Oxygen Reduces Accumulation of Type IV Collagen in Endothelial Cell Subcellular Matrix via Oxidative Stress

Abstract: 

Anchorage-dependent cells in culture attach initially to proteins adsorbed to the culture substrate from the medium, and produce and deposit a subcellular matrix during the course of the cultivation. The aim of this study was to determine whether the concentration of O₂ in the culture atmosphere affects the accumulation of type IV collagen and laminin under human endothelial-cell monolayers. Enzyme-linked immunoassays on decellularized polystyrene substrates showed less type IV collagen, but not less laminin, under cells incubated in the standard atmosphere (5% CO₂ in air, i.e., ∼20% O₂) compared to an atmosphere of 5% O₂ and 5% CO₂ in N₂. Type IV collagen accumulation was inhibited via oxidative stress, because the inhibitory effect of 20% O₂ was antagonized by antioxidant ascorbic acid, and mimicked by prooxidant pyrogallol and exogenous H₂O₂. Measurements of endogenous H₂O₂ accumulation demonstrated that endothelial cells partially adapt to the high O₂ concentration. These results may have implications in endothelium modeling in vitro and in engineering of endothelial cell sheets and endothelialized vascular grafts.