Background-Coronary atherosclerotic disease remains the leading cause of death in the Western world. Although the exact sequence of events in this process is controversial, reactive oxygen and nitrogen species (RS) likely play an important role in vascular cell dysfunction and atherogenesis. Oxidative damage to the mitochondrial genome with resultant mitochondrial dysfunction is an important consequence of increased intracellular RS. Methods and Results-We examined the contribution of mitochondrial oxidant generation and DNA damage to the progression of atherosclerotic lesions in human arterial specimens and atherosclerosis-prone mice. Mitochondrial DNA damage not only correlated with the extent of atherosclerosis in human specimens and aortas from apolipoprotein E Ϫ/Ϫ mice but also preceded atherogenesis in young apolipoprotein E Ϫ/Ϫ mice. Apolipoprotein E Ϫ/Ϫ mice deficient in manganese superoxide dismutase, a mitochondrial antioxidant enzyme, exhibited early increases in mitochondrial DNA damage and a phenotype of accelerated atherogenesis at arterial branch points. Key Words: atherosclerosis Ⅲ muscle, smooth Ⅲ antioxidants R eactive species (RS) define a collective grouping of reactive oxygen and nitrogen species that can alter the biological functions of essential molecules such as lipids, proteins, and DNA. Numerous studies have linked excess RS generation with vascular lesion formation and functional defects. [1][2][3] This association has been reported for various RS models and species. 4 -6 A role for RS in atherogenesis is supported by epidemiological evidence of links between common risk factors for coronary artery disease and increased levels of RS. [7][8][9] Among the extensively studied intracellular systems capable of generating RS in vascular cells are the NADH/NADPH oxidase, xanthine oxidase, lipoxygenase, and cyclooxygenase systems. 6,10 -12 Mitochondria are biologically important sources and targets for RS. 13,14 However, their role as mediators of oxidative disease processes such as atherogenesis has not been examined. We recently reported that exposure of vascular cells to RS in vitro results in preferential mitochondrial DNA (mtDNA) damage and dysfunction and that mtDNA damage is a very sensitive marker for RS-mediated cellular effects. 15 In addition to the potential role of mtDNA damage as a marker of ambient oxidative stress, oxidative damage to the mitochondrion can lead to decreased oxidative energetic capacity (via impaired oxidative phosphorylation) and increased generation of intracellular RS. 15-17 Thus, we hypothesized that mitochondrial dysfunction accentuates atherosclerosis by modulating the phenotype of vascular cells and that measurements of mtDNA damage reflect RS-mediated atherosclerosis risk. Conclusions-MitochondrialUsing human aortic specimens and a murine model of early atherogenesis (the apolipoprotein E null, apoE Ϫ/Ϫ ), we examined the correlation between mtDNA damage and atherogenesis and sought to determine whether mtDNA damage is a cause or an effect in this pr...

The mechanisms by which reactive species (RS) participate in the development of atherosclerosis remain incompletely understood. The present study was designed to test the hypothesis that RS produced in the vascular environment cause mitochondrial damage and dysfunction in vitro and, thus, may contribute to the initiating events of atherogenesis. DNA damage was assessed in vascular cells exposed to superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite. In both vascular endothelial and smooth muscle cells, the mitochondrial DNA (mtDNA) was preferentially damaged relative to the transcriptionally inactive nuclear beta-globin gene. Similarly, a dose-dependent decrease in mtDNA-encoded mRNA transcripts was associated with RS treatment. Mitochondrial protein synthesis was also inhibited in a dose-dependent manner by ONOO(-), resulting in decreased cellular ATP levels and mitochondrial redox function. Overall, endothelial cells were more sensitive to RS-mediated damage than were smooth muscle cells. Together, these data link RS-mediated mtDNA damage, altered gene expression, and mitochondrial dysfunction in cell culture and reveal how RS may mediate vascular cell dysfunction in the setting of atherogenesis.

Background-A shared feature among cardiovascular disease risk factors is increased oxidative stress. Because mitochondria are susceptible to damage mediated by oxidative stress, we hypothesized that risk factors (secondhand smoke and hypercholesterolemia) are associated with increased mitochondrial damage in cardiovascular tissues. Methods and Results-Atherosclerotic lesion formation, mitochondrial DNA damage, protein nitration, and specific activities of mitochondrial proteins in cardiovascular tissues from age-matched C57 and apoE Ϫ/Ϫ mice exposed to filtered air or secondhand smoke were quantified. Both secondhand smoke and hypercholesterolemia were associated with significantly increased mitochondrial DNA damage and protein nitration. Tobacco smoke exposure also resulted in significantly decreased specific activities of mitochondrial enzymes. The combination of secondhand smoke and hypercholesterolemia resulted in increased atherosclerotic lesion formation and even greater levels of mitochondrial damage. Conclusions-These

Transforming growth factor (TGF)-beta 1, whose gene is located on mouse chromosome 7, has been proposed to be involved in skin carcinogenesis. In the study presented here, we demonstrated that single topical treatments with different types of tumor promoters, i.e., the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA, 2 micrograms); the non-protein kinase C activators anthralin (22.6 micrograms), benzoyl peroxide (20 mg), and cumene hydroperoxide (1.2 mg); the first-stage tumor promoters 4-O-methyl-TPA (500 micrograms) and A23187 (166 micrograms); and the second-stage tumor promoter mezerein (2 micrograms) produced transient induction of TGF-beta 1 mRNA in SSIN (inbred SENCAR) mouse skin. The time of maximum induction varied from 3 to 12 h; the relative extent of induction was ranked as cumene hydroperoxide > benzoyl peroxide > anthralin > TPA > 4-O-methyl-TPA > mezerein > A23187. These findings suggested that TGF-beta 1 mRNA induction is a common response of skin to several types of complete and stage-specific promoters; however, the extent of induction did not correlate with the reported hyperplastic activity of single applications of these promoters. We also demonstrated that TGF-beta 1 mRNA expression in papillomas of SENCAR mice generally correlated with expression levels of cyclin D1, another gene on chromosome 7, and with stage of tumor progression. TGF-beta 1 mRNA expression was constitutively elevated in most squamous cell carcinomas from either initiation-promotion or complete carcinogenesis protocols. Cell lines established from carcinomas also overexpressed TGF-beta 1 mRNA. Immunohistochemical staining of tissue sections of normal and TPA-treated skin revealed the presence of extracellular TGF-beta 1 protein in the dermis and intracellular TGF-beta 1 protein in the epidermis, especially in the suprabasal layers. The staining patterns of papillomas varied, with 62 +/- 13% of the tissue showing strong intracellular staining but only 25 +/- 8% of the connective tissue staining for extracellular TGF-beta 1. Variable staining patterns were also found in carcinomas; some areas stained heavily for both the intracellular and extracellular forms of TGF-beta 1. Overall, 28 +/- 6% of the tissue of the 12 analyzed carcinomas stained for the intracellular form and 18 +/- 5% for the extracellular form of TGF-beta 1.

The goal of this study was to compare the response of mouse epidermal keratinocytes (MEKs) and human epidermal keratinocytes (HEKs) to 12-O-tetradecanoylphorbol-13-acetate (TPA) with respect to the activation and downregulation of protein kinase C (PKC), the expression of c-jun and c-fos, and the expression and induction of ornithine decarboxylase (ODC) activity. Keratinocytes from adult CD-1 mice and from discarded adult human skin were grown in primary culture in a high-calcium serum-free medium that supported proliferation and differentiation. Immunoblotting of freshly isolated and cultured MEKs and HEKs for isozymes of protein kinase C revealed that fresh HEKs contained PKC alpha, PKC beta, and PKC delta; no PKC gamma, PKC epsilon, or PKC zeta were detected. In fresh MEKs, PKC alpha, PKC beta, PKC delta, and PKC zeta were observed, but not PKC gamma or PKC epsilon. After 2 wk in culture, the isozyme profiles of MEKs and HEKs were similar except that PKC gamma was noticeably present in HEK cultures. Activation of partially purified total PKC by TPA was similar in freshly isolated and cultured MEKs and HEKs, indicating that the two species were similar in this regard and that 2 wk of culture did not alter this characteristic. When MEK and HEK cultures were treated with TPA for 3 h, less than 30% of the control level of PKC activity was detected, indicating that TPA-induced downregulation of PKC was similar in MEKs and HEKs. After treatment with TPA, MEK cultures produced a large induction of both c-jun and c-fos mRNA by 60 min, as determined by northern blot analysis, and a large induction of ODC mRNA and enzyme activity by 6 h. TPA treatment of cultured HEKs, however, did not induce ODC activity; in fact, less activity, compared with that of control cultures, was observed. Northern blot analysis also revealed no increase in c-jun, c-fos, and ODC mRNA in HEKs. However, c-jun and c-fos mRNA and both ODC mRNA and enzyme activity were induced in HEKs fed growth factors after several days of deprivation. This suggests that the lack of ODC induction by TPA in HEKs is probably due to species differences in downstream steps in PKC signal transduction.