The Increase in Hemoglobin Concentration With Altitude Differs Between World Regions and Is Less in Children Than in Adults.

To compensate for decreased oxygen partial pressure, high-altitude residents increase hemoglobin concentrations [Hb]. The elevation varies between world regions, posing problems in defining cutoff values for anemia or polycythemia. The currently used altitude adjustments (World Health Organization [WHO]), however, do not account for regional differences. Data from The Demographic and Health Survey (DHS) Program were analyzed from 32 countries harboring >4% of residents at altitudes above 1000 m. [Hb]-increase, (ΔHb/km altitude) was calculated by linear regression analysis. Tables show 95% reference intervals (RIs) for different altitude ranges, world regions, and age groups. The prevalence of anemia and polycythemia was calculated using regressions in comparison to WHO adjustments. The most pronounced Δ[Hb]/km was found in East Africans and South Americans while [Hb] increased least in South/South-East Asia. In African regions and Middle East, [Hb] was decreased in some altitude regions showing inconsistent changes in different age groups. Of note, in all regions, the Δ[Hb]/km was lower in children than in adults, and in the Middle East, it was even negative. Overall, the Δ[Hb]/km from our analysis differed from the region-independent adjustments currently suggested by the WHO resulting in a lower anemia prevalence at very high altitudes. The distinct patterns of Δ[Hb] with altitude in residents from different world regions imply that one single, region-independent correction factor for altitude is not be applicable for diagnosing abnormal [Hb]. Therefore, we provide regression coefficients and reference-tables that are specific for world regions and altitude ranges to improve diagnosing abnormal [Hb].

Hemoglobin is an oxygen-dependent glutathione buffer adapting the intracellular reduced glutathione levels to oxygen availability.

Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown. This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O2 saturation to 50% or less observed in healthy humans while at high altitude, or in red blood cell suspensions results in rising of the intraerythrocytic GSH level that is proportional to the reduction in Hb O2 saturation. This effect was not caused by the stimulation of GSH de novo synthesis or its release during deglutathionylation of Hb's cysteines. Using isothermal titration calorimetry and in silico modeling, we observed the non-covalent binding of four molecules of GSH to oxy-Hb and the release of two of them upon deoxygenation. Localization of the GSH binding sites within the Hb molecule was identified. Oxygen-dependent binding of GSH to oxy-Hb and its release upon deoxygenation occurred reciprocally to the binding and release of 2,3-bisphosphoglycerate. Furthermore, noncovalent binding of GSH to Hb moderately increased Hb oxygen affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide an advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation.

Hypoxia Aggravates Inhibition of Alveolar Epithelial Na-Transport by Lipopolysaccharide-Stimulation of Alveolar Macrophages.

Inflammation and hypoxia impair alveolar barrier tightness, inhibit Na- and fluid reabsorption, and cause edema. We tested whether stimulated alveolar macrophages affect alveolar Na-transport and whether hypoxia aggravates the effects of inflammation, and tested for involved signaling pathways. Primary rat alveolar type II cells (rA2) were co-cultured with rat alveolar macrophages (NR8383) or treated with NR8383-conditioned media after stimulation with lipopolysaccharide (LPS; 1 µg/mL) and exposed to normoxia and hypoxia (1.5% O2). LPS caused a fast, transient increase in TNFα and IL-6 mRNA in macrophages and a sustained increase in inducible nitric oxide synthase (NOS2) mRNA in macrophages and in rA2 cells resulting in elevated nitrite levels and secretion of TNF-α and IL-6 into culture media. In normoxia, 24 h of LPS treated NR8383 decreased the transepithelial electrical resistance (TEER) of co-cultures, of amiloride-sensitive short circuit current (ISCΔamil); whereas Na/K-ATPase activity was not affected. Inhibition was also seen with conditioned media from LPS-stimulated NR8383 on rA2, but was less pronounced after dialysis to remove small molecules and nitrite. The effect of LPS-stimulated macrophages on TEER and Na-transport was fully prevented by the iNOS-inhibitor L-NMMA applied to co-cultures and to rA2 mono-cultures. Hypoxia in combination with LPS-stimulated NR8383 totally abolished TEER and ISCΔamil. These results indicate that the LPS-stimulation of alveolar macrophages impairs alveolar epithelial Na-transport by NO-dependent mechanisms, where part of the NO is produced by rA2 induced by signals from LPS stimulated alveolar macrophages.

Adeno-Associated Virus-Mediated Gene Transfer of Inducible Nitric Oxide Synthase to an Animal Model of Pulmonary Hypertension.

Pulmonary hypertension (PH) is characterized by progressive obstruction of pulmonary arteries owing to inflammatory processes, cellular proliferation, and extracellular matrix deposition and vasoconstriction. As treatment options are limited, we studied gene transfer of an inducible nitric oxide synthase (iNOS) using adeno-associated virus (AAV) vectors specifically targeted at endothelial cells of pulmonary vessels in a murine model of PH. Adult mice were intravenously injected with AAV vectors expressing iNOS. Mice were subjected to hypoxia for 3 weeks and killed afterward. We found elevated levels of iNOS both in lung tissue and pulmonary endothelial cells in hypoxic controls that could be further increased by AAV-mediated iNOS gene transfer. This additional increase in iNOS was associated with decreased wall thickness of pulmonary vessels, less macrophage infiltration, and reduced molecular markers of fibrosis. Taken together, using a tissue-targeted approach, we show that AAV-mediated iNOS overexpression in endothelial cells of the pulmonary vasculature significantly decreases vascular remodeling in a murine model of PH, suggesting upregulation of iNOS as promising target for treatment of PH.

In Vitro Erythropoiesis at Different pO2 Induces Adaptations That Are Independent of Prior Systemic Exposure to Hypoxia.

Hypoxia is associated with increased erythropoietin (EPO) release to drive erythropoiesis. At high altitude, EPO levels first increase and then decrease, although erythropoiesis remains elevated at a stable level. The roles of hypoxia and related EPO adjustments are not fully understood, which has contributed to the formulation of the theory of neocytolysis. We aimed to evaluate the role of oxygen exclusively on erythropoiesis, comparing in vitro erythroid differentiation performed at atmospheric oxygen, a lower oxygen concentration (three percent oxygen) and with cultures of erythroid precursors isolated from peripheral blood after a 19-day sojourn at high altitude (3450 m). Results highlight an accelerated erythroid maturation at low oxygen and more concave morphology of reticulocytes. No differences in deformability were observed in the formed reticulocytes in the tested conditions. Moreover, hematopoietic stem and progenitor cells isolated from blood affected by hypoxia at high altitude did not result in different erythroid development, suggesting no retention of a high-altitude signature but rather an immediate adaptation to oxygen concentration. This adaptation was observed during in vitro erythropoiesis at three percent oxygen by a significantly increased glycolytic metabolic profile. These hypoxia-induced effects on in vitro erythropoiesis fail to provide an intrinsic explanation of the concept of neocytolysis.

Exposure to 16 h of normobaric hypoxia induces ionic edema in the healthy brain.

Following prolonged exposure to hypoxic conditions, for example, due to ascent to high altitude, stroke, or traumatic brain injury, cerebral edema can develop. The exact nature and genesis of hypoxia-induced edema in healthy individuals remain unresolved. We examined the effects of prolonged, normobaric hypoxia, induced by 16 h of exposure to simulated high altitude, on healthy brains using proton, dynamic contrast enhanced, and sodium MRI. This dual approach allowed us to directly measure key factors in the development of hypoxia-induced brain edema: (1) Sodium signals as a surrogate of the distribution of electrolytes within the cerebral tissue and (2) Ktrans as a marker of blood-brain-barrier integrity. The measurements point toward an accumulation of sodium ions in extra- but not in intracellular space in combination with an intact endothelium. Both findings in combination are indicative of ionic extracellular edema, a subtype of cerebral edema that was only recently specified as an intermittent, yet distinct stage between cytotoxic and vasogenic edemas. In sum, here a combination of imaging techniques demonstrates the development of ionic edemas following prolonged normobaric hypoxia in agreement with cascadic models of edema formation.

Absence of neocytolysis in humans returning from a 3-week high-altitude sojourn.

AIMS: Total haemoglobin mass (tot-Hb) increases during high-altitude acclimatization. Normalization of tot-Hb upon descent is thought to occur via neocytolysis, the selective destruction of newly formed erythrocytes. Because convincing experimental proof of neocytolysis is lacking, we performed a prospective study on erythrocyte survival after a stay at the Jungfraujoch Research Station (JFJRS; 3450 m). METHODS: Newly formed erythrocytes of 12 male subjects (mean age 23.3 years) were age cohort labelled in normoxia (110 m) and during a 19-day high-altitude sojourn by ingestion of 13 C2- and 15 N-labelled glycine respectively. Elimination dynamics for erythrocytes produced in normoxia and at high altitude were measured by isotope ratio mass spectrometry of haem, by determining tot-Hb, reticulocyte counts, erythrocyte membrane protein 4.1a/4.1b ratio and by mathematical modelling. RESULTS: Tot-Hb increased by 4.7% ± 2.7% at high altitude and returned to pre-altitude values within 11 days after descent. Elimination of 13 C- (normoxia) and 15 N- (high altitude) labelled erythrocytes was not different. Erythropoietin levels and counts of CD71-positive reticulocytes decreased rapidly after descent. The band 4.1a/4.1b ratio decreased at altitude and remained low for 3-4 days after descent and normalized slowly. There was no indication of haemolysis. CONCLUSION: We confirm a rapid normalization of tot-Hb upon descent. Based on the lack of accelerated removal of age cohorts of erythrocytes labelled at high altitude, on patterns of changes in reticulocyte counts and of the band 4.1a/4.1b ratio and on modelling, this decrease did not occur via neocytolysis, but by a reduced rate of erythropoiesis along with normal clearance of senescent erythrocytes.

Chronic rhinosinusitis with nasal polyps is associated with impaired TMEM16A-mediated epithelial chloride secretion.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is one of the most common chronic disorders with limited therapeutic options. However, the pathogenesis of CRSwNP remains poorly understood. OBJECTIVE: We sought to determine the role of abnormalities in nasal epithelial ion transport in primary epithelial cultures and patients with CRSwNP. METHODS: We studied epithelial ion transport and transcript levels of the Cl- channels cystic fibrosis transmembrane conductance regulator and transmembrane protein 16A (TMEM16A) in human primary nasal epithelial cultures of patients with CRSwNP and healthy controls. Furthermore, we determined expression levels of proinflammatory cytokines that have been implicated in the regulation of epithelial ion channels (IL-1ß, INF-γ, TNF-α, IL-13) and studied effects of the key TH2 signaling molecule IL-13 in CRSwNP and control nasal epithelial cultures. Finally, we measured in vivo nasal potential difference to compare epithelial ion transport in patients with CRSwNP and controls. RESULTS: Bioelectric studies demonstrated that Ca2+-activated Cl- secretion was reduced in CRSwNP versus control nasal epithelial cultures. Transcript levels of IL-13 and the Ca2+-activated Cl- channel TMEM16A were increased in CRSwNP cultures. Stimulation with IL-13 increased TMEM16A expression further and restored Ca2+-activated Cl- secretion in CRSwNP cultures. Nasal potential difference measurements demonstrated reduced Ca2+-activated Cl- transport in patients with CRSwNP versus controls. CONCLUSIONS: This study demonstrates that TMEM16A-mediated Ca2+-activated Cl- secretion is reduced in primary nasal epithelial cultures and nasal epithelia of patients with CRSwNP. Our data suggest that the Ca2+-activated Cl- channel TMEM16A may be implicated in the pathogenesis and serve as a novel therapeutic target in patients with CRSwNP.

Geographical ancestry affects normal hemoglobin values in high-altitude residents.

Increasing the hemoglobin (Hb) concentration is a major mechanism adjusting arterial oxygen content to decreased oxygen partial pressure of inspired air at high altitude. Approximately 5% of the world's population living at altitudes higher than 1,500 m shows this adaptive mechanism. Notably, there is a wide variation in the extent of increase in Hb concentration among different populations. This short review summarizes available information on Hb concentrations of high-altitude residents living at comparable altitudes (3,500-4,500 m) in different regions of the world. An increased Hb concentration is found in all high-altitude populations. The highest mean Hb concentration was found in adult male Andean residents and in Han Chinese living at high altitude, whereas it was lowest in Ethiopians, Tibetans, and Sherpas. A lower plasma volume in Andean high-altitude natives may offer a partial explanation. Indeed, male Andean high-altitude natives have a lower plasma volume than Tibetans and Ethiopians. Moreover, Hb values were lower in adult, nonpregnant females than in males; differences between populations of different ancestry were less pronounced. Various genetic polymorphisms were detected in high-altitude residents thought to favor life in a hypoxic environment, some of which correlate with the relatively low Hb concentration in the Tibetans and Ethiopians, whereas differences in angiotensin-converting enzyme allele distribution may be related to elevated Hb in the Andeans. Taken together, these results indicate different sensitivity of oxygen dependent control of erythropoiesis or plasma volume among populations of different geographical ancestry, offering explanations for differences in the Hb concentration at high altitude.

The role of hypoxia-induced modulation of alveolar epithelial Na+- transport in hypoxemia at high altitude.

Reabsorption of excess alveolar fluid is driven by vectorial Na+-transport across alveolar epithelium, which protects from alveolar flooding and facilitates gas exchange. Hypoxia inhibits Na+-reabsorption in cultured cells and in-vivo by decreasing activity of epithelial Na+-channels (ENaC), which impairs alveolar fluid clearance. Inhibition also occurs during in-vivo hypoxia in humans and laboratory animals. Signaling mechanisms that inhibit alveolar reabsorption are poorly understood. Because cellular adaptation to hypoxia is regulated by hypoxia-inducible transcription factors (HIF), we tested whether HIFs are involved in decreasing Na+-transport in hypoxic alveolar epithelium. Expression of HIFs was suppressed in cultured rat primary alveolar epithelial cells (AEC) with shRNAs. Hypoxia (1.5% O2, 24 h) decreased amiloride-sensitive transepithelial Na+-transport, decreased the mRNA expression of α-, ß-, and γ-ENaC subunits, and reduced the amount of αßγ-ENaC subunits in the apical plasma membrane. Silencing HIF-2α partially prevented impaired fluid reabsorption in hypoxic rats and prevented the hypoxia-induced decrease in α- but not the ßγ-subunits of ENaC protein expression resulting in a less active form of ENaC in hypoxic AEC. Inhibition of alveolar reabsorption also caused pulmonary vasoconstriction in ventilated rats. These results indicate that a HIF-2α-dependent decrease in Na+-transport in hypoxic alveolar epithelium decreases alveolar reabsorption. Because susceptibles to high-altitude pulmonary edema (HAPE) have decreased Na+-transport even in normoxia, inhibition of alveolar reabsorption by hypoxia at high altitude might further impair alveolar gas exchange. Thus, aggravated hypoxemia might further enhance hypoxic pulmonary vasoconstriction and might subsequently cause HAPE.

Iron metabolism in high-altitude residents.

Residents at high altitude cope with decreasing inspiratory oxygen partial pressure by stimulating erythropoiesis. The increase in hemoglobin levels requires high amounts of additional iron supplied from the diet. Here, we review available data on how iron metabolism adapts when living in a hypoxic environment. Our analysis reveals that long-term adaptation to high altitude enables healthy individuals to maintain their iron stores within the physiological range despite elevated requirements for erythropoiesis. However, in vulnerable populations with increased iron demand (e.g., pregnant women or exercising individuals), iron stores are less likely to be replenished quickly when living at high altitude. Future studies need to address whether different ethnicities have acquired genetic mechanisms to adapt to the elevated iron demand for erythropoiesis at high altitude.

Preserved right ventricular function but increased right atrial contractile demand in altitude-induced pulmonary hypertension.

PURPOSE: Ascent to high altitude increases right ventricular (RV) afterload and decreases myocardial energy supply. This study evaluates physiologic variables and comprehensive echocardiographic indices of RV and right atrial (RA) function following rapid ascent to high altitude. METHODS: Fifty healthy volunteers actively ascended from 1130 to 4559 m in < 22 h. All participants underwent 2D echocardiography during baseline examination at low altitude (424 m) and at three study time-points (7, 20 and 44 h) after arrival at high altitude. In addition to systolic pulmonary artery pressure (sPAP), comprehensive 2D planimetric-, tissue Doppler- and speckle-tracking-derived strain indices of RA and RV function were obtained. RESULTS: sPAP increased from baseline (24 ± 4 mmHg) to the first altitude examination (39 ± 8 mmHg, p < 0.001) and remained elevated during the following 44 h. Global RV function did not change. RA reservoir strain showed a trend towards increase from baseline (50.2 ± 12.1%) to the first altitude examination (53.8 ± 11.0%, p = 0.07) secondary to a significant increase of RA contraction strain (19.2 ± 6.4 vs. 25.4 ± 9.6%, p < 0.001). Volumetric RA data largely paralleled RA strain results and RA active emptying volume was increased throughout the 44 h stay at high altitude. CONCLUSION: Active and rapid ascent of healthy individuals to 4559 m is associated with an increased contractile performance of the RA that compensates for the increased workload of the RV.

Rapid Ascent to 4559 m Is Associated with Increased Plasma Components of the Vascular Endothelial Glycocalyx and May Be Associated with Acute Mountain Sickness.

Background: The stress of high altitude alters vascular permeability, which may be related to structural changes in the endothelial glycocalyx. We aimed to study these changes by measuring plasma concentrations of several glycocalyx components upon exposure to high altitude. Methods: Plasma collected from 17 subjects at low altitude (423 m) and at three time points (7, 20, and 44 hours) after rapid ascent to high altitude (4559 m) were evaluated for concentrations of three glycocalyx components: syndecan-1, intercellular adhesion molecule-1 (ICAM-1), and heparan sulfate. Vital signs and echocardiographic measurement of systolic pulmonary artery pressure (sPAP) and cardiac output were also obtained at low and high altitudes. Results: Mean age of the study population was 35.5 ± 11.2 years with a body mass index of 22.7 ± 2.5 kg/m2. Concentrations of ICAM-1 and heparan sulfate increased from baseline to 7 hours after arrival at high altitude; the ICAM-1 rise persisted at 20 hours. Syndecan-1 concentrations were increased only at 44 hours. Increased ICAM-1 concentrations correlated with sPAP and peripheral edema. Elevations in heparan sulfate appeared to correlate with acute mountain sickness (AMS). Conclusions: Levels of circulating glycocalyx components increase after exposure to high altitude and may correlate with AMS. Measuring plasma concentrations of various glycocalyx components could serve as a useful tool for further evaluation of vascular endothelial injury and repair in illness at high altitude.

Genetic Predisposition to High-Altitude Pulmonary Edema.

Background: Exaggerated pulmonary arterial hypertension (PAH) is a hallmark of high-altitude pulmonary edema (HAPE). The objective of this study was therefore to investigate genetic predisposition to HAPE by analyzing PAH candidate genes in a HAPE-susceptible (HAPE-S) family and in unrelated HAPE-S mountaineers. Materials and Methods: Eight family members and 64 mountaineers were clinically and genetically assessed using a PAH-specific gene panel for 42 genes by next-generation sequencing. Results: Two otherwise healthy family members, who developed re-entry HAPE at 3640 m during childhood, carried a likely pathogenic missense mutation (c.1198T>G p.Cys400Gly) in the Janus Kinase 2 (JAK2) gene. One of them progressed to a mild form of PAH at the age of 23 years. In two of the 64 HAPE-S mountaineers likely pathogenic variants have been detected, one missense mutation in the Cytochrome P1B1 gene, and a deletion in the Histidine-Rich Glycoprotein (HRG) gene. Conclusions: This is the first study identifying an inherited missense mutation of a gene related to PAH in a family with re-entry HAPE showing a progression to borderline PAH in the index patient. Likely pathogenic variants in 3.1% of HAPE-S mountaineers suggest a genetic predisposition in some individuals that might be linked to PAH signaling pathways.