Human adaptation to high altitude: a review of convergence between genomic and proteomic signatures

Proteomics for high-altitude acclimatization

HA acclimatization consists of integrated physiological, biochemical, and molecular changes in native lowlanders for quickly adapting to compromised oxygen availability. Individual acclimatization to hypobaric hypoxia depends on external or environmental factors (rate of ascent, mode of high-altitude induction, severity, and duration of hypoxic stress) and individual response (including cellular oxygen transport, intensity oxygen consumption, metabolic characteristics, and behavioral reactions). These combined factors determine whether the body successfully acclimatizes to HA or is overwhelmed by hypobaric hypoxia and develops pathologies. The following section will discuss lowlander response to acute exposure followed by chronic exposure to HA.

Plasma and serum proteomics

Studies reporting human exposure to short-term hypoxia exposure (30 min and 9 h).

have either used simulated chambers [34, 53] or real HA exposure (H. [66, 67] to study blood plasma and serum proteins after exposure. Evaluating the effect of acute hypoxia exposure of 30 min in a simulated altitude (2400 m or 8,000 ft) equivalent to cabin pressure during airline travel, Hinkelbein et al. studied the serum proteome of 10 volunteers (9 males and 1 female) using 2D DIGE (2- Dimensional Differential Gel Electrophoresis) followed by MALDI-TOF (Matrix Assisted Laser Desorption/Ionization- Time of Flight). The authors identified 10 proteins, out of which higher levels of 6 proteins, including apolipoprotein E (APOE), complement C1q subcomponent subunit precursor A (C1QA), complement C1q subcomponent subunit precursor B (C1QB), clusterin precursor (CLU), prothrombin (F2) and glyceraldehyde 3 phosphate dehydrogenase GAPDH) and lower level of 4 proteins comprising albumin (ALB), phosphoglycerate kinase 1 (PGK1), catalase (CAT) and carbonic anhydrase 1 (CA1) were observed as compared to pre-exposure levels.

Two independent studies exposed lowlanders to 9 h of hypobaric hypoxia and screened the volunteers based on LLQ (Lake Louise Questionnaire) scoring for AMS resistance. Using the discovery plasma proteomics approach, the authors identified proteins associated with AMS resistance and thus facilitating high-altitude acclimatization [53, 66, 67]. Julian et al. exposed 20 healthy volunteers (17 men and 3 women) living near Denver, Colorado (1609 m) for 9 h of hypobaric hypoxia (4875 m; 425 mmHg) in a hypobaric chamber [53]. Using pooled plasma samples of AMS-resistant volunteers with two different nano-liquid chromatography–tandem mass spectrometry platforms, the authors identified 350 proteins or protein fragments for both the studied groups. 19 proteins were identified (fold change ≥ 1.8) between pre-ascent and post-9 h hypoxia exposure. Out of these 19 proteins, 8 proteins, including serum amyloid P component (SAMP), insulin-like growth factor-binding protein complex acid-labile subunit (IGFALS), complement component 7 (C7), mannose-binding protein C (MBL2), apolipoprotein C4 (APOC4), serum amyloid A4 protein (SAA4), apolipoprotein C1 (APOC1) and von Willebrand factor (VWF) were unique to AMS-resistant group. Extending this 9-h hypoxia exposure protocol to real high-altitude exposure, Lu et al. exposed healthy lowlanders to 3800 m height and identified AMS-resistant and AMS-susceptible volunteers using Lake Louise Questionnaire Scoring [66, 67]. Comparing the post-ascent plasma proteome with the pre-ascent proteome, the authors identified 89 proteins (fold change > 1.5) for AMS-susceptible patients, out of which 17 proteins were upregulated and 72 were downregulated. Similarly, for AMS-resistant volunteers, the authors identified 421 proteins, out of which 61 were upregulated and 360 downregulated proteins. Interestingly, AMS-resistant volunteers exhibit lower plasma levels of 10 key TCA cycle metabolic enzymes, namely, pyruvate dehydrogenase alpha 1 (PDHA1), dihydrolipoyl dehydrogenase (DLD), ATP-citrate synthase (ACLY), aconitase (ACON), isocitrate dehydrogenase (IDH1, IDH2, IDH3A, IDH3B), 2-oxoglutarate dehydrogenase (OGDH), dihydrolipoyl lysine-residue succinyltransferase (DLST), succinyl-CoA ligase (SUCLG1, SUCLG2, and SUCLA2), succinate dehydrogenase (SDHA, SDHB), and malate dehydrogenase (MDH2). 11qIn addition, lower plasma levels of proteins belonging to the ribosome (Rps2p, Rps9p, Rps24p, Rpl15p) and proteasome (PSMC3, PSMA3, PSMD12, PSMD1, PSMD2, PSMD11, PSMA7, PSMC4, PSMB1, PSMA2, PSMA1, PSMC5, PSMA6, PSMD7) pathway were also identified (Table 1).

We have picked out several molecular pathways for AMS-resistant and -positive volunteers (Table 1). We recognized that EIF2 signaling, regulation of EIF4 and p70SCK signaling, mTOR signaling, sirtuin signaling pathway, TCA cycle, and gluconeogenesis are predicted to be activated in AMS-positive volunteers. For AMS-resistant volunteers, molecular pathways like activation of the complement system, and acute-phase response signaling are predicted to be activated in IPA-based pathway mining, LXR/RXR activation, coagulation system, and intrinsic prothrombin activation pathway. These results reinforce that suppression of energy-generating pathways and energy-consuming protein translation and elongation pathways play a crucial role during the early phase of high-altitude acclimatization.

The chronic exposure (days to weeks) strategy has been used to study the acclimatization response of lowlanders to high altitude [27, 59, 74, 104]. In order to identify serum biomarkers for high-altitude acclimatization at sea level, Yang et al. compared serum peptidome of high-altitude acclimatized (LLQ ≤ 3) and ill (LLQ ≥ 3) lowlanders [104] prior to high-altitude exposure. Using a comparative analysis of serum peptides at sea level, the authors have identified two upregulated serum peptide peaks (m/z values: 1061.9 and 1088.33 and one downregulated serum peptide peak (m/z value: 4057.63 corresponding to regions of proteins inter-α trypsin inhibitor heavy chain H4 (ITIH4, inter-α trypsin inhibitor heavy chain H1 fragment (ITIH1, and isoform 1 of fibrinogen α chain precursor (FGA, respectively, for high-altitude acclimatized lowlanders as compared to ill lowlanders. Subsequent ELISA-based studies also corroborated significant higher serum levels of ITIH1 (range 3.69–5.0 ng/mL and ITIH4 (range, 4.82–6.64 ng/mL in high-altitude acclimatized group as compared to ill group at sea level. ITIH1 and ITIH4 belong to inter-alpha-trypsin inhibitors (ITI, a family of plasma protease inhibitors contributing to extracellular matrix stability by covalent linkage to hyaluronan [15]. They accumulate in the vascular endothelium and might have a role in stabilizing endothelial cells and ECM damaged by high-altitude hypoxia. The third protein, fibrinogen, is a plasma glycoprotein that participates in the final phase of blood coagulation. Epidemiological studies have demonstrated that increased circulating levels of fibrinogen are a significant risk factor for cardiovascular disease, and fibrinogen biosynthesis is upregulated during hypobaric hypoxia [17, 95]. The studies of Padhy et al. and Gangwar et al. reported plasma proteome alterations of healthy lowlanders at high altitude (3500 m after 1, 4, and 7 days of exposure [27, 74]. These two studies have reported upregulation of acute-phase response proteins (SAA, AGP2, IGFBP1, ORM1, APCS, FGA, FGB, and FGG), complement pathway proteins, and inflammatory proteins (CRP, S100A8, and S100A9) in acclimatizing lowlanders. In addition, the authors have identified altered expression of several apolipoproteins (APOAI, APOAII, APOAIV, APOAV, APOB, APOCIII, APOCIV, APOD, APOE, APOH, APOL, and APOM) and associated lipoprotein pathway proteins (LCAT, CETP, and PLTP). Interestingly, the acclimatization process also results in higher plasma levels of KNG (kininogen), the precursor protein of the KKS (kallikrein–kinin system) and the KKS pathway proteins plasma KLKB1 (kallikrein) and BK (bradykinin). Activation of KKS pathway and resultant higher levels of BK modulates eNOS-mediated enhanced NO production promoting vasodilation and oxygen delivery, a process critical for hypoxia acclimatization [75]. In contrast, decreasing plasma ANGT (angiotensinogen) levels on day 7 compared to day 1 and day 4 of high-altitude exposure also supports activation of the vasodilation process during high-altitude acclimatization [27].

To understand the effect of prolonged exposure (3 months) to high altitude on lowlanders, Pooja et al. used a TMT (Tandem mass tags)-based proteomics approach to decipher plasma proteome level alterations after 3 months of high-altitude exposure [60]. The authors identified higher plasma levels of several apolipoproteins like APOAII, APOB, APOCI, APOCIII, APOE, and APOL, promoting dyslipidemia. In contrast, lower plasma levels of cytoskeletal proteins like PROF-1, ACTG1, TLN1, MYH9, and ACTN1 were identified after prolonged exposure. The molecular pathways associated with the altered proteome include plasma lipoprotein assembly, chylomicron assembly, chylomicron remodeling, VLDL (Very low-density lipoprotein) clearance, plasma lipoprotein clearance, and chylomicron clearance.

We prepared a comprehensive list of proteins identified for each high-altitude exposure time point (9 h, 1 day, 4 days, 7 days, and 90 days) and subjected it to IPA-based pathway analysis (Additional file 1). It is important to note we identified LXR/RXR activation as the common pathway for all the exposure time points. LXRs (Liver X receptors) represent a subset of the nuclear receptor superfamily that is regulated by oxidized forms of cholesterol (oxysterols) and intermediate products of the cholesterol biosynthetic pathway [49, 50]. LXRs form obligate heterodimers with RXRs (Retinoid X receptors), which are members of the nuclear receptor superfamily that can be regulated by 9-cis-retinoic acid (9cRA) and long-chain polyunsaturated fatty acids. LXR/RXR heterodimers regulate their target genes by recognizing specific LXR-response elements (LXRE) consisting of two direct hexanucleotide repeats separated by four nucleotides [98]. LXR/RXR heterodimers induce the expression of genes that mediate cholesterol efflux from cells to bile [81]. Activation of the LXR/RXR pathway is essential for regulating cholesterol homeostasis in macrophages, which can accumulate massive amounts of cholesterol during disease settings such as atherosclerosis [93]. A recent plasma proteomics investigation has reported dyslipidemia in lowlanders during a long-term stay at a high altitude, further supporting LXR/RXR activation during hypobaric hypoxia [59]. We have also identified acute-phase response and nitric oxide and reactive oxygen species production in macrophage pathways for all high-altitude exposure time points except the 30-min time point. Activation of these pathways at high altitude indicates inflammation and concomitant increase in acute-phase proteins, which might have adverse implications [25].

Urine and saliva

HA exposure also modulates the urinary protein profiles depending upon the altitude of ascending low landers (Fig. 2, Table 1). Studying urinary proteome of HIGH CARE-2008 study volunteers at sea level, HA (3500 m, Namche Bazaar) and very HA (5400 m, Mount Everest base camp), Mainini et al. identified six peptides (m/z 1165, 1683, 1953, 2194, 4298, 4757) differentially expressed in hypobaric hypoxia at high or very HA compared to the sea level [69]. These peptides identified two proteins, uromodulin (UMOD) and a1-antitrypsin (A1AT), corresponding to m/z 1683 and 2194, and levels were significantly reduced at both the studied altitudes. It is noteworthy that higher serum A1AT level is a risk factor for HAPE. Even minor increases in levels of serum A1AT are associated with the development of arterial hypertension and increased cardiovascular disease [14, 77]. These studies suggest that the upregulation of A1AT inhibits the activity of the kallikrein–kinin system that promotes HA acclimatization and favors the renin–angiotensin system leading to systemic vasoconstriction and hypertension [74]. Hence, downregulation of uromodulin and A1AT probably promotes nitric oxide availability, and vasodilation promotes HA acclimatization in low landers (Additional file 1).

The saliva proteome holds an excellent promise for HA research as it is a noninvasive body fluid and can be effectively used in devising effective strategies for the screening of HA fitness and early diagnosis of HA-induced pathologies. The salivary proteome of lowlanders was studied at 13,700 ft after 7-day post-ascent using 2D-gel electrophoresis [47]. By comparing with sea level saliva proteome, the authors identified a higher abundance of apoptosis-inducing factor-2 (AIFM2), cystatin S (CST4), cystatin SN (CST1), and carbonic anhydrase 6 (CA6) proteins for healthy lowlanders at HA. In contrast, a lower abundance of glycolysis-associated protein alpha-enolase was observed, indicating curtailed glycolysis corroborating the plasma proteomics observations for HA [48]. In addition, a lower abundance of immunoglobulin receptor and prolactin-inducible protein was observed, further substantiating the utility of saliva proteins for HA research. Subsequently, using an iTRAQ-based proteomics strategy, the authors compared the sea level saliva proteome with HA (4420 m) exposed saliva proteomes after 7, 30, and 120 days of stay. A total of 67 proteins were identified from saliva, out of which 56 (4 upregulated and 52 downregulated), 46 (10 upregulated and 36 downregulated), and 48 (7 upregulated and 41 downregulated) proteins were identified for 7, 30, and 120 days of high stay, respectively. Using IPA-based pathway analysis, the authors identified LXR/RXR activation, acute-phase response signaling, and production of reactive oxygen species and nitric oxide in macrophages as significant pathways (Additional file 1). In corroboration with previous salivary proteomics study, the authors reported lower enolase and prolactin-inducible protein (PIP) levels in all the study groups, suggesting curtailed glycolysis even after 120 days of stay at HA. Most notably, saliva proteomics studies also identified LXR/RXR pathway activation, corroborating plasma proteomics studies (Additional file 1). In contrast, the molecular production pathway of reactive oxygen species and nitric oxide in macrophages was downregulated across all exposure time points (days 7, 30, and 120). These studies also add to the growing consensus that lipid and energy are the key regulators of high-altitude acclimatization.

Muscle

Hypoxia exposure can have opposite effects on the regulation of muscle mass according to both degrees of hypoxia and exposure duration [22]. Macroscopic and microscopic muscle measurements after a sustained (8–12 weeks) sojourn at altitudes above 5,000 m demonstrated a reduction in lean lower limb mass of 10–15%, accompanied by a 20–25% decrease in mitochondrial volume density [36]. These morphological changes are associated with a proportional drop in muscle oxidative-enzyme activity and a relatively small reduction in glycolytic enzyme activities [37, 39, 61].

To investigate lowlanders muscle adaptation to HA, Vigano A et al. studied the muscle proteome after 7–9 days of exposure to HA (Margherita Hut, 4559 m) using 2D DIGE and MS. Separating muscle proteins on 4–7 and 6–11 pH range gels, the authors could identify altered abundance in 122 protein spots differentially changed in all subjects, among which 89 were decreased, and 33 were increased [94]. Out of these spots, 56 proteins were identified by MS, in which the abundance of 54 proteins was decreased while increased for 2. Further analysis revealed that proteins involved in iron transport (Serotransferrin), tricarboxylic acid (TCA) cycle, oxidative phosphorylation (2-oxoglutarate dehydrogenase (ODGH2), malate dehydrogenase (MDH1), and aconitate hydratase (ACO2), and oxidative stress responses (superoxide dismutase 1 (SOD1), glutathione S-transferase P (GSTP1), glutathione S-transferase Mu 2 (GSTM2), glutathione S-transferase Omega (GSTO1), peroxiredoxin 2 (PRDX2), peroxiredoxin 6 (PRDX6), protein deglycase 1 (DJ-1), cytoplasmic carbonic anhydrase 3 (CA-III), heat-shock protein beta 1 (HSPβ1), mitochondrial heat-shock protein 60 (HSP60) were significantly decreased during hypoxia exposure.

These results were further substantiated by independent research groups studying human muscle response to simulated hypoxia (Fraction of inspired oxygen, FiO2 -14.1%, 3,200 m for 15 days) and the Mount Everest Expedition (19 days at 5300 m altitude and 66 days up to 8848 m) [61, 21, 62]. Chronic exposure to hypobaric hypoxia results in an average 30% drop in muscular creatine kinase and glycolytic enzyme abundance. Similarly, the protein abundance of most enzymes of the tricarboxylic acid cycle and oxidative phosphorylation was reduced at HA, and the decrease was more evident at extreme altitudes. The elongation factor-2 alpha levels decrease at HA, indicating decreased protein translation, whereas increased heat-shock cognate 71 kDa protein levels were observed, indicating chaperone-mediated autophagy. HA exposure also damaged sarcomere structures, evidenced by increased protein levels of catalase and biliverdin reductase and decrement of voltage-dependent anion channels 1 and 2 and of myosin-binding protein C [19]. Chronic exposure of lowlanders to extreme altitude (6400 m and beyond) resulted in a decrease in muscle mitochondria density with loss of almost three fourth subsarcolemmal mitochondria in contrast to native Tibetans. Correspondingly, lower levels of the transcriptional coactivator PGC-1α also suggest downregulation of mitochondrial biogenesis with a concomitant decrease in expression of electron transport chain complexes I (NADH coenzyme Q oxidoreductase) and IV (cytochrome C oxidase) and mitochondrial uncoupling protein 3 (UCP3) levels [61]. Our IPA-based analysis identified the downregulation of glycolysis and gluconeogenesis pathways in muscle proteome during prolonged exposure (16 days and above) to extreme altitude. Our analysis also identified downregulation of HIF1α (hypoxia-inducible factor 1, Alpha) signaling pathway, sirtuin signaling pathway, and mitochondrial dysfunction across all exposure time points (Additional file 1). Our present observations corroborate that prolonged exposure of lowlanders to extreme altitudes decreases muscle energy-generating pathways, protein synthesis, mitochondrial biogenesis, and increased autophagy leading to muscle loss.

Proteomic signatures for indigenous highlanders

A limited number of proteomics investigations have been performed to decipher plasma proteome alterations in Tibetan and Ladakhi highlanders [20, 75]. Using a TMT-based proteomics approach, Du et al. investigated alterations of Tibetan plasma proteome and identified changes in 137 proteins. The authors identified higher levels of CCL18, C9, PF4, MPO, and S100A9 while the levels of HRG and F11 decreased. These altered proteins participated in molecular functions like complement and coagulation cascades, antioxidative stress, and glycolysis [20] (Fig. 1). Investigating plasma proteome alterations of Ladakhi highlanders, Padhy et al. reported alterations in 36 plasma proteins belonging to blood pressure regulation (TETN, FIBB, AMBP, A2MG, FIBG, ANT3), acute-phase response (ITIH4, FETUA, ANGT, AACT), complement activation (C3, C4A, C1S), lipoproteins (APOE, APOM, APOL1, APOAIV), antioxidant proteins (A1BG, GPX3) and iron transport proteins (TRFE) [76]. The study identified lower plasma levels of angiotensinogen (ANGT) and angiotensin 2 (ANG II) with concomitant high levels of NO metabolites facilitates high-altitude adaptation of indigenous Ladakhi highlanders.

Fig. 1figure 1

Top 15 Canonical pathways associated with differentially regulated plasma proteins identified for high-altitude native Ladakhi population. Pathway mining was performed using Ingenuity Pathway Analysis tool (https://www.qiagen.com/ontent-databases/ingenuity-pathway-analysis/)

There is only one report on muscle proteomics on Tibetan highlanders. Gelfi et al. investigated the alterations in muscle proteome of native Tibetan highlanders permanently living at high altitude with second-generation Tibetans born at living at low altitude [29]. The authors also used muscle samples of lowland Nepal volunteers as a control. Using 2D-gel electrophoresis and mass spectrometry, the authors identified alterations in 7 proteins GSTP1-1, ECH (Enoyl CoA Hydratase), GAPDH, LDH, PGAM2, NADH ubiquinone oxidoreductase, and myoglobin. Lower levels of glycolytic proteins GAPDH and LDH were observed for Tibetan highlanders. In comparison, 400% higher levels of ECH, a member of fatty acid beta-oxidation pathway, NADH ubiquinone oxidoreductase, a member of mitochondria respiratory chain, and 380% higher levels of GSTP1-1was observed. These results indicate that native Tibetan highlanders are protected from ROS-induced tissue damage and have developed specific metabolic adaptations in the muscle tissue (Fig. 2).

Fig. 2figure 2

Top 15 Canonical pathways associated with differentially regulated plasma proteins identified for high-altitude native Tibetan population. Pathway mining was performed using Ingenuity Pathway Analysis tool (https://www.qiagen.com/ontent-databases/ingenuity-pathway-analysis/)

Genomic signatures for human adaptation to high altitude

Populations residing in three major high-altitude regions have been studied for population-specific genomic adaptation signatures: The Himalayas, including the Tibetan plateau, the Andean Altiplano, and the Ethiopian Highlands. Recent single-nucleotide polymorphism and whole-genome sequencing studies combined with statistical methods for detecting evidence of natural selection have compared genomes of high-altitude populations with similar low-altitude populations in an attempt to identify the genomic regions, genes, and genetic variants that exhibit signals of positive selection [12, 85]. Interestingly, the genes identified for these populations to date majorly belong to oxygen-sensing pathways under the regulation of hypoxia-inducible factor (HIF). In contrast, several other genes involved in the regulation of vascular control, metabolic hemostasis, redox homeostasis, and erythropoiesis, in general, have also been identified. In the present study, we have catalogued all the genes reported to date for experiencing positive selection.

Himalayan signatures

Studies comparing high-altitude Himalayan genomes (Tibetan and Sherpa) with matched low landers have identified a positive selection of EPAS1 (endothelial PAS domain protein 1), which encodes the HIF-2α subunit of HIF complex, and EGLN1, which encodes PHD2, members of the HIF-oxygen-sensing pathway [9, 12, 85, 105]. Population-based genetic studies have identified adaptive evolution of EPAS1 and EGLN1 sequence variants that were significantly associated with the decreased hemoglobin phenotype, which is unique to Tibetans and Sherpas as compared to Andeans [9, 51, 52, 64, 78, 85, 102, 103]. Notably, an EPAS1 haplotype whose frequency is strongly correlated with altitude in the Himalayan populations has been suggested to have been acquired from an extinct hominin species, Denisovans [32, 43]. Studying polygenic adaptation of high-altitude Himalayan populations, Gnecchi-Ruscone et al. have reported a positive selection of many genes, including COL11A1, COL11A2, ESR1 LAMC1, LAMC2, ITGA6, ITGA1 and ITGA2 in Tibetans and Sherpas [30]. Multiple studies examining of positive selection of Himalayan populations, including Tibetan, Sherpa, and Nepalese genomes for hypoxia adaptation, have reported candidate gene loci like PPARA, HBB, MTHFR, SLC52A3, ANKH, ZNF532, COL4A4, MKL1, and GRB2 genes to name a few (Additional file 1: Fig. S4, Table S2), [3, 4, 103] (Additional file

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