Global proteome analysis indicates STAT3 modulates lipid
metabolism associated inflammatory signaling
Since redox stress management was ruled out as the main molecular event,
we investigated the global plasma proteome using iTRAQ-LC-MS/MS (Table
S2, Supplementary information ) to ascertain the molecular events.
In all groups, 312 proteins were identified and quantified. In HD3, 32
proteins were up-regulated and 42 were down-regulated. In HD7, 48
proteins were up-regulated and 36 proteins were down-regulated. In HD14,
60 proteins (the highest) were up-regulated and 31 proteins (the lowest)
were down-regulated (Fig. 4a). This is in contrast to the lung proteome
where across all groups, down-regulation of protein expression was
dominant. The most significant molecular and cellular processes across
groups were the same along with the same proteins involved in those
processes (Fig. 4b). Cellular movement, Cell death and survival and
Cell-to-cell signaling and interaction were also observed to be
significant in the lung proteome. Protein synthesis and cellular
compromise are unique to the plasma proteome. The trends of protein
expression in the global proteome across HD3, HD7 and HD14 (relative to
Normoxia) was pictured using hierarchical clustergram (Fig. 4c). The
most significant canonical pathways were Acute phase response signaling
(APRS), LXR/RXR Activation, FXR/RXR Activation, Coagulation system and
Complement system (Fig. 4d). LXR/RXR remained down-regulated throughout
the hypoxia exposure. Coagulation system remained up-regulated
throughout the environmental hypoxia exposure. Complement system is
up-regulated sequentially in HD7 and HD14 after being neutral in HD3.
FXR/RXR remains non-directional throughout the environmental hypoxia
exposure. APRS, quite interestingly, is strongly up-regulated in HD3,
gets down-regulated at HD7. In HD14, it becomes up-regulated again.
Since STAT3 is a regulatory protein for Acute phase response, we
investigated the APRS with overlaid expression data from our plasma
dataset (Fig. 5a). Furthermore, an integrated network created using the
top five canonical pathways (Fig. 5b) to identify the upstream
regulators. We observed an interplay of RXR with HNF4A, complement
proteins, apolipoproteins and coagulation proteins. This was indicative
of perturbations in lipid metabolism associated with perturbations in
inflammatory signaling and coagulation cascades during extended
exposures to low pO2. To further verify these
perturbations (in addition to their links with acute phase response) and
assess antioxidant status we performed immunoblotting of proteins like
glutathione peroxidase 3 (GPx3), plasminogen, C3, C4b, transthyretin
(TTR), apolipoproteins A1 and H (Fig. 5c). GPx-3 was observed to be
higher in HD3, similar to Normoxia in HD7 while HD14 showed lowest
levels. C3 levels sequentially increased across the groups, reaching
highest levels at HD14. C4b sequentially increased in HD3 and HD7,
reaching its highest levels. In HD14, C4b levels were similar to HD3.
Ttr levels decreased in HD3 and remained nearly constant in HD7 and
HD14. Apo A1 levels were progressively and notably lower across HD3 to
HD14 as compared to control group. Apo H levels were notably low
throughout the environmental hypoxia exposure. Overall, upon increasing
durations of low pO2 exposures, particularly past day 7,
there is increased inflammatory signaling, declining antioxidant
reserves and adverse effects on lipid metabolism.
Indicators of cytoskeletal
remodeling and inflammation in altitude acclimatized humans
The real-world manifestation of lowered pO2 exposure for
extended durations is observed during high altitude explorations. We
observed that during extended exposure to low pO2 in SD
rats, lipid metabolism was favored while TCA cycle was subdued (Fig. 3c
& d; RXR and MDH) indicating perturbed systemic energy homeostasis.
However, results also indicate lowered HDL levels (incumbent on Apo A1
expression) as exposure of duration to lower pO2increased (Fig. 5c). We found that it was associated with the lung
cytoskeletal re-arrangements (Fig. 3d). The perturbed energy homeostasis
resulted in innate immune activation, mostly inflammatory signaling,
which was observed via acute phase response signaling (Fig. 5). Based on
these aspects, we selected the following proteins that were specific for
a signaling network/molecular process: alpha-1-antitrypsin, cofilin-1
and S100A8. These may not be
called translated markers owing to the different time points at altitude
as well as a different altitude zone as compared to the experimental
conditions of the SD rats. The small sample size may also be considered
a disqualification for the same. However, the results obtained (Fig. 6)
do provide qualitative hints at the major processes that may be further
analyzed for finding biomarkers of altitude acclimatization/disease and
therapeutic targets in human samples.
Since innate immune activation and calcium associated proteins were
observed in rat plasma (Table S1; Supplementary information), we
measured S100A8 in human plasma (Fig. 6a). S100A8 inhibits neutrophil
activation and adhesion and also modulates inflammatory processes
(Raquil, Anceriz, Rouleau, & Tessier, 2008). It is a component of
calprotectin (Yui, Nakatani, & Mikami, 2003), a fecal biomarker of
gastrointestinal inflammation in irritable bowel syndrome (Van Rheenen,
Van de Vijver, & Fidler, 2010). There were no major changes throughout
HAD7, HAD30 and HAD120 as compared to Normoxia. This is indicative that
innate immune processes, particularly inflammatory processes, are
well-balanced in individuals acclimatized to low pO2levels at high altitude. Next, we evaluated cofilin-1 levels in human
plasma (Fig. 6b). Along with Lim kinase, cofilin signaling is essential
for remodeling of cytoskeleton (Danen et al., 2005; Wang, Eddy, &
Condeelis, 2007). Higher levels were observed in HAD7 suggesting
increased cytoskeletal re-modeling corroborated by increased redox
stress in HD3 in rats. Its near Normoxic levels in HAD30 and HAD120
suggest a balanced impetus for actin dynamics. However, till day 7
cofilin-1 levels are higher than control group. To delve further into
this aspect, alpha-1-antitrypsin (Tables S1 and S2; Supplementary
information) was also assayed in human plasma (Fig. 6c). It was observed
in the Acute phase response signaling in SD rats. It is known to inhibit
elastase and thus prevents lung tissue damage during various conditions
(Brantly et al., 1988; Garratt et al., 2016; Gøtzsche & Johansen,
2016). In a previous study, we had shown that Acute phase response
signaling is a homologous pathway between rat and human exposed to
hypoxia (Paul, Bhargava, & Ahmad, 2017). Alpha-1-antitrypsin levels
were investigated in human plasma based on our findings that
cytoskeletal re-arrangement was taking place in the rat lung. We
observed that plasma alpha-1-antitrypsin levels are lowered throughout
the high altitude exposure in humans till HAD30 suggesting increased
chances of lung tissue damage/re-modeling. However, it’s increased
levels at HAD120 suggest better adjusted lung cytoskeletal dynamics in
agreement with recovering cofilin-1 levels.