Effects of different hypoxia degrees on endothelial cell cultures-Time course study

Introduction: Exposure of the endothelial cells to hypoxia, the decrease in oxygen supply can trigger an endothelial response. This response is involved in inflammatory diseases, tumorigenesis, and also with the micro vascular damage associated with aging. The aim of our study was to determine the hypoxia/re-oxygenation induced response in vitro, using human umbilical vein endothelial cells (HUVEC) cultures, at different time points with focus on cell viability, apoptosis oxidative stress and angiogenesis stimulation.

Materials and methods: Cells were exposed to 10%, 5% or 0% O₂ for 6h, 12h, and 24h. Viability was measured through colorimetry, apoptosis - annexin V-FITC staining, DNA lesions (γH₂AX), endothelial activation (sICAM1), angiogenesis (HIF1α), oxidative stress (malondialdehyde, superoxidismutase and NFκB activation) were determined by ELISA, Western Blot and spectrophotometry.

Results and discussion: Hypoxia decreased viability, increased apoptosis, oxidative stress, endothelial activation and angiogenesis, depending on O₂ concentration and time exposure. Short exposures to 5% and 10% O₂, efficiently activated anti-apoptotic mechanisms through NFκB activation, HIF1α and γH₂AX related DNA damage repair pathways. However, severe hypoxia and longer exposures to mild hypoxia induced high oxidative stress related damage and eventually led to apoptosis, through strong increases of HIF1α and accumulating DNA lesions.