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Hypoxia promotes oxidative base modifications in the pulmonary artery endothelial cell VEGF gene
Author(s) -
Grishko Valentina,
Solomon Marie,
Breit Jeffrey F.,
Killilea David W.,
LeDoux Susan P.,
Wilson Glenn L.,
Gillespie Mark N.
Publication year - 2001
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.00-0755fje
Subject(s) - hypoxia (environmental) , vegf receptors , oxidative phosphorylation , pulmonary artery , gene , microbiology and biotechnology , chemistry , cancer research , cardiology , medicine , biology , oxygen , biochemistry , organic chemistry
Hypoxia, a stimulus for angiogenesis and vascular remodeling, has been proposed to use reactive oxygen species as second messengers in signal transduction. This contention remains controversial, in part because of vagaries associated with fluorescence‐based methods of free‐radical detection. We took a different approach. Rat main pulmonary artery endothelial cells (PAECs) were cultured in hypoxia for up to 48 h, and, with dichlorofluorescein fluorescence to detect free‐radical production, we used quantitative Southern blot and ligation‐mediated PCR analyses to search for oxidative modifications in the mitochondrial genome and in the nuclear vascular endothelial cell growth factor (VEGF) gene. In accord with previous studies in other cell types, we found that acute hypoxic exposure promoted time‐dependent dichlorofluorescein fluorescence in PAECs. Quantitative Southern blot analysis showed that although hypoxia failed to alter mitochondrial DNA integrity, prominent oxidative lesions occurred in a 5.0‐kb sequence of the VEGF promoter. Using ligation‐mediated PCR to map the modifications at single nucleotide resolution, we found clusters of oxidized bases in a VEGF promoter sequence that included the AP‐1 and HIF‐1 response elements. These actions of hypoxia differed from exogenous xanthine oxidase, which obliterated the mitochondrial genome but failed to erode integrity of the VEGF promoter. Our observations indicate that hypoxia promotes an oxidant stress in main PAECs as detected by oxidative base modifications in the nuclear VEGF gene. The presence of hypoxia‐induced, oxidative base modifications in functionally significant sequences within the VEGF promoter suggests new concepts for mechanisms by which reactive oxygen species participate in hypoxic signal transduction.