Discussion
In a preceding study, we observed redox homeostasis to be the single causal event that determined the overall response of both lung and plasma proteome to acute low pO2 exposure (Paul et al., 2018). In the case of extended environmental hypoxia exposure, redox homeostasis was not the defining impetus, after day 3. Our initial investigations into biochemical redox parameters in both lung and plasma samples revealed ROS and TBARS to be near normoxic values in both HD7 and HD14. Even NOx levels confirmed to Normoxic levels in HD14 lung tissues and plasma. The overall antioxidant defenses in both lung and plasma were adept at disposing off excess radicals. TAC trends were opposing in lung and plasma. While lung showed the highest TAC values in HD3 with subsequent lowering till HD14, plasma had lowest TAC levels in HD3 with subsequent increases till HD14. Plasma had nearly similar total GSH concentrations across groups. However, lung tissue saw increased GSH concentrations in HD3 and HD7 with normalized GSH concentrations in HD14. SOD and catalase levels in both lungs and plasma had similar activity levels across all experimental groups relative to cata. We also failed to observe any redox stress/homeostasis mediated differential trends in the redox specific PCR array in both lung and plasma of the experimental groups (Fig. S1 and S3,Supplementary information ). Thus, after 3 days of exposure to environmental hypoxia, we may conclude that a systemic redox homeostasis, at least at the level of proteome, is achieved. Sudden lowering of pO2 (observed during rapid ascent to altitude) is a causal factor of acute altitude illnesses (Barry & Pollard, 2003; Fiore, Hall, & Shoja, 2010) and redox stress being an underlying molecular event of acute altitude illnesses (Damian Miles Bailey, Bärtsch, Knauth, & Baumgartner, 2009; Damian M Bailey & Davies, 2001), slow graded lowering of pO2 in this study may be aiding redox homeostasis. Probably due to the slower ascent rates (250 m/min), even in presence of redox stress (noticeably till HD3), longer hypoxic durations enabled the organism to attenuate its effects. Such an effect is the uneven expression of housekeeping proteins which was observed in a previous study concerning rapid ascent to altitude (Paul et al., 2018). In this study, abrogation of such effects was observed in GAPDH expression profile (Fig. 3c & d). Given the susceptibility of housekeeping proteins’ expression to rapid extreme hypoxia exposure, they are pivotal in deciphering whether an organism is adapting to hypoxia.
Further exploration of the lung proteome revealed that most of the differentially expressed proteins were involved in cytoskeletal re-arrangements. The major canonical pathways were Remodeling of epithelial adherens junctions, Actin cytoskeleton signaling and Signaling by Rho-family GTPases. As most of the differentially expressed lung proteins were down-regulated, especially in HD3, we observed the directional canonical pathways to be either neutral (Remodeling of epithelial adherens junctions) or down-regulated (Actin cytoskeleton signaling, Signaling by Rho-family GTPases). Although Clathrin mediated endocytosis signaling and Epithelial adherens junction signaling did not show any trends, their presence among the top canonical pathways implicated lipid derived processes and internalization of protein channels as two features to be further investigated. Further since both clathrin coated pits and adherens junctions are cell surface features, extended low pO2 exposure is thus managed by modulation of lung cell surface features. Adherens junction formation is closely linked to biochemical, mechanical and cytoskeletal events, particularly Actin cytoskeleton.
The overall downregulation pathways like Actin cytoskeleton signaling points to a bias towards internalization of clathrin coated pits and lowered turnover of adherens junction assembly/disassembly in order to maintain homeostasis considering the lowered energy status of hypoxic tissues/cells. Actin cytoskeleton signaling is mainly involved in modulation of cell shape and external structures in response to external stimuli (Schmidt & Hall, 1998). Actin cytoskeleton signaling has been previously reported to be associated with integrins (DeMali, Wennerberg, & Burridge, 2003; Wu & Dedhar, 2001), Rho-family (Maekawa et al., 1999; Winter et al., 2001) and even T-cell activation (Dustin & Cooper, 2000; Valitutti, Dessing, Aktories, Gallati, & Lanzavecchia, 1995). Integrin based signaling is also involved in modulation of signaling by Rho-family GTPases, another significantly up-regulated canonical pathway in the lung, via Rac and Cdc42 (DeMali et al., 2003). Further Actin cytoskeleton signaling via Integrin beta 1 may also affect the phosphorylation trends through the action of Tyrosine kinases (Kornberg, Earp, Turner, Prockop, & Juliano, 1991). The interplay between Rho-GTPase family, actin cytoskeleton and epithelial adherens junction remodeling suggests increased cell movement, formation of cell-cell junctions and may indicate structural changes in the lungs to withstand the lower ambient partial pressure. Stress failure of pulmonary vasculature is implicated in development of HAPE (Stream & Grissom, 2008; West et al., 1995; West & Mathieu-Costello, 1992). Since these rats can be considered acclimatized in the absence of any mortality or signs of altitude illnesses, the above canonical pathways can be speculated to modify the lung cellular structures in such a manner as to prevent stress failure in alveolar capillaries. As in the previous study (Paul et al., 2018), STAT3 was observed to be an upstream regulator present in lung tissue and affecting the global proteome. RXR was also observed to show differential signaling.
In the plasma proteome, we observed APRS signaling and LXR/RXR Activation as the two most significant canonical pathways. STAT3 is an up-stream regulator of APRS while RXR is involved in LXR/RXR Activation pathway. In the plasma, STAT3 via APRS triggered perturbations in multiple apolipoproteins (also downstream of LXR/RXR mediated signaling), complement proteins (components of Complement system) and proteins associated with coagulation cascades. APRS is activated in HD3 and HD14 while its down-regulated in HD7. This could be due to the lesser degree of activation of Coagulation system in HD7. The consistently lower levels of Protein C and Protein S during extended low pO2 exposure (Fig. S6, Supplementary information ) indicate increased clotting tendency. This is corroborated by previous reports suggesting exaggerated occurrence of thrombosis during extended low pO2 exposure at high altitude involving low levels of Protein C & protein S (Anand, Jha, Saha, Sharma, & Adya, 2001; Boulos, Kouroukis, & Blake, 1999; Fujimaki, Matsutani, Asai, Kohno, & Koike, 1986; Le Roux, Larmignat, Marchal, & Richalet, 1992; Nair et al., 2008). Complement system shows sequential up-regulation across groups with HD14 showing highest degree of up-regulation (Fig. S7). However, across the key complement proteins like C3 and C4b (Fig. 5c), we observed a very slight increase in HD14 suggesting minimal chances of a strong complement cascade. In our analysis, we observed that APRS signaling proteins provide the most comprehensive clues regarding survival of rats at low pO2. We observed antioxidants (thioredoxin, glutathione peroxidase); transporters (transthyretin); metabolic proteins (Apo A1) and inflammatory proteins (C3) to be indicative of survival at low pO2 for SD rats.
Based on the processes that were observed to be perturbed in the rats exposed to extended durations of low pO2, we evaluated particular proteins known to be associated with these processes in humans exposed to chronic durations at altitude. These proteins were essentially qualitative indicators for the specific process. We observed that acute phase signaling was observed to be perturbed. Thus, we assessed alpha-1-antitrypsin in human plasma. It serves as a connector between acute phase response and cytoskeletal remodeling cascades. We observed cytoskeletal re-modeling in rat and assessed cofilin-1 in human plasma. Similarly, S100A8 was assessed in human plasma as innate immune activation was observed in rat. Each of the above three proteins that were measured using ELISA were picked from the LC-MS/MS dataset (Tables S1 & S2, Supplementary information ) owing to their crucial roles in the molecular processes that were perturbed by extended exposure to low pO2 during high altitude stay.