Elevated Levels of Oxidative DNA Damage and DNA Repair Enzymes in Human Atherosclerotic Plaques Wim Martinet, PhD; Michiel W.M. Knaapen, PhD; Guido R.Y. De Meyer, PharmD, PhD; Arnold G. Herman, MD, PhD; Mark M. Kockx, MD, PhD Background—The formation of reactive oxygen species is a critical event in atherosclerosis because it promotes cell proliferation, hypertrophy, growth arrest, and/or apoptosis and oxidation of LDL. In the present study, we investigated whether reactive oxygen species–induced oxidative damage to DNA occurs in human atherosclerotic plaques and whether this is accompanied by the upregulation of DNA repair mechanisms. Methods and Results—We observed increased immunoreactivity against the oxidative DNA damage marker 7,8-dihydro- 8-oxo-2'-deoxyguanosine (8-oxo-dG) in plaques of the carotid artery compared with the adjacent inner media and nonatherosclerotic mammary arteries. Strong 8-oxo-dG immunoreactivity was found in all cell types of the plaque including macrophages, smooth muscle cells, and endothelial cells. As shown by competitive ELISA, carotid plaques contained 16029 8-oxo-dG residues/10 5 dG versus 31 8-oxo-dG residues/10 5 dG in mammary arteries. Single-cell gel electrophoresis showed elevated levels of DNA strand breaks in the plaque. The overall number of apoptotic nuclei was low (1% to 2%) and did not correlate with the amount of 8-oxo-dG immunoreactive cells (90%). This suggests that initial damage to DNA occurs at a sublethal level. Several DNA repair systems that are involved in base excision repair (redox factor/AP endonuclease [Ref 1] and poly(ADP-ribose) polymerase 1 [PARP-1]) or nonspecific repair pathways (p53, DNA-dependent protein kinase) were upregulated, as shown by Western blotting and immunohisto- chemistry. Overexpression of DNA repair enzymes was associated with elevated levels of proliferating cell nuclear antigen. Conclusions—Our findings provide evidence that oxidative DNA damage and repair increase significantly in human atherosclerotic plaques. (Circulation. 2002;106:927-932.) Key Words: atherosclerosis  oxidative stress  apoptosis N umerous studies have linked excess generation of reac- tive oxygen species (ROS) with cellular damage and atherogenesis. 1,2 Although this notion is widely held, thor- ough factual evidence is lacking. ROS have been implicated in a variety of distinct cellular processes, including initiation of gene expression and promotion of cell proliferation, hypertrophy, growth arrest, and/or apoptosis. 1,2 On the other hand, ROS are involved in oxidation of LDL, which is considered a fundamental step in the initiation and progres- sion of atherosclerosis. It is also tempting to speculate that ROS may have some deleterious effects on DNA. Indeed, ROS can provoke extensive oxidative DNA damage, DNA strand breaks, and chromosomal aberrations. 3 Significant damage to DNA resulting from endogenous free radical attack has already been suggested to contribute to the pathol- ogy of cancer 4 and several neurodegenerative diseases. 5,6 A growing body of evidence indicates that oxidative DNA damage is also a prominent feature of atherosclerotic plaques. 7–9 In light of this, we have recently described elevated levels of oxidative DNA damage and repair in the thoracic aorta of cholesterol-fed rabbits. 10 The aim of the present study was to investigate whether oxidative DNA damage occurs in human atherosclerotic plaques and whether DNA repair mechanisms are upregulated in response to DNA damage. Methods Carotid Endarterectomy Specimens Human carotid endarterectomy specimens (n=13) were obtained from patients with a carotid stenosis 70%, as demonstrated by digital subtraction angiography and duplex ultrasonography. The specimens were opened along their longitudinal axis. Half of the specimen was fixed in 4% formalin within 2 minutes after surgical removal. The other half was gently pressed against an agarose-coated slide to examine DNA strand breaks by the alkaline single-cell gel electrophoresis assay (see below). Tissue was frozen in liquid nitrogen to be used as cryosections for RNA/DNA extraction and for Western blotting. Complete longitudinal sections of formalin-fixed, paraffin-embedded specimens contained the inner wall of the distal Received March 28, 2002; revision received May 31, 2002; accepted May 31, 2002. From the Division of Pharmacology, University of Antwerp, Wilrijk, Belgium (W.M., G.R.Y.D.M., A.G.H., M.M.K.); HistoGeneX, Edegem, Belgium (M.W.M.K.); and the Cardiovascular Translational Research Institute Middelheim Antwerp (CATRIMA), Antwerp, Belgium (M.M.K.). Correspondence to Dr Mark M. Kockx, Department of Pathology, AZ Middelheim, Lindendreef 1, B-2020 Antwerp, Belgium. E-mail mark.kockx@uia.ua.ac.be © 2002 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org DOI: 10.1161/01.CIR.0000026393.47805.21 927 Downloaded from http://ahajournals.org by on June 2, 2020