Human beings experience compromised oxygen delivery at high altitudes (≥ 2,500 m) due to decreased atmospheric pressure and partial pressure of oxygen. This condition of hypobaric hypoxia is the unavoidable, unmodifiable, and uniform environmental stress for everyone at any given altitude. In addition, extreme cold, solar radiation, and aridity are other major stressors for lowlanders at high altitudes, at least for the first few days upon the arrival. Lowlanders elicit an integrated physiological (including respiratory and cardio-pulmonary) and hematological response for acclimatization to high altitude (Houston and Riley, 1947; Bartsch and Gibbs, 2007; Naeije, 2010). It is generally accepted that increased ventilation and hemoconcentration facilitate near normalization of the arterial oxygen content of lowlanders after an approximately 7-day residence at a high altitude (Muza et al., 2010). More recently, several proteins and biochemical pathways have been reported facilitating high altitude acclimatization (Padhy et al., 2016; Tang et al., 2018; Gangwar et al., 2019; Pooja et al., 2020). Failure in such responses may lead to high altitude illnesses, ranging from acute mountain sickness (AMS) to severe and life-threatening forms like high altitude pulmonary edema (HAPE), high altitude cerebral edema (HACE), thromboembolism, and high altitude polycythemia (HAPC) (Reynafarje et al., 1959; Hackett and Roach, 2001; Gallagher and Hackett, 2004; Palmer, 2010; Gupta and Ashraf, 2012). Both animal and human studies have reported that hypobaric hypoxia-induced oxidative stress and inflammation are important factors for the genesis of these high altitude maladies (Hartmann et al., 2000; Sarada et al., 2008; Himadri et al., 2010; Siervo et al., 2014; Boos et al., 2016; Pena et al., 2020a; Pham et al., 2021).

More than 40 million people including thousands of army personnel, government officials, miners, pilgrims, trekkers, and porters visit high altitude regions annually and are at risk of suffering from altitude illness and potentially dying from it (Moore, 2001; Basnyat, 2018). The working conditions and duration of high altitude residence differ among lowlanders depending on their professional requirements. On average trekkers, porters, sportsperson, pilgrims, and tourists spend few days to weeks at high altitudes whereas military personnel, diplomats, miners, and scientists spend months to years at high altitudes. Both acute and chronic exposure to high altitude induces several metabolic alterations and adjustments (Murray, 2016; Murray et al., 2018). More specifically, hypobaric hypoxia exposure alters lipid profiles (Gangwar et al., 2019) inducing hypercholesterolemia and hypertriglyceridemia (Mohanna et al., 2006; Sherpa et al., 2011; Vats et al., 2013) and associated cardiovascular disorders above 3,000 m (Virues-Ortega et al., 2009; Mallet et al., 2021). In addition, extended stay of young and healthy lowlanders at high and extreme altitudes is reportedly associated with a higher risk of spontaneous vascular thrombosis (Anand et al., 2001), massive infarcts, and stroke (Jha et al., 2002). Hence, studying lowlander molecular response above 3,000 m and extended residence period (weeks to months) is a pertinent scientific endeavor that can save both money and manpower.

Blood plasma is a highly accessible sample for monitoring the health status of an individual (Geyer et al., 2016), and high-throughput plasma proteome analysis is a popular omics method to investigate system-level protein alterations that may be rooted from basic science or clinical perspective (Pernemalm and Lehtio, 2014; Ignjatovic et al., 2019). Several research groups including ours have used plasma proteomics for gaining insight into high altitude acclimatization (Ahmad et al., 2011, 2013; Levett et al., 2011, 2015; Julian et al., 2014; Yang et al., 2014; Lu et al., 2018; Wang et al., 2019) and high altitude disorders like AMS (Julian et al., 2014; Lu et al., 2018), HAPE (Ahmad et al., 2011; Yang et al., 2014), polycythemia (Wang et al., 2019), and CMS (Zhang et al., 2021). Despite these studies, no information is available for plasma proteome level alterations of lowlanders during prolonged (months) stay at high altitude. Hence, we sought to investigate global plasma proteome alterations of lowlanders during a 3-month stay at 4,176 m as compared to 7 days stay at high altitude and sea level. In addition, we have also monitored physiological indices, proinflammatory cytokines, and lipid profiles. To the best of our knowledge, this is the first plasma proteomics investigation reporting molecular pathways perturbed during prolonged exposure to high altitude. We have further used biochemical estimation, ELISA studies, and Western blot analysis to validate our proteomics observations. Our present results indicate that acute exposure to high altitude induces dyslipidemia among lowlanders chronic exposure.

The present study reports the physiological and plasma proteome level changes for lowlanders during short- and long-term stay at high altitudes. Comparing the short-term and long-term plasma proteomes with sea level plasma proteome, the present study highlights activation of proatherogenic lipoproteins at high altitude that exacerbates during longer stay durations.

The arterial oxygen saturation level of lowlanders decreased upon acute exposure to high altitude (HA-D7) and increased significantly after long-term stay (HA-D150), though the levels were lower than sea level. This signifies healthy acclimatization to HA (Calbet et al., 2003; Soria et al., 2016) as severe and prolonged oxygen desaturation can lead to AMS (Mandolesi et al., 2014). As expected, the hemoglobin levels also increased significantly at high altitudes and persistently high levels were maintained irrespective of the duration of the stay at HA. Both the systolic and diastolic blood pressure along with heart rate increased upon HA exposure and was more pronounced during a long-term stay at HA. This is majorly due to massive activation of the sympathetic nervous system, contributed by noradrenaline despite enhanced hemoglobin levels and concomitant improved arterial oxygen content (Bartsch and Gibbs, 2007; Hainsworth et al., 2007).

Our TMT-based plasma proteomics studies identified a differential abundance of 377 proteins in HA-D7 and HA-D150 as compared to sea level. Using a cut-off of 0.8-fold for downregulation and 1.2-fold for upregulation, we identified 75 and 56 down-regulated proteins for HA-D7 and HA-D150 groups respectively. Similarly, we identified 62 and 79 upregulated proteins for HA-D7 and HA-D150 groups respectively. Bioinformatics analysis revealed alterations in several lipoprotein-associated pathways like plasma lipoprotein assembly, VLDL clearance, chylomicron assembly, plasma lipoprotein clearance, and chylomicron clearance. We observed higher levels of lipoproteins like APOB, APOCI, APOCIII, APOE, and APOL for both HA-D7 and HA-D150 in accordance with our previous studies (Gangwar et al., 2019). Interestingly, we observed higher levels of Apo-B for both HA-D7 and HA-D150 representing higher levels of low-density lipoprotein (LDL). Findings from several large studies indicate that elevated triglyceride (TG) levels along with increased levels of small dense LDL particles with concomitantly decreased levels of HDL cholesterol are often a component of atherogenic dyslipidemia (Reiner, 2017). We also observed similar high levels of APOCI, the endogenous inhibitor of cholesterol ester transfer protein (CETP) that limits the exchange of lipids has reportedly enhanced the risk of atherosclerosis (Westerterp et al., 2007). Our plasma proteomics and western blot studies identified higher APOCIII levels in high-altitude exposed groups. APOCIII is now recognized as a key regulator in severe hypertriglyceridemia due to its inhibition of lipoprotein lipase (LPL) and hepatic lipase. ApoCIII gain of function mutations are associated with atherosclerosis and coronary heart disease (CHD) and contribute to the development of hypertriglyceridemia, whereas loss of function mutations are associated with lower levels of plasma triglycerides and attenuation of vascular inflammatory processes (Pollin et al., 2008; Tg et al., 2014; Natarajan et al., 2015; Rocha et al., 2017).

Lipid profiling revealed higher levels of cholesterol, triglycerides, LDL, and lower levels of HDL were observed for HA-D150 as compared to HA-D7 and sea level groups (Table 2). It is noteworthy that sea-level values for total cholesterol, triglycerides (TGs), and LDL were within the physiological range, increased after high altitude exposure, and levels were further elevated during the prolonged stay. We also observed a moderate correlation of diastolic blood pressure (DBP) with LDL (R-squared = 0.46, p = 0.04) for HA-D7 group. Persistent hypoxia at high altitude induces HIF-1α that upregulates stearoyl-CoA desaturase (SCD)-1 in the sterol regulatory element-binding protein (SREBP)-1c pathway resulting in increased hepatic de novo TGs synthesis (Siques et al., 2020). In contrast, we observed the lowest HDL levels for HA-D150. High altitude exposure compromises HDL maturation and alters the levels of HDL associated proteins limiting its protective functions (Gangwar et al., 2019). Consequently, ratios of LDL/HDL, cholesterol/HDL, and AIP, potential risk factors for coronary artery disease (CAD) were elevated for HA-D150. Both animal and human studies have reported that hypoxia increases plasma triglycerides by decreasing tissue uptake (Barnholt et al., 2006; Siques et al., 2014). Epidemiological studies have reported a high prevalence of hypercholesterolemia and low HDL levels for indigenous high land native populations of Peru (Mohanna et al., 2006), Chile (Santos et al., 2001), and Tibet (Sherpa et al., 2011). These studies along with our current observation suggest that extended stay at high altitude results in dyslipidemia and elevates proatherogenic lipoprotein levels.

There is accumulating evidence that HA exposure is associated with an inflammatory response and associated endothelial activation/dysfunction (Hartmann et al., 2000; Bruno et al., 2014; Boos et al., 2016) that aggravates many forms of cardiovascular diseases (Riley and Gavin, 2017; Parati et al., 2018). Healthy young lowlanders with no preexisting risk factors develop massive infarct and ischemic stroke during a prolonged stay at HA (above 4,270 m) (Jha et al., 2002). Acclimatized lowlanders exhibit increased levels of coronary risk factors after 15–18 months stay at the Indian trans-Himalayan Ladakh region (altitude more than 3,500 m) (Dhar et al., 2018). Hence, we measured proinflammatory cytokines IL-6, CRP, TNFα, and oxidized LDL (oxLDL) levels in all three groups. The levels of IL-6 increased after altitude exposure (HA-D7) and levels were further increased after prolonged exposure (HA-D150). Studying cytokines after high altitude exposure, Hartmann et al. have reported increased IL-6 levels indicating considerable inflammation (Hartmann et al., 2000). Increased IL-6 level serves as an independent predictor of AMS (Boos et al., 2016) and plays a role in the pathogenesis of HAPE (Kubo et al., 1998). Similar higher levels of TNFα were observed after high altitude exposure and the highest levels were recorded for the HA-D150 group. Studying inflammatory cytokines in BAL fluid of HAPE patients, Kubo et al. have reported higher TNFα levels (Kubo et al., 1998) supporting our present observations. We also observed increased CRP levels for HA-D7 and the levels subsequently decreased for HA-D150 but remained higher than the sea level. Increased CRP levels have been reported for both acute (Hartmann et al., 2000; Gangwar et al., 2019) and prolonged high altitude exposure (Hu et al., 2016). Elevated levels of plasma CRP are associated with the risk of atherosclerotic events in general populations and show a predictive value even in terms of secondary prevention (Libby and Ridker, 2004; Calabro et al., 2009). Hence, we measured oxLDL levels that contribute to atherosclerotic plaque formation and progression by several mechanisms, including the induction of endothelial cell activation and dysfunction, macrophage foam cell formation, and smooth muscle cell migration and proliferation (Pirillo et al., 2013; Poznyak et al., 2020). We observed significantly higher oxLDL levels after a prolonged stay at high altitudes as compared to HA-D7 and sea level. Our present observations of elevated proinflammatory cytokines and oxLDL levels after prolonged exposure to high altitude indicate vascular inflammation leading to a proatherosclerotic state. A recent study evaluating long-term chronic intermittent hypobaric hypoxia-induced right ventricular hypertrophy has reported upregulation of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a major OxLDL receptor on endothelial cell surface supports our current observations (Pena et al., 2020b).

We identified CA1 and CA2 as the topmost upregulated proteins for HA-D150, on average 2-fold higher than the HA-D7 group. These two are a member of the CA family that reversibly catalyzes the hydration of CO₂ to form HCO3-, which then rapidly binds to calcium ions to form calcium carbonate (Supuran, 2008). This well-known reaction is involved in a range of physiologic processes, ranging from CO₂ metabolism to cell proliferation and glucose/lipid metabolism (Gilmour, 2010). It is important to note that acetazolamide and other related CA inhibitors have been effectively used for the prevention and treatment of AMS and remain the standard of care for this indication (Swenson, 2006; Nieto Estrada et al., 2017). While there exists no difference between red blood cell CA activity between lowlanders and native highlanders (Gamboa et al., 2000), increased activity has been reported in patients with obstructive sleep apnea (OSA) (Hoff et al., 2020). Inhibition of CA activity curtails inflammation and experimental hypertension (Hudalla et al., 2019) suggesting that CAs are involved in the inflammation and vascular calcification (Adeva-Andany et al., 2015; Yuan et al., 2019). Though our results do not provide any direct evidence for CA-mediated vascular inflammation and calcification, their profound upregulation and existence of a proatherogenic propensity during long-term stay at high altitude suggests a possible link between CA and high altitude-induced dyslipidemia.

The present study is limited by studying only plasma samples and blood parameters for understanding the body response to hypoxia, whereas at organ and tissue level the response is more complex. All global plasma proteomics studies are constrained by a high dynamic range of plasma proteins, and even after several high abundance protein depletion steps, it is not possible to detect very low abundance proteins that may be of critical importance. We have studied only male volunteers for 3 months in the present study for specific reasons. A vast number of studies have reported sexual dimorphism for cardiovascular response and disorders particularly due to the sex hormone estrogen (Hester et al., 2019; Horiuchi et al., 2019; Hou et al., 2019; Shen et al., 2020; Ndzie Noah et al., 2021). It is important to note that estrogen levels widely vary between both the sex (Khosla et al., 1998), and cardiac adaptive responses significantly vary between males and females during chronic hypoxia (Bohuslavova et al., 2010). Hence to minimize variations, women were consciously excluded from the present study. However, such proteomics studies with women and longer durations of high altitude stay (6 months or more) could provide additional molecular information.

In summary, the present study reports elevated levels of SpO₂, HR, SBP, and DBP after long-term exposure to high altitude as compared to short-term exposure whereas the hemoglobin levels remained the same. Global plasma proteomics studies revealed upregulation of several apolipoproteins, and subsequent bioinformatics analysis revealed perturbation in assembly, remodeling, and clearance of plasma lipoproteins including VLDL and chylomicrons. In corroboration, we also observed higher TC, TGs, and LDL after a prolonged stay at high altitude indicating dyslipidemia. These observations were further supported by higher levels of inflammatory cytokines IL-6, TNFα, CRP as well as oxLDL. These cumulative results indicate persistent vascular inflammation after long-term exposure to high altitude leading to dyslipidemia and a proatherosclerotic condition.

The original contributions presented in the study are publicly available. This data can be found here: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD028070.

The studies involving human participants were reviewed and approved by Ethics Committee of Defence Institute of Physiology and Allied Sciences (IEC/DIPAS/B2/1). The patients/participants provided their written informed consent to participate in this study.

NK designed the experiments and wrote the manuscript with inputs from P. P and VS performed the experiments. RM, KR, UP, and NK collected samples at sea level and high altitude as well as recorded physiological parameters. P, RV, and NK analyzed the data. All authors contributed to the article and approved the submitted version.

This study was supported by Defence Research and Development Organisation (Project number DIP-263).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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