Hypoxic Conditions Modulate HREC

Human retinal endothelial cells (HRECs) were procured from Cell Systems (Kirkland, WA, USA) and cultured, as previously described^{38 (link),39 (link)}, in Microvascular Endothelial Cell Growth Medium-2 Bullet Kit (Lonza, Walkersville, MD, USA; Catalog No. CC-3202 EGM-2 MV), which includes base medium and growth kit supplements, containing human fibroblast growth factor (hFGF), human vascular endothelial growth factor (hVEGF), human insulin-like growth factor (hIGF-1), human epidermal growth factor (hEGF), and 5% fetal bovine serum (FBS). HRECs of passage 3–6 were seeded in 100 mm Petri dishes until they reached 90% confluency, then the medium was changed to fresh medium containing 5% FBS, but without growth factors, that had either mannitol (25 mM), which was the osmotic control group, or HG (25 mM) for 5 days. Hypoxia (Hyp) was introduced 24 h before cell harvesting by incubating HRECs in a 2% O₂ humidified hypoxic chamber with concurrent 5% CO₂.