Hypoxic Signature of High Altitude Acclimatization: A Gene Expression Study

Introduction

High altitude region presents an environment of hypoxia and cold. Indian Air Force and Army Aviation Corps routinely undertake flight to high altitude and engage in different operations wherein exposure to the harsh environment is inescapable. On arrival at altitude, a number of physiological changes occur through process of acclimatization which ultimately enables the body to function optimally in low oxygen environment. These physiological responses are complex and involve a range of mechanisms occurring within minutes of oxygen sensing resetting a cascade of biosynthetic and physiological events within the cellular milieu [1].

During the initial phase of ascent to HA, most sojourners experience symptoms of acute mountain sickness (AMS) characterized by headache, nausea, vomiting, giddiness, anorexia leading to hypophagia, sleep disturbance and adverse psychological effects (secondary), muscular weakness and depression [2].

High altitude pulmonary edema (HAPE) is a severe form of altitude sickness that generally occurs within 6 to 48 hours of ascent beyond a height of 2500 to 4000 m. Genetic predisposition and individual susceptibility in cases of HAPE has been postulated [3]. Mechanism of high altitude acclimatization and/or maladaptation still remains unclear. Gene expression responses of circulating leukocytes can potentially provide an early warning of threat they discover and have the potential to be used diagnostically for direct sampling of sites of infection or other disease processes. The present investigation aimed at studying the gene expression profile in individuals who were inducted to high altitude to identify changes related to high altitude exposure and understand the mechanism of acclimatization.

Material and Methods

24 male low landers (weight-63.7±6 kg, age-27.7±6 years) were included in the study who were studied at sea level (Chandigarh at 0700h before breakfast) and thereafter at high altitude (Leh, Jammu and Kashmir, AMSL 3650 m). Samples were also collected from HAPE patients (n=6) admitted in the hospital at Leh and age matched control subjects who did not develop HAPE (n=4). Verbal information on the experimental protocol and procedures were given to the subjects after which the subjects gave their informed, written consent to participate. The study conformed to Institute Ethical guidelines. Lake Luoise score was determined for each subject for assessment of AMS and seven volunteers who developed AMS were excluded. Samples were treated anonymously throughout the analysis. Blood samples were directly collected through a scalp vein set (Beckton Dickinson) in PAXgene Blood RNA tubes containing a stabilizing fluid (PreAnalytix, Qiagen).

Results

Of the 3800 sequences in the gene array, about 297 transcripts showed expression on the 3.8 K array (expressed in at least one condition), 64 transcripts expressed in all three conditions, 49 transcripts expressed in at least 2 conditions and 184 transcripts expressed in only condition. About 89 transcripts showed a change in gene expression after acute induction to altitude and were protein coding type. The differentially regulated genes belonged to both biological functions and cellular component. Seventy three gene transcripts had a decreased expression and about fifteen transcripts were upregulated under the high altitude hypoxic stress (Table 1). Genes of G-protein coupled receptor protein signaling pathway were down regulated on altitude induction: these included guanine nucleotide binding protein (GNA11) and regulator of G-protein signaling 11 (RS11).

Calcitonin/calcitonin-related polypeptide (CGRP), angiotensin II receptor type 2 (ATGR2), olfactory receptor family 6 (OR6A2P) and gonadotropin releasing hormone receptor (GRHR) were also downregulated. Among the other downregulated genes were present genes for cyclic nucleotide gated channel (CNG1) and mitochondrial solute carrier family 25 (ARALAR1). Genes involved in RNA processing, regulation of transcription, RNA processing/catabolism (PLAGL2), (APP1), zinc finger proteins (ZNF124, MZF1), retinoic acid receptor (FAR), genes involved in mRNA cleavage (RNS4), RNA splicing gene [DEAH (Asp-Glu- Ala_His) box polypeptide 16] (DBP2), mRNA capping RNA (guanine-7-methyltransferase) were also downregulated. Also downregulated was dualspecificity tyrosine (Y) phosphorylated kinases (DYRK5, DYRK2). Genes involved in defence response like interferon alpha 14 (MGC125756), apolipoproein H (APOB) andforkhead box N1 (FKHL20) were downregulated on exposure to high altitude. Transcripts involved in cell adhesion like calcium/calmodulin dependent serine protein kinase (LIN2) and scavenger receptor class F (SREC) were downregulated. Antigen presenting major histocompatibility complex class 1 (HLA-JY3 or D6S204), blood coagulation factor glycoprotein V (CD42d) and neurotransmitter synapsin II (SYNII) were also downregulatedon high altitude induction.

Upregulated transcripts on altitude induction were for various binding molecules viz., heme binding (hemoglobin alpha 1), hemoglobin alpha 2 (HBA1), GTP binding (ADP-ribosylation factor like 4A, ARIA), GTP binding septin 5 (H5), RNA binding ribosomal protein L3 type (RPL3L), protein binding (syntaxinlA, STX1A), parathymosin (PTMS) which is known to be involved in cellular defense response, transporter activity related to excretion (aquaporin 5, AQP5), gene involved in carbohydrate metabolism (ST8 alpha-n-acetyl-neuraminide alpha 2,8 sialytransferase, GD3S), cytochrome c oxidase subunit Via polypeptide involved in electron transport (COX6AH), cell adhesion molecule protein tyrosine phosphatase receptor (PTPSIGMA), actin related protein 2/3 complex involved in actin related polymerization (ARC20) as well as chromosome 10 open reading frame 116 of unknown biological function. The prominent functional clusters were regulation of apoptosis, T cell activation, oxygen transport, neurotransmitter secretion, regulation of blood pressure, regulation of body fluid levels, cell-cell signaling, transcripts of calcium ion binding etc (Table 2). The pathways which were found to be affected were antigen processing and presentation (hsa04612), h_ctlPathway: CTL mediated immune response against target cells, GnRH signaling pathway (hsa04912), vascular smooth muscle contraction (hsa04270), ubiquitin mediated proteolysis (hsa04120), regulation of actin cytoskeleton (hsa04810), calcium signaling pathway (hsa04020), neuroactive ligand-receptor interaction (hsa04080) and cytokine-cytokine receptor interaction (hsa04060) (Table 3).

In individuals with HAPE, thirty one transcripts were down regulated and fourteen transcripts were upregulated when compared to URR. In resistant control samples, twenty six genes were down regulated and eighteen genes were upregulated compared to URR. Although the pattern of gene expression was distinct in the three groups, there was overlapping also (Fig 1). Genes like alpha 2-HS glycoproein (AHSG), neurotansmiter transporter (SLC6A2), ADAM metallopeptidase domain 12 (ADAM12), UDP- glucose ceramideglycosyltransferase (UGCG) gonadotropin releasing hormone receptor (GNRHR), solute carrier family 6 (SLC6A2), protein coupled receptor CD3 antigen (T3E), aquaporin 2 (AQP2), mitochodrial ribosomal protein L49 (MRPL49), ATP binding cassette sub family C (CFTR/MRP), member 6 (ABCC6), lymphocyte cytosolic protein 1 (LCP1), distal less homeobox 3 (DLX3), keratin 13 (KRT13), a transmembrane glycoprotein A33 (GPA33) major histocompatibility complex class I C (HLA-C) adenine phosphorybosyltransferase (APRT) were more pronounced in HAPE than in resistant controls. Downregulated transcripts in HAPE were lysyl oxidase-like 1 (LOXL1), Wiskott-Aldrich syndrome protein interacting protein (WSPIP), pancreatic popeptide (PPY), hepatic transcription factor 1 (TCF1), actin gama 2 (ACTG2), solute carrier family 30 (zinc transporter) (SLC30A3), protein tyrosine phosphatase receptor type S (PTPRS) and protein tyrosine phosphatase receptor type N (PPRN).

Close

Fig 1.

Hierarchical clustering of gene expression from individuals who developed HAPE labeled with Cy3 compared to Universal Reference RNA labeled with Cy5 (Group I), matched controls who did not developed HAPE labeled with Cy3 compared to Universal Reference RNA labeled with Cy5 (Group II) and individuals at sea level labeled with Cy3 and at high altitude after acclimatization labeled with Cy5 (Group III).

Export to PPT

Discussion

Low cellular oxygen tension (hypoxia) is a feature of high altitude. An integral part of the human cellular response to hypoxia is changes in gene expression [6, 7]. Till date, more than 100 genes have been identified that show a change in expression during hypoxic exposure, including a number of genes that are thought to be part of a nonspecific cellular response to stress. In the present study, about 89 transcripts showed a change in gene expression on the 3.8 K gene array after acute induction to altitude and were protein coding type. High altitude hypoxia appears to have a substantial down regulatory effect on transcript expression in peripheral blood cells. The functional clusters of apoptosis, oxygen transport, neurotransmitter secretion, regulation of blood pressure, regulation of body fluid levels, cell-cell signaling, transcripts of calcium ion binding were evident of an hypoxic signature of altitude acclimatization. The pathways which were found to be affected were antigen processing and presentation (hsa04612), h_ctlPathway: CTL mediated immune response against target cells, GnRH signaling pathway (hsa04912), vascular smooth muscle contraction (hsa04270), ubiquitin mediated proteolysis (hsa04120), regulation of actin cytoskeleton (hsa04810), calcium signaling pathway (hsa04020), neuroactive ligand-receptor interaction (hsa04080) and cytokine-cytokine receptor interaction (hsa04060).

It has been reported that continuous residence at moderate heights (2,000-2,500 m) tends to improve oxygen transport capacity by an erythropoietin-induced increase in the hematocrit [8]. An increase in hemoglobin concentration augments maximal 0₂ consumption (V0_2max) and enhances exercise performance [9]. In the present study, increase in expression of hemoglobin alpha 1 and hemoglobin alpha 2 was noted on acute altitude induction. The result of the present study suggests that cellular response to hypoxia at the level of transcript expression is quite broad, although it may also be more specific to hypoxia than generally appreciated. Fink and colleagues [10] by applying DNA array technology and real-time PCRin a variety of human hepatocyte cell lines identified several previously unrecognized hypoxia-responsive genes; it was also seen that hypoxic exposure without reoxygenation led to an overall decrease in the number of transcripts expressed by cells, although increase in expression of heat shock proteins was not observed. In a recent study on effect of hypoxia on gene expression in HepG2 cells, it was shown that gene expression was broad, had a significant component of down regulation, and included a relatively small number of genes whose response was independent of cell and stress type [11].

Profiles of gene expression patterns are helping to define the complex biological processes associated with both health and disease in vivo. Microarrays can identify changes in gene expression that can be used as biomarkers of environmental and any other stress related exposure and their early effect and can provide information on mechanisms of various biological processes. DNA arrays have increased substantially in power and complexity and application of late-generation arrays would enable identification of more hypoxia- responsive genes. Gene expression is often stochastic [12] because most genes exist at single or low copy number in a cell. Some genes are expressed at high levels and others at low levels. It is now possible to track mRNA expression in a single cell with single molecule sensitivity in real time dynamics providing mechanistic insight into macromolecules [13]. Such kind of real time assays together with other emerging single molecule techniques [14] will yield further insight into not only gene expression and but many other fundamental biological processes. Understanding of this biological phenomenon will strategize therapeutic approaches for combating the harsh environment as well as perform better under the circumstances.