An Oxygen–Glucose Deprivation Model with GasPak for Studying ER Stress–Ischemia Interactions in Human Endothelial Cells

During ischemia, endothelial cells' integrity is compromised, as a consequence, blood–barrier homeostasis is disrupted. Therefore, the structural and functional preserva-tion 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 straightfor-ward 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 hours of oxygen–glucose deprivation (OGD) in glucose-free medium with rapidly induced hypoxia. Hy-poxia, oxygen consumption, cell viability, apoptosis, and ER stress markers (BiP/GRP78, PERK, ATF6, IRE1/XBP1s, CHOP) were assessed by RT-qPCR and Western blot. The mod-el enables quantification of metabolic stress (OCR), as well as evaluation of viability loss, membrane integrity, apoptotic commitment, and discrimination between ER stress reso-lution versus maladaptation. Overall, GasPak EZ Pouch Systems provide a reproducible and practical in vitro platform to study ischemic injury down to its mechanistic details of ER-mitochondria signalling. They give the opportunity to evaluate therapeutic approach-es 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 interven-tions aiming at preserving endothelial barrier function, in various ischemic contexts.