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Mitochondrial DNA damage in vascular endothelial cells exposed to shear stress
Author(s) -
Galbraith Valerie K,
Giedt Randy J,
Panduri Vijji,
Van Houten Bennett,
Alevriadou B Rita
Publication year - 2009
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.23.1_supplement.638.1
Subject(s) - peroxynitrite , dna damage , mitochondrial dna , microbiology and biotechnology , oxidative stress , dna , reactive oxygen species , chemistry , nuclear dna , superoxide , biophysics , biology , biochemistry , gene , enzyme
Superoxide radicals (O 2 = ), hydrogen peroxide (H 2 O 2 ) and other reactive oxygen species (ROS) produced by endothelial cells (ECs) play an important role in cell (dys)function. We recently showed that, during cultured EC exposure to shear stress, nitric oxide (NO), produced by nitric oxide synthase, diffuses to the mitochondria and reacts with O 2 − , produced by the electron transport chain (ETC), resulting in peroxynitrite (ONOO − ) formation and inhibition of the ETC complex activities. EC incubation with H 2 O 2 or ONOO − is known to induce mitochondrial DNA (mtDNA) damage. Hence, we hypothesized that the endogenous shear‐induced ROS may cause mtDNA damage. DNA damage was assessed by a quantitative PCR assay that relies on the principle that oxidative DNA lesions inhibit DNA polymerases. DNA damage was quantified by comparing the relative efficiency of amplification of large DNA fragments (12.2 kb for DNA polymerase β gene; 8.9 kb for mtDNA) and normalizing it to the amplification of smaller fragments. EC exposure to shear stress (10 dynes/cm 2 ) for 1 h caused a 2‐fold increase in mtDNA damage (1.1±0.4 lesions/10 kb) compared to nuclear. At 6 h, there was a complete loss of nuclear DNA damage, but mtDNA damage persisted. Shear followed by incubation with H 2 O 2 had an additive effect on mtDNA damage. Our goal is to examine the time/dose dependency of mtDNA damage on shear stress, and reveal how the DNA damage affects cell function. Funding by NIH HL91417.