42 ISHIDA T et al. Circulation Journal REVIEW Official Journal of the Japanese Circulation Society http://www.j-circ.or.jp Role of DNA Damage in Takafumi Ishida, MD, PhD; Mari Ishida, MD, PhD; Satoshi Tashiro, MD, PhD; Masao Yoshizumi, MD, PhD; Yasuki Kihara, MD, PhD

Patients with some progeroid syndromes, such as , exhibit atherosclerotic cardiovascular disease (CVD) at a young age as a manifestation of premature aging. Recent studies have revealed that most progeroid syndromes are caused by genetic defects in specific molecules involved in the DNA damage response, a corner- stone of genome stability. Ionizing radiation is one of the most potent genotoxic stimuli and causes various kinds of DNA damage. Further, there is increasing evidence that therapeutic radiation treatments can cause cardiovascular complications. Here, we describe the DNA damage and subsequent response, review recent advances in the un- derstanding of the molecular basis of progeroid syndromes (especially those syndromes that involve CVD), review the pathological and epidemiological analysis of radiation-induced CVD, and discuss the possible role of DNA dam- age and the DNA damage response in the pathogenesis of atherosclerotic CVD. (Circ J 2014; 78: 42 – 50)

Key Words: ; DNA repair; ; ;

rogeroid syndromes are rare genetic disorders that reca- duce the formation of pyrimidine dimers,8 and IR can also in- pitulate physiological aging at an accelerated rate. In duce oxidation of DNA bases, single-strand breaks (SSBs) and P 1995, Yosef Shiloh’s group identified ATM, the double-strand breaks (DSBs). Chemotherapeutic agents can mutated in ataxia-.1 The following year, the gene cause a variety of different DNA lesions, including alkylation responsible for Werner syndrome, WRN, which encodes a he- of bases, covalent links between bases of the same DNA strand licase, was identified by positional cloning.2 Since the time of (intrastrand crosslinks) or of different DNA strands (inter- those discoveries, the links between the DNA repair system and strand crosslinks), SSBs and DSBs. Cigarette smoke contains progeroid syndromes have been a major research focus in the genotoxic components and can cause a wide variety of DNA field of molecular genetics, gerontology and oncology. Most adducts and oxidative DNA damage. ROS, which are produced of these syndromes are caused by in encoding in the course of normal cellular metabolism, electron leaks in DNA repair proteins, and therefore, these syndromes involve the electron transport chains, various enzymes, IR and UV, can genome instability. Growing evidence suggests that molecules induce base modifications, abasic sites, protein-DNA adducts, that mediate the response to DNA damage and the subsequent intra-/interstrand DNA crosslinks,9 SSBs and DSBs. Mis-in- cellular outcomes of this process can also contribute to the corporation and erroneous insertion and deletion of bases can development of cardiovascular disease.3–7 Elucidation of the also arise during DNA replication and recombination, as well role of DNA damage and the DNA damage response in car- as during the repair of some forms of DNA damage.10 diovascular disease (CVD), especially in age-related athero- sclerosis is of particular interest. DNA Damage Response and Its Cellular Consequences Cells have an evolutionally conserved pathway, termed the DNA-damage response (DDR), that senses, transduces the DNA Damage and DNA Damage Response signal of, and repairs the damage with the goal of maintaining Genotoxic Stimuli and Types of DNA Damage genomic integrity. This repair response can arrest the cell cycle The human genome is under constant attack by endogenous in order to avoid propagating damaged DNA into daughter and exogenous factors, and the protection and faithful repair cells. Generally, DSBs are detected by the MRE11-RAD50- of genomic DNA is critical for cell survival. DNA damage is NBS1 (MRN) complex, which activates the primary kinase, caused by a wide variety of environmental agents [eg, ionizing ATM (ataxia-telangiectasia mutated), whereas SSBs are de- radiation (IR), (UV) radiation from sunlight, and tected by RPA and the RAD9-RAD1-HUS1 (9-1-1) complex, numerous genotoxic chemicals] and by the byproducts of nor- which recruit another primary kinase ATR (ataxia-telangiecta- mal cellular metabolism [eg, (ROS) sia and Rad3-related). ATM/ATR phosphorylate mediators and products of lipid peroxidation]. IR and UV light can in- and downstream kinases, including H2AX, CHK1/2, , and

Received September 30, 2013; revised manuscript received November 28, 2013; accepted December 1, 2013; released online December 12, 2013 Department of Cardiovascular Medicine (T.I., Y.K.), Department of Cardiovascular Physiology and Medicine (M.I., M.Y.), Graduate School of Biomedical and Health Sciences; Department of Cellular Biology, Research Institute of Radiation Biology and Medicine (S.T.), Hiroshima University, Hiroshima, Japan Mailing address: Takafumi Ishida, MD, PhD, Department of Cardiovascular Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. E-mail: [email protected] ISSN-1346-9843 doi: 10.1253/circj.CJ-13-1194 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

Circulation Journal Vol.78, January 2014 DNA Damage and CVD 43

BRCA1, to transduce DDR signaling.11 Multiple repair pathways have evolved to counteract DNA damage and each is directed to a specific type of lesion. To date, 5 major DNA repair pathways have been identified: (BER), nucleotide excision repair (NER), mis- match repair, (HR) and nonho- mologous end joining (NHEJ). If the DNA lesions are properly repaired, the cells resume normal proliferation after transient cell-cycle arrest. If the DNA damage is severe and/or remains unrepaired, the cells undergo or . DDR modulates many other cellular responses, including and chromatin re- modeling.

Repair of Single-Strand DNA Damage BER is the primary DNA repair pathway that corrects single lesion or small alteration of bases, such as oxidation, alkyla- tion, deamination and SSBs. The repair process occurs as fol- lows: (1) excision of a damaged or inappropriate base, (2) inci- sion at the resulting abasic site, (3) clean-up of termini to permit repair synthesis and/or nick ligation, (4) gap-filling, and (5) sealing of the final, remaining DNA nick.12,13 NER is a more complex process involved in the removal of a lesion containing oligonucleotides and is a particularly im- portant repair system for the removal of bulky DNA lesions.14 There are 2 types of pathway depending on the manner in which DNA damage is recognized: global genome repair (GGR) or transcription-coupled repair (TCR). GGR scans the genome constantly and repairs damage in inactive nontranscribed genes Figure 1. Repair of double-strand breaks (DSB) by nonho- mologous end joining (NHEJ). DSB are first detected by throughout the genome. By contrast, TCR is initiated when Ku70/80 subunits, which recruit the DNA-PK catalytic subunit RNA polymerase stalls at a lesion during active transcription. and assemble DNA-PK holoenzyme. The specific endonucle- In either system, after identification of a damaged site, the re- ases are recruited and process the damaged DNA ends. pair proteins are recruited to the site to confirm the presence of DNA polymerase fills in the gaps and the ligase seals the damage, excise the damaged DNA, and then fill in the gap. DNA nicks.

Repair of Double-Strand Breaks DSBs are life-threatening lesions that are repaired by a complex network of multiple DNA repair pathways. To date, at least 4 mammalian cells, the HR process is largely limited to the S- independent pathways are known to repair DSBs: HR, NHEJ, phase and to the repair of specific DNA lesions.16 By contrast, alternative-NHEJ, and single-strand annealing.15 Among these terminally differentiated cells or cells in the G0 or G1 phase pathways, NHEJ and HR are evolutionarily conserved DNA rely on NHEJ rather than HR. repair pathways and maintain genomic stability in response to 16 DSBs. The main differences in these pathways are the re- Progeroid Syndromes/ quirement for DNA homology (template) on the sister chroma- tid in HR, and the accuracy of the repair. In mammalian cells, Genomic Instability Syndromes NHEJ is the predominant pathway and is utilized throughout As mentioned before, most progeroid syndromes are caused the cell cycle.16 NHEJ repairs DNA damage by simply rejoin- by mutations in the genes encoding DNA repair proteins or by ing the ends of the DNA, and this process does not use a tem- mutations in genes encoding protein components of the nucle- plate to synthesize new DNA at the damaged sites (Figure 1). ar lamina. It is noteworthy that some syndromes predomi- Thus, NHEJ is less accurate and may give rise to some dele- nantly manifest with senescence, others are associated with tions (ie, error-prone repair). Important proteins involved in predisposition to , and some have both phenotypes (sum- NHEJ include the Ku70/80 heterodimer, DNA-dependent pro- marized in the Table). Some syndromes are of particular inter- tein kinase, catalytic subunit (DNA-PKCS), and the DNA li- est to the study of CVD, because affected patients exhibit gase IV/X-ray repair cross-complementing protein 4 (Xrcc4) premature atherosclerosis, dyslipidemia and . complex.13 HR is essential for preservation of genomic integrity. Mul- Werner Syndrome tiple regulatory mechanisms have evolved to ensure that HR Werner syndrome (WS) is a rare autosomal recessive disease occurs at the correct timing and in the correct context. HR re- that is highly prevalent in Japan.18 It is characterized by pre- quires a sister chromatid for template purposes (Figure 2). A mature onset of the signs of aging, which includes graying and central player in HR is Rad51, which scans the genome for an loss of hair, bilateral , -like changes, intact copy of the broken DNA on the sister chromatid.17 Dur- short stature, thin limbs, atherosclerosis, diabetes, osteoporo- ing HR, the missing information on the broken strand is copied sis, and a high incidence of .18,19 Symptoms ap- from the sister chromatid, so the damage is repaired without pear during the second or third decade of life, and the median loss of genetic information. HR can only take place in dividing age of death of affected patients is 46 years.20 The major cells, because of the necessity of a sister chromatid. In most causes of death in patients with WS are myocardial infarction

Circulation Journal Vol.78, January 2014 44 ISHIDA T et al.

Figure 2. Repair of DNA double-strand break (DSB) by homologous recombination (HR). See the main text for details. DSB are recog- nized by the Mre11–Rad50–Nbs1 complex, which is recruited to the DSB site to generate a single-stranded DNA by resection. The sin- gle-stranded ends are bound by Rad51 which polymerizes and searches the homology, and then invades the homologous template. DNA polymerases use the intact copy to re-synthe- size the deleted DNA sequences and DNA li- gases join the synthesized fragments.

(MI), stroke, and malignancies. Several lines of research have shown that cells derived from The gene underlying WS, WRN, was identified by positional WS patients and WRN knock-down cells are hypersensitive to cloning in 1996.2 The WRN protein contains a RecQ-type genotoxic reagents and display various types of chromosomal domain at the central regions and a 3’→5’ exonucle- abnormalities. These observations support the notion that the ase domain at the N-terminal region. This protein also shows WRN protein plays an important role in DNA repair.22,24 In- single-strand annealing activity, which means WRN is in- deed, WRN plays a pivotal role in DNA replication. Several volved in DNA replication, DNA repair, telomere mainte- studies suggest that WRN promotes recovery from replication nance, and transcription.21,22 Most mutations associated with fork stalling by preventing SSBs from being converted into WS lead to truncation of the WRN protein, but other mutations DSBs, regressing the fork, allowing bypass formation, and result in instability of the protein.19,23 resolving DNA structures at fragile sites. Further, WRN rap-

Circulation Journal Vol.78, January 2014 DNA Damage and CVD 45

Table. Progeroid Syndromes and Related Diseases Disease Genetic defect Affected cellular process Clinical phenotype Mean lifespan Ataxia-telangiectasia (AT*) ATM DSB repair (HR, NHEJ) Cerebellar ataxia 20 years Telangiectasia Immunodeficiency Cancer predisposition (Premature aging) (Insulin resistance) Hutchinson-Gilford progeria A Nuclear organization Premature aging 13 years syndrome (HGPS*) DSB repair Growth retardation Transcription Atherosclerosis No cancer predisposition Werner syndrome (WS*) WRN (RecQ helicase) DSB repair (HR, NHEJ) Premature aging ~50 years DNA replication Short stature Telomere maintenance Thin limbs Transcription Atherosclerosis Diabetes Cancer predisposition BLM (RecQ helicase) HR Growth retardation 27 years DNA replication UV sensitivity Immunodeficiency Cancer predisposition Rothmund-Thomson syndrome RecQL4 (RecQ helicase?) Unknown Premature aging Normal? Growth retardation Poikiloderma Cancer predisposition CSA, CSB Transcription-coupled NER UV sensitivity 12 years BER? Mental retardation Premature aging Kiphosis No cancer predisposition *AT, HGPS, and WS are discussed in detail in the main text. BER, base excision repair; DSB, double-strand breaks; HR, homologous recombination; NER, nucleotide excision repair; NHEJ, non-homolo- gous end joining; UV, ultraviolet.

idly accumulates at DSB sites. WRN-knockdown cells display findings suggest that WRN plays an important role in telomere increased amounts of DSB and enhanced DDR.25 These cells maintenance and that telomere attrition is a key mechanism in are sensitive to DSB-inducing reagents, such as hydroquinone. the pathogenesis of WS. As mentioned before, DSBs are repaired either by HR or It is noteworthy that WS, Bloom’s syndrome, and Rothmund- NHEJ. Several studies have shown that WRN is involved in Thomson syndrome exhibit some different clinical features but HR in concert with BRCA1, Rad51, and Rad52. WRN protein share overlapping phenotypes, despite the fact that the defec- also interacts with Ku and DNA-PKcs, which are key mole- tive proteins in all of these syndromes belong to the RecQ- cules involved in NHEJ. Additionally, in cells from patients like DNA helicase family. For example, patients with Bloom’s with WS, extensive deletion is generated at ends joined by syndrome do not show premature aging, whereas patients NHEJ.26 Collectively, these findings suggest that WRN plays with WS and Rothmund-Thomson syndrome do. In addition, a major role in DSB repair both by HR and NHEJ. Finally, Bloom’s syndrome and Rothmund-Thomson syndrome are several lines of evidence suggest that WRN also plays a major associated with sun-sensitivity, whereas this phenomenon is role in BER. For example, WS cells and WRN-knockdown not common in patients with WS (Table). Functional analysis cells accumulate more oxidative DNA damage when compared of these might provide further insight into the mo- with control cells; oxidative DNA damage is corrected by lecular mechanisms of genomic instability and the clinical BER, and WRN-knockdown cells display a defect in BER.27,28 manifestation of these helicase-associated syndromes. Telomeres are markedly shortened in WS cells, and the introduction of telomerase extends lifespan and reduces chro- Ataxia-Telangiectasia mosomal aberration in these cells.29–31 Double-null mice for Ataxia-telangiectasia is an autosomal recessive disorder1 that WRN and the telomerase RNA component (TERC), a compo- is characterized by progressive cerebellar ataxia because of nent of the telomerase complex, recapitulate clinical features neuronal degeneration, telangiectasia (dilated blood vessels) of WS patients, such as diabetes, cataracts, , hair in the conjunctivae and skin, immunodeficiency, predisposi- graying, alopecia and premature death,32,33 whereas WRN tion to lymphoreticular malignancies, premature aging, growth deficiency alone does not lead to premature aging.34 These retardation, and insulin resistance. Interestingly, heterozygotes

Circulation Journal Vol.78, January 2014 46 ISHIDA T et al. for the ataxia-telangiectasia mutated (ATM) allele have an mice show increased DNA damage and defects in DDR.46 increased risk of death from ischemic heart disease compared Foci of 53BP1 and the amount of γH2AX (phosphorylated with noncarriers.35 histone H2AX) are increased at basal state in HGPS cells and Ataxia-telangiectasia is caused by mutations in ATM, which tissue samples. In addition, ATM, ATR, and their downstream encodes the ATM protein. ATM is a serine/threonine kinase kinases, CHK2 and CHK1, are activated in HGPS cells, and and has significant homology to phosphatidylinositol 3-kinase these cells have elevated sensitivity to genotoxic reagents, (PI3K). Upon DSB induction, ATM rapidly localizes to DNA indicating that abnormally processed prelamin A causes an damage sites, and the kinase activity increases. ATM phos- increase in DNA damage, especially DSBs.45,47,48 Recruitment phorylates itself, MRN, CHK2, DNA-PK, p53, 53BP1, and of DDR proteins to DNA damage sites is delayed and sus- BRCA1, all of which are involved in DDR, including cell- tained after genotoxic stress. Thus, it is likely that HGPS cells cycle checkpoint, DNA repair, apoptosis, and senescence. possess some defects in the DNA repair machinery, especially Among these downstream targets of ATM, p53 modulates the for DSBs, whereas checkpoint signaling is preserved or even transcription of many target genes, and plays a key role in activated in these cells.47,49,50 The details regarding these de- DSB-induced cell senescence, apoptosis and cell-cycle arrest. fects in DNA repair have not been elucidated, and therefore, Experiments with ATM, p53-double-null mice suggest that it is largely unknown how causes abnormal DDR and there are also ATM-independent, p53-dependent pathways for the progeroid phenotype. Interestingly, a similar nuclear de- cell senescence and apoptosis.36,37 Cells from AT patients formity, and increased progerin and DSBs are also observed display a defective response to DSBs, increased chromosomal in normally aged individuals, implicating progerin in physio- breakages, sensitivity to IR, and cell-cycle checkpoint defects. logical aging.51 Thus, ATM likely functions as a trigger molecule for DDR and thereby promotes the maintenance of genomic stability and a reduction in the risk of cancer and other diseases. Role of DNA Damage in Atherosclerosis DNA Damage in Atherosclerotic Plaques Hutchinson-Gilford Progeria Syndrome Studies using human samples and animal models suggest that Hutchinson-Gilford progeria syndrome (HGPS) is a rare and atherosclerotic plaques contain accumulated DNA damage and severe premature aging syndrome. The incidence of this disor- activated DNA damage response elements. Mahmoudi et al der is approximately 1 in 4,000,000 live births. HGPS is char- reported that human atherosclerotic plaques have more DSBs acterized by symptoms of advanced aging, including severe and activation of ATM when compared with normal tissue.52 growth retardation, alopecia, lipodystrophy, scleroderma, de- Similarly, oxidative DNA damage is also increased with the creased joint mobility, osteolysis, and progressive atheroscle- marker for BER in human atherosclerotic plaques.53 These find- rosis. Death of affected children occurs at approximately 13 ings suggest that accumulation of DNA damage in atheroscle- years of age, mostly from MI or stroke. Other features of rotic plaques is mediated, at least in part, by ROS. Increased aging, such as malignancies, diabetes, and neural degeneration, apoptosis and senescence, which are possible consequences of are not common.38,39 the DNA damage response, have also been demonstrated in In most cases, HGPS is caused by a single de novo point the cellular components of atherosclerotic lesions.3,54,55 in the LMNA gene, which encodes lamin A/C.40,41 Lamin A is a major component of the nuclear lamina and is Atherosclerosis in Progeroid Syndromes thought to play a major role in maintaining the integrity of the As mentioned earlier, the main causes of death of WS patients and in the regulation of transcription and are MI and stroke. Considerable atherosclerotic plaque with replication. Lamin A is translated from LMNA as a precursor calcium deposition is present in the coronary arteries and aorta prelamin A. Prelamin A is modified several times, including of patients with WS.56,57 The precise mechanism by which farnesylation, methylation, and cleavage twice by the endo- WRN mutation results in accelerated atherosclerosis in the peptidase, ZMPSTE24. Mature lamin A does not contain the affected individuals remains unclear. Recently, Okabe et al farnesylated Cys residue. The most common mutation found reported that metabolic disorders, such as diabetes mellitus, in HGPS is G608G (GGC>GGT) within 11. Although hyperlipidemia, and hypertension, are closely related to athero- this single-base substitution is silent in terms of the coding sclerotic vascular disease in patients with WS.58 In addition, sequence, it activates a cryptic splice site and causes the re- experiments using a mouse model of WS, in which the helicase moval of 150 nucleotides. The resultant mutant prelamin A is domain of WRN is deleted, showed increases in visceral fat, internally deleted near the C-terminus, and is called progerin. and of fasting triglyceride and cholesterol levels followed by Progerin lacks the second cleavage site, and as a result, cannot insulin resistance, all of which closely recapitulate the pheno- be correctly processed and remains farnesylated. Fibroblasts type of human metabolic syndrome.59 Thus, metabolic abnor- from HGPS patients show nuclear abnormalities, such as blebs malities may be related to accelerated atherosclerosis in WS. of the nuclear envelope, thickening of the nuclear lamina, and Abnormalities in the immune system, extracellular matrix, and clustering of nuclear pores.40–42 Mice lacking ZMPSTE24 also the coagulation system may also be involved in accelerated exhibit progeroid phenotypes and these nuclear abnormalities, atherosclerosis and subsequent fatal vascular events in patients whereas mice expressing nonfarnesylated prelamin A do not with WS.18,60 The direct effects of functional defects on vascu- exhibit progeria,43,44 suggesting that premature aging and nu- lar cells, such as vascular smooth muscle cells (VSMCs) and clear abnormality in HGPS patients are, at least in part, result- endothelial cells, have not been clearly demonstrated in pa- ing from accumulation of farnesylated progerin. Of note, p53 tients with WS. target genes are markedly upregulated in Zmpste24-deficient Human ATM heterozygotes have increased risk of death mice, and organismal accelerated aging and cellular senes- from ischemic heart disease.35 Studies using mouse ATM cence phenotypes are partially recovered by p53 deficiency, heterozygotes have also shown features of metabolic syn- which suggests activation of p53 plays pivotal role in the ac- drome, including increased visceral fat, insulin resistance, and celerated aging observed in HGPS.45 high blood pressure.4,61 ATM deficiency promotes atheroscle- Importantly, cells from HGPS patients and Zmpste24–/– rosis in apoE–/– mice, which was attenuated by transplantation

Circulation Journal Vol.78, January 2014 DNA Damage and CVD 47 of the bone marrow of wild-type mice. Tissues from ATM+/– radiation accelerates atherosclerosis. For example, incidental showed increased mitochondrial DNA damage and ROS, and exposure of the heart to IR during the course of radiotherapy reduced oxidative phosphorylation, as well as increased nucle- for breast cancer increases the rate of coronary artery dis- ar DNA damage. Although the precise mechanisms are not ease,70,71 and women with preexisting coronary risk factors fully elucidated, it appears that acceleration of atherosclerosis have greater absolute increases in the risk of coronary artery by ATM deficiency involves direct effects on vascular wall disease from radiotherapy than women without those risk fac- cells and disturbance of systemic metabolism resulting from tors. Similarly, radiation therapy for brain tumors or for head- both nuclear and mitochondrial DNA damage and increased and-neck increases the risk of stroke.72 ROS.4,61–63 The Life Span Study, a cohort study of atomic bomb survi- Similar to WS, atherosclerotic CVD, such as MI and stroke, vors in Hiroshima and Nagasaki for more than 50 years, dem- is the main cause of death in patients with HGPS. However, the onstrated that radiation doses above 0.5 Gy increase the risk of histological features of vascular change vary among the pub- heart disease and stroke.70,73 The estimated increase in risk for lished studies,38 probably because the diagnosis of HGPS in heart disease is 14%/Gy, which is higher than that seen in those these few studies was not consistently based on genetic testing, women who underwent radiotherapy for breast cancer. The and therefore, non-HGPS cases may have been included in the Adult Health Study, which consists of biennial examinations patient populations. Collectively, the vascular lesions of HGPS of approximately 15% of the Life Span Study cohort members can be characterized as those of conventional atherosclerosis, since 1958, has shown radiation dose-related increase in the except that VSMCs are lost in the media, and the lipid core and prevalence of hypertension, elevated serum cholesterol, and inflammation are not as profound. The thickened intima and aortic arch calcification.74–76 In addition, studies of atomic thinned media in patients with HGPS is generally hypocellular, bomb survivors have revealed that radiation also causes persis- possibly because of degeneration of VSMCs and replacement tent increases of inflammatory response markers, such as inter- by fibrosis.64–66 These features may be caused by the absence leukin 6, CRP, and interferon γ, and alteration of T- and B-cell of other overt risk factors, such as diabetes or hyperlipidemia composition.77,78 Chronic inflammation, as well as increased (in contrast to WS) and are reminiscent of vascular changes in blood pressure and serum cholesterol, may partially be respon- the elderly. Recently, Olive et al5 described adventitial fibrosis sible for the increased rate of cardiovascular events in atomic in the arteries from 2 patients with genetically determined bomb survivors. HGPS. The classic features of atherosclerosis, such as necrotic Experimental studies have provided insights into the mech- core, foam cells, and foci of chronic inflammation were ob- anisms of accelerated atherosclerosis in response to radiation. served in those samples. In addition, progerin was detected in Stewart et al showed that a single exposure to 14 Gy of radia- the media, plaques, and adventitia of the coronary arteries (and tion resulted in acceleration of atherosclerosis, as characterized preferentially in VSMCs and fibroblasts) in HGPS patients. A by a high proportion of macrophages and granulocytes and by mouse model of HGPS exhibited severe loss of VSMCs and intraplaque hemorrhage, in apoE–/– mice.79 Fractionated radia- diminished response to a nitric oxide donor, sodium nitroprus- tion (2 Gy ×20) had similar effects,80 and studies suggest that side, and these findings mimic the vascular abnormalities seen radiation-induced atherosclerosis is characterized by accelera- in HGPS patients.67 The role of thrombosis in cardiovascular tion of initial lesions and exacerbation of local inflammation. events in patients with HGPS is unclear. No obvious occlusion These clinical and experimental studies support the notion by thrombus has been documented, although healed plaque that IR has direct and harmful effects on the arterial wall. How- rupture was observed.5,64 In contrast, total or subtotal occlusion ever, there is no firm evidence that definitively proves that by unruptured plaque or fibrous intima has been document- DNA strand breaks and/or the subsequent DDR are involved ed.5,68 Further pathohistological studies are required to fully in the acceleration of atherosclerosis, despite the fact that ra- characterize the vascular lesions of HGPS. diation doses in the context of radiation therapy or the atomic Recently, 2 laboratories have generated induced pluripotent bomb are sufficient to induce DNA damage. IR produces ROS, stem cells (iPSCs) from fibroblasts that were isolated from and therefore, it can induce vascular via oxidative HGPS patients. A study by Zhang et al demonstrated that stress. Further in vivo and in vitro studies are needed to eluci- progerin is expressed selectively in iPSC-derived mesenchy- date the precise molecular mechanisms by which radiation mal stem cells, VSMCs, and fibroblasts, but not in endothelial accelerates atherosclerosis and increases the risk of coronary cells or iPSCs.69 These lineages exhibit nuclear deformity, an artery disease and stroke after radiotherapy. increase in DSBs, and premature senescence in response to hypoxia or other stressors. Liu et al reported similar findings and a shorter telomere length in VSMCs derived from HGPS- Other CVD Associated With DNA Damage iPSCs compared with those derived from control iPSCs.50 HGPS patients display several other cardiovascular abnormali- Additionally, they showed that progerin interacts with DNA- ties. Thickening and calcification of the aortic and mitral valves PKcs, a key enzyme involved in DSB repair. The amounts of are common, and studies have found that progerin accumulates DNA-PKcs, and its regulatory subunits, Ku70/Ku80, are down- within these altered structures. Other abnormalities include regulated in HGPS-VSMCs. A clinical study by Meredith et general hypertrophy of the left ventricle, interstitial fibrosis, al showed that flow-mediated dilatation in HGPS patients was and patchy MI. The fibrosis, which is prominent in the endo- comparable to that in normal controls, indicating that endothe- cardium, may be caused both by local ischemia and altered lial function is preserved in HGPS.39 Together, these findings expression of the components of extracellular matrix.5,38,39,66 suggest that mesenchymal-lineage cells are selectively af- Cardiac fibrosis and aortic stenosis are observed in a mouse fected in HGPS and that VSMCs are the main target of proger- model of WS. Interestingly, the level of hydrogen peroxide is in accumulation. increased in both the serum and the cardiac tissue of these mice, which is followed by accumulation of oxidative DNA Radiation-Induced CVD damage in the heart.59 The incidence of aortic stenosis or car- IR causes various type of DNA damage, including SSBs, diomyopathy is uncertain in WS patients. DSBs, and base damage. Accumulating evidence suggests that Besides accelerating atherosclerosis, IR can cause pericar-

Circulation Journal Vol.78, January 2014 48 ISHIDA T et al.

Figure 3. Possible role of DNA damage/DDR in the development of atherosclerosis. Cardiovascular risk factors, organismal aging, and ionizing radiation increase DNA damage in vascular wall cells. Accumulated DNA damage because of incomplete or incorrect repair leads to persistent DDR, telomere attrition, and genomic instability, which drive the cells to undergo senescence and apop- tosis. Senescent cells secrete proinflammatory cytokines and chemokines, and also do not function normally. These phenomena accelerate atherosclerosis and/or increase plaque instability.

ditis, pericardial fibrosis, and myocardial fibrosis.70 Some senescence, apoptosis and dysfunction over time. Addition- studies suggest that valvular disease is also induced by radia- ally, the cells that senesce because of persistent DNA damage tion.81,82 response secrete proinflammatory cytokines, chemokines, growth factors, and proteases, termed the senescence-associ- ated secretory phenotype (SASP), which fuels chronic inflam- Conclusions and Future Perspectives mation.83 These phenomena may accelerate atherosclerosis DNA damage and the consequences of the DNA damage re- and/or increase plaque vulnerability (Figure 3).84,85 It is likely sponse (eg, cellular senescence and apoptosis) are present in that these cellular events target other tissues, including adipose atherosclerotic plaques. Studies of progeroid syndromes sug- tissue, as well as the vasculature.86,87 Thus, DNA damage/ gest that accumulated DNA damage causes persistent activa- DNA damage response could be therapeutic targets for age- tion of the DNA damage response, telomere attrition, and related CVD. DNA damage and progerin levels are increased genome instability, which, in turn, lead to progressive cellular in the normally aged vasculature, although the mechanisms by

Circulation Journal Vol.78, January 2014 DNA Damage and CVD 49 which progerin increases DNA damage and/or alters DNA 344. repair remain to be elucidated. Further characterization of the 23. Moser MJ, Oshima J, Monnat RJ Jr. WRN mutations in Werner syn- defective proteins in progeroid syndromes that contribute to drome. Hum Mutat 1999; 13: 271 – 279. 24. Singh DK, Ahn B, Bohr VA. Roles of RECQ helicases in recombina- the DNA damage response will provide insight into the mo- tion based DNA repair, genomic stability and aging. Biogerontology lecular mechanisms underlying progression of atherosclerosis 2009; 10: 235 – 252. with aging. 25. Galvan N, Lim S, Zmugg S, Smith MT, Zhang L. Depletion of WRN enhances DNA damage in HeLa cells exposed to the benzene me- tabolite, hydroquinone. Mutat Res 2008; 649: 54 – 61. Acknowledgments 26. Oshima J, Huang S, Pae C, Campisi J, Schiestl RH. 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