Rationale: Chronic exposure to hypoxia is associated with elevated sympathetic nervous activity and reduced vascular function in lowlanders, and Andean highlanders suffering from excessive erythrocytosis (EE); however, the mechanistic link between chronically elevated sympathetic nervous activity and hypoxia-induced vascular dysfunction has not been determined. Objective: To determine the impact of heightened sympathetic nervous activity on resistance artery endothelial-dependent dilation (EDD), and endothelial-independent dilation, in lowlanders and Andean highlanders with and without EE. Methods and Results: We tested healthy lowlanders (n=9) at sea level (344 m) and following 14 to 21 days at high altitude (4300 m), and permanent Andean highlanders with (n=6) and without (n=9) EE at high altitude. Vascular function was assessed using intraarterial infusions (3 progressive doses) of acetylcholine (ACh; EDD) and sodium nitroprusside (endothelial-independent dilation) before and after local α+β adrenergic receptor blockade (phentolamine and propranolol). Intraarterial blood pressure, heart rate, and simultaneous brachial artery diameter and blood velocity were recorded at rest and during drug infusion. Changes in forearm vascular conductance were calculated. The main findings were (1) chronic hypoxia reduced EDD in lowlanders (changes in forearm vascular conductance from sea level: ACh1: −52.7±19.6%, ACh2: −25.4±38.7%, ACh3: −35.1±34.7%, all P ≤0.02); and in Andeans with EE compared with non-EE (changes in forearm vascular conductance at ACh3: −36.4%, P =0.007). Adrenergic blockade fully restored EDD in lowlanders at high altitude, and normalized EDD between EE and non-EE Andeans. (2) Chronic hypoxia had no effect on endothelial-independent dilation in lowlanders, and no differences were detected between EE and non-EE Andeans; however, EID was increased in the non-EE Andeans after adrenergic blockade ( P =0.012), but this effect was not observed in the EE Andeans. Conclusions: These data indicate that chronic hypoxia reduces EDD via heightened α-adrenergic signaling in lowlanders and in Andeans with EE. These vascular mechanisms have important implications for understanding the physiological consequences of acute and chronic high altitude adaptation.

Erythrocytosis, or increased production of red blood cells, is one of the most well-documented physiological traits that varies within and among in high-altitude populations. Although a modest increase in blood O2-carrying capacity may be beneficial for life in highland environments, erythrocytosis can also become excessive and lead to maladaptive syndromes such as Chronic Mountain Sickness (CMS).

Monge’s disease (chronic mountain sickness (CMS)) is a maladaptive condition caused by chronic (years) exposure to high-altitude hypoxia. One of the defining features of CMS is excessive erythrocytosis with extremely high hematocrit levels. In the Andean population, CMS prevalence is vastly different between males and females, being rare in females. Furthermore, there is a sharp increase in CMS incidence in females after menopause. In this study, we assessed the role of sex hormones (testosterone, progesterone, and estrogen) in CMS and non-CMS cells using a well-characterized in vitro erythroid platform. While we found that there was a mild (nonsignificant) increase in RBC production with testosterone, we observed that estrogen, in physiologic concentrations, reduced sharply CD235a+ cells (glycophorin A; a marker of RBC), from 56% in the untreated CMS cells to 10% in the treated CMS cells, in a stage-specific and dose-responsive manner. At the molecular level, we determined that estrogen has a direct effect on GATA1, remarkably decreasing the messenger RNA (mRNA) and protein levels of GATA1 (p < 0.01) and its target genes (Alas2, BclxL, and Epor, p < 0.001). These changes result in a significant increase in apoptosis of erythroid cells. We also demonstrate that estrogen regulates erythropoiesis in CMS patients through estrogen beta signaling and that its inhibition can diminish the effects of estrogen by significantly increasing HIF1, VEGF, and GATA1 mRNA levels. Taken altogether, our results indicate that estrogen has a major impact on the regulation of erythropoiesis, particularly under chronic hypoxic conditions, and has the potential to treat blood diseases, such as high altitude severe erythrocytosis.

Excessive erythrocytosis (EE; hemoglobin concentration [Hb] ≥21 g/dL in adult males) is associated with increased cardiovascular risk in highlander Andeans. We sought to quantify shear stress and assess endothelial function via flow-mediated dilation (FMD) in male Andeans with and without EE. We hypothesized that FMD would be impaired in Andeans with EE after accounting for shear stress and that FMD would improve after isovolemic hemodilution. Brachial artery shear stress and FMD were assessed in 23 male Andeans without EE (age: 40±15 years [mean±SD]; Hb<21 g/dL) and 19 male Andeans with EE (age: 43±14 years; Hb≥21 g/dL) in Cerro de Pasco, Peru (4330 m). Shear stress was quantified from Duplex ultrasound measures of shear rate and blood viscosity. In a subset of participants (n=8), FMD was performed before and after isovolemic hemodilution with blood volume replaced by an equal volume of human serum albumin. Blood viscosity and Hb were 48% and 23% higher (both P <0.001) and FMD was 28% lower after adjusting for the shear stress stimulus ( P =0.013) in Andeans with EE compared to those without. FMD was inversely correlated with blood viscosity ( r 2 =0.303; P<0.001) and Hb ( r 2 =0.230; P =0.001). Isovolemic hemodilution decreased blood viscosity by 30±10% and Hb by 14±5% (both P <0.001) and improved shear stress stimulus-adjusted FMD from 2.7±1.9% to 4.3±1.9% ( P =0.022). Hyperviscosity, high Hb, or both, actively contribute to acutely reversible impairments in FMD in EE, suggesting that this plays a pathogenic role in the increased cardiovascular risk.

At high altitude Andean region, hypoxia-induced excessive erythrocytosis (EE) is the defining feature of Monge’s disease or chronic mountain sickness (CMS). At the same altitude, resides a population that has developed adaptive mechanism(s) to constrain this hypoxic response (non-CMS). In this study, we utilized an in vitro induced pluripotent stem cell model system to study both populations using genomic and molecular approaches. Our whole genome analysis of the two groups identified differential SNPs between the CMS and non-CMS subjects in the ARID1B region. Under hypoxia, the expression levels of ARID1B significantly increased in the non-CMS cells but decreased in the CMS cells. At the molecular level, ARID1B knockdown (KD) in non-CMS cells increased the levels of the transcriptional regulator GATA1 by 3-fold and RBC levels by 100-fold under hypoxia. ARID1B KD in non-CMS cells led to increased proliferation and EPO sensitivity by lowering p53 levels and decreasing apoptosis through GATA1 mediation. Interestingly, under hypoxia ARID1B showed an epigenetic role, altering the chromatin states of erythroid genes. Indeed, combined Real-time PCR and ATAC-Seq results showed that ARID1B modulates the expression of GATA1 and p53 and chromatin accessibility at GATA1/p53 target genes. We conclude that ARID1B is a novel erythroid regulator under hypoxia that controls various aspects of erythropoiesis in high-altitude dwellers.

Rationale Excessive erythrocytosis (EE; Hb≥21g/dL in men and ≥19g/dL in women) is the main sign of Chronic Mountain Sickness (CMS), a maladaptive clinical syndrome prevalent in Andean and other high‐altitude populations. Although the pathophysiological mechanism for EE is still controversial, chronic hypoxemia is accepted as its underlying stimulus. Although EE is common to all CMS subjects, most studies have shown similar mean values for serum erythropoietin compared to healthy highlanders (HH). We have previously shown that plasma concentration of the soluble Epo receptor (sEpoR), an endogenous Epo antagonist, is decreased in highlanders with CMS, which would lead to higher Epo circulating availability and contribute to EE. However, it is also possible that erythroid progenitor cells of CMS subjects are more sensitive to Epo, and have a stronger proliferative response than HH cells exposed to similar circulating Epo concentration. Thus, the aim of the present study was to determine the hypoxic proliferative response of erythroid progenitor cells (BFU‐E) of CMS and HH to equal rhEpo concentration. Methods SamplesThirty‐six highlanders (CMS, n=17; HH, n=19) from Cerro de Pasco, Peru, at 4340m, were recruited for the study. A small blood sample was obtained for hematocrit determination and CMS score was assessed. A 10ml blood sample was taken in heparin‐coated BD Vacutainer tubes for peripheral blood mononuclear cells (PBMCs) isolation. Two additional 6ml blood samples were also obtained in clot‐activator‐coated tubes for Epo and iron profile determination. Serum was separated by centrifugation and stored in liquid N2 until analysis. CulturePBMCs were isolated using Histopaque 1077 (Sigma) and were cultured in Methocult H‐4534 medium (Stemcell Technologies) at a final density of 2.5×105cells/ml in the presence of 3U/ml of rhEpo (Stemcell Technologies) for 14 days. Cultures were maintained at 37°C in a Biospherix X3 hood set for cellular hypoxia (1%O2, 5% CO2). BFU‐E identification and countColony identification and count were performed at days 7 and 10 using an inverted microscope (LS 560, Lumascope) inside the hypoxic hood at a magnification of 20X. On day 14 cultures were removed from the hood and analyzed using an AxioZoom Stereomicroscope (Carl Zeiss Microscopy) at a magnification of 10X. Data analysisStudent's t‐test for equal and unequal variances was applied as parametric test of comparison and Wilcoxon as nonparametric to evaluate differences between CMS and HH. Results As expected, Hct and CMS score were significantly higher in the CMS group (Table 1). Mean serum Epo concentration was higher in CMS subjects (12.79±2.8 vs 26.8±8.1pg/ml, p<0.05) due to two participants showing very high Epo values (high‐Epo CMS highlanders, as previously described). Percentage of change in BFU‐E number from day 7 to day 14 was larger in the CMS group (87.84±27.2 vs 258.6±69.12%, Figure 1B), and CMS BFU‐E colonies occupied a larger total area compared to HH (4.31±0.5 vs 6.4±0.5mm2, Figure 1A). Conclusion Our findings show th...

Excessive erythrocytosis [EE; hemoglobin concentration (Hb) ≥ 21 g/dL in adult men] is a maladaptive high-altitude pathology associated with increased cardiovascular risk and reduced reactive hyperemia flow-mediated dilation (FMD); however, whether a similar impairment occurs in response to more commonly encountered sustained increases in shear stress [sustained stimulus (SS)-FMD] over a range of overlapping stimuli is unknown. We characterized SS-FMD in response to handgrip exercise in Andeans with and without EE in Cerro de Pasco, Peru (4,330 m). Andean highlanders with EE ( n = 17, Hb = 23.2 ± 1.2 g/dL) and without EE ( n = 23, Hb = 18.7 ± 1.9 g/dL) performed 3 min of rhythmic handgrip exercise at 20, 35, and 50% of maximum voluntary contraction (MVC). Duplex ultrasound was used to continuously record blood velocity and diameter in the brachial artery, and blood viscosity was measured to accurately calculate shear stress. Although baseline shear stress did not differ, Andeans with EE had 22% lower shear stress than Andeans without at 50% MVC ( P = 0.004). At 35 and 50% MVC, SS-FMD was 2.1 ± 2.0 and 2.8 ± 2.7% in Andeans with EE compared with 4.1 ± 3.4 and 7.5 ± 4.5% in those without ( P = 0.048 and P < 0.001). The stimulus-response slope (∆shear stress vs. ∆diameter) was lower in Andeans with EE compared with Andeans without ( P = 0.028). This slope was inversely related to Hb in Andeans with EE ( r2 = 0.396, P = 0.007). A reduced SS-FMD in response to small muscle mass exercise in Andeans with EE indicates a generalized reduction in endothelial sensitivity to shear stress, which may contribute to increased cardiovascular risk in this population. NEW & NOTEWORTHY High-altitude excessive erythrocytosis (EE; hemoglobin concentration ≥ 21 g/dL) is a maladaptation to chronic hypoxia exposure and is associated with increased cardiovascular risk. We examined flow-mediated dilation (FMD) in response to sustained elevations in shear stress achieved using progressive handgrip exercise [sustained stimulus (SS)-FMD] in Andean highlanders with and without EE at 4,330 m. Andeans with EE demonstrated lower SS-FMD compared with those without. Heightened hemoglobin concentration was related to lower SS-FMD in Andeans with EE.

Wales and member of the Neurovascular Research Laboratory led by Professor Damian Bailey where he completed his PhD. Damian M. Bailey is a Royal Society Wolfson Research Fellow and Professor of Physiology and Biochemistry at the University of South Wales. He is Editor-in-Chief of Experimental Physiology and Chair of the Life Sciences Working Group to the European Space Agency. The Neurovascular Research Laboratory takes an integrated translational approach to investigate how free radicals and associated reactive oxygen/nitrogen species control substrate delivery to the brain across the clinical spectrum of human health and disease.