instilled rat lungs. S-ketamine was administered IV over 6 h (loading dose: 20 mg/kg, followed by 20 mg/kg/h), or intratracheally by addition to the instillate (75 µg/ml). ALI was induced by IV lipopolysaccharide (LPS; 7 mg/kg). Interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant (CINC)-3 were measured by ELISA in plasma and bronchoalveolar lavage fluid. Isolated rat alveolar type-II cells were exposed to S-ketamine (75 µg/ml) and/or LPS (1 mg/ml) for 6 h, and transepithelial ion transport was measured as short circuit current (ISC). AFC was 27±5% (mean±SD) over 60 min in control rats and was unaffected by IV S-ketamine. Tracheal S-ketamine reduced AFC to 18±9%. In LPS-treated rats, AFC decreased to 16±6%. This effect was not enhanced by IV S-ketamine. LPS increased IL-6 and CINC-3 in plasma and bronchoalveolar lavage fluid. In alveolar type-II cells, S-ketamine reduced ISC by 37% via a decrease in amiloride-inhibitable sodium transport. Continuous administration of IV S-ketamine does not affect rat AFC even in endotoxin-induced ALI. Tracheal application with direct exposure of alveolar epithelial cells to S-ketamine decreases AFC by inhibition of amiloride-inhibitable sodium transport.

Who Gets High Altitude Pulmonary Edema And Why

This paper focuses on high altitude pulmonary edema (HAPE) that occurs in individuals who are free of any pre-existing disease. An exaggerated hypoxic pulmonary vasoconstriction (HPV) is a hallmark of susceptibility to HAPE. In addition, a low hypoxic ventilatory response and defective sodium-dependent absorption of water from the alveoli may contribute to HAPE-susceptibility. However, excessive pulmonary artery hypertension appears to be crucial for the development of HAPE, since lowering pulmonary artery pressure by drugs, such as nifedipine or tadalafil (phospho-diesterase-5-inhibitor), will in most cases prevent HAPE. There is increasing evidence that the excessive pulmonary artery pressure response in HAPE-susceptible individuals is due to a reduced NO bioavailability. HAPE-susceptible individuals show an endothelial dysfunc- tion in the systemic circulation in hypoxia. Lower levels of exhaled NO in hypoxia before and during HAPE in susceptible individuals suggest that this abnormality also occurs in the lungs and polymorphisms of the eNOS gene are associated with susceptibility to HAPE in the Indian and Japanese population.

Neocytolysis: How to Get Rid of the Extra Erythrocytes Formed by Stress Erythropoiesis Upon Descent From High Altitude

Neocytolysis is the selective destruction of those erythrocytes that had been formed during stress-erythropoiesis in hypoxia in order to increase the oxygen transport capacity of blood. Neocytolysis likely aims at decreasing this excess amount of erythrocytes and hemoglobin (Hb) when it is not required anymore and to decrease blood viscosity. Neocytolysis seems to occur upon descent from high altitude. Similar processes seem to occur in microgravity, and are also discussed to mediate the replacement of erythrocytes containing fetal hemoglobin (HbF) with those having adult hemoglobin (HbA) after birth. This review will focus on hypoxia at high altitude. Hemoglobin concentration and total hemoglobin in blood increase by 20–50% depending on the altitude (i.e., the degree of hypoxia) and the duration of the sojourn. Upon return to normoxia hemoglobin concentration, hematocrit, and reticulocyte counts decrease faster than expected from inhibition of stress-erythropoiesis and normal erythrocyte destruction rates. In parallel, an increase in haptoglobin, bilirubin, and ferritin is observed, which serve as indirect markers of hemolysis and hemoglobin-breakdown. At the same time markers of progressing erythrocyte senescence appear even on reticulocytes. Unexpectedly, reticulocytes from hypoxic mice show decreased levels of the hypoxia-inducible factor HIF-1α and decreased activity of the BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), which results in elevated mitochondrial activity in these cells. Furthermore, hypoxia increases the expression of miR-21, which inhibits the expression of catalase and thus decreases one of the most important mechanisms protecting against oxygen free radicals in erythrocytes. This unleashes a series of events which likely explain neocytolysis, because upon re-oxygenation systemic and mitochondrial oxygen radical formation increases and causes the selective destruction of those erythrocytes having impaired anti-oxidant capacity.

Intravenous S-ketamine does not inhibit alveolar fluid clearance in septic rats with acute lung injury: 12AP3-7

Background and Goal of Study: S-ketamine is frequently used for analgosedation, especially during sepsis. We have previously shown that intratracheally administered S-ketamine inhibits alveolar fluid clearance (AFC)1. The present study investigated whether intravenous (i.v.) S-ketamine also inhibits AFC and whether its ef fect is af fected by lipopolysaccharide (LPS)-induced lung injury which might favor the appearance of S-ketamine at the alveolar surface. Materials and Methods: AFC was measured in fluid-instilled lungs of anesthetized rats using a 2% dextran-500 solution containing fluorescein isothiocyanate-labeled dextran as alveolar volume marker. S-ketamine was either administered i.v. over 6 h (loading dose: 20 mg/kg, followed by 20 mg/kg/h) or added to the instillate (75 μg/ml). To induce lung injury, lipopolysaccharide (LPS; 7 mg/kg) was injected i.v.. Interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant (CINC)-3 in plasma and bronchoalveolar lavage fluid (BALF) were measured by ELISA. In other experiments isolated rat alveolar type II cells were exposed to S-ketamine (75 μg/ml) for 6 h and/or to the sodium channel blocker amiloride (100 μM), and transepithelial transport indicated by short circuit currents (ISC) was measured in Ussing chambers. Data are Mean±SEM. Level of significance p< 0,05. Results: AFC was 27±2% af ter 60 min in control rats and was not af fected by i.v. S-ketamine. In contrast, LPS decreased AFC to 16±2% (p< 0,01). In rats treated with LPS and i.v. S-ketamine AFC was 19±2% (p< 0,05 vs. control, p=0,28 vs. LPS). LPS caused an increase in plasma IL-6 and CINC-3 af ter 6 h (each p< 0.05). This increase was accompanied by an increase of IL-6 and CINC-3 in BALF (p< 0.05). Tracheally administered S-ketamine reduced AFC to 18±3% (p< 0,05). In isolated alveolar type II cells 6 h S-ketamine decreased ISC by 36% (p< 0,01), an ef fect related to a decrease in amiloridesensitive sodium transport. Conclusion: These findings show that direct exposure of the rat alveolar epithelium to S-ketamine, either by tracheal administration or by administration to isolated alveolar epithelial cells, decreases amiloride-sensitive sodium transport and AFC. In contrast, S-ketamine applied intravenously does not af fect AFC, even when the alveolar permeability was increased by LPS. Thus, S-ketamine does not seem to be disadvantageous for patients with pulmonary edema. References: 1. Berger MM, Pitzer B, Zügel S et al. Anesth Analg 2010;111:164-70