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Hydrogen Peroxide Induces Up-Regulation of Fas in Human Endothelial Cells

Toshimitsu Suhara^*, Keisuke Fukuo^1,*, Tomosada Sugimoto, Shigeto Morimoto^*, Takeshi Nakahashi^*, Shigeki Hata^*, Masumi Shimizu^* and Toshio Ogihara^*

^* Department of Geriatric Medicine, Osaka University Medical School, Suita, Osaka, Japan; and 2nd Department of Oral Anatomy, Okayama University Dental School, Okayama, Japan

Hydrogen peroxide (H₂O₂), an oxidant generated by inflammatory cells, is an important mediator of injury of endothelial cells (ECs). Here we show that H₂O₂ induces up-regulation of the expression of Fas, a death signal, in human ECs in culture. Flow cytometric analysis with a mAb against human Fas showed that incubation for 24 h with H₂O₂ induced a dose-dependent increase in the level of Fas in ECs. Coincubation with catalase, which rapidly degrades H₂O₂, inhibited H₂O₂-induced up-regulation of Fas. H₂O₂ also induced a dose-dependent increase in Fas mRNA level. A significant increase in Fas mRNA levels was observed from 6 h after stimulation with H₂O₂. Vanadate, a protein phosphatase inhibitor, significantly enhanced Fas mRNA and protein levels in H₂O₂-treated ECs. On the other hand, genistein, a tyrosine kinase inhibitor, inhibited H₂O₂-induced Fas mRNA expression. Furthermore, a flow cytometric method with propidium iodide staining and electron microscopic analysis showed that incubation with an agonistic Ab against Fas (anti-Fas IgM) induced apoptosis in H₂O₂-treated cells. These findings suggest that H₂O₂ induces up-regulation of Fas in ECs and that activation of protein tyrosine kinase may be involved in the mechanism of H₂O₂-induced Fas expression. Therefore, Fas-mediated apoptosis may have a pathologic role in H₂O₂-induced EC injury and thereby provide a new therapeutic target.

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