A GasPak-Based Ischemia Model for Studying ER Stress–Ischemia Interactions in Human Endothelial Cells

During ischemia, endothelial cell integrity is compromised, as a consequence, blood barrier homeostasis is disrupted. Therefore, the structural and functional preservation of endothelial cells is paramount when trying to improve outcomes after ischemic injury. Endoplasmic reticulum (ER) stress is increasingly recognized as a key player in ischemic injury through unfolded protein response (UPR) signalling, and its crosstalk with mitochondrial death pathways. This study provides a cost-effective and straightforward method to delve into the relationship between ER stress and ischemia in human microvascular endothelial cells-1 (HMEC-1). HMEC-1 was exposed to 8 h of oxygen-glucose deprivation (OGD) in glucose-free medium with rapidly induced hypoxia. Hypoxia, oxygen consumption, cell viability, apoptosis, and ER stress markers (BiP/GRP78, PERK, ATF6, IRE1/XBP1s, CHOP) were assessed by RT-qPCR and Western blot. Cell viability decreased by approximately 33% following OGD, while CHOP expression increased ~4-fold, indicating significant ER stress induction. The model enables quantification of metabolic stress (OCR), as well as evaluation of viability loss, membrane integrity, apoptotic commitment, and discrimination between ER stress resolution versus maladaptation. Overall, GasPak EZ Pouch Systems provide a reproducible and practical in vitro platform to study ischemic injury down to the mechanistic details of ER-mitochondria signalling. They give the opportunity to evaluate therapeutic approaches that target ER homeostasis to limit apoptosis and/or recovery of metabolic function after ischemia. This method could allow rapid screening of ER stress-modulating interventions aimed at preserving endothelial barrier function, in various ischemic contexts.