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.