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Age-dependent accumulation of mitochondrial DNA deletions in the aortic root of atherosclerosis-prone apolipoprotein E-knockout mice Feng Tiana , Junan Lib , Xin-wen Liua , Tan-jun Tonga , Zong-yu Zhanga,* a b
Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Peking University Research Center on Aging, Beijing, China College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 July 2015 Received in revised form 2 November 2015 Accepted 3 November 2015 Available online xxx
Purpose: To investigate whether mitochondrial DNA (mtDNA) damage, specifically deletion, contributes to the development of atherosclerosis or is simply a secondary effect of the primary factors causing atherosclerosis. Materials and methods: mtDNA deletion was detected by PCR in the aortic root of atherosclerosis-prone C57BL/6J apolipoprotein (Apo) E gene deficient (/) mice and control C57BL/6J mice at different ages. Atherosclerotic plaques in the Apo E/ mice were assessed using frozen sections of the aortic root. The protein levels of COX III and 8-oxoguanine glycosylase (OGG1) were determined. Results: while mtDNA deletions accumulated significantly in mice as young as 2- month-old, atherosclerotic plaques were not detected until mice were 6 months old or older, suggesting that mtDNA deletion occurs prior to the formation of atherosclerotic plaques in the aortic root of these mice. Moreover, the expression levels of mtDNA-encoded COX III protein in both 2-month-old and 16-monthold C57BL/6J ApoE/ mice were significantly lower than those in C57BL/6J mice (p < 0.05). Additionally, the protein level of 8-oxoguanine glycosylase (OGG1), a mitochondrial enzyme that functions in DNA excision repair, decreased with age in these mice, indicating that age-related down-regulation of mtDNA excision repair also contributes to atherosclerosis in C57BL/6J ApoE/ mice. Conclusion: These results reveal that mtDNA deletions occur during the early “initiation” stage of atherosclerosis in C57BL/6J ApoE/ mice and have the potential to promote atherosclerosis. ã 2015 Published by Elsevier Ireland Ltd.
Keywords: Mitochondrial DNA deletion C57BL/6J ApoE/ mice Atherosclerosis
1. Introduction Numerous studies have demonstrated that mitochondrial DNA (mtDNA) damage accumulates with aging and such accumulation is involved in organism senescence and the development of cardiovascular diseases such as atherosclerosis and myocardial infarction (Greaves & Turnbull, 2009; Greaves, Reeve, Taylor, & Turnbull, 2012; Lee & Wei, 2012; Phillips, Simpkius, & Roby, 2014; Yu, Mercer, & Bennett, 2012). Due to the lack of protective histones and its close proximity to the electron transfer chain in the inner mitochondrial membrane, mtDNA is particularly vulnerable to the damaging effects of reactive oxygen species (ROS), which are generated during oxidative phosphorylation in mitochondria and potently induce mutations (single- and double-stranded breaks, DSBs) and deletions in mtDNA as well as chromosomal DNA (Mercer et al., 2010; Mikhed, Daiber, & Steven, 2015). Interestingly,
* Corresponding author. Fax: +86 10 8280 2931. E-mail address: [email protected] (Z.-y. Zhang).
mtDNA encodes a number of key components of mitochondrial respiratory chains, such as cytochrome oxidases I, II, and III (COX I– III) (Sugie & Nishino, 2002) and mtDNA damage may even downregulate or up-regulate these genes and disrupt oxidative phosphorylation, leading to further increased ROS production and mtDNA damage (Mikhed et al., 2015). Arguably, a positive feedback mechanism exists between mtDNA damage and diseases associated with increased ROS production. From this perspective, mtDNA damage could contribute to some cardiovascular diseases as an “initiating” and/or “promoting” factor. mtDNA damage, especially mtDNA deletion, is frequently observed in human atherosclerosis in both circulating and vessel wall cells and is associated with mitochondrial dysfunction (Fishbein & Fishbein, 2015; Madamanchi & Runge, 2007; Shah & Mahoudi, 2015; Sobenin et al., 2015). However, it remains unknown whether DNA damage is directly involved in the development of atherosclerosis or is a secondary consequence of atherosclerosis risk factors, such as smoking, hypercholesterolemia, diabetes, and hypertension, all of which bring about increased ROS production in mitochondria.
http://dx.doi.org/10.1016/j.archger.2015.11.004 0167-4943/ ã 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: F. Tian, et al., Age-dependent accumulation of mitochondrial DNA deletions in the aortic root of atherosclerosis-prone apolipoprotein E-knockout mice, Arch. Gerontol. Geriatr. (2015), http://dx.doi.org/10.1016/j.archger.2015.11.004
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Apolipoprotein (Apo) E is an essential ligand for the uptake and clearance of atherogenic lipoproteins, such as very low density lipoproteins (VLDLs) and chylomicrons. It is known that mice is a species normally resistant to the development of atherosclerosis. However, when mice with inactivated Apo E gene are given diets that are normal, high in fat, or rich in cholesterol, the plasma cholesterol concentration in these mice significantly increases, which leads to rapid and severe atherosclerosis. The pathophysiology of atherosclerosis in Apo E-deficient mice (ApoE/) parallels that in humans with respect to vascular distribution, cellular components, and overall lipid content of the lesions (Zhang, Reddick, Piedrahita, & Maeda, 1992). Therefore, ApoE/ mice are regarded as one of the best models for studying the development of atherosclerosis (Fetterman et al., 2013; Weigartner et al., 2015; Yi, Xu, Kim, Kim, & Maeda, 2010). In the present study, we investigated the involvement of mtDNA deletion in the development of atherosclerosis using atherosclerosis-prone C57BL/6J ApoE/ mice.
COX III gene. The polymerase chain reaction (PCR) mixture contained 5 ml 10 PCR buffer, 4 ml dNTPs (10 mM each), 1 ml mtDNA-f1, 1 ml mtDNA-f2, 1 ml mtDNA-r (54 mM), 2–3 units Taq DNA polymerase (Huamei Biotech, Beijing, China) and 1 ml mtDNA template (0.1 mg) in a total volume of 50 ml. The reaction mixtures were incubated for 5 min at 93 C, followed by 30 cycles of 30 s at 93 C, 1 min at 55 C, and 1 min at 72 C. After amplification, 10 ml of each PCR mixture was loaded onto a 1% agarose gel for electrophoretic separation, and mtDNA bands were visualized and analyzed with an Image Master VDS System (Amersham Pharmarcia, Piscataway, NJ, USA). Both target bands were recovered from the gel using a QIAquick Gel Extraction Kit (Qiagen, Valenica, CA, USA) and subjected to direct sequencing on an ABI377A DNA automated sequencer (Applied Biosystems Inc., Foster City, CA, USA). The PCR amplification product was identified by Hind III digestion (Huamei Biotech) and then loaded onto a 1% agarose gel for separation. 2.4. Protein extraction from aortic roots
2. Materials and methods 2.1. ApoE/ mice The apolipoprotein E gene-deficient mouse (ApoE/) is an excellent animal model for atherosclerosis research because it exhibits severe hyperlipidemia and a plaque distribution pattern similar to that of human atherosclerosis. ApoE/ mice were first generated at Rockefeller University and the University of North Carolina in 1992 (Zhang et al., 1992) and were introduced to the Peking University Health Science Center in 1997. Three groups of C57BL/6J ApoE/ mice (n = 18) and C57BL/6J mice (n = 18), including 2-month-old (2m), 6-month-old (6m) and 16-monthold (16m) mice, were housed in the Department of Laboratory Animal Science, Peking University Health Science Center under clean conditions at 22 2 C with 55 5% humidity and were maintained on a commercial diet with tap water ad libitum. Lights were on from 7:00 AM to 7:00 PM. Both male and female mice were selected randomly for the experiments. All research protocols were approved by the Animal Welfare Committee of Peking University Health Science Center (LA2013-18, approved in February 25, 2013). After decapitation of the animals at the indicated times, aortic roots were removed immediately for subsequent analyses or frozen in liquid nitrogen for future studies. 2.2. Histological analysis Aortic roots from ApoE/ mice were rapidly frozen in liquid nitrogen, and frozen sections were prepared as previously described (Zhang et al., 2004). The sections were stained with Oil Red O to visualize the formation of atherosclerotic plaques. 2.3. mtDNA isolation and identification of deletion mtDNA was extracted from the aortic root samples as previously described (Zeng et al., 1999). The following primers were used for mtDNA amplification: mtDNA-f1 (forward primer sequence 1), 50 -AGTATCATGCTGCGGCTTCA-30 ; mtDNA-f2 (forward primer sequence 2), 50 -GCAAAGATGCTTCCGAATGC-30 ; and mtDNA-r (reverse primer sequence), 50 -TACCAAGGCCACCACACTCC-30 . MtDNA-f1 and mtDNA-r were used to amplify mtDNA without deletions, and the amplicon was 526 bp. MtDNA-f2 and mtDNA-r were used to amplify mtDNA with deletions, and the amplicon was 455 bp (3867-bp deletion). Of note, this 3867-bp DNA fragment proposed to be deleted during aging contains the
After one wash with ice-cold phosphate-buffered saline (PBS), the aortic roots were minced in liquid nitrogen and then transferred into a plastic homogenizer containing protein extraction buffer (10 mM Tris–HCl, 150 mM NaCl, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 0.2% NP-40, and 1 mg/ml Aprotinin, pH 8.0). After homogenization, the tissue samples were centrifuged at 3,000 rpm for 5 min. Subsequently, the supernatant was transferred to a new Eppendorf tube, and the DNA in the supernatant was fragmented by four sonication cycles of 4 s each with a 20-s interval between each cycle. Finally, the samples were centrifuged at 10,000 g, 4 C for 10 min, and the supernatants were kept at 20 C for further studies. 2.5. Western blot Equal amounts (50 mg) of proteins were separated on 12% SDSPAGE gels and then transferred to a nitrocellulose membrane using a Trans-Blot1 SD system (Bio-Rad, Hercules, CA, USA). The nitrocellulose membrane was washed three times using TBST buffer (20 mM Tris–HCl, 150 mM NaCl, 0.5% Tween-20, pH 7.5) and subsequently blocked with TBST containing 5% nonfat dry milk at 4 C for 1 h. Upon incubation with rabbit anti-OGG1 polyclonal antibody (ADI, San Antonio, TX, USA) (1:2,000 dilution), mouse anti-COX III polyclonal antibody (molecular targeting) (1:2,000 dilution), or goat anti-b-actin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:2,000 dilution) at 4 C overnight, the membrane was washed five times with TBST and probed with a horseradish peroxidase (HRP)-conjugated goat antirabbit immunoglobulin G (IgG), goat anti-mouse IgG, or rabbit anti-goat IgG (ZhongShan Golden Bridge, Beijing, China) (1:5,000 dilution). Immunoblots were visualized by adding SuperSignal West Pico Chemiluminescent Substrates (Pierce, Rockford, IL, USA), and digitized images of the blots were analyzed with the ImageMaster VDS software (Amersham Pharmarcia). Mouse b-actin was used as the reference for normalization. 2.6. Statistical analyses All data in this study were statistically analyzed using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to evaluate age-related changes in protein levels of COX III, and OGG1 in C57BL/6J ApoE/ mice and C57BL/ 6J mice, and the differences in these parameters between the two types of mice were subjected to two-way ANOVA. Values of p < 0.05 were considered to indicate significance.
Please cite this article in press as: F. Tian, et al., Age-dependent accumulation of mitochondrial DNA deletions in the aortic root of atherosclerosis-prone apolipoprotein E-knockout mice, Arch. Gerontol. Geriatr. (2015), http://dx.doi.org/10.1016/j.archger.2015.11.004
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3. Results 3.1. Atherosclerosis plaques are formed in 6-month and older C57BL/ 6J-ApoE/ mice The formation of atherosclerotic plaques is a well-known pathological/histological marker of aging. Hence, we first investigated the formation of atherosclerotic plaques in the aortic roots of C57BL/6J ApoE/ mice using histological staining. As shown in Fig. 1, distinct atherosclerotic plaques were observed in the aortic roots of 6-month-old and 16-month-old C57BL/6J ApoE/ mice (data not shown), but not in the aortic roots of 2-month-old C57BL/ 6J ApoE/ mice (data not shown). In comparison, atherosclerotic plaques were not detected in the corresponding tissues of C57BL/6J mice at all tested ages (2, 6, and 16 months). Apparently, aging in C57BL/6J ApoE/ mice occurred much earlier than that in C57BL/ 6J mice (6 months vs>16 months). Since both C57BL/6J ApoE/ and C57BL/6J mice were fed a normal diet, our results indicate that even on a normal diet, C57BL/6J Apo E/ mice exhibits accelerated aging in comparison with their C57BL/6J counterparts. 3.2. mtDNA deletion occurs in 2-month C57BL/6J-ApoE/ mice and the extent of such deletion increases with age Because mtDNA damage, a molecular event occurring during the early stage of aging, is frequently observed in atherosclerosis, we investigated mtDNA deletion, the major type of mitochondrial DNA damage, in C57BL/6J ApoE/ mice using well-established PCR assays (Tian, Tong, Zhang, McNutt, & Liu, 2009). In these assays, an amplicon of 526 bp was generated from intact mtDNA, whereas a truncated mtDNA template (with a 3867-bp mtDNA deletion) led to an amplicon of 455 bp. Furthermore, the 526-bp amplicon from intact mtDNA harbors a Hind III restriction site, which leads to fragments of 310 bp and 190 bp, respectively, upon Hind III digestion. Contrary to this, this Hind III site is absent in the 455-bp amplicon. Hence, the status of mtDNA can be determined from different Hind III-digestion patterns of the amplicons. As shown in Fig. 2A, upon Hind III digestion, 455-bp products were present in the PCR mixtures containing template DNA from aortic roots of 2-month-old C57BL/6J ApoE/ mice, even though the major products were 310 bp and 190 bp. These results indicate that mtDNA deletion occurred in 2-month-old C57BL/6J ApoE/ mice to certain extent. Notably, more 455-bp products were detected in PCR mixtures containing template DNA from the aortic roots of 6and 16-month-old C57BL/6J ApoE/ mice, indicating that the extent of mtDNA deletion increases with aging in C57BL/6J ApoE/ mice. In contrast, 455-bp products were only detected in PCR mixtures containing template DNA from the aortic roots of 16-
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month-old C57BL/6J mice and not template DNA from 2-monthold C57BL/6J mice (Fig. 2B). These results are consistent with our previous findings that the formation of atherosclerotic plaques occurs earlier in C57BL/6J ApoE/ mice than in C57BL/6J mice. 3.3. The mRNA and protein levels of COX I, not COX III, in the C57BL/6JApoE/ mice increase with aging COX III is a key component of the mitochondrial respiratory chain encoded by mtDNA. Interestingly, the COX III gene is located in the 3867-bp fragment of mtDNA frequently deleted during aging in mice including C57BL/6J mice. Deletion of this COX IIIcontaining fragment of mtDNA was observed in aortic roots of C57BL/6J ApoE/ mice as young as 2-month-old (Fig. 2). Therefore, it is rational to evaluate the impact of accelerated aging in C57BL/6J ApoE/ mice on the expression of COX III protein. As shown in Fig. 3, the expression levels of COX III protein in both 2-month-old and 16-month-old C57BL/6J ApoE/ mice were significantly lower than their corresponding values in C57BL/ 6J mice at the same ages (p < 0.05), indicating that accelerated aging in Apo E-knockout mice significantly down-regulates the expression of COX III even in mice as young as 2-month-old. Presumably, such down-regulation of COX III could impair the oxidative phosphorylation process in mitochondria thus promoting aging. Notably, the expression levels of COX III protein in both 2month-old and 16-month-old C57BL/6J ApoE/ mice were comparable (p > 0.05). Apparently, this finding is not fully consistent with previous observations showing that mtDNA deletion accumulates during aging (Tian et al., 2009). While the molecular mechanisms underlying such “inconsistency” remain to be elucidated, it is worthwhile to note that changes at the protein level may not always or sufficiently represent the changes at the DNA level. For example, upon aging, the transcription of COX III might be enhanced through unknown feedback mechanisms, which may partially “compensate” the loss of COX III gene and lead to compromised impact on COX III protein. 3.4. The levels of OGG1 mRNA and protein in C57BL/6J-ApoE/ mice significantly decrease with aging It is well known that oxidative damage of mtDNA is monitored by the mitochondrial anti-oxidation systems and its DNA repair systems (Gredilla, Bohr, & Stevnsner, 2010; Chen et al., 2011), in which OGG1 plays a critical role. 8-oxyoguanine is the most common base modification occurring in oxidative damage of mtDNA, and OGG1 specifically recognizes and excises such modification for DNA repair (Lyama & Wilson, 2013). Previous
Fig. 1. Atherosclerotic plaques in the aortic root of C57BL/6J ApoE/ and C57BL/6J mice. (A) Oil Red O staining of the aortic root of a 6-month-old C57BL/6J ApoE/ mouse (100). (B) Oil Red O staining of the aortic root of a 6-month-old C57BL/6J mouse (100).
Please cite this article in press as: F. Tian, et al., Age-dependent accumulation of mitochondrial DNA deletions in the aortic root of atherosclerosis-prone apolipoprotein E-knockout mice, Arch. Gerontol. Geriatr. (2015), http://dx.doi.org/10.1016/j.archger.2015.11.004
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Fig. 2. Accumulation of mtDNA deletion in C57BL/6J ApoE/ mice. mtDNA deletions were evaluated using PCR and Hind III fragmentation as previously described (Tian et al., 2009). (A) mtDNA deletion in C57BL/6J ApoE/ mice of different ages. (B) mtDNA deletion in C57BL/6J mice of different ages. M1, 50-bp DNA ladder.
studies in our laboratory as well as by other groups have demonstrated that down-regulation of OGG1, especially mitochondrial OGG1, is strongly associated with aging-induced mtDNA damage in SAM-P/8 mice, a species with accelerated senescence (Tian et al., 2009). To further explore the mechanism(s) underlying accumulated mtDNA deletion in C57BL/6J ApoE/ mice, we evaluated the protein levels of OGG1 in these mice. OGG1 protein levels significantly decreased between 2 and 16 months in C57BL/ 6J ApoE/ mice (p < 0.05), and the OGG1 protein levels in C57BL/ 6J ApoE/ mice were significantly lower than those in C57BL/6J mice of the same age (p < 0.05; Fig. 4). Therefore, the expression of OGG1 is negatively associated with aging in C57BL/6J ApoE/ mice. Although the level of OGG1 protein evaluated in this study is that for “total” OGG1 protein, which includes cytoplasmic, nuclear, and mitochondrial OGG1, our previous studies showed that aginginduced down-regulation of OGG1 in SAM-P/8 mice has the greatest impact on mitochondrial OGG1 expression (Tian et al., 2009). Hence, it is likely that mitochondrial OGG1 decreases with age in the aortic root of C57BL/6J ApoE/ mice.
4. Discussion Atherosclerosis is characterized by the development of a fibrofatty lesion in the artery wall involving a variety of different cell types, such as vascular smooth muscle cells (VSMC), monocyte-derived macrophages, lymphocytes, dendritic cells and platelets (Gray & Bennett, 2011; Yu & Bennett, 2014; Sobenin et al., 2015). In the past decade, increasing evidence has shown that damages in both genomic and mitochondrial DNAs occur prevalently in VSMCs and inflammatory cells in atherosclerotic plaques, suggesting that there could be a causative association between DNA damage and atherosclerosis (Gray & Bennett, 2011; Sobenin et al., 2015). However, the role(s) of DNA damage, especially mtDNA damage in the initiation and progression of atherosclerosis remains highly debated. It is still unknown whether mtDNA damage is simply a by-product of those atherosclerosis-promoting risk factors or mtDNA damage itself promotes atherosclerosis (Gray & Bennett, 2011). Apparently, our results support the involvement of mtDNA deletion in promoting atherosclerosis. As shown in Fig. 1, the formation of atherosclerotic
Fig. 3. Changes in COX III expression in the aortic root of C57BL/6J ApoE/ mice of different ages. Total proteins from the aortic roots of C57BL/6J ApoE/ and C57BL/6J mice were separated by SDS-PAGE and then blotted against anti-COX III antibody. b-actin was used as an internal control. (A) COX III expression in C57BL/6J ApoE/ and C57BL/6J mice of different ages. (B) b-actin expression in C57BL/6J ApoE/ and C57BL/6J mice of different ages; (C) densitometric scanning analysis of the the COX III/b-actin intensity ratio. #, p < 0.05, 2- and 16-month-old C57BL/6J ApoE/ mice vs 2- and 16-month-old C57BL/6J mice, respectively.
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Fig. 4. Changes in the expression of OGG1 protein in the aortic root of C57BL/6J ApoE/ mice of different ages. Total proteins from the aortic roots of C57BL/6J ApoE/ and C57BL/6J mice were subjected to immunoblotting against anti-OGG1 antibody. b-actin was used as an internal control. (A) OGG1 expression in C57BL/6J ApoE/ and C57BL/ 6J mice of different ages; (B) b-actin expression in C57BL/6J ApoE/ and C57BL/6J mice of different ages; (C) densitometric scanning analysis of the OGG1/b-actin intensity ratio. *, p < 0.05, 16-month-old C57BL/6J ApoE/ mice vs2-month-old C57BL/6J ApoE/ mice; #,p < 0.05, 2- and 16-month-old C57BL/6J ApoE/ mice vs 2- and 16month-old C57BL/6J mice, respectively.
plaques was observed only in 6- and 16-month-old, but not 2month-old, C57BL/6J ApoE/ mice, implying that mtDNA deletion, a molecular event, occurs earlier in the aging process than the formation of atherosclerotic plaques, a histological event (2 vs 6 months). This notion is further supported by the fact that only mtDNA deletion, not the formation of atherosclerotic plaques, was observed in the aortic roots of 16-month-old C57BL/6J mice. Our results are consistent with recent studies demonstrating that mtDNA deletion could be an early event in atherosclerosis. It has been demonstrated recently that DNA damage links mitochondrial dysfunction to atherosclerosis and metabolic syndrome in ATM (+/)/ApoE (/) mice (Mercer et al., 2010; Schneider et al., 2006). While ATM(+/)/ApoE (/) mice developed accelerated atherosclerosis, hypertension, obesity, and other metabolic abnormalities, increased mtDNA deletions and reduced mitochondrial oxidative phosphorylation were also observed in these mice. These results indicate that mtDNA deletion is sufficient in ATM (+/)/ApoE (/) mice to induce mitochondrial dysfunction, resulting in a metabolic syndrome phenotype that promotes atherosclerosis. It has also been reported that mtDNA damage occurred early in the vessel wall in ApoE knockout mice before significant atherosclerosis developed (Yu et al., 2013). Additionally, studies on patients with coronary artery disease have shown that there is a significant higher incidence and extent of a common mtDNA deletion (mtDNA4977) in blood cells and atherosclerotic lesions of these patients (Botto et al., 2005). DNA damage in atherosclerosis includes deletions, single strand breaks, double strand breaks, base modification (8-oxo-G) and
mis-pairing (Gray & Bennett, 2011). In addition to the accumulation of mtDNA deletion, we also observed decreased expression of OGG1 at both mRNA and protein levels (Fig. 4). OGG1, especially mitochondrial OGG1, functions to recognize and repair 8-oxo-G modification, and its down-regulation has been found to be strongly associated with aging-induced mtDNA damage (Tian et al., 2009). Hence, it is likely that 8-oxo-G modification also accumulates in the aortic roots of C57BL/6J ApoE/ mice and contributes to the development of atherosclerosis. In summary, we investigated the involvement of mtDNA deletion in the development of atherosclerosis using atherosclerosis-prone C57BL/6J ApoE/ mice. Our results show that mtDNA deletion accumulated in the aortic root of C57BL/6J ApoE/ mice occurred prior to the formation atherosclerotic plaques, supporting that mtDNA deletion (mtDNA damage, in general) may represent a major casual factor in both the initiation and progression of atherosclerosis in this animal model. Since the pathophysiology of atherosclerosis in ApoE knockout mice parallels that in humans with respect to vascular distribution, cellular components, and overall lipid content of the lesions (Zhang et al., 1992), our findings provide novel insights into understanding human atherosclerosis. However, in regard to the complex nature of atherosclerosis in molecular biology and pathology, many questions about the involvement mtDNA damage in this disease remain and need to be answered by future studies. For example, while there is a growing body of evidence demonstrating that ROSmediated mtDNA damages are associated with atherogenesis in mice and human (Balliner et al., 2000, 2002; Barry-Lane et al.,
Please cite this article in press as: F. Tian, et al., Age-dependent accumulation of mitochondrial DNA deletions in the aortic root of atherosclerosis-prone apolipoprotein E-knockout mice, Arch. Gerontol. Geriatr. (2015), http://dx.doi.org/10.1016/j.archger.2015.11.004
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2001; Ding et al., 2013; Fetterman et al., 2013; Mikhed et al., 2015), it has been reported that mtDNA damage can promote atherosclerosis in ROS-independent manners (Yu et al., 2013). Evidently, it is essential to evaluate the ROS levels in cytoplasm and mitochondrial in the aortic roots of C57BL/6J ApoE/ mice to understand the molecular mechanisms underlying the atherosclerosis-promoting role of mtDNA damage. Moreover, it has been reported that in addition to mtDNA deletion, there are a number of mtDNA mutations (such as A155G, G3256T, T3336C, G13523A, and G15059A) present in atherosclerotic lesions and their levels of heteroplasmy are significantly higher than those in normal control tissues (Sazonova et al., 2009; Sobenin, Sazonova, Postnov, Bobryshev, & Orekhov, 2012). It has been suggested that these mtDNA mutations may interrelate with the structural alterations to mitochondria in atherosclerosis (Sobenin, Sazonova, Postnov, Bobryshev, & Orekhov, 2013). From this perspective, a comprehensive analysis of mtDNA alterations, including deletion, mutation and changes in copy number, will provide more insights into understanding the correlation between mtDNA damage and atherosclerosis. In addition, the formation of atherosclerosis plaques is a histological event, and many atherosclerosis-related molecular events occur before the formation of histologically detectable atherosclerosis plaques, such as p53 expression and apoptosis, telomerase activity and telomere length in VSMC and macrophages (Aviv et al., 2001; Bennett, Macdonald, Chan, Boyle, & Weissberg, 1998; Costopoulos, Liew, & Bennett, 2008; Okuda et al., 2000; Wu et al., 2000). The association(s) between mtDNA alteration and these atherosclerosis-related molecular events are crucial to elucidate the roles of mtDNA alterations in the initiation and progression of atherosclerosis. Conflict of interest statement The authors declaimed no interest of conflict. Acknowledgments This work was supported by grants from the National Basic Research Programs of China, Nos. 2013CB530801 and 2012CB911203 and the National Natural Science Foundation of China, No. 81170319. References Aviv, H., Khan, M. Y., Skurnick, J., Okuda, K., Kimura, M., Gardner, J., et al. (2001). Age dependent aneuploidy and telomere length of the human vascular endothelium. Atherosclerosis, 159, 281–287. Balliner, S. W., Patterson, C., Yan, C., Doan, R., Burow, D., Young, C., et al. (2000). Hydrogen peroxide and peroxynitrite induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circulation Research, 86, 960–966. Balliner, S. W., Patterson, C., Knight-Lozano, C. A., Burow, D. L., Conklin, C. A., Hu, Z., et al. (2002). Mitochondrial integrity and function in atherosclerosis. Circulation, 106, 544–549. Barry-Lane, P. A., Patterson, C., van der Merwe, M., Hu, Z., Hollad, S. M., & Yeh, E. T. (2001). p47phox is required for atherosclerotic lesion progression in ApoE(/) mice. The Journal of Clinical Investigation, 108, 1513–1522. Bennett, M. R., Macdonald, K., Chan, S. W., Boyle, J. J., & Weissberg, L. (1998). Cooperative interactions between RB and p53 regulate cell proliferation, cell senescence, and apoptosis in human vascular smooth muscle cells from atherosclerotic plaques. Circulation Research, 82, 704–712. Botto, N., Berti, S., Manfredi, S., Al-Jabri, A., Federici, C., Clerico, A., et al. (2005). Detection of mtDNA with 4977 deletion in blood cells nad atherosclerotic lesions of patients with coronary artery disease. Mutation Research, 570, 81–88. Chen, B., Zhong, Y., Peng, W., Sun, Y., Hu, Y. J., Yang, Y., et al. (2011). Increased mitochondrial DNA damage and decreased base excision repair in the auditory cortex of D-galactose-induced aging rats. Molecular Biology Reports, 38, 3635–3642. Costopoulos, C., Liew, T. V., & Bennett, M. (2008). Aging and atherosclerosis: mechanisms and therapeutic options. Biochemical Pharmacology, 75, 1251–1261.
Ding, Z., Liu, S., Wang, X., Khaidakov, M., Dai, Y., & Mehta, J. L. (2013). Oxidant stress in mitochondrial DNA damage, autophagy and inflammation in atherosclerosis. Scientific Reports, 3(1077) . Fetterman, J. L., Pompilius, M., Westbrook, D. G., Uyeminami, D., Brown, J., Pinkerton, K. E., et al. (2013). Developmental exposure to second-hand smoke increases adult atherogenesis and alters mitochondrial DNA copy number and deletions in apoE (/) mice. PLoS One, 8, e66835. Fishbein, M. C., & Fishbein, G. A. (2015). Atheriosclerosis: facts and fancy. Cardiovascular Pathology, S1054–8807, 84–88. Gray, K., & Bennett, M. (2011). Role of DNA damage in atherosclerosis-Bystander or participant? Biochemical Pharmacology, 82, 693–700. Greaves, L. C., & Turnbull, D. M. (2009). Mitochondrial DNA mutations and aging. Biochimica et Biophysica Acta, 1790, 1015–1020. Greaves, L. C., Reeve, A. K., Taylor, R. W., & Turnbull, D. M. (2012). Mitochondrial DNA and disease. Journal of Pathology, 226, 274–286. Gredilla, R., Bohr, V. A., & Stevnsner, T. (2010). Mitochondrial DNA repair and association with aging–an update. Experimental Gerontology, 45, 478–488. Lee, H. C., & Wei, Y. H. (2012). Mitochondria and aging. Advances in Experimental Medicine and Biology, 942, 311–327. Lyama, T., & Wilson, D. M. (2013). DNA repair mechanisms in dividing and nondividing cells. DNA Repair, 12, 620–636. Madamanchi, N. R., & Runge, M. S. (2007). Mitochondrial dysfunction in atherosclerosis. Circulation Research, 100, 460–473. Mercer, J. R., Cheng, K.-K., Figg, N., Gorenne, I., Mahmoudi, M., Griffin, J., et al. (2010). DNA damage links mitochondrial dysfunction to atherosclerosis and the metabolic syndrome. Circulation Research, 107, 1021–1031. Mikhed, Y., Daiber, A., & Steven, S. (2015). Mitochondrial oxidative stress, mitochondrial DNA damage and their role in age-related vascular dysfunction. International Journal of Molecular Sciences, 16, 15918–15953. Okuda, K., Khan, M. Y., Skurnick, J., Kimura, M., Aviv, H., & Aviv, A. (2000). Telomere attrition of the human abdominal aorta: relationships with age and atherosclerosis. Atherosclerosis, 152, 391–398. Phillips, N. R., Simpkius, J. W., & Roby, R. K. (2014). Mitochondrial DNA deletion in Alzheimer’s brains. A review. Alzheimer’s & Dementia, 10, 393–400. Sazonova, M., Budnikov, E., Khasanova, Z., Sobenin, I., Postnov, A., & Orekhov, A. (2009). Studies of the human arotic intima by a direct quantitative assay of mutant alles in the mitochonadrial genome. Atherosclerosis, 2004, 184–190. Shah, N. R., & Mahoudi, M. (2015). The role of DNA damage and repair in atherosclerosis: a review. Journal of Molecular and Cellular Cardiology, 86, 147–157. Schneider, J. G., Finck, B. N., Ren, J., Standley, K. N., Takagi, M., Maclean, K. H., et al. (2006). ATM-dependent suppression of stress signaling reduces vascular disease in metabolic syndrome. Cell Metabolism, 4, 377–389. Sobenin, I. A., Sazonova, M. A., Postnov, A. Y., Bobryshev, Y. V., & Orekhov, A. N. (2012). Mitochondrial mutations are associated with atherosclerotic lesions in the human arota. Clinical and Developmental Immunology 83464. Sobenin, I. A., Sazonova, M. A., Postnov, A. Y., Bobryshev, Y. V., & Orekhov, A. N. (2013). Changes of mitochondria in atherosclerosis: possible determinant in the pathogenesis of the disease. Atherosclerosis, 227, 283–288. Sobenin, I. A., Zhelankin, A. V., Mitrofanov, K. Y., Sinyov, V. V., Bobryshev, Y. V., & Orekhov, A. N. (2015). Mitochondrial aging: Focus on mitochondrial DNA damage in atherosclerosis-a mini-review. Gerontology, 61, 343–349. Sugie, K., & Nishino, I. (2002). Complex IV(cytochrome c oxidase). Nippon Rinsho— Japanese Journal of Clinical Medicine, 60, 490–494. Tian, F., Tong, T. J., Zhang, Z. Y., McNutt, M. A., & Liu, X. W. (2009). Age-dependent down-regulation of mitochondrial 8-oxoguanine DNA glycosylase in SAM-P/8 mouse brain and its effect on brain aging. Rejuvenation Research, 12, 209–215. Weigartner, O., Huscle, C., Schott, H. F., Speer, T., Bohm, M., Miller, C. M., et al. (2015). Vascular effects of oxysteroids and oxyphytosteroids in ApoE/ mice. Atherosclerosis, 240, 73–91. Wu, K., Higashi, N., Hansen, E. R., Lund, M., Bang, K., & Thestrup-Pedersen, K. (2000). Telomerase activity is increased and telomere length shortned in T cells from blood of patients with atopic dermatitis and psoriasis. The Journal of Immunology, 165, 4742–4747. Yi, X., Xu, L., Kim, K., Kim, H. S., & Maeda, N. (2010). Genetic reduction of lipoic acid synthase expression modestly increases atherosclerosis in male, but not in female, appolioprotein E-deficient mice. Atherosclerosis, 211, 424–430. Yu, E., & Bennett, M. R. (2014). Mitochondrial DNA damage and atherosclerosis. Trends in Endocrinology & Metabolism, 25, 481–487. Yu, E., Mercer, J., & Bennett, M. (2012). Mitochondria in vascular disease. Cardiovascular Research, 95, 173–182. Yu, E., Calvert, P. A., Mercer, J. R., Harrison, J., Baker, L., Figg, N. L., et al. (2013). Mitochondrial DNA damage can promote atherosclerosis independently of reactive oxygen species through effects on smooth muscle cells and monocytes and correlates with higher-risk plaques in humans. Circulation, 128, 702–712. Zeng, Z., Zhang, Z. Y., Yu, H., Corbley, M. J., Tang, Z., & Tong, T. J. (1999). Mitochondrial DNA deletions are associated with ischemia and aging in Balb/c mouse brain. Journal of Cellular Biochemistry, 73, 545–553. Zhang, S. H., Reddick, R. L., Piedrahita, J. A., & Maeda, N. (1992). Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science, 258, 468–471. Zhang, S., Gong, Z., Chen, W., Hu, H., Zheng, W., & Zhu, M. (2004). Frozen section staining method improvement and application in whole blood vessels and the heart of laboratory animal. Chinese Journal of Histochemistry and Cytochemistry, 13, 127–128.
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Age-dependent accumulation of mitochondrial DNA deletions in the aortic root of atherosclerosis-prone apolipoprotein E-knockout mice Feng Tiana , Junan Lib , Xin-wen Liua , Tan-jun Tonga , Zong-yu Zhanga,* a b
Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Peking University Research Center on Aging, Beijing, China College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 July 2015 Received in revised form 2 November 2015 Accepted 3 November 2015 Available online xxx
Purpose: To investigate whether mitochondrial DNA (mtDNA) damage, specifically deletion, contributes to the development of atherosclerosis or is simply a secondary effect of the primary factors causing atherosclerosis. Materials and methods: mtDNA deletion was detected by PCR in the aortic root of atherosclerosis-prone C57BL/6J apolipoprotein (Apo) E gene deficient (/) mice and control C57BL/6J mice at different ages. Atherosclerotic plaques in the Apo E/ mice were assessed using frozen sections of the aortic root. The protein levels of COX III and 8-oxoguanine glycosylase (OGG1) were determined. Results: while mtDNA deletions accumulated significantly in mice as young as 2- month-old, atherosclerotic plaques were not detected until mice were 6 months old or older, suggesting that mtDNA deletion occurs prior to the formation of atherosclerotic plaques in the aortic root of these mice. Moreover, the expression levels of mtDNA-encoded COX III protein in both 2-month-old and 16-monthold C57BL/6J ApoE/ mice were significantly lower than those in C57BL/6J mice (p < 0.05). Additionally, the protein level of 8-oxoguanine glycosylase (OGG1), a mitochondrial enzyme that functions in DNA excision repair, decreased with age in these mice, indicating that age-related down-regulation of mtDNA excision repair also contributes to atherosclerosis in C57BL/6J ApoE/ mice. Conclusion: These results reveal that mtDNA deletions occur during the early “initiation” stage of atherosclerosis in C57BL/6J ApoE/ mice and have the potential to promote atherosclerosis. ã 2015 Published by Elsevier Ireland Ltd.
Keywords: Mitochondrial DNA deletion C57BL/6J ApoE/ mice Atherosclerosis
1. Introduction Numerous studies have demonstrated that mitochondrial DNA (mtDNA) damage accumulates with aging and such accumulation is involved in organism senescence and the development of cardiovascular diseases such as atherosclerosis and myocardial infarction (Greaves & Turnbull, 2009; Greaves, Reeve, Taylor, & Turnbull, 2012; Lee & Wei, 2012; Phillips, Simpkius, & Roby, 2014; Yu, Mercer, & Bennett, 2012). Due to the lack of protective histones and its close proximity to the electron transfer chain in the inner mitochondrial membrane, mtDNA is particularly vulnerable to the damaging effects of reactive oxygen species (ROS), which are generated during oxidative phosphorylation in mitochondria and potently induce mutations (single- and double-stranded breaks, DSBs) and deletions in mtDNA as well as chromosomal DNA (Mercer et al., 2010; Mikhed, Daiber, & Steven, 2015). Interestingly,
* Corresponding author. Fax: +86 10 8280 2931. E-mail address: [email protected] (Z.-y. Zhang).
mtDNA encodes a number of key components of mitochondrial respiratory chains, such as cytochrome oxidases I, II, and III (COX I– III) (Sugie & Nishino, 2002) and mtDNA damage may even downregulate or up-regulate these genes and disrupt oxidative phosphorylation, leading to further increased ROS production and mtDNA damage (Mikhed et al., 2015). Arguably, a positive feedback mechanism exists between mtDNA damage and diseases associated with increased ROS production. From this perspective, mtDNA damage could contribute to some cardiovascular diseases as an “initiating” and/or “promoting” factor. mtDNA damage, especially mtDNA deletion, is frequently observed in human atherosclerosis in both circulating and vessel wall cells and is associated with mitochondrial dysfunction (Fishbein & Fishbein, 2015; Madamanchi & Runge, 2007; Shah & Mahoudi, 2015; Sobenin et al., 2015). However, it remains unknown whether DNA damage is directly involved in the development of atherosclerosis or is a secondary consequence of atherosclerosis risk factors, such as smoking, hypercholesterolemia, diabetes, and hypertension, all of which bring about increased ROS production in mitochondria.
http://dx.doi.org/10.1016/j.archger.2015.11.004 0167-4943/ ã 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: F. Tian, et al., Age-dependent accumulation of mitochondrial DNA deletions in the aortic root of atherosclerosis-prone apolipoprotein E-knockout mice, Arch. Gerontol. Geriatr. (2015), http://dx.doi.org/10.1016/j.archger.2015.11.004
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Apolipoprotein (Apo) E is an essential ligand for the uptake and clearance of atherogenic lipoproteins, such as very low density lipoproteins (VLDLs) and chylomicrons. It is known that mice is a species normally resistant to the development of atherosclerosis. However, when mice with inactivated Apo E gene are given diets that are normal, high in fat, or rich in cholesterol, the plasma cholesterol concentration in these mice significantly increases, which leads to rapid and severe atherosclerosis. The pathophysiology of atherosclerosis in Apo E-deficient mice (ApoE/) parallels that in humans with respect to vascular distribution, cellular components, and overall lipid content of the lesions (Zhang, Reddick, Piedrahita, & Maeda, 1992). Therefore, ApoE/ mice are regarded as one of the best models for studying the development of atherosclerosis (Fetterman et al., 2013; Weigartner et al., 2015; Yi, Xu, Kim, Kim, & Maeda, 2010). In the present study, we investigated the involvement of mtDNA deletion in the development of atherosclerosis using atherosclerosis-prone C57BL/6J ApoE/ mice.
COX III gene. The polymerase chain reaction (PCR) mixture contained 5 ml 10 PCR buffer, 4 ml dNTPs (10 mM each), 1 ml mtDNA-f1, 1 ml mtDNA-f2, 1 ml mtDNA-r (54 mM), 2–3 units Taq DNA polymerase (Huamei Biotech, Beijing, China) and 1 ml mtDNA template (0.1 mg) in a total volume of 50 ml. The reaction mixtures were incubated for 5 min at 93 C, followed by 30 cycles of 30 s at 93 C, 1 min at 55 C, and 1 min at 72 C. After amplification, 10 ml of each PCR mixture was loaded onto a 1% agarose gel for electrophoretic separation, and mtDNA bands were visualized and analyzed with an Image Master VDS System (Amersham Pharmarcia, Piscataway, NJ, USA). Both target bands were recovered from the gel using a QIAquick Gel Extraction Kit (Qiagen, Valenica, CA, USA) and subjected to direct sequencing on an ABI377A DNA automated sequencer (Applied Biosystems Inc., Foster City, CA, USA). The PCR amplification product was identified by Hind III digestion (Huamei Biotech) and then loaded onto a 1% agarose gel for separation. 2.4. Protein extraction from aortic roots
2. Materials and methods 2.1. ApoE/ mice The apolipoprotein E gene-deficient mouse (ApoE/) is an excellent animal model for atherosclerosis research because it exhibits severe hyperlipidemia and a plaque distribution pattern similar to that of human atherosclerosis. ApoE/ mice were first generated at Rockefeller University and the University of North Carolina in 1992 (Zhang et al., 1992) and were introduced to the Peking University Health Science Center in 1997. Three groups of C57BL/6J ApoE/ mice (n = 18) and C57BL/6J mice (n = 18), including 2-month-old (2m), 6-month-old (6m) and 16-monthold (16m) mice, were housed in the Department of Laboratory Animal Science, Peking University Health Science Center under clean conditions at 22 2 C with 55 5% humidity and were maintained on a commercial diet with tap water ad libitum. Lights were on from 7:00 AM to 7:00 PM. Both male and female mice were selected randomly for the experiments. All research protocols were approved by the Animal Welfare Committee of Peking University Health Science Center (LA2013-18, approved in February 25, 2013). After decapitation of the animals at the indicated times, aortic roots were removed immediately for subsequent analyses or frozen in liquid nitrogen for future studies. 2.2. Histological analysis Aortic roots from ApoE/ mice were rapidly frozen in liquid nitrogen, and frozen sections were prepared as previously described (Zhang et al., 2004). The sections were stained with Oil Red O to visualize the formation of atherosclerotic plaques. 2.3. mtDNA isolation and identification of deletion mtDNA was extracted from the aortic root samples as previously described (Zeng et al., 1999). The following primers were used for mtDNA amplification: mtDNA-f1 (forward primer sequence 1), 50 -AGTATCATGCTGCGGCTTCA-30 ; mtDNA-f2 (forward primer sequence 2), 50 -GCAAAGATGCTTCCGAATGC-30 ; and mtDNA-r (reverse primer sequence), 50 -TACCAAGGCCACCACACTCC-30 . MtDNA-f1 and mtDNA-r were used to amplify mtDNA without deletions, and the amplicon was 526 bp. MtDNA-f2 and mtDNA-r were used to amplify mtDNA with deletions, and the amplicon was 455 bp (3867-bp deletion). Of note, this 3867-bp DNA fragment proposed to be deleted during aging contains the
After one wash with ice-cold phosphate-buffered saline (PBS), the aortic roots were minced in liquid nitrogen and then transferred into a plastic homogenizer containing protein extraction buffer (10 mM Tris–HCl, 150 mM NaCl, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 0.2% NP-40, and 1 mg/ml Aprotinin, pH 8.0). After homogenization, the tissue samples were centrifuged at 3,000 rpm for 5 min. Subsequently, the supernatant was transferred to a new Eppendorf tube, and the DNA in the supernatant was fragmented by four sonication cycles of 4 s each with a 20-s interval between each cycle. Finally, the samples were centrifuged at 10,000 g, 4 C for 10 min, and the supernatants were kept at 20 C for further studies. 2.5. Western blot Equal amounts (50 mg) of proteins were separated on 12% SDSPAGE gels and then transferred to a nitrocellulose membrane using a Trans-Blot1 SD system (Bio-Rad, Hercules, CA, USA). The nitrocellulose membrane was washed three times using TBST buffer (20 mM Tris–HCl, 150 mM NaCl, 0.5% Tween-20, pH 7.5) and subsequently blocked with TBST containing 5% nonfat dry milk at 4 C for 1 h. Upon incubation with rabbit anti-OGG1 polyclonal antibody (ADI, San Antonio, TX, USA) (1:2,000 dilution), mouse anti-COX III polyclonal antibody (molecular targeting) (1:2,000 dilution), or goat anti-b-actin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:2,000 dilution) at 4 C overnight, the membrane was washed five times with TBST and probed with a horseradish peroxidase (HRP)-conjugated goat antirabbit immunoglobulin G (IgG), goat anti-mouse IgG, or rabbit anti-goat IgG (ZhongShan Golden Bridge, Beijing, China) (1:5,000 dilution). Immunoblots were visualized by adding SuperSignal West Pico Chemiluminescent Substrates (Pierce, Rockford, IL, USA), and digitized images of the blots were analyzed with the ImageMaster VDS software (Amersham Pharmarcia). Mouse b-actin was used as the reference for normalization. 2.6. Statistical analyses All data in this study were statistically analyzed using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to evaluate age-related changes in protein levels of COX III, and OGG1 in C57BL/6J ApoE/ mice and C57BL/ 6J mice, and the differences in these parameters between the two types of mice were subjected to two-way ANOVA. Values of p < 0.05 were considered to indicate significance.
Please cite this article in press as: F. Tian, et al., Age-dependent accumulation of mitochondrial DNA deletions in the aortic root of atherosclerosis-prone apolipoprotein E-knockout mice, Arch. Gerontol. Geriatr. (2015), http://dx.doi.org/10.1016/j.archger.2015.11.004
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3. Results 3.1. Atherosclerosis plaques are formed in 6-month and older C57BL/ 6J-ApoE/ mice The formation of atherosclerotic plaques is a well-known pathological/histological marker of aging. Hence, we first investigated the formation of atherosclerotic plaques in the aortic roots of C57BL/6J ApoE/ mice using histological staining. As shown in Fig. 1, distinct atherosclerotic plaques were observed in the aortic roots of 6-month-old and 16-month-old C57BL/6J ApoE/ mice (data not shown), but not in the aortic roots of 2-month-old C57BL/ 6J ApoE/ mice (data not shown). In comparison, atherosclerotic plaques were not detected in the corresponding tissues of C57BL/6J mice at all tested ages (2, 6, and 16 months). Apparently, aging in C57BL/6J ApoE/ mice occurred much earlier than that in C57BL/ 6J mice (6 months vs>16 months). Since both C57BL/6J ApoE/ and C57BL/6J mice were fed a normal diet, our results indicate that even on a normal diet, C57BL/6J Apo E/ mice exhibits accelerated aging in comparison with their C57BL/6J counterparts. 3.2. mtDNA deletion occurs in 2-month C57BL/6J-ApoE/ mice and the extent of such deletion increases with age Because mtDNA damage, a molecular event occurring during the early stage of aging, is frequently observed in atherosclerosis, we investigated mtDNA deletion, the major type of mitochondrial DNA damage, in C57BL/6J ApoE/ mice using well-established PCR assays (Tian, Tong, Zhang, McNutt, & Liu, 2009). In these assays, an amplicon of 526 bp was generated from intact mtDNA, whereas a truncated mtDNA template (with a 3867-bp mtDNA deletion) led to an amplicon of 455 bp. Furthermore, the 526-bp amplicon from intact mtDNA harbors a Hind III restriction site, which leads to fragments of 310 bp and 190 bp, respectively, upon Hind III digestion. Contrary to this, this Hind III site is absent in the 455-bp amplicon. Hence, the status of mtDNA can be determined from different Hind III-digestion patterns of the amplicons. As shown in Fig. 2A, upon Hind III digestion, 455-bp products were present in the PCR mixtures containing template DNA from aortic roots of 2-month-old C57BL/6J ApoE/ mice, even though the major products were 310 bp and 190 bp. These results indicate that mtDNA deletion occurred in 2-month-old C57BL/6J ApoE/ mice to certain extent. Notably, more 455-bp products were detected in PCR mixtures containing template DNA from the aortic roots of 6and 16-month-old C57BL/6J ApoE/ mice, indicating that the extent of mtDNA deletion increases with aging in C57BL/6J ApoE/ mice. In contrast, 455-bp products were only detected in PCR mixtures containing template DNA from the aortic roots of 16-
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month-old C57BL/6J mice and not template DNA from 2-monthold C57BL/6J mice (Fig. 2B). These results are consistent with our previous findings that the formation of atherosclerotic plaques occurs earlier in C57BL/6J ApoE/ mice than in C57BL/6J mice. 3.3. The mRNA and protein levels of COX I, not COX III, in the C57BL/6JApoE/ mice increase with aging COX III is a key component of the mitochondrial respiratory chain encoded by mtDNA. Interestingly, the COX III gene is located in the 3867-bp fragment of mtDNA frequently deleted during aging in mice including C57BL/6J mice. Deletion of this COX IIIcontaining fragment of mtDNA was observed in aortic roots of C57BL/6J ApoE/ mice as young as 2-month-old (Fig. 2). Therefore, it is rational to evaluate the impact of accelerated aging in C57BL/6J ApoE/ mice on the expression of COX III protein. As shown in Fig. 3, the expression levels of COX III protein in both 2-month-old and 16-month-old C57BL/6J ApoE/ mice were significantly lower than their corresponding values in C57BL/ 6J mice at the same ages (p < 0.05), indicating that accelerated aging in Apo E-knockout mice significantly down-regulates the expression of COX III even in mice as young as 2-month-old. Presumably, such down-regulation of COX III could impair the oxidative phosphorylation process in mitochondria thus promoting aging. Notably, the expression levels of COX III protein in both 2month-old and 16-month-old C57BL/6J ApoE/ mice were comparable (p > 0.05). Apparently, this finding is not fully consistent with previous observations showing that mtDNA deletion accumulates during aging (Tian et al., 2009). While the molecular mechanisms underlying such “inconsistency” remain to be elucidated, it is worthwhile to note that changes at the protein level may not always or sufficiently represent the changes at the DNA level. For example, upon aging, the transcription of COX III might be enhanced through unknown feedback mechanisms, which may partially “compensate” the loss of COX III gene and lead to compromised impact on COX III protein. 3.4. The levels of OGG1 mRNA and protein in C57BL/6J-ApoE/ mice significantly decrease with aging It is well known that oxidative damage of mtDNA is monitored by the mitochondrial anti-oxidation systems and its DNA repair systems (Gredilla, Bohr, & Stevnsner, 2010; Chen et al., 2011), in which OGG1 plays a critical role. 8-oxyoguanine is the most common base modification occurring in oxidative damage of mtDNA, and OGG1 specifically recognizes and excises such modification for DNA repair (Lyama & Wilson, 2013). Previous
Fig. 1. Atherosclerotic plaques in the aortic root of C57BL/6J ApoE/ and C57BL/6J mice. (A) Oil Red O staining of the aortic root of a 6-month-old C57BL/6J ApoE/ mouse (100). (B) Oil Red O staining of the aortic root of a 6-month-old C57BL/6J mouse (100).
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Fig. 2. Accumulation of mtDNA deletion in C57BL/6J ApoE/ mice. mtDNA deletions were evaluated using PCR and Hind III fragmentation as previously described (Tian et al., 2009). (A) mtDNA deletion in C57BL/6J ApoE/ mice of different ages. (B) mtDNA deletion in C57BL/6J mice of different ages. M1, 50-bp DNA ladder.
studies in our laboratory as well as by other groups have demonstrated that down-regulation of OGG1, especially mitochondrial OGG1, is strongly associated with aging-induced mtDNA damage in SAM-P/8 mice, a species with accelerated senescence (Tian et al., 2009). To further explore the mechanism(s) underlying accumulated mtDNA deletion in C57BL/6J ApoE/ mice, we evaluated the protein levels of OGG1 in these mice. OGG1 protein levels significantly decreased between 2 and 16 months in C57BL/ 6J ApoE/ mice (p < 0.05), and the OGG1 protein levels in C57BL/ 6J ApoE/ mice were significantly lower than those in C57BL/6J mice of the same age (p < 0.05; Fig. 4). Therefore, the expression of OGG1 is negatively associated with aging in C57BL/6J ApoE/ mice. Although the level of OGG1 protein evaluated in this study is that for “total” OGG1 protein, which includes cytoplasmic, nuclear, and mitochondrial OGG1, our previous studies showed that aginginduced down-regulation of OGG1 in SAM-P/8 mice has the greatest impact on mitochondrial OGG1 expression (Tian et al., 2009). Hence, it is likely that mitochondrial OGG1 decreases with age in the aortic root of C57BL/6J ApoE/ mice.
4. Discussion Atherosclerosis is characterized by the development of a fibrofatty lesion in the artery wall involving a variety of different cell types, such as vascular smooth muscle cells (VSMC), monocyte-derived macrophages, lymphocytes, dendritic cells and platelets (Gray & Bennett, 2011; Yu & Bennett, 2014; Sobenin et al., 2015). In the past decade, increasing evidence has shown that damages in both genomic and mitochondrial DNAs occur prevalently in VSMCs and inflammatory cells in atherosclerotic plaques, suggesting that there could be a causative association between DNA damage and atherosclerosis (Gray & Bennett, 2011; Sobenin et al., 2015). However, the role(s) of DNA damage, especially mtDNA damage in the initiation and progression of atherosclerosis remains highly debated. It is still unknown whether mtDNA damage is simply a by-product of those atherosclerosis-promoting risk factors or mtDNA damage itself promotes atherosclerosis (Gray & Bennett, 2011). Apparently, our results support the involvement of mtDNA deletion in promoting atherosclerosis. As shown in Fig. 1, the formation of atherosclerotic
Fig. 3. Changes in COX III expression in the aortic root of C57BL/6J ApoE/ mice of different ages. Total proteins from the aortic roots of C57BL/6J ApoE/ and C57BL/6J mice were separated by SDS-PAGE and then blotted against anti-COX III antibody. b-actin was used as an internal control. (A) COX III expression in C57BL/6J ApoE/ and C57BL/6J mice of different ages. (B) b-actin expression in C57BL/6J ApoE/ and C57BL/6J mice of different ages; (C) densitometric scanning analysis of the the COX III/b-actin intensity ratio. #, p < 0.05, 2- and 16-month-old C57BL/6J ApoE/ mice vs 2- and 16-month-old C57BL/6J mice, respectively.
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Fig. 4. Changes in the expression of OGG1 protein in the aortic root of C57BL/6J ApoE/ mice of different ages. Total proteins from the aortic roots of C57BL/6J ApoE/ and C57BL/6J mice were subjected to immunoblotting against anti-OGG1 antibody. b-actin was used as an internal control. (A) OGG1 expression in C57BL/6J ApoE/ and C57BL/ 6J mice of different ages; (B) b-actin expression in C57BL/6J ApoE/ and C57BL/6J mice of different ages; (C) densitometric scanning analysis of the OGG1/b-actin intensity ratio. *, p < 0.05, 16-month-old C57BL/6J ApoE/ mice vs2-month-old C57BL/6J ApoE/ mice; #,p < 0.05, 2- and 16-month-old C57BL/6J ApoE/ mice vs 2- and 16month-old C57BL/6J mice, respectively.
plaques was observed only in 6- and 16-month-old, but not 2month-old, C57BL/6J ApoE/ mice, implying that mtDNA deletion, a molecular event, occurs earlier in the aging process than the formation of atherosclerotic plaques, a histological event (2 vs 6 months). This notion is further supported by the fact that only mtDNA deletion, not the formation of atherosclerotic plaques, was observed in the aortic roots of 16-month-old C57BL/6J mice. Our results are consistent with recent studies demonstrating that mtDNA deletion could be an early event in atherosclerosis. It has been demonstrated recently that DNA damage links mitochondrial dysfunction to atherosclerosis and metabolic syndrome in ATM (+/)/ApoE (/) mice (Mercer et al., 2010; Schneider et al., 2006). While ATM(+/)/ApoE (/) mice developed accelerated atherosclerosis, hypertension, obesity, and other metabolic abnormalities, increased mtDNA deletions and reduced mitochondrial oxidative phosphorylation were also observed in these mice. These results indicate that mtDNA deletion is sufficient in ATM (+/)/ApoE (/) mice to induce mitochondrial dysfunction, resulting in a metabolic syndrome phenotype that promotes atherosclerosis. It has also been reported that mtDNA damage occurred early in the vessel wall in ApoE knockout mice before significant atherosclerosis developed (Yu et al., 2013). Additionally, studies on patients with coronary artery disease have shown that there is a significant higher incidence and extent of a common mtDNA deletion (mtDNA4977) in blood cells and atherosclerotic lesions of these patients (Botto et al., 2005). DNA damage in atherosclerosis includes deletions, single strand breaks, double strand breaks, base modification (8-oxo-G) and
mis-pairing (Gray & Bennett, 2011). In addition to the accumulation of mtDNA deletion, we also observed decreased expression of OGG1 at both mRNA and protein levels (Fig. 4). OGG1, especially mitochondrial OGG1, functions to recognize and repair 8-oxo-G modification, and its down-regulation has been found to be strongly associated with aging-induced mtDNA damage (Tian et al., 2009). Hence, it is likely that 8-oxo-G modification also accumulates in the aortic roots of C57BL/6J ApoE/ mice and contributes to the development of atherosclerosis. In summary, we investigated the involvement of mtDNA deletion in the development of atherosclerosis using atherosclerosis-prone C57BL/6J ApoE/ mice. Our results show that mtDNA deletion accumulated in the aortic root of C57BL/6J ApoE/ mice occurred prior to the formation atherosclerotic plaques, supporting that mtDNA deletion (mtDNA damage, in general) may represent a major casual factor in both the initiation and progression of atherosclerosis in this animal model. Since the pathophysiology of atherosclerosis in ApoE knockout mice parallels that in humans with respect to vascular distribution, cellular components, and overall lipid content of the lesions (Zhang et al., 1992), our findings provide novel insights into understanding human atherosclerosis. However, in regard to the complex nature of atherosclerosis in molecular biology and pathology, many questions about the involvement mtDNA damage in this disease remain and need to be answered by future studies. For example, while there is a growing body of evidence demonstrating that ROSmediated mtDNA damages are associated with atherogenesis in mice and human (Balliner et al., 2000, 2002; Barry-Lane et al.,
Please cite this article in press as: F. Tian, et al., Age-dependent accumulation of mitochondrial DNA deletions in the aortic root of atherosclerosis-prone apolipoprotein E-knockout mice, Arch. Gerontol. Geriatr. (2015), http://dx.doi.org/10.1016/j.archger.2015.11.004
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2001; Ding et al., 2013; Fetterman et al., 2013; Mikhed et al., 2015), it has been reported that mtDNA damage can promote atherosclerosis in ROS-independent manners (Yu et al., 2013). Evidently, it is essential to evaluate the ROS levels in cytoplasm and mitochondrial in the aortic roots of C57BL/6J ApoE/ mice to understand the molecular mechanisms underlying the atherosclerosis-promoting role of mtDNA damage. Moreover, it has been reported that in addition to mtDNA deletion, there are a number of mtDNA mutations (such as A155G, G3256T, T3336C, G13523A, and G15059A) present in atherosclerotic lesions and their levels of heteroplasmy are significantly higher than those in normal control tissues (Sazonova et al., 2009; Sobenin, Sazonova, Postnov, Bobryshev, & Orekhov, 2012). It has been suggested that these mtDNA mutations may interrelate with the structural alterations to mitochondria in atherosclerosis (Sobenin, Sazonova, Postnov, Bobryshev, & Orekhov, 2013). From this perspective, a comprehensive analysis of mtDNA alterations, including deletion, mutation and changes in copy number, will provide more insights into understanding the correlation between mtDNA damage and atherosclerosis. In addition, the formation of atherosclerosis plaques is a histological event, and many atherosclerosis-related molecular events occur before the formation of histologically detectable atherosclerosis plaques, such as p53 expression and apoptosis, telomerase activity and telomere length in VSMC and macrophages (Aviv et al., 2001; Bennett, Macdonald, Chan, Boyle, & Weissberg, 1998; Costopoulos, Liew, & Bennett, 2008; Okuda et al., 2000; Wu et al., 2000). The association(s) between mtDNA alteration and these atherosclerosis-related molecular events are crucial to elucidate the roles of mtDNA alterations in the initiation and progression of atherosclerosis. Conflict of interest statement The authors declaimed no interest of conflict. Acknowledgments This work was supported by grants from the National Basic Research Programs of China, Nos. 2013CB530801 and 2012CB911203 and the National Natural Science Foundation of China, No. 81170319. References Aviv, H., Khan, M. Y., Skurnick, J., Okuda, K., Kimura, M., Gardner, J., et al. (2001). Age dependent aneuploidy and telomere length of the human vascular endothelium. Atherosclerosis, 159, 281–287. Balliner, S. W., Patterson, C., Yan, C., Doan, R., Burow, D., Young, C., et al. (2000). Hydrogen peroxide and peroxynitrite induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circulation Research, 86, 960–966. Balliner, S. W., Patterson, C., Knight-Lozano, C. A., Burow, D. L., Conklin, C. A., Hu, Z., et al. (2002). Mitochondrial integrity and function in atherosclerosis. Circulation, 106, 544–549. Barry-Lane, P. A., Patterson, C., van der Merwe, M., Hu, Z., Hollad, S. M., & Yeh, E. T. (2001). p47phox is required for atherosclerotic lesion progression in ApoE(/) mice. The Journal of Clinical Investigation, 108, 1513–1522. Bennett, M. R., Macdonald, K., Chan, S. W., Boyle, J. J., & Weissberg, L. (1998). Cooperative interactions between RB and p53 regulate cell proliferation, cell senescence, and apoptosis in human vascular smooth muscle cells from atherosclerotic plaques. Circulation Research, 82, 704–712. Botto, N., Berti, S., Manfredi, S., Al-Jabri, A., Federici, C., Clerico, A., et al. (2005). Detection of mtDNA with 4977 deletion in blood cells nad atherosclerotic lesions of patients with coronary artery disease. Mutation Research, 570, 81–88. Chen, B., Zhong, Y., Peng, W., Sun, Y., Hu, Y. J., Yang, Y., et al. (2011). Increased mitochondrial DNA damage and decreased base excision repair in the auditory cortex of D-galactose-induced aging rats. Molecular Biology Reports, 38, 3635–3642. Costopoulos, C., Liew, T. V., & Bennett, M. (2008). Aging and atherosclerosis: mechanisms and therapeutic options. Biochemical Pharmacology, 75, 1251–1261.
Ding, Z., Liu, S., Wang, X., Khaidakov, M., Dai, Y., & Mehta, J. L. (2013). Oxidant stress in mitochondrial DNA damage, autophagy and inflammation in atherosclerosis. Scientific Reports, 3(1077) . Fetterman, J. L., Pompilius, M., Westbrook, D. G., Uyeminami, D., Brown, J., Pinkerton, K. E., et al. (2013). Developmental exposure to second-hand smoke increases adult atherogenesis and alters mitochondrial DNA copy number and deletions in apoE (/) mice. PLoS One, 8, e66835. Fishbein, M. C., & Fishbein, G. A. (2015). Atheriosclerosis: facts and fancy. Cardiovascular Pathology, S1054–8807, 84–88. Gray, K., & Bennett, M. (2011). Role of DNA damage in atherosclerosis-Bystander or participant? Biochemical Pharmacology, 82, 693–700. Greaves, L. C., & Turnbull, D. M. (2009). Mitochondrial DNA mutations and aging. Biochimica et Biophysica Acta, 1790, 1015–1020. Greaves, L. C., Reeve, A. K., Taylor, R. W., & Turnbull, D. M. (2012). Mitochondrial DNA and disease. Journal of Pathology, 226, 274–286. Gredilla, R., Bohr, V. A., & Stevnsner, T. (2010). Mitochondrial DNA repair and association with aging–an update. Experimental Gerontology, 45, 478–488. Lee, H. C., & Wei, Y. H. (2012). Mitochondria and aging. Advances in Experimental Medicine and Biology, 942, 311–327. Lyama, T., & Wilson, D. M. (2013). DNA repair mechanisms in dividing and nondividing cells. DNA Repair, 12, 620–636. Madamanchi, N. R., & Runge, M. S. (2007). Mitochondrial dysfunction in atherosclerosis. Circulation Research, 100, 460–473. Mercer, J. R., Cheng, K.-K., Figg, N., Gorenne, I., Mahmoudi, M., Griffin, J., et al. (2010). DNA damage links mitochondrial dysfunction to atherosclerosis and the metabolic syndrome. Circulation Research, 107, 1021–1031. Mikhed, Y., Daiber, A., & Steven, S. (2015). Mitochondrial oxidative stress, mitochondrial DNA damage and their role in age-related vascular dysfunction. International Journal of Molecular Sciences, 16, 15918–15953. Okuda, K., Khan, M. Y., Skurnick, J., Kimura, M., Aviv, H., & Aviv, A. (2000). Telomere attrition of the human abdominal aorta: relationships with age and atherosclerosis. Atherosclerosis, 152, 391–398. Phillips, N. R., Simpkius, J. W., & Roby, R. K. (2014). Mitochondrial DNA deletion in Alzheimer’s brains. A review. Alzheimer’s & Dementia, 10, 393–400. Sazonova, M., Budnikov, E., Khasanova, Z., Sobenin, I., Postnov, A., & Orekhov, A. (2009). Studies of the human arotic intima by a direct quantitative assay of mutant alles in the mitochonadrial genome. Atherosclerosis, 2004, 184–190. Shah, N. R., & Mahoudi, M. (2015). The role of DNA damage and repair in atherosclerosis: a review. Journal of Molecular and Cellular Cardiology, 86, 147–157. Schneider, J. G., Finck, B. N., Ren, J., Standley, K. N., Takagi, M., Maclean, K. H., et al. (2006). ATM-dependent suppression of stress signaling reduces vascular disease in metabolic syndrome. Cell Metabolism, 4, 377–389. Sobenin, I. A., Sazonova, M. A., Postnov, A. Y., Bobryshev, Y. V., & Orekhov, A. N. (2012). Mitochondrial mutations are associated with atherosclerotic lesions in the human arota. Clinical and Developmental Immunology 83464. Sobenin, I. A., Sazonova, M. A., Postnov, A. Y., Bobryshev, Y. V., & Orekhov, A. N. (2013). Changes of mitochondria in atherosclerosis: possible determinant in the pathogenesis of the disease. Atherosclerosis, 227, 283–288. Sobenin, I. A., Zhelankin, A. V., Mitrofanov, K. Y., Sinyov, V. V., Bobryshev, Y. V., & Orekhov, A. N. (2015). Mitochondrial aging: Focus on mitochondrial DNA damage in atherosclerosis-a mini-review. Gerontology, 61, 343–349. Sugie, K., & Nishino, I. (2002). Complex IV(cytochrome c oxidase). Nippon Rinsho— Japanese Journal of Clinical Medicine, 60, 490–494. Tian, F., Tong, T. J., Zhang, Z. Y., McNutt, M. A., & Liu, X. W. (2009). Age-dependent down-regulation of mitochondrial 8-oxoguanine DNA glycosylase in SAM-P/8 mouse brain and its effect on brain aging. Rejuvenation Research, 12, 209–215. Weigartner, O., Huscle, C., Schott, H. F., Speer, T., Bohm, M., Miller, C. M., et al. (2015). Vascular effects of oxysteroids and oxyphytosteroids in ApoE/ mice. Atherosclerosis, 240, 73–91. Wu, K., Higashi, N., Hansen, E. R., Lund, M., Bang, K., & Thestrup-Pedersen, K. (2000). Telomerase activity is increased and telomere length shortned in T cells from blood of patients with atopic dermatitis and psoriasis. The Journal of Immunology, 165, 4742–4747. Yi, X., Xu, L., Kim, K., Kim, H. S., & Maeda, N. (2010). Genetic reduction of lipoic acid synthase expression modestly increases atherosclerosis in male, but not in female, appolioprotein E-deficient mice. Atherosclerosis, 211, 424–430. Yu, E., & Bennett, M. R. (2014). Mitochondrial DNA damage and atherosclerosis. Trends in Endocrinology & Metabolism, 25, 481–487. Yu, E., Mercer, J., & Bennett, M. (2012). Mitochondria in vascular disease. Cardiovascular Research, 95, 173–182. Yu, E., Calvert, P. A., Mercer, J. R., Harrison, J., Baker, L., Figg, N. L., et al. (2013). Mitochondrial DNA damage can promote atherosclerosis independently of reactive oxygen species through effects on smooth muscle cells and monocytes and correlates with higher-risk plaques in humans. Circulation, 128, 702–712. Zeng, Z., Zhang, Z. Y., Yu, H., Corbley, M. J., Tang, Z., & Tong, T. J. (1999). Mitochondrial DNA deletions are associated with ischemia and aging in Balb/c mouse brain. Journal of Cellular Biochemistry, 73, 545–553. Zhang, S. H., Reddick, R. L., Piedrahita, J. A., & Maeda, N. (1992). Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science, 258, 468–471. Zhang, S., Gong, Z., Chen, W., Hu, H., Zheng, W., & Zhu, M. (2004). Frozen section staining method improvement and application in whole blood vessels and the heart of laboratory animal. Chinese Journal of Histochemistry and Cytochemistry, 13, 127–128.
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