Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-14
2001-11-27
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024300, C536S023500
Reexamination Certificate
active
06322974
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of physiology and molecular biology. More specifically, the present invention relates to DNA damage and the effects of DNA damage on atherosclerosis.
2. Description of the Related Art
Reactive oxygen species (reactive oxygen species) have been suggested to play a critical role in the pathogenesis of atherosclerotic lesions (1-6), but the underlying mechanisms have not yet been elucidated. For example, reactive oxygen species-mediated mechanisms are likely to be a significant factor in the oxidation of LDL (ox-LDL), a key event in atherogenesis (3,7,8). Studies have shown that both superoxide (O
2
−
) and peroxynitrite (peroxynitrite; formed from O
2
−
+nitric oxide) are capable of oxidizing LDL(9-11). Hence, reactions involving nitric oxide and/or O
2
−
are believed to play a critical role in the pathogenesis of atherosclerotic lesions and impaired vascular function (i.e. endothelial cell dysfunction), with the actions of their oxidizing products (H
2
O
2
, peroxynitrite) not yet well defined.
The mitochondrion is a major source of cellular reactive oxygen species (O
2
−
), which are formed during electron transport (12-16). These reactive oxygen species are capable of preferentially damaging the mitochondrial membranes and proteins (17-19), affecting key cell functions, including mitochondrial respiration, which, if altered, leads to increased reactive oxygen species production (20-22), mediating lipid peroxidation (23, 24) and DNA damage (25, 26). Because mitochondrial oxidative phosphorylation (OXPHOS) capacities decline as mitochondrial DNA (mtDNA) damage and mutations accumulate with age (6, 27-29), mitochondrial damage and reactive oxygen species generation may act as catalysts for age-related degenerative disease, such as coronary artery disease (CAD). It was hypothesized that free radicals generated within the endothelial and smooth muscle cell environment mediate mitochondrial damage within these cells, establishing a vicious cycle of further reactive oxygen species generation and mitochondrial damage leading to vascular cell dysfunction.
Coronary atherosclerotic heart disease is the leading cause of death in the Western world. Although there is considerable controversy about the exact sequence of events leading to coronary atherosclerotic heart, there is growing evidence that atherosclerotic lesions result from factors mediated by reactive oxygen species. Macrophages recognize and internalize ox-LDL via “scavenger” receptors, becoming foam cells. Accumulation of these foam cells is associated with long-term changes in vascular physiology, including smooth muscle cell migration and proliferation, synthesis of extracellular matrix proteins, and further endothelial cell dysfunction, all core components of atherosclerotic plaques. Similarly, many of the risk factors for coronary atherosclerotic heart are related to increased reactive oxygen species production (i.e. smoking and hypercholestermia). Within the artery, reactive oxygen species can be induced by metabolic processes (mitochondrial oxidative phosphorylation), cytokine or growth factor activation, macrophage or neutrophil stimulation (inflammatory response), and the reaction of nitric oxide with superoxide to yield peroxynitrite, which in turn, generates singlet oxygen and hydroxyl radicals. Hence, while there are a variety of processes that are important for atherogenesis, reactive oxygen species-mediated mechanisms and their effects are among the most significant.
Numerous studies have implicated the mitochondria as a vulnerable target for reactive oxygen species. The association of the mitochondrial DNA with the matrix side of the inner membrane make it susceptible to membrane disturbances, and a potential target for electrophiles generated in the membrane. Aside from its close association with the inner membrane and OXPHOS, additional factors which make the mitochondrial DNA sensitive to damage are the lack of protective histone and non-histone proteins, and its limited DNA repair capacity. Previous studies have shown that the mitochondria are susceptible to reactive oxygen species mediated damage, manifested in extensive lipid peroxidation and mitochondrial DNA damage. Specifically, it has been shown that reactive oxygen species treatment of endothelial cells results in preferential mitochondrial DNA damage, decreased mitochondrial DNA transcripts, and mitochondrial OXPHOS dysfunction.
The prior art is deficient in methods of measuring oxidative stress that contributes to atherogenesis. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention demonstrates that reactive oxygen species mediates mitochondrial damage and dysfunction in human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMC) in vitro. DNA damage, gene expression, and mitochondrial protein synthesis were assessed in cells treated with H
2
O
2
, peroxynitrite , and O
2
−
. The mitochondrial DNA in both cell types was more susceptible to acute doses of reactive oxygen species relative to the nuclear DNA, and was associated with a decrease (40%-60%) in mitochondrial encoded polypeptide OXPHOS transcripts (ND2 and Cytochrome b). Mitochondrial protein synthesis was inhibited with peroxynitrite treatment and reactive oxygen species-exposed cells also had significantly decreased ATP levels and mitochondrial respiration (complex II), consistent with the notion that reactive oxygen species impair mitochondrial function. The present invention reveals a link between oxidative mitochondrial DNA damage, altered gene expression, and mitochondrial dysfunction in vitro, thus, demonstrating that oxidative cell injury and mitochondrial damage play a role in vascular dysfunction and atherogenesis.
By virtue of the notion that the mitochondrial DNA is more susceptible to reactive oxygen species-mediated damage, and because increased oxidative stress is believed to play a role in the early events of atherogenesis, the present invention demonstrates that the mitochondrial DNA in aortic tissues destined to become atherosclerotic has increased damage. For this, the levels of mitochondrial DNA damage sustained in aortic tissues from a hypercholestermic mouse model for atherosclerosis (the apolipoprotein E null mouse) was compared to healthy, age-matched control mice. Assessment of DNA damage found that the aortic tissues from the apoE mice had significantly increased mitochondrial DNA damage before and after the development of pathologically detectable lesions (relative to healthy controls). Additionally, mitochondrial DNA damage increased with age in all mice, however, only the apoE mice had significantly increased (p<0.05) levels of damage associated with age. By contrast, diet correlated with the level of mitochondrial DNA damage in only the 10 week old c57B1mice, with the western diet associated with increased damage. Finally, it was found that decreased dietary protein was significantly (P<0.05) related with decreased mitochondrial DNA damage in the aortas from both apoE and control mice. There were no clear patterns of damage associated with the &bgr;-globin locus, a marker of nuclear DNA damage. Histochemical analysis of aortas from each group revealed the presence of atherosclerotic lesions in only the aged apoE mice on chow (4% fat) or western (21% fat) diets. As expected, lipid peroxides and cholesterol levels were significantly increased in the apoE mice relative to age-matched c57B1 controls (p<0.05). However, lipid peroxide levels did not increase significantly in the apoE on the higher fat western diet compared to the chow diet. Consequently, these data suggest: 1) that mitochondrial DNA damage occurs prior to, or simultaneous with, atherosclerotic lesion development in a mouse model of atherosclerosis in vivo; 2) aortic mitochondrial DNA damage increases with age in vivo, 3) the apoE genotype confers a gr
Ballinger Scott W.
Runge Marschall S.
VanHouten Bennett
Adler Benjamin Aaron
Houtteman Scott W.
Research Development Foundation
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