ACTIVATION AND INJURY OF ENDOTHELIAL CELLS BY HYPOXIA/REOXYGENATION. EFFECTS OF EXTRACELLULAR pH
Disturbance of blood flow due to occlusion or spasm of blood vessels leads to hypoxia and acidosis, and its subsequent restoration leads to reoxygenation and normalization of pH (realkalinization) in ischemic sites of the vascular bed. Effects of hypoxia/reoxygenation on activation and injury of cultured human endothelial cells were investigated in the present study. Cells were subjected to hypoxia (2% О<sub>2</sub>, 5% СО<sub>2</sub>, 93% N<sub>2</sub>) for 24 hours and subsequent reoxygenation (21% О<sub>2</sub>, 5% СО<sub>2</sub>, 74% N<sub>2</sub>) for 5 hours. Reoxygenation was performed at different pH of the culture medium - 6.4, (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete realkalinization respectively). Hypoxia only slightly (by ~30%) increased the amount of cell adhesion molecule ICAM-1 on the cell surface, while reoxygenation enhanced its expression more than twofold. The reoxygenation effect depended on the acidity of the medium - ICAM-1 increase was more pronounced at pH 7.0 than at pH 6.4 and 7.4. Hypoxia as well as reoxygenation did not induce expression of two other cell adhesion molecules - VCAM and E-selectin. Incubation of cells under hypoxic condition, but not subsequent reoxygenation, stimulated secretion of von Willebrand factor and increased its concentration in the culture medium more than fourfold. The percentage of cells containing a marker of apoptosis, activated caspase-3, was increased by ~50% by hypoxia as well as by hypoxia/reoxygenation. Maximal levels were achieved when reoxygenation was performed at pH 7.0. The data indicate that hypoxia/reoxygenation stimulates proinflammatory activation (ICAM-1 expression) and apoptosis (activation of caspase-3) of endothelial cells, and the pH of the culture medium affects both these processes.