Activation and Damage of Endothelial Cells upon Hypoxia/Reoxygenation. Effect of Extracellular pH

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O. A. Antonova, S. A. Loktionova, Yu. A. Romanov, O. N. Shustova, M. V. Khachikian, and A. V. Mazurov^*

Russian Cardiology Research and Production Complex, 3-ya Cherepkovskaya ul. 15a, 121552 Moscow, Russia; fax: (495) 414-6735; E-mail: calab@cardio.ru

^* To whom correspondence should be addressed.

Received July 22, 2008; Revision received November 5, 2008

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Disturbances of blood flow upon vascular occlusions and spasms result in hypoxia and acidosis, while its subsequent restoration leads to reoxygenation and pH normalization (re-alkalization) in ischemic sites of the vascular bed. The effect of hypoxia/reoxygenation on activation and stimulation of apoptosis in cultured human endothelial cells was studied. The cells were subjected to hypoxia (2% O₂, 5% CO₂, 93% N₂) for 24 h followed by reoxygenation (21% O₂, 5% CO₂, 74% N₂) for 5 h. Reoxygenation was carried out at different pH—6.4 (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete re-alkalization, respectively). Hypoxia only slightly (by ~30%) increased the cell adhesion molecule ICAM-1 content on the cell surface, whereas reoxygenation more than doubled its expression. The reoxygenation effect depended on the medium acidity, and ICAM-1 increase was more pronounced at pH 7.0 compared to that at pH 6.4 and 7.4. Neither hypoxia nor reoxygenation induced expression of two other cell adhesion molecules, VCAM and E-selectin. Incubation of cells under hypoxic conditions but not reoxygenation stimulated secretion of von Willebrand factor and increased its concentration in the culture medium by more than 4 times. The percentage of cells containing apoptosis marker, activated caspase-3, was increased by approximately 1.5 times upon hypoxia as well as hypoxia/reoxygenation. Maximal values were achieved when reoxygenation was performed at pH 7.0. These data show that hypoxia/reoxygenation stimulate pro-inflammatory activation (ICAM-1 expression) and apoptosis (caspase-3 activation) of endothelial cells, and the extracellular pH influences both processes.

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KEY WORDS: endothelium, hypoxia, reoxygenation, apoptosis, cell adhesion molecules, von Willebrand factor, extracellular pH

DOI: 10.1134/S0006297909060030

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