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Hypoxia blunts angiogenic signaling and upregulates the antioxidant system in elephant seal endothelial cells

Kaitlin N Allen, Julia María Torres-Velarde, Juan Manuel Vazquez, Diana D Moreno-Santillan, Peter H Sudmant, José Pablo Vázquez-Medina

Posted on: 13 September 2023

Preprint posted on 3 July 2023

Diving into Molecular Defenses: How elephant seals protect themselves from oxygen poisoning during deep dives

Selected by Sarah Young-Veenstra

Background

Elephant seals (Mirounga angusitrostris) routinely dive to hunt active prey for durations up to an hour. To make such intense activity possible without access to oxygen, seals undergo specialized physiological changes when diving, such as bradycardia and vasoconstriction. Despite these abilities, the extreme hypoxia that seals experience during their dives has the potential to take a significant pathological toll.

Most mammals resort to reversing mitochondrial transport under extreme hypoxic conditions as a means to fuel their body’s activity in the absence of oxygen. This reversal produces succinate, which functions as an alternative energetic substrate. However, this succinate accumulation becomes dangerous upon return to normoxia, when mitochondrial transport returns to normal and oxidizes the succinate, resulting in the production of dangerous reactive oxygen species (ROS). This ROS production should cause oxidative damage, but no such damage is seen in seals, suggesting they must have some defense against hypoxia-induced pathologies.

Glutathione (GSH), a major antioxidant in animals, may be at the center of seals’ defense against hypoxia-induced oxidative damage. Indeed, marine mammals possess high GSH levels both in tissues and in circulation, and diving mammals display positive selection for, and duplications of, genes along the GSH metabolic pathway. However, whether the dynamics of GSH expression are hypoxia dependent (i.e., whether GSH plays any role during submersion and dive recovery) is unknown. Molecular changes during active dives have not previously been assessed due to the infeasibility of taking biological samples from seals mid-dive. Allen and her team from the University of California Berkely developed a novel primary cell culture system, wherein they isolated arterial endothelial cells from elephant seal placentas, which allowed them to study the cellular response to hypoxia. The goal of their study was to investigate the real-time molecular changes during an elephant seal’s dive.

Key Findings

The research team dove into measuring multiple biological responses to hypoxia in arterial endothelial cells of the elephant seal. Furthermore, they conducted the same measurements on human arterial endothelial cells in order to gauge which aspects of the hypoxia response are specialized to the diving mammal. They found that elephant seals decrease their inflammatory signalling capacity, inhibit the typical mammalian hypoxia signalling pathway to prevent angiogenesis and reliance on glycolysis, accumulate glutathione (GSH) and increase expression of GSH metabolic genes, and suggest that promotion of GSH metabolism effectively combats oxidative damage.

Elephant seals limit inflammation to maintain critical blood supply

Hypoxia induced an immediate anti-inflammatory response in both elephant seal and human cells by downregulating the Tumor Necrosis Factor (TNF) signalling pathway, which promotes cell proliferation. However, the overall anti-inflammatory effect was more pronounced in seal cells, which also downregulated additional cell-proliferation signalling pathways (Transforming Growth Factor [TGF-β] and Nuclear Factor [NF-kB]) (Figure 1). Decreasing the body’s inflammatory signaling capacity under hypoxic conditions likely limits the vasodilation potential during dives, thereby reducing blood flow to peripheral tissues and conserving blood to oxygenate essential organs such as the brain and heart.

Figure 1. Short-term hypoxia modulates inflammatory signaling in seal cells. Reactome pathway enrichment for genes differently expressed at all early time points. (A,B) Functional interaction networks and select enriched pathways for seal (A) and human (B) genes differentially expressed  at all early time points. Figure reproduced from Allen et al. (2023), bioRxiv with author permission.

Elephant seals dissociate from the typical mammalian hypoxia response

A typical mammalian response to hypoxia, as evidenced here in the human cells, sees increased angiogenesis and an increased reliance on glycolysis for anaerobic energy provision (Figure 3B). These whole-organism level modulations are promoted on a molecular level through the Hypoxia Inducible Factor (HIF)-1 regulatory pathway. The protein coding gene HIF-1α, commonly referred to as the “master regulator” of the hypoxia response, is stabilized in response to hypoxia exposure, which sets off a signalling cascade that ultimately induces biological changes that maintain oxygen homeostasis, namely angiogenesis and a metabolic shift to glycolytic pathways. Interestingly, elephant seal cells stabilized HIF-1α more rapidly than human cells (Figure 2), but seals neither promoted angiogenesis nor shifted to glycolytic metabolism (Figure 3A). The lack of HIF-1 downstream effects suggests that HIF-1α stabilization is decoupled from vascular homeostasis in elephant seals, thereby inhibiting the HIF-1 pathway from promoting biological shifts that may render the vascular system vulnerable to oxidative damage.

Figure 2. Hypoxia rapidly stabilizes HIF-1α in seal endothelial cells. Fold change in HIF-1α protein abundance compared to normoxic baseline in endothelial cells exposed to 1% O2 for up to 6 h, n=3. † p<0.05 versus control (seal); * p<0.05 versus control (human). Figure reproduced from Allen et al. (2023), bioRxiv with author permission.

 

Figure 3. Seal cells delay angiogenic signaling in response to hypoxia exposure. (A, B) Top three transcription factors predicted from genes differently expressed at 30 min, 60 min, or 6 h versus control for (A) seal and (B) human cells. Figure reproduced from Allen et al. (2023), bioRxiv with author permission.

Elephant seals may anaerobically metabolize polyamines to generate energy

Succinate levels did not increase in human cells exposed to hypoxia but increased 40% in elephant seal cells after 6 hours of hypoxia exposure (Figure 4). This difference in succinate levels between seals and humans experiencing hypoxia is consistent with the human cells, but not the seal cells, needing to rely on glycolysis during hypoxia, as seals may use succinate as their energetic substrate instead. Interestingly, 40% is a relatively mild succinate accumulation, and suggests that the source of the succinate is not reversal of mitochondrial transport. The authors pose that a more likely cause of the elephant seal’s succinate accumulation is polyamine processing, and that this pathway may be advantageous. Indeed, under hypoxic conditions, elephant seals upregulated expression of several genes along the GSH metabolic pathway, some of which also play a role in polyamine synthesis. Polyamines regulate mitochondrial respiration by modulating the pyruvate dehydrogenase complex. Additionally, the polyamine putrescine can be further converted into succinate, which may go on to fuel oxidative phosphorylation as well as competitively inhibit the enzymes that hydrolyze HIF-1α, potentially accounting for the increased hypoxia sensitivity of HIF-1α in elephant seal cells relative to human cells.

Figure 4. Concerted changes in gene expression in response to hypoxia in seal and human cells. (A) GSH content relative to each species’ baseline. Lettering indicates intraspecific changes among treatments. (B) Percent change in intracellular succinate concentration in response to hypoxia exposure. * p<0.05 versus species baseline. Figure reproduced from Allen et al. (2023), bioRxiv with author permission.

Glutathione is likely the crux of oxidative damage control in elephant seals

Increased expression of GSH metabolic genes was unique to the elephant seal cells and correlated with elevated GSH levels in seal cells relative to human cells. Such metabolic genes included glutamate-cysteine ligase, which catalyzes the rate limiting step of GSH biosynthesis, and glutathione synthetase, another a key enzyme in GSH metabolism. Furthermore, elephant seals demonstrated sustained GSH production throughout the duration of hypoxia exposure.

Conclusion

This study suggests that the elephant seal’s affinity for GSH metabolism may be at the center of their defense against oxidative pathology, not only due to the antioxidative properties of GSH, but also because metabolic genes along the GSH biosynthetic pathway have a dual function in synthesizing polyamines, which ultimately produce succinate to fuel oxidative phosphorylation under hypoxic conditions.

 

Why I Chose This Paper

This preprint points out an interesting physiological dichotomy in the diving physiology of seals that I had never come across before. Understanding how seals cope with routine hypoxic dives while, seemingly contradictorily, protecting themselves against superoxide production is an intriguing avenue of research and is also potentially relevant to other species as a rapidly changing climate threatens many species with increased instances of hypoxia. As the researchers report, there are several avenues where human endothelial cells employ different pathways from the elephant seal’s, presumably reflecting the lack of adaptation to routinely coping with hypoxia. Furthering knowledge as to how the seal’s specialized pathways effectively fight against pathological effects of hypoxia, and then learning which species are capable of employing similar pathways, may be important to predicting which species may be more vulnerable to impending climate change and consequent hypoxic zones.

Questions for the Authors and Future Directions

  1. In what way do you think reducing the vasodilation potential is affiliated with preventing oxidative pathology?
  2. Although the 40% succinate accumulation observed in elephant seals is more mild than you might expect from ETC reversal, do you suspect that the observed levels are generally utilized before surfacing from a dive (i.e., would even these levels have a chance to be oxidized into ROS)?
  3. Why do you think humans did not appear to accumulate any succinate under hypoxia? Might humans not be reversing the ETC either?
  4. Based on this study, an interesting continuation would investigate the synthesis and processing of polyamines, specifically putrescine, during hypoxia and reoxygenation in elephant seals to test whether this pathway does, in fact, 1) modify pyruvate dehydrogenase function to provide succinate during elephant seals’ dives and 2) competitively inhibit HIF-1α.

Tags: cell culture, free radicals, gene expression, homeostasis, phocid, physiology

doi: https://doi.org/10.1242/prelights.35529

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Author's response

Kaitlin Allen shared

1) In what way do you think reducing the vasodilation potential is affiliated with preventing oxidative pathology?
Seals are really dependent on maintaining vasoconstriction during diving as a way to prevent excessive oxygen consumption by “non-active” tissues. There’s a tradeoff here, though – by limiting blood flow (and thus oxygen delivery) to tissues, they are effectively creating ischemia/reperfusion events that we typically see as pathophysiological during adverse cardiovascular events. The seals likely suppress angiogenesis due to a lack of increased demand during low oxygen exposure.
 
2) Although the 40% succinate accumulation observed in elephant seals is more mild than you might expect from ETC reversal, do you suspect that the observed levels are generally utilized before surfacing from a dive (i.e., would even these levels have a chance to be oxidized into ROS)?
This is a great question, and it’s hard to know! In our experiment, we measured succinate in cells that never saw reoxygenation, so in theory that should reflect what’s occurring within the dive itself. Even in cases where succinate does accumulate during reverse electron transport it’s still useful as a metabolic fuel, but there is an associated ROS “burst” that can be damaged during reperfusion/reoxygenation.
3) Why do you think humans did not appear to accumulate any succinate under hypoxia? Might humans not be reversing the ETC either?
Since we see fairly mild increases in succinate concentration in the seal cells, it’s possible ETC reversal is not occurring in either species. Overall, I think it’s likely that succinate’s role here is based in signaling rather than metabolism directly.
 
4) Based on this study, an interesting continuation would investigate the synthesis and processing of polyamines, specifically putrescine, during hypoxia and reoxygenation in elephant seals to test whether this pathway does, in fact, 1) modify pyruvate dehydrogenase function to provide succinate during elephant seals’ dives and 2) competitively inhibit HIF-1α.
Definitely! I think the big open question here is what is HIF-1 really doing in these cells if we see that it is both highly sensitive/responsive to hypoxia, and also not kicking off the typical pathways we associate as being a critical component of the endothelial cell response to hypoxia.

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