Evaluating the Molecular Basis for Acute Mountain Sickness: Hypoxia Response Gene Expression Patterns in Warfighters and Murine Populations

ABSTRACT

Acute mountain sickness (AMS) is an illness that affects many individuals at altitudes above 2,400 m (8,000 ft) resulting in decreased performance. Models that provide quantitative estimates of AMS risk are expanding, but predictive genetic models for AMS susceptibility are still under investigation. Thirty-four male U.S. Army Soldier volunteers were exposed to baseline, 3,000 m, 3,500 m, or 4,500 m altitude conditions in a hypobaric chamber and evaluated for onset of AMS symptoms. In addition, mice were evaluated at extreme hypoxia conditions equivalent to 7,600 m. Real-time polymerase chain reaction hypoxia response array was used to identify 15 genes that were activated in Soldiers and 46 genes that were activated in mice. We identified angiopoietin-like 4 (ANGPTL4) as a gene that is significantly activated in response to hypoxia (5.8-fold upregulated at 4,500 m in humans). The role of ANGPTL4 in high-altitude response has not been explored. Pretreatment of mice with fenofibrate, an ANGPTL4-activating pharmaceutical, had a considerable effect on overall hypoxia response gene expression and resulted in significantly decreased cerebral edema following exposure to hypoxia. Activation of ANGPTL4 may protect against cerebral edema by inhibiting vascular endothelial growth factor and therefore serve as a potential target for AMS prevention.

INTRODUCTION

Foreign contingency operations frequently require United States military action in mountainous environments at moderate (1,500 m–3,500 m) to high altitudes (3,501 m–5,500 m).¹ Acute mountain sickness (AMS) is an illness that affects many individuals at altitudes above 2,400 m (8,000 ft) resulting in decreased performance.² In addition, some altitude-sensitive individuals develop potentially fatal pulmonary or cerebral edema. A number of predictive models of AMS have been published that correlate altitude, rate of ascent, and physical activity to risk of AMS within populations.³ However, there are no standard laboratory tests to identify individuals that may be at risk for altitude-related illness other than previous incidence.

AMS is characterized by headache, nausea, vomiting, dizziness, decreased energy, and insomnia.⁴ Symptoms generally appear 6 to 12 hours after arrival at altitude with faster ascent and higher elevation resulting in greater incidence and severity of AMS.^3,5 Individuals with a previous history of AMS are more likely to develop AMS during subsequent ascents compared to individuals who have ascended to altitude previously without developing AMS but factors such as age, race, training, and body mass index are not correlated with AMS susceptibility.^5,–7 Numerous studies indicate that some individuals are more susceptible to AMS than others which supports the hypothesis of a definable genetic basis for AMS susceptibility.^8,–10

There is no consensus regarding the pathophysiological mechanism of AMS, but increased cerebral vascular leakiness is considered by some to be a contributing factor.^2,12 Magnetic resonance imaging has provided evidence for mild cerebral edema in response to hypoxia and current studies have correlated increased blood–brain barrier permeability to high-altitude cerebral edema but the role of cerebral permeability in AMS susceptibility is still under investigation.^13,14

The goals of this study were to (1) determine the incidence of AMS in U.S. Army Soldier populations at different elevations, (2) use focused “hypoxia signaling response” gene expression analysis techniques to evaluate gene expression patterns at various altitudes, and (3) use the gene expression data from humans and mice to identify potential markers of AMS susceptibility and targets for advanced prophylaxis.

MATERIALS AND METHODS

Human Study Population

Informed consent was obtained from all subjects, as reviewed and approved by a human subject institutional review board. Investigators adhered to policies for protection of human subjects as prescribed in Army Regulations 70-25 and U.S. Army Medical Research and Materiel Command Regulation 70-25. The research was conducted in adherence with the provisions of 45 Code of Federal Regulations Part 46. Thirty-four male volunteers with an average age of 22.2 ± 1.1 years and weight of 82.3 ± 2.3 kg were exposed to simulated altitude conditions in a hypobaric chamber ranging from baseline (55 m) to 4,500 m (3,000 m, 3500 m, or 4,500 m) for 24 hours. Blood samples were obtained from individuals at baseline and altitude (morning following 24 hours). The diagnosis of AMS was determined by AMS-Cerebral (AMS-C) score from the Environmental Symptoms Questionnaire.¹⁵

DNA and RNA Isolation

Genomic DNA and RNA were isolated from human and mouse whole blood samples using the PAXgene system (Qiagen, Valencia, California) per the manufacturer's instructions and stored at −20°C. PAXgene blood RNA tubes contain a proprietary reagent composition that allows for RNA stabilization as well as minimal ex vivo RNA transcription to facilitate reliable analysis of gene expression profiles.¹⁶

Human Study: Real-Time Quantitative PCR Array and Data Analysis

Polymerase chain reaction (PCR) array was conducted to compare hypoxia response gene expression patterns at 3,000 m, 3,500 m, and 4,500 m. Gene expression profiles were determined using RNA samples taken from individual AMS positive Soldiers at altitude and compared to RNA samples taken from the same individual at baseline. Baseline RNA samples were collected before exposure to altitude conditions. PCR array was conducted using an RT² Profiler Human Hypoxia Signaling Pathway PCR Array (Catalog no. PAHS-032A; SABiosciences, Frederick, Maryland) according to the manufacturer's instructions. This array allowed for simultaneous analysis of 84 genes known to be involved in human hypoxia-related signaling and the physiological response to hypoxia. The cDNA was prepared by reverse transcription using the RT² PCR Array First Strand kit (SA Biosciences) as recommended by the manufacturer. Real-time PCR (RT-PCR) was performed using a Bio-Rad iCycler iQ5 Multicolor Real-Time PCR Detection System (Hercules, California). Briefly, 25 μL of PCR array reaction mixture (1,350 μL SuperArray RT² qPCR Master Mix solution, 102 μL first strand cDNA synthesis reaction, and 1,248 μL dd H₂O) was loaded into each well of each PCR array plate. PCR amplification of cDNA was performed under the following conditions: initial denaturation for 10 minutes at 95°C for one cycle, followed by 40 cycles of 15 seconds at 95°C, and 1 minute at 60°C. Threshold and baseline values were set manually according to the manufacturer's instructions. The resulting threshold cycle values for groups of 3 to 4 individuals for each altitude and baseline were transferred into a data analysis template spreadsheet and uploaded onto the manufacturer's Web site (http://www.sabiosciences.com/per/arrayanalysis.php) for analysis. The mRNA expression level of each gene was normalized using the expression of five housekeeping genes (β-actin, β-2 microglobulin, glyceraldehyde-3-phosphate dehydrogenase, β-glucuronidase, and heat shock protein 90 α [cytosolic] class B member 1). The relative expression of each gene compared to the baseline altitude control group was calculated on the manufacturer's Web site using the ΔΔCT method. A fold change (FC) in gene expression ≥2.5 was determined to be significant relative to baseline controls (confidence interval [CI] > 95%).

Mouse Study Population

All animal research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations related to animals under an Institutional Animal Care and Use Committee approved animal protocol. All experiments involving animals were performed according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Ed 8.¹⁷ Ten-week-old female C57BL/6 mice were exposed to normobaric hypoxia at 8% oxygen for 24 hours or were kept at normal atmospheric oxygen (normoxia). Hypoxia was achieved using a Colorado Altitude Training hypoxia system (Colorado Altitude Training, Louisville, Colorado). Mice were gradually brought from normoxia to 8% oxygen over a 3-hour period (beginning in the morning with 8% hypoxia achieved by mid-day). Mice had free access to food and water during the experiment. Mice were taken from hypoxia and immediately anesthetized for the analysis of vascular permeability by fluorescein assay.

Mouse Study: Real-Time Quantitative PCR Array and Data Analysis

To compare hypoxia response gene expression patterns between humans and mice and to evaluate the effect of fenofibrate pretreatment on hypoxia response gene expression in mice, PCR array was conducted in mice that were exposed to hypoxia (8% oxygen) or normoxia (21% oxygen) for 24 hours. Fenofibrate pretreatment was performed in mice by IP injection of 0.2 mL total volume fenofibrate (2.5 mg/mouse) solubilized in a solution of 10% dimethyl sulfoxide, 10% ethanol, 20% Solutol HS 15 (Sigma-Aldrich, St. Louis, Missouri), and 60% water. All mice were weighed before injection to ensure ∼25 g body mass for a final dose concentration of ∼100 mg/kg. Fenofibrate injections were given 24 hours before and again 1 hour before hypoxia exposure. PCR array was conducted using an RT² Profiler Mouse Hypoxia Signaling Pathway PCR Array (Catalog no. PAMM-032ZA, SABiosciences) according to the manufacturer's instructions. Reaction conditions and data analysis were identical to the human study. A FC in gene expression ≥3.2 was determined to be significant relative to baseline controls (CI > 95%).

Determination of Cerebral Vascular Permeability in Mice by Fluorescein Assay

Sodium fluorescein (MW 376.3) is a fluorescent tracer that does not cross an intact blood–brain barrier and has been used to assay vascular permeability of brain vessels.^12,18 Pretreatment with fenofibrate was performed to evaluate relative cerebral vascular permeability following exposure to hypoxia. To quantify vascular permeability of brain vessels, female 8- to 12-week-old C57BL/6 mice (10-week-old average) were anaesthetized and 500 μL of sodium fluorescein (Sigma-Aldrich) at a concentration of 20 mg/mL in phosphate buffered saline (PBS) was injected IP immediately after 24 hours of hypoxic or normoxic exposure. One hour later, mice were euthanized by injection with Euthasol (pentobarbital sodium and phenytoin sodium) (Virbac AH, Fort Worth, Texas). Perfusion with excess PBS through the left heart ventricle was used to remove residual fluorescent tracer from the vascular bed. Subsequently, both brain hemispheres were removed, weighed, and frozen at −20°C. To assess fluorescence, brain hemispheres were homogenized in PBS and spun at 1,000 × g for 15 minutes at 4°C. Soluble proteins were then precipitated from the supernatants using equal volume ethanol and samples were spun again at 20,000 × g for 20 minutes at 4°C. Supernatant fluorescence was measured at 485 nm at an excitation wavelength of 330 nm using a Nanodrop Spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware). Relative fluorescent units (rfu) were determined as fluorescence per mg wet weight of tissue.

RESULTS

Human AMS Susceptibility Study

Human volunteers were exposed to simulated altitude conditions ranging from baseline (55 m) to a maximum of 4,500 m. AMS-C scores were recorded for individuals 6 times during altitude exposure. Any peak AMS-C score ≥0.7 was considered positive for AMS. The observed frequency of AMS in the human study population was 7/12 (58%) at 3,000 m, 6/11 (55%) at 3,500 m, and 11/11 (100%) at 4,500 m.

Human Gene Expression Study

Gene expression levels for hypoxia response genes were determined using RNA samples taken at baseline and after 24-hour exposure to simulated altitude produced by hypobaric chamber (Table I). The atmospheric pressure and relative available oxygen concentrations (expressed as a percentage of available oxygen compared to sea level) for each altitude were as follows: baseline control 55 m (1.0 atm, 21% O₂), 3,000 m (0.71 atm, 15% O₂), 3,500 m (0.66 atm, 14% O₂), and 4,500 m (0.59 atm, 12% O₂). Expression values ≥ 2.5-fold change in gene regulation over baseline was determined to represent significant changes in gene expression for the human study (CI > 95%).

RT-PCR array identified differentially expressed genes in all of the groups that were surveyed compared to baseline. Two genes at 3,000 m, 3 genes at 3,500 m, and 14 genes at 4,500 m were found to be significantly upregulated in response to hypoxia in AMS positive Soldiers (Table I). At 3,000 m, significantly upregulated genes included angiopoietin-like-4 (ANGPTL4) and pyruvate kinase, muscle (PKM2). At 3,500 m, significantly upregulated genes included ANGPTL4, adenosine A2b receptor (ADORA2B), and mitogen-activated protein kinase kinase kinase 1 (MAP3K1). As expected, the greatest number of significantly upregulated hypoxia response genes was observed in the 4,500 m group. A total of 14 genes were ≥2.5-fold upregulated at 4,500 m compared to baseline (Table I). None of the hypoxia response genes in our analysis were significantly downregulated at any altitude. The greatest change in expression among all of the human genes in our analysis was observed for ANGPTL4, which was 5.8-fold upregulated at 4,500 m. ANGPTL4 was consistently upregulated across all groups at 3,000 m, 3,500 m, and 4,500 m. ANGPTL4 was upregulated in an altitude-dependent manner with increased expression corresponding to increased altitude.

Mouse Gene Expression Study

Hypoxia response gene expression patterns were evaluated for mice that were exposed to normobaric hypoxia (8% oxygen) compared to ambient air (21% O₂) at a baseline altitude of 143 m (Fig. 1; Table II). Hypoxia response genes were clearly activated in mice exposed to 8% hypoxia (equivalent to 7,600 m altitude) for 24 hours. Forty-six hypoxia response genes were ≥3.2-fold upregulated in response to hypoxia alone. The 95% CI for the mouse study was slightly higher than the human study (95% CI ≥ 3.2 FC). The mouse Angptl4 gene, homologous to the most highly upregulated gene from the human expression analysis, was 38-fold upregulated in hypoxic mice compared to controls. Additional highly upregulated genes (≥20-fold upregulation) included aldolase A, fructose-bisphosphate (Aldoa) 72-fold, ankyrin repeat domain 37 (Ankrd37) 23-fold, annexin A2 (Anxa2) 75-fold, basic helix-loop-helix family member e40 (Bhlhe40) 20-fold, BCL2/adenovirus E1B-interacting protein 3-like (Bnip3l) 34-fold, B-cell translocation gene 1 antiproliferative (Btg1) 26-fold, eukaryotic translation initiation factor 4E-binding protein 1 (Eif4ebp1) 20-fold, and coagulation factor X (F10) 22-fold.

Fenofibrate is a fibrate class drug that activates gene expression through the peroxisome proliferator–activated receptor α (PPARα) element.^19,20 We selected fenofibrate for further analysis because this drug is a potent activator of PPARα-mediated gene expression and fenofibrate has been shown to increase blood plasma levels of truncated ANGPTL4 protein in humans.²¹ Fenofibrate pretreatment alone resulted in measureable changes in gene expression for normoxic mice (Table II). Of the 84 genes in our hypoxia response analysis, 14 genes were found to be at least 3.2-fold upregulated, whereas 14 genes were at least 3.2-fold downregulated relative to controls. However, the degree of variation in differential gene expression in response to fenofibrate pretreatment was much less pronounced than the changes in fold regulation that were observed with hypoxic mice. The most highly upregulated genes in response to normoxic fenofibrate pretreatment were Aldoa (28-fold), Angptl4 (7-fold), and Anxa2 (19-fold).

The combination of fenofibrate pretreatment and exposure to 8% hypoxia appeared to synergistically increase the expression of certain hypoxia response genes (Table II). Fifty-seven of the genes in our panel were at least 3.2-fold upregulated in fenofibrate pretreated mice following exposure to 8% oxygen for 24 hours. Of the 46 hypoxia response genes that were upregulated by hypoxia alone, 27 genes demonstrated enhanced activation in mice that were pretreated with fenofibrate before hypoxia exposure. The “fenofibrate effect” on gene upregulation was most pronounced for Aldoa (72-fold with hypoxia alone compared to 134-fold with fenofibrate pretreatment before hypoxia exposure), Angptl4 (38-fold upregulation with hypoxia alone compared to 54-fold with fenofibrate pretreatment), Anxa2 (increased from 75-fold to 151-fold with fenofibrate pretreatment), and F10 (increased from 22-fold to 44-fold).

Fluorescein Assay

To simulate exposure to high altitude, mice were exposed to normobaric hypoxia (8% oxygen) for a period of 24 hours. We used sodium fluorescein as a marker for direct visualization of hypoxia-induced vascular leakage.¹² Sodium fluorescein was injected IP and allowed to enter circulation over the course of 1 hour as described.^22,23

The average rfu from the brains of mice that were exposed to 8% oxygen was 1.9-fold higher than mice that were kept in normal atmospheric oxygen (Fig. 2). Average fluorescence for control mice was 25 ± 8 rfu/mg (n = 9). Average fluorescence increased to 47 ± 7 rfu/mg (n = 9) following 24-hour exposure to 8% oxygen in mice that were not pretreated with fenofibrate. Average fluorescence for mice that were pretreated with fenofibrate before 24-hour hypoxia exposure remained similar to normoxic control mice. The average fluorescence for pretreated normoxic mice was 24 ± 9 rfu/mg (n = 9) compared to 25 ± 10 rfu/mg for fenofibrate pretreated hypoxic mice (n = 9).

DISCUSSION

The purpose of this study was to determine hypoxia signaling pathway gene expression patterns in Soldiers that were exposed to various “operationally relevant” altitude conditions. “Operationally relevant” refers to altitudes at which ground-based military operations may be expected to occur.¹ Since rodent models are commonly used in hypoxia research, we included the murine model to evaluate similarities and differences in gene expression patterns between humans and mice. The mouse model also allowed for evaluation of gene expression in extremely hypoxic conditions. As expected, numerous genes were activated in response to hypoxia. The greatest change in expression among Soldiers was observed for ANGPTL4, which was 5.8-fold upregulated at 4,500 m. The ANGPTL4 gene was of particular interest because of its high level of activation at operationally relevant altitudes as well as the known relationships between ANGPTL4 protein and modulators of human hypoxia response such as vascular endothelial growth factor (VEGF). In addition, ANGPTL4 has been shown to play an integral role in the regulation of angiogenesis and hypoxia-induced vascular permeability.^24,25

We identified diverse sets of genes at 3,000 m, 3,500 m, and 4,500 m that were ≥2.5-fold upregulated relative to controls in humans. Certain genes of interest, such as ANGPTL4, were upregulated in a “dose-dependent” (altitude-dependent) manner. Interestingly, many of the other hypoxia response genes in the PCR array panel did not appear to be activated at the altitudes we examined. These results suggest that dramatic changes in the differential expression of many hypoxia response genes, at least those genes that are detectable in human whole blood after 24 hours, are not occurring at operationally relevant altitudes in Soldiers. Our results do not rule out the differential expression of additional hypoxia response genes in different tissue types. In addition, our analysis focused on steady state changes in gene expression after 24-hour exposure to altitude conditions, which would not allow for the identification of early transient changes in gene expression.

Hypoxia response gene expression in mice was dramatically more pronounced compared to the human subjects. Although the human subjects were evaluated across a range of “military relevant” altitudes in a hypobaric chamber, mice were exposed to hypoxia equivalent to approximately 7,600 m (8% oxygen). For reference, the summit of Mount Everest in the Mahalangur Himalaya mountain range is 8,848 m. Close to 100% of a human population would be expected to exhibit AMS symptoms at 7,600 m with a significantly increased risk for development of more severe high altitude cerebral and pulmonary edema without a substantial acclimatization process.²⁶ As described by Schoch et al, the 8% oxygen hypoxic conditions were necessary to increase cerebral vascular permeability in mice to a level measurable by fluorescein assay.¹²

In response to 8% hypoxia alone, we observed 46 genes of interest that were ≥3.2-fold upregulated in mice. None of the genes in the 84 gene hypoxia signaling pathway panel were downregulated compared to normoxic controls. The homolog for the most highly upregulated gene from the human study, ANGPTL4, was also notably activated in hypoxic mice (38-fold upregulated). Of the 13 genes that were activated at 4,500 m in humans, 8 were also found to be upregulated in mice—the exceptions being Map3k1, Nfkb1, Per1, Serpine1, and Vegfa. Conversely, the two most highly upregulated genes from the mice study Aldoa and Anxa2 were not significantly upregulated in our Soldier population at 4,500 m.

ANGPTL4 has been well characterized as a pleiotrophic protein with functions in lipid metabolism, glucose homeostasis, inhibition of angiogenesis, and vascular permeability (Fig. 3).^27,29 The ANGPTL4 protein exhibits a modular structure with an N-terminal secretion signal, two coiled-coil domains, and a fibrinogen-like domain.³⁰ The protein inhibits vascular permeability and angiogenesis likely through modulation of VEGF.²⁴ VEGFA is a glycosylated mitogen that specifically acts on endothelial cells and has various effects, including mediating increased vascular permeability, inducing angiogenesis, vasculogenesis, endothelial cell growth, promoting cell migration, and inhibiting apoptosis.³¹ VEGF is a central player in the molecular response to hypoxia and the protein was originally identified as a “vascular permeability factor.” Several studies support the importance of VEGF in hypoxia response and cerebral vascular leakiness.¹²

The transcriptional regulation of ANGPTL4 gene expression is known to be controlled by glucocorticoid response elements and peroxisome proliferation activated receptor elements (PPAR) (Fig. 3).^21,27 All three classes of PPAR agonists, α, β/δ, and γ have all been shown to increase ANGPTL4 expression through the gene's PPAR element.²¹ Specific PPAR agonists include fenofibrate and thiazolidinediones such as rosiglitazone and pioglitazone. ANGPTL4 has been shown to be upregulated in response to hypoxia, fasting, and fenofibrate.^21,33 Interestingly, ANGPTL4 is also known to be upregulated in response to dexamethasone.²⁷ Dexamethasone is a potent synthetic glucocorticoid receptor agonist and a well established treatment for AMS and high-altitude cerebral edema.

In this study, mice were pretreated with fenofibrate before hypoxia exposure. Fenofibrate was selected because of its role as a PPAR agonist and activator of ANGPTL4 expression. A final fenofibrate dose of ∼100 mg/kg was used in the mice for this study. For comparison, a typical human dose for fenofibrate ranges from 48 to 145 mg/day. Selection of an appropriate fenofibrate dose range for AMS prevention in humans will be an important consideration for future studies.

In a recent whole-genome microarray study, ANGPTL4 was identified as a significantly upregulated gene in response to fenofibrate treatment in human hepatocytes.³⁴ An advantage of targeted gene expression profiling compared to whole-genome microarray is the ability to focus on particular biological pathway genes of interest when the relevant pathways are already known. Although a primary advantage of whole-genome microarray is the wealth of gene expression information obtained across the transcriptome, the large amounts of data generated by whole-genome approaches require systems biology–based computational methods to produce comprehensible information and chip-based microarray results are often confirmed by RT-PCR. The end results of systems based analytics are typically the identification of biological pathways and representative genes within those pathways that appear to be differentially expressed in response to a given variable. When the biological pathways of interest are already known, such as the case with hypoxia response, a focused pathway array is a better analytical tool.

Targeted gene expression profiling allows for both hypothesis testing and the generation of new avenues for research. Thorough analysis of the genes identified in this study, to include polymorphic variation analysis of the identified genes of interest, may better elucidate the relationship between these genes and AMS susceptibility.³⁵ In addition, the downregulation of hypoxia response genes by fenofibrate in the absence of hypoxia is of particular interest and represents an interesting opportunity for further study. A decrease in baseline levels of hypoxia response proteins may significantly influence the overall physiological response to altitude. The most highly downregulated genes were Epo (7-fold), Hif3a (9-fold), Nos3 (13-fold), and Pgf (15-fold). Also of interest was the downregulation of Vegfa (5-fold) in mice following fenofibrate pretreatment. Decreased steady state levels of VEGF may contribute to decreased cerebral vascular permeability following fenofibrate pretreatment.

Experimental evidence supports a genetic basis for susceptibility and resistance to altitude-related illness, but additional work is needed to better elucidate the impact of individual genes, the synergistic role of multigene polymorphisms and the molecular pathways involved.^36,–38 The results from the present study suggest that pretreatment with fenofibrate will reduce cerebral vascular permeability following 24-hour exposure to 8% hypoxia in mice. Fenofibrate is generally well tolerated in humans and the “potential” for fenofibrate to serve as a novel pretreatment strategy for AMS susceptible Warfighters slated for rapid deployment to mountainous environments is compelling.

ACKNOWLEDGMENTS

This project was funded by in part by the Defense Medical Research and Development Program Proposal Number D61_I_10_J5_178 and the U.S. Army.

REFERENCES

Author notes