Validation of Differentially Expressed Proteins by Western Blot Analysis

We selected eight proteins Ttr, Prdx-2, Gpx-3, ApoA-I, Hp, Apo-E, Fetub and Nme, and further verified whether the expression patterns of proteins observed in 2-DE gels paralleled those validated by Western blot analysis. Band intensity was measured using Quantity One 1-D Analysis Software version 4.6.7 BIO-RAD, and the intensity ratio corresponding to β–tubulin band was calculated. Both Apo-E and Fetub were down-regulated in plasma after exposure to acute hypobaric hypoxia (Figure 4). The protein levels of Fetub increased gradually from 6 to 12 hr but down-regulated at 24 hr after acute hypobaric hypoxia. The expression of ApoA-I, Hp, Nme and Ttr increased gradually from 6 to 24 hr after acute hypoxia (Figure 4). Both Prxd-2 and Gpx-3 were up-regulated in plasma after acute hypobaric hypoxia and remained at high levels (Figure 4). The expression patterns of the selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis.

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Figure 4. Validation of protein change patterns at different time points by Western blot analysis of proteins Ttr, Prdx-2, Gpx-3, Apo A -1, Hp, Apo-E, Fetub and Nme.

Total plasma proteins (40 µg/lane) were separated by SDS-PAGE and probed with primary antibodies against these eight proteins.

https://doi.org/10.1371/journal.pone.0098027.g004

Regulation of Apolipoproteins in Plasma after Hypobaric Hypoxia

We found several apolipoproteins to be regulated in plasma of rat exposed to hypobaric hypoxia. These proteins function primarily as lipid binding proteins to transport lipids from the intestine to the liver and from the liver to tissues, including adipocytes, lung, heart, muscle, and breast tissues. In recent studies, apolipoproteins have been shown to have a clinical importance because of their roles in endothelial protection, anti-oxidation, and anti-inflammation [14], [15]. Here, we evidence the differential expression of apolipoproteins A-I, M, E, A-IV and H at different time points in plasma after acute hypobaric hypoxia. Specifically, apolipoprotein A-I (spot no.10) belong to the ApoA-I/A4/E protein family and is primarily produced in the liver and the intestine. ApoA-I can be found in the extracellular space and, being a structural component of high density lipoprotein (HDL), takes part in cholesterol absorption. ApoA-I up-regulation is associated with breast and lung cancer [16], [17]. Interestingly, studies provide new evidence supporting the notion that HDL plays a protective role in the lung. ABCA1, which interacts with lipid-poor ApoA-I, was earlier shown to be essential for maintaining normal lipid composition and architecture of the lung as well as respiratory physiology [18]. There is emerging evidence for the critical role of ApoA-I in protecting pulmonary artery and airway function as well as in preventing inflammation and collagen deposition in the lung [19]. More recently, proteomic studies revealed the anti-inflammatory role of ApoA-I in HAPE patients [20]. Here, we report ApoA-I expression increased gradually from 6 to 24 hr after acute hypoxia suggesting the anti-inflammatory role of ApoA-I. Spot 11 was identified as apolipoprotein M (Apo-M) which is produced in the liver and secreted in plasma. Apo-M is a member of the lipocalin family, a group of proteins with a characteristic coffee filter-like structure and a hydrophobic binding pocket [21]. It is a negative acute-phase protein that decreases during infection and inflammation [22]. Apo-M has also been shown to have antioxidant properties, protecting low density lipoprotein (LDL) against oxidative injury [23]. Plasma levels of Apo-M decreased gradually from 6 to 24 hr and the function of Apo-M was likely to be impaired during acute hypoxia, suggesting the decreased antioxidant activity of HDL. Apo-E (spot no. 17) is a mediator for binding, internalizing, and metabolism of lipoprotein particles. In addition to its role in lipoprotein metabolism, Apo-E may have other functions such as antioxidant [24], anti-platelet aggregation [25], anti-proliferative effects [26], and immunomodulation properties [27]. In this study, Apo-E was found to be down-regulated in plasma concentration after acute hypoxia. This could be related to the decreased antioxidant activity of Apo-E. Apolipoprotein A-IV (ApoA-IV, spot no. 18) is a glycoprotein that circulates freely or in association with chylomicrons and HDLs [31]. ApoA-IV has several proposed physiological roles, including lipid transport, lipoprotein metabolism, and control of food intake and gastric functions [28]. Most recently, the findings in studies of transgenic mice have supported a role for ApoA-IV in protection against atherosclerosis [29], [30]. Recent in vitro studies have supported an antioxidant action of Apo A-IV [31]. In this study, we found that ApoA-IV was down-regulated in the plasma after acute hypoxia. Additionally, apolipoprotein H also named as β-2 glycoprotein-1 (Apo-H, spot no.21) binds to various kinds of negatively charged substances, including heparin, phospholipids, and dextran sulfate. Apo-H may prevent activation of the intrinsic blood coagulation cascade by binding to phospholipids on the surface of damaged cells. Apo-H is synthesized in liver and secreted into the plasma. High levels of pathogenic Apo-H antibody in turn could cause hypercoagulation and venous and arterial thrombosis, and is clinically relevant to anti phospholipid syndrome [32]. Apo-H was found to be down-regulated in plasma after acute hypobaric hypoxia in this study.

Regulation of Acute Phase Proteins in Plasma After Hypobaric Hypoxia

In general, concentrations of various positive and negative regulators of acute phase proteins (APP) increase or decrease in response to inflammation [33]. As observed in plasma of normobaric and hypobaric hypoxia exposed rats, six APP were regulated. We found enhanced levels of positive acute phase regulators, such that Alpha-1-antiproteinase and haptoglobin in plasma after acute hypobaric hypoxia. Alpha-1-antiproteinase (spot no.13) is a serine protease inhibitor (serpin) that functions as an antitrypsin as well as an antithrombin [34]. Its primary targets include elastase, plasmin and thrombin. A1AT protects the connective tissues (elastin) from inflammatory enzymes such as elastase in the lungs and pulmonary system, as well as helps to prevent blood coagulation. Haptoglobin (Hp, spot no. 16) is produced mainly in the liver and normally released in the blood during acute and chronic inflammation [35]. In addition to transporting hemoglobin to the liver, thus facilitating hemoglobin catabolism and preventing tissue injury, several functions have been assigned to Hp, including anti-oxidant activity, angiogenesis, and the host defense response to infection and inflammation [36]–[37]. It can be inferred that A1AT and Hp might play important role in defense in hypobaric hypoxia characterized by inflammation and accumulated hemoglobin. Tansthyretin (Ttr, spot no. 3, 4, 5), also called prealbumin, is a beta-sheet rich protein. It is synthesized predominantly by hepatic parenchymal cells. It behaves as a tetramer and binds to retinol binding protein (RBP) and thyroxin in plasma [38]. Because Ttr is a tryptophan-rich protein, the protein is used as a useful marker for nutrition assessment (NST). However, Ttr is also an anti-acute phase protein, and its levels are influenced by various conditions, such as inflammation and infection. A persistent low level of serum transthyretin is predictive of lethality, whereas increased levels were associated with improved ventilator performances. In this study, we found that Ttr was up-regulated in the plasma of rats exposed to hypobaric hypoxia. Alpha-1-macroglobulin (A1m, spot 14, 15) is a glycoprotein secreted in plasma. This protein presumably plays a role in the regulation of the immune system, and is involved in defending tissue against oxidation by reactive oxygen species and heme [39], [40]. A1m is known to be present in association with macromolecules (immunoglobulin A, albumin, and prothrombin), as well as in a free monomeric form protein [41]. This protein is considered to be a marker of renal insufficiency, and to reflect the overall inflammatory status in patients with arterial hypertension and normal renal function [42]. Recent studies have shown the medical use of alpha-1-microglobulin as an antioxidant in the treatment of diseases related to oxidative stress, infection, inflammation, ischemia-reperfusion, prophylaxis or conditions associated with the presences of free radicals and/or free haemoglobin [43]. In this study, the plasma level of A1m was up-regulated after acute hypobaric hypoxia. Another reported negative APP, fetuin (also called α-2-HS-glycoprotein) which was usually expressed at a decreased level in acute inflammatory condition caused by major surgical procedures, trauma, and burns [44], showed lower plasma levels in rat exposed to hypobaric hypoxia. Additionally, fetuin has been shown to function as an acute phase anti-inflammatory mediator that is critical to regulating the innate immune response following tissue injury. We also observed regulation of complement C4 (spot no. 24) which plays a central role in the activation of the classical pathway of the complement system. The complement system, composed of over 30 proteins that act in concert to protect the host against invading organisms, initiates inflammation and tissue injury [45]. Complement activation promotes chemotaxis of inflammatory cells and generates proteolytic fragments that enhance phagocytosis by neutrophils and monocytes. Collard et al. showed complement activation by endothelial cells in vitro in response to hypoxia/reoxygenation [46], providing a possible link between endothelial oxidant generation and neutrophil adhesion and activation. In this study, complement C4 was down-regulated in plasma of rat after acute hypobaric hypoxia. Retinol binding protein (RBP 4, spot no.12) is a vitamin-A transport protein that acts as an adipokine when secreted from adipose tissue. RBP 4 down-regulates GLUT4 [47], the insulin-activated glucose transporter responsible for translocation of glucose into both muscle and fat cells [48], and has also recently been shown to induce expression and secretion of pro-inflammatory cytokines in primary human macrophages known to induce insulin resistance [49]. Recent studies have demonstrated that during the onset of a stress reaction of medium severity, the reduction of RBP plasma levels by half releases more retinol in free form into the extracellular space in amounts corresponding to about 10 times the normal free concentration [50]. In this study, we also found that RBP was down-regulated after hypobaric hypoxia. The low plasma concentration of RBP might result from the declining synthesis of RBP in rat livers.

Regulation of Binding Proteins in Plasma After Hypobaric Hypoxia

Vitamin D-binding protein (VDBP, spot no. 19) is a multifunctional protein found in body fluids and on the surface of many cell types. In plasma, it carries vitamin D sterols and prevents polymerization of actin by binding its monomers. VDBP also associates with membrane-bound immunoglobulin on the surface of B lymphocytes and with IgG Fc receptors on the membranes of T lymphocytes, suggesting its possible role in the immunopathogenesis and progression of the disease [51]. Besides serving as a transporter of vitamin D, VDBP also plays an important role in response to tissue injury, in which VDBP can be converted to a macrophage activating factor (VDBP-MAF) to stimulate macrophages [52]. Another role of VDBP in response to injury is to scavenge for vascular and extracellular actin as a result of cellular necrosis, and VDBP has been shown to be in lower circulating concentrations in the presence of inflammatory or necrotic diseases [51], [53]. Furthermore, VDBP is also implicated in a numerous diseases, including chronic obstructive pulmonary disease, cancer, and trauma [54]–[56]. In present study, we found that the plasma concentration of VDBP was increased significantly, suggesting the protective role in defense in hypobaric hypoxia.

Role of Antioxidant and Oxidative Stress Associated Proteins during Hypoxia

In this study, three cellular antioxidants, thioredoxin domain containing protein 12, peroxiredoxin-2 and glutathione peroxidase 3 were identified in the plasma of rats treated with hypobaric hypoxia. Thioredoxin domain containing protein 12 (TXNDC12, spot no. 22) belongs to the thioredoxin super family. Members of this super family possess a thioredoxin fold with a consensus active-site sequence (CxxC) and have roles in redox regulation, defense against oxidative stress, refolding of disulfide-containing proteins, and regulation of transcription factors [57]. In this study, TXNDC12 was up-regulated, suggesting the protective of TXNDC in the cellular defense against oxidative stress caused by hypobaric hypoxia. Peroxiredoxins (PRDXS) are H₂O₂ scavenging antioxidant proteins and six mammalian isoforms (I–VI) have been identified. They are small proteins (22–27 kDa) and all have the common thioredoxin CxxC motif and catalyze peroxide detoxification [58]. Increased expression of different peroxiredoxins has been reported in lung cancer, alveolitis, and hypoxic conditions [59]. Peroxiredoxin-2 (PRDX2, spot no. 8) belongs to a ubiquitous PRX family, which has been shown to have multiple functions such as enhancing natural killer cell activity [60], increasing cell resistance to oxidative stress [61], regulating transcription activator protein [62], protecting erythrocytes against oxidative stress [63], and anti- HIV activity [64]. In light of its multifaceted biological functions, the increased plasma level of PRDX2 might play a multifunctional role during the acute inflammation induced by hypobaric hypoxia. Glutathione peroxidase 3 (GPX-3, spot no. 9) functions in response to oxidative damage by catalyzing the reduction of hydrogen peroxide, lipid peroxides, and organic hydroperoxide. Rousseau et al. reported the increase in plasma glutathione peroxidase activity as a potential indicator of hypoxic stress in breath-hold diving [65]. Interestingly recent study from our group showed that ‘hypoxia-sensitive’ animals fail to increase GPX3 after acute hypobaric hypoxia [66]. Despite significant difference in experimental conditions employed in these two studies (exposure time and altitude), it is likely that the upregulation of GPX-3 could play a role in acclimatization to hypobaric hypoxia. Sulfonate conjugation is an important pathway in the metabolism of a variety of endogenous and exogenous compounds, including estrogens and other mammary carcinogens. This reaction is catalyzed by SULTs, 3 a superfamily of multifunctional enzymes including six cytosolic SULTs that have been identified in human tissues. Sulfotransferase 1A1 (SULT 1A, spot no: 23) is one of the most important members in this enzyme family due to its extensive tissue distribution and abundance. This enzyme has a substantially higher activity than other SULTs in catalyzing the sulfonation of 4-nitrophenol, a commonly used assay in biochemical pharmacogenetic studies for testing the activity of thermostable phenol SULTs [67]. SULT1A1 catalyzes the sulfonation of estrogens to form water-soluble and biologically inactive estrogen sulfates, reducing the level of estrogen exposure in their target tissues [68]. The mutation in the SULT1A1 gene would affect an individual’s capacity to efficiently sulfate endogenous compounds, drugs and xenobiotics, and consequently result in an individual’s susceptibility to cancer [69]. Yunfei et al. reported SULT 1A1 polymorphism as a predisposition factor for lung cancer [70]. In this study, Sulfotransferase 1A1 was up-regulated in plasma of hypobaric hypoxia treated rats. Other proteins detected include nucleoside diphosphate kinase (NME, spot no: 26), a multifunctional enzyme involved in the maintenance of the cellular pools of nucleoside triphosphate and in transcriptional regulation [71]. Nucleoside diphosphate kinase has been shown to undergo S-thiolation or disulfide cross-linking under conditions of oxidative stress, which could have implications in function switching [71]–[73]. In this study, NME was up-regulated, suggesting the protective role against the oxidative stress induced by hypobaric hypoxia.

In this experiment, candidate proteins corresponding to spots 6, 7 and 27 could not be detected in the database. They may be novel proteins, or else they may be small fragments of some proteins, as can be suggested from their low molecular weight. The ESI-MS/MS study of these spots may lead us to identify these novel proteins.

Study Limitations

We have noted some important limitations of our study. First limitation is that the protein quantification is based on only 2D-gels. Most of the proteins have multiple isoforms that differ in electrophoretic mobility. We could not estimate all the isoforms of the identical protein in 2D-gel, and this may affect the accuracy of the single protein quantification. Second limitation of the 2D gel-based proteomic approach is the variable validity of the protein identification. Some gel spots may contain more than one protein. The identification of proteins from peptide sequence is calculated as a high probability. Therefore, the validity of the approach has to be confirmed by different methods. However, since only a few of the respective antibodies against the observed proteins are currently available, it remains necessary to confirm the validity of our gel-based protein expression data by antibody-based techniques (e.g Western blotting), whenever antibodies become available. Third, although our work has provided some important clues, further studies are still needed to elucidate the detailed roles of these differentially expressed proteins during acute hypobaric hypoxia exposure. In addition to this, while we feel that altitude decompression sickness is unlikely, we cannot exclude the possibility that some of the findings may be related to the secondary effects of bubbles in tissues or blood. Exclusion of this possibility will require normobaric hypoxic exposures.

Collection and Isolation of Plasma Samples

Male Adult male Sprague-Dawley rats (n = 36) weighing approximately 220±10 g were used for the study. Animals were maintained in the animal house facility of the Defence Institute of Physiology and Allied Science (DIPAS) with a 12-hr light/dark cycle and were provided with food and water at ad libitum. To study the effects of acute hypobaric hypoxia exposure, 36 male Sprague-Dawley rats (220±10 g) were randomly divided into four groups (I to IV; n = 9). Group I served as the normoxia group maintained in standard environment. Groups II, III and IV served as hypoxia groups where the rats were exposed to simulated hypobaric hypoxia for 6 h, 12 h and 24 h respectively, at 25,000 ft (7620 m, 282 mm Hg, 37.60 kPa) in a specially designed animal decompression chamber where altitude could be maintained by reducing the ambient barometric pressure and temperature and humidity could be precisely controlled. The temperature and humidity was maintained at 28±2°C and 60±5% respectively. The rate of ascent to altitude was maintained at the rate of 300 m/min and it took a period of 20–25 min to reach the desired altitude. Being a slow rate of ascent in altitude (gradual decrease in ambient pressure), it is less likely to induce decompression induced gas bubbles during exposure. All the experimental protocol and animal care was approved by the ethical committee of the Defence Institute of Physiology and Allied Sciences (27/1999/CPCSEA) in accordance with the guidelines of “Committee for the Purpose of Control and Supervision of Experiments on Animals” of Govt. of India. Blood was withdrawn from the ocular vein immediately after 6, 12 and 24 h hypoxia. To prepare plasma, anticoagulants (either EDTA, heparin or sodium citrate), were added to the blood samples immediately after the blood was drawn to prevent clotting (EDTA plasma: 10 mL containing approximately 1.7 mg potassium EDTA; heparin plasma: 5 mL containing heparin 1 vial; sodium citrate plasma: 10 mL containing 1 mL of 0.118 mol/L (3.2%) citrate solution). The specimens were then centrifuged at 1500 g for 10 min/4°C to avoid hemolysis, decanted and transferred into Eppendorf tubes as aliquots. To each 1.0 mL plasma aliquots, 10 µl of protease inhibitor were added to obtain the reproducible results by 2-DE analysis. Proteases used are less active at lower temperature; therefore it is recommended that the plasma be prepared at as low a temperature as possible. The plasma samples were stored at –80°C until further analysis. For optimal reproducibility, plasma proteins from each of the nine sample pairs of rats (hypoxia and controls) were processed together throughout the 2-DE procedure. The protein concentration in plasma was determined using Bradford reagent.

Acetone/TCA Precipitation

A 100 µl of plasma sample was diluted with 900 µl of 10% TCA in acetone. The mixture was incubated overnight at−20°C and centrifuged at 15000 g, 4°C for 10 min. The supernatant was removed and 1000 µl of 90% ice-cold acetone were added to wash the pellet. The sample was incubated at −20°C for 10 min and centrifuged as above. The acetone containing supernatant was removed and the pellet was air dried. For 2-D gel electrophoresis, the protein pellet was suspended in 100 µl of lysis buffer as described earlier [74]. The protein sample was stored frozen at −20°C until analysis.

First-dimensional IEF Using the Protean IEF Cell

Total protein content in plasma samples was determined by Bradford assay and employed bovine albumin standards. Immobilized linear pH gradient strips (17 cm, pH 5–8, Biorad ) were rehydrated with the individual plasma samples, 500 µg of protein, in 300 µl of a improved rehydration buffer solution as described earlier (13) containing 7 M urea, 2 M thiourea, 1.2%, w/v CHAPS, 0.4% w/v ABS-14, 20 mM dithiothreitol (DTT), 0.25%, v/v, pH 3–10 ampholytes and 0.005% w/v bromophenol blue (BPB), for 18 h without current (in-gel passive rehydration). After rehydration, the focusing tray was renewed to remove any proteins not absorbed into the strip. IEF was conducted using a Protean IEF Cell (Bio-Rad) at 20°C as follow: 250 V for 1 h (slow ramping), changing the wicks every 30 min (to assist removal of ionic contaminants), 1000V for 1 h, linear ramping 10 000 V to over 3 h and a constant of 10 000V until approximately 60 kVh was reached. Strips were removed and stored at –80°C until run on the second dimension.

Second-dimensional Electrophoresis

For 2-DE analysis, individual samples from each animal group (Groups I-IV; n = 9) were run thrice (9×3 = 27 gels). Prior to SDS-PAGE, the IPG strips were equilibrated twice for 15 min with gentle shaking. The first equilibration solution contained 50 mM of Tris-HCl, pH 8.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS, 1% w/v DTT and 0.01% w/v BPB. In the second equilibration solution, DTT was replaced with 2.5% (w/v) iodoacetamide. The equilibrated IPG strips were slightly rinsed with milli-Q water, blotted to remove excess equilibration buffer and then applied to SDS-PAGE gels (20 cm × 20 cm × 1 mm 8–19% polyacrylamide (30% (w/v ) acrylamide:0.8% (w/v) bis-acrylamide, 37.5∶1 stock) using a PROTEAN II XL system (Bio Rad) at 10 mA per gel for 30 min followed by 35 mA per gel for 12 h until the dye front had run off the edge of the 2-D gel.

Staining and Imaging

After electrophoresis, proteins were visualized by modified silver staining procedure compatible with MS [75]. The gels were fixed in 50% v/v methanol, 12% v/v acetic acid and 0.05% v/v formaldehyde for at least 2 h. The fixed gels were rinsed with 50% v/v ethanol three times for 20 min each, then again sensitized with 0.02% w/v sodium thiosulfate followed by three washings with milli-Q water each for 20 s. The gels were immersed in 0.1% w/v silver nitrate and 0.075% v/v formaldehyde for 20 min and rinsed with milli-Q water twice for 20 s each. It was developed with 6% sodium carbonate and 0.05% v/v formaldehyde. Finally, the reaction was terminated by fixing with 50% v/v methanol and 12% v/v acetic acid. The stained gels were imaged using an Investigator ProPic II Genomics Solutions. Gel images were exported as 16-bit-gray scale TIFF files that were imported into 2Dprogenesis analysis software package. The principles of measuring intensity values by 2D analysis software are similar to those of densitometric measurement. After the background subtraction the protein spots were automatically defined and quantified with the feature detection algorithm. Spot intensities were expressed as relative volumes in percentages (% volume) by integrating the OD of each pixel in the spot area (vol) and dividing with the sum volumes of all spots detected in the gel. A master gel was prepared for each group, after considering one gel from each animal of the group, using Progenesis Software, thus representing plasma proteome of each individual group. In the master gel each spot is assigned a unique number. The quality of the match made by the computer was critically evaluated in each case, and necessary editions and corrections were done manually. Initially, protein spots with significant changes test (p≤0.05, one –way ANOVA) in a consistent direction (increase or decrease) were cut for identification, according to the method of Turko et al. [76]. Mostly, only spots that are statistically different between control and experimental groups were subjected to in-gel trypsin digestion for subsequent analysis by mass spectrometery.

MS identification of Proteins

Western Blot

The protein quantification of transthyretin, peroxiredoxin-2, glutathione peroxidise, Apo A-I, haptoglobin, Apo-E, fetuin–B and Nucleoside diphosphate kinase B were selected to be validated by Western-blot analysis because the expression changes of these proteins were more obvious than that of the other proteins and the obtaining of their antibodies was convenient. Briefly, plasma samples were first diluted 10 times by 1 X PBS, and then total proteins (40 µg) were separated by SDS-PAGE and electro-blotted to nitrocellulose membrane. After being blotted with 5% nonfat-dried milk in 1 X TBST (25 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20) overnight, membranes were incubated with primary antibodies for 2 h, followed by secondary antibody for another hour. All these experiments were conducted at room temperature. The immunocomplexes were visualized by chemiluminescence using the chemiluminescent peroxidase substrate kit (Sigma-Aldrich, St. Louis Mo 63103, USA). The quantification of protein was done by densitometric digital analysis of protein bands (TIFF image) using Quantity One 1-D Analysis Software version 4.6.7 (BIO-RAD, Hercules, CA). Each protein band was normalized to the corresponding β–tubulin band. The results of western blot are representations of three independent experiments (Mean ± SEM). Statistical analyses were performed using GraphPad Prism (Version 5) one- way ANOVA with pair-wise multiple comparison procedures (student-Newman-keuls method ), and a p-value of <0.05 was considered significant.