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Thrombin induces oxidative mitochondrial (mt) DNA damage through a membrane NADPH oxidase in pulmonary microvascular endothelial cells
Author(s) -
Swiger Brad M.,
AlMehdi AbuBakr,
Gillespie Mark N.
Publication year - 2006
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/fasebj.20.4.a669-d
Subject(s) - mitochondrial ros , mitochondrial dna , reactive oxygen species , nadph oxidase , mitochondrion , oxidative stress , thrombin , oxidative phosphorylation , biology , microbiology and biotechnology , chemistry , biochemistry , gene , platelet , immunology
Recent evidence suggests that mtDNA mutations may play a role in the pathogenesis of atherosclerosis ( Mutat. Res ., 570: 80, 2005). Though oxidative damage to mtDNA is a precursor to mtDNA mutations, the source of oxidants leading to mtDNA damage in ECs is unclear. Thrombin and other inflammatory mediators incriminated in development of atherosclerosis are known to utilize reactive oxygen species (ROS) generated from a membrane‐bound NADPH oxidase as second messengers. In this study, we tested the hypothesis that thrombin‐induced ROS production causes mtDNA damage in pulmonary microvascular endothelial cells. Fluorescence microscopy with 10‐acetyl‐3,7‐dihydroxyphenoxazine (Amplex® Red) and 5‐(and‐6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate (CM‐H 2 DCFDA) showed that thrombin caused ROS production that was sensitive to diphenyliodonium (DPI), thus supporting involvement of NADPH oxidase. In addition, quantitative Southern blot analyses indicated that thrombin increased the equilibrium lesion density of oxidative lesions within the mitochondrial genome. Like the thrombin‐induced ROS production, the increase in mtDNA lesion density also was suppressed by DPI. These findings raise the possibility that ROS used as second messengers in inflammatory signaling damage mtDNA in ECs thus leading to formation of mtDNA mutations that contribute to atherogenesis. Supported by NIH