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Proteomics reveals lowering oxygen alters cytoskeletal and endoplasmatic stress proteins in human endothelial cells

A proteomic approach was applied to explore the signalling pathways elicited by lowering O2 in endothelial cells. Endothelial cells isolated from native umbilical cords were subjected to 21, 5, or 1% O2 for 24 h. 2‐D PAGE was performed and candidate proteins were identified using LC‐MS/MS. Lowering of O2 from 21 to 5% induced upregulation of cofilin‐1, cyclophilin A, tubulin and tubulin fragments, a fragment of glucose‐regulated protein 78 (Grp78) and calmodulin. The upregulation of Grp78 suggested that ER stress proteins were altered and indeed Grp94 and caspase 12 expression were increased in cells exposed to 5% O2. The presence of ER stress is also supported by findings of blunted caffeine‐evoked ER calcium release in cells exposed to 5 and 1% O2. Exposure to 1% O2 caused increases in cofilin‐1, cyclophilin A, and caspase 12 as well as a decrease of β‐actin, but it did not alter the expression of calmodulin, tubulin, Grp78, and Grp94. Incubation with CoCl2, a stabilizer of the hypoxia‐inducible factor, increased the expression of several of the proteins. The present investigations reveal that lowering O2, probably in part through hypoxia‐inducible factor, alter the expression of a series of proteins mainly involved in cytoskeletal changes (e.g. cofilin‐1, tubulin, and β‐actin) and in ER stress/apoptosis (e.g. Grp78/94, caspase 12, and cyclophilin A).