Primary Endothelial Cells from Seals Mount a Rapid and Sustained Response to Acute Hypoxia

Abstract

Marine mammals tolerate repeated bouts of hypoxemia while diving, with up to 90% of blood oxygen stores depleted in some species. Accordingly, tissues such as the endothelium experience acute fluctuations in oxygen availability. In terrestrial mammals, hypoxia/reoxygenation events of similar magnitude contribute to inflammatory signaling, reactive oxygen species (ROS) generation, and endothelial dysfunction in pathological settings. Marine mammals, however, appear well protected against oxidant stress and inflammation despite repeated exposure to hypoxia/reoxygenation, though the specific cellular mechanisms driving this protection remain unknown. We isolated endothelial cells (ECs) from placental arteries from northern elephant seals (Mirounga angustirostris), a pinniped species noted for its extreme diving capacity. Seal primary EC cultures proliferate, express the canonical EC markers PECAM‐1 and VE‐cadherin, and take up acetylated LDL. Seal ECs are metabolically active in adherent culture and respond to mitochondrial inhibition and uncoupling and pharmacological NADPH oxidase activation, demonstrating utility as a live, functionally relevant system in which to study the biochemical drivers of hypoxia/reoxygenation tolerance in diving seals. Seal ECs exposed to 1% O 2 for up to 6 hours upregulated HIF‐1α protein, with maximal expression occurring within 15 minutes (68‐fold increase over normoxic levels). Human ECs matched this magnitude (73‐fold increase) though not until 1 hour, after which expression of HIF‐1α rapidly and progressively declined (22‐fold above normoxic levels at 6 hours). In contrast, seal ECs maintained elevated HIF‐1α protein expression (52‐fold) at 6 hours. Pharmacological stimulation with menadione, an inducer of redox cycling, significantly increased oxidant generation in human (5‐fold, p=0.027) but not seal ECs (2.5‐fold, p=0.18), as did acute exposure to hypoxia/reoxygenation. Together, our data show that seal ECs mount a rapid, sustained response to hypoxia /reoxygenation events, and that these cells are a functional system in which to study the mechanisms underlying innate hypoxia and oxidative stress tolerance in diving mammals.Support or Funding InformationNational Science Foundation GRFP