Ionizing radiation and heart risks

Accepted Manuscript Title: Ionizing radiation and heart risks Author: Souparno Bhattacharya Aroumougame Asaithamby PII: DOI: Reference:

S1084-9521(16)30042-8 http://dx.doi.org/doi:10.1016/j.semcdb.2016.01.045 YSCDB 1956

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Seminars in Cell & Developmental Biology

Received date: Revised date: Accepted date:

27-10-2015 7-1-2016 29-1-2016

Please cite this article as: Bhattacharya Souparno, Asaithamby Aroumougame.Ionizing radiation and heart risks.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2016.01.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ionizing Radiation and Heart Risks Souparno Bhattacharya and Aroumougame Asaithamby*

Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75390.

*Corresponding author Division of Molecular Radiation Biology Department of Radiation Oncology University of Texas Southwestern Medical Center Dallas, Texas 75390 Phone: 214-648-5175 E-mail: [email protected] Fax: 214-648-5995

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Highlights   

Ionizing radiation exposure contributes to the development of cardiovascular diseases. Persistent reactive oxygen species may promote radiation induced heart complications. Alleviation of reactive oxygen species may prevent radiation induced cardiotoxicity.

Abstract Cardiovascular disease and cancer are the two leading causes of morbidity and mortality worldwide. As advancements in radiation therapy (RT) have significantly increased the number of cancer survivors, the risk of radiation-induced cardiovascular disease (RICD) in this group is a growing concern. Recent epidemiological data suggest that accidental or occupational exposure to low dose radiation, in addition to therapeutic ionizing radiation, can result in cardiovascular complications. The progression of radiation-induced cardiotoxicity often takes years to manifest but is also multifaceted, as the heart may be affected by a variety of pathologies. The risk of cardiovascular disease development in RT cancer survivors has been known for 40 years and several risk factors have been identified in the last two decades. However, most of the early work focused on clinical symptoms and manifestations, rather than understanding cellular processes regulating homeostatic processes of the cardiovascular system in response to radiation. Recent studies have suggested that a different approach may be needed to refute the risk of cardiovascular disease following radiation exposure. In this review, we will focus on how different radiation types and doses may induce cardiovascular complications, highlighting clinical manifestations and the mechanisms involved in the pathophysiology of radiation-induced cardiotoxicity. We will finally discuss how current and future research on heart development and homeostasis can help reduce the incidence of RICD. Abbreviations: RT- Radiation therapy; IR- Ionizing radiation; LET- Linear energy transfer; RICDRadiation induced cardiovascular disease; DDR- DNA damage response; DSB- Double strand breaks; ROS- Reactive oxygen species; CAD- Coronary artery disease; CHFCongestive heart failure; HZE- High-charge and -energy; CT- Computed tomography Keywords: Ionizing radiation, heart disease, DNA damage, reactive oxygen species, cardiomyocyte renewal; Space Radiation

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1. Background Cardiovascular disease is the world’s leading cause of mortality [1], affecting over 62 million people in the United States alone [2], and accounting for 31.5% percent of all deaths worldwide [3]. Cancer survivors who underwent high dose radiation therapy (RT) are known to be at risk of developing cardiovascular disease later in life [4]. In addition to relatively high doses of therapeutic ionizing radiation (IR), occupational or accidental exposure to low IR doses also increases the risk of nonischemic heart disease [5, 6]. The pathophysiological basis of heart failure lies in the inability of the adult heart to regenerate the lost or damaged myocardium following an injury. For a long time, a consensus in the cardiovascular biology field was that the adult mammalian heart does not effectively regenerate itself once development is complete [7]. However, recent reports have suggested that limited cardiomyocyte turnover occurs after the proliferation of pre-existing cardiomyocytes. Bergman et al. performed an elegant set of experiments using a retrospective birth dating technique to determine cardiomyocyte renewal. By systematically measuring the integrated

14C

from nuclear bomb tests during cold war in

the genomic DNA of human myocardial cells, they concluded that the rate of cardiomyocyte turnover in the adult human heart is age-dependent, with rates of around 1% at the age of 25 and around 0.5% after 40 years of age [8]. While this turnover rate is insufficient for complete heart regeneration after an injury, it contributes to the slow but constant replacement of dead or damaged myocytes under normal physiological conditions. The human heart can replace about 50% of cardiomyocytes throughout its lifespan. Therefore, inhibition of cardiomyocyte turnover can result in heart failure, even in the absence of increased cell death. Events or factors responsible for the regulation of cardiomyocyte renewal are also poorly understood. Over the years, several direct and indirect regulators of cardiomyocyte cell cycle have been identified, including E2F1, ErbB4, mir-17-92, mir590, mir-199a, mir-15 family, c-Myc, CDK2, cyclinG1, Meis-1, insulin like growth factor1, Rb-p130 and hippo pathway effector Yap [7-17]. While all these factors contribute to the regulation of cardiomyocyte renewal to varying degrees, the upstream signal implicated 3

in permanent cell-cycle arrest of cardiomyocytes has remained unknown. Recent work by Puente et al. identified increased oxidative stress as the upstream factor limiting cardiomyocyte proliferation [9]. Their data showed that after birth metabolism switches from glycolysis to oxidative metabolism to meet an increased demand for energy. The result of this metabolic switch is enhanced oxidative stress which ultimately leads to an increased amount of DNA damage, culminating in cardiomyocytes exiting the cell cycle [9]. This finding indicates that DNA damage response (DDR) signaling plays a pivotal role in regulating the proliferation potential of adult cardiomyocytes.

In this article, we will briefly introduce the concept of IR and how the heart can be exposed to it. We will discuss the known effects of IR exposure on the cardiovascular system and the possible mechanisms driving the pathophysiology of radiation-induced cardiovascular toxicity. We will also explore the contribution of DNA damage and oxidative stress to disease development. Finally, we will briefly discuss how our current understanding of ROS signaling can be exploited to reduce radiation induced heart risks.

2. Ionizing radiation and DNA damage response signaling

Ionizing radiation (IR) is a type of electromagnetic wave or particle that removes tightly bound electrons from an atom, causing it to become ionized. The most common unit of measurement of IR is the gray (Gy) which quantifies the “absorbed dose”. The Gy unit is defined as 1 joule of initial energy per kilogram of tissue. However, to compare the biological effectiveness of different IR types exhibiting the same absorbed dose, another unit called Sievert (Sv) measuring the “effective dose” is used. While 1 Gy of x-ray or γray is equal to 1 Sv, relationships involved in higher energy radiations like proton or alpha radiation are more complex (e.g. 1 Gy of proton radiation is equivalent to 10 Sv) [Sources and Effects of Ionizing Radiation, UNSCEAR report to general assembly, 2000].

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DNA damage is the most important effect of radiation. Most IR-induced DNA lesions are base modifications such as 8-OxoG [10]. Additionally, IR causes DNA breaks, 1 Gy IR induces ~1,000 single-strand (SSBs) and 35 double-strand (DSBs) breaks per cell in vitro/in silico [11]. Although DSBs only constitute a minority of DNA lesions induced by IR, they can lead to cytotoxic, mutagenic and carcinogenic effects if unrepaired or misrepaired [12-14]. To ensure genomic integrity, cells have evolved sophisticated mechanisms to repair DSBs. The two major DSB repair pathways are non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ is an error-prone repair pathway that can occur throughout all cell cycle phases, whereas HR is an error-free pathway that predominantly occurs in late S and G2 phases.

In response to DNA breaks, cells initiate a series of signaling pathways, namely DDR signaling, mediated by members of the phosphatidylinositol 3-kinase (PI3K)-like kinase (PIKK) family, ATM, ATR (ataxia telangiectasia and Rad3-related protein), and DNA-PK (DNA-dependent kinase). ATM plays a critical role in DNA damage signaling originating at DSBs, whereas ATR responds to single-stranded DNA (ssDNA) regions associated with replication forks. DNA-PK is directly involved in DNA repair by promoting DSB religation through NHEJ. DDR signaling contributes to repair of DNA breaks, transient cell cycle arrest, transcriptional and post transcriptional activation of a wide array of genes and, under certain circumstances, it triggers programmed cell death. Faithful repair of DNA lesions and activation of DDR signaling are critical for cellular survival and prevention of genomic instability.

In addition to directly inducing nuclear DNA damage, IR exposure leads to increased production of reactive oxygen species (ROS) that may damage nucleic acids, proteins and lipids [Radiobiology for the Radiologist 7th Edition by Hall, Eric J., Giaccia, Amato]. Furthermore, ROS levels continue to rise even after initial radiation exposure leading to cellular oxidative stress [15]. Radiation-induced oxidative stress may also spread from targeted cells to bystander neighboring cells [16]. Radiation exposure also affects other cellular organelles [17, 18]. Examples of this influence include mitochondrial mass and 5

function changes in gene and protein expression [19]. Radiation also results in excess production of ROS by mitochondria which can be deleterious for cells, resulting in various diseases [20]. Thus, IR induces damage in nuclear and extranuclear DNA (e.g. mitochondria) not only by direct deposition of energy but also by excess production of ROS. Enhanced ROS levels induce cellular oxidative stress even long after radiation exposure, often limiting cellular proliferation and inducing cell death or apoptosis.

3. Ionizing radiation and cardiovascular complications

Humans are continually exposed to ionizing radiation (IR) from different sources, including ubiquitous background radiation, during routine medical procedures and radiotherapy (Table 1). Due to the advent of medical imaging techniques in the last twenty years, the average annual effective dose per individual in the U.S. population (excluding RT) has increased by a factor of 1.7 [5]. A fundamental law of radiobiology (“Law of Bergonie and Tribondeau”, 1906) states that highly differentiated organs with low mitotic activity are radioresistant. The heart is regarded as the prototype radioresistant organ because of its limited proliferative capacity and abundance of postmitotic cardiac myocytes. However, the heart and blood vessels are actually not radio resistant and are considered among the most dose-limiting organs [21, 22]. Indeed, radiation-induced heart disease occurs due to both occupational and non-occupational radiation exposures (Table 2). 3.1. Atomic bomb survivors: Most of the information regarding accidental low dose radiation exposure and cardiovascular disease derives from studies performed among Japanese atomic bomb survivors in Hiroshima and Nagasaki. The radiation from the Hiroshima (235U) and the Nagasaki (239Pu) atomic bombs was comprised of neutrons and γ-rays, with survivors absorbing as much as 4 Gy of radiation [23]. A life span study conducted among atomic bomb survivors, who had received an acute single dose of 1-2 Gy, showed increased mortality from myocardial infarction 40 years after radiation exposure. The risk of cardiovascular disease-related death increased by 17% per Gy of whole body irradiation dose received (range 0-4 Gy) [24]. Another independent study by 6

Yamada et al. found a positive dose response relationship between the radiation dose and incidence of myocardial infarction in radiation-exposed survivors below 40 years of age [25]. According to this study, 16% of myocardial infarctions could be attributed to radiation exposure from the atomic bomb. Most of these cases are result of exposure to radiation doses >1 Gy. Another study carried out in atomic bomb survivors reported that exposure to radiation as low as 0.5-2 Gy may increase the risk of stroke, heart disease and other circulatory diseases [6].

3.2. Occupational radiation exposure: A study conducted among radiologists and nuclear power reactor workers in the United States indicated that occupational exposure to low doses of radiation increases the risk of circulatory disease-related mortality [26, 27]. These findings were supported by two more independent studies; first, the Chernobyl nuclear power plant accident in 1986 which released radiation in the environment coming from different radionuclides, most notably

131I, 137Cs, 90Sr,

resulting

into β- and γ-ray emission. The average dose received by the Russian workers involved in the cleanup of the accident was 10-150 mSv. These workers were found to be at increased risk of ischemic heart disease and cardiovascular disease [28]. Second, a significant association between dose and mortality from ischemic heart disease was also found among employees of British Nuclear fuels [29]. Finally, a recent report by Little et al. measured mortality risks for circulatory disease from exposure to low-level IR and found a positive correlation between circulatory disease-related mortality and low to moderate doses of IR exposure [30].

3.3. Medical radiation exposure: Medical imaging for diagnostic purposes (computed tomography, interventional radiology, and nuclear medicine) results in significant background radiation exposure (3.0 mSv which is 48% of total US background radiation; 6.4 Gy in 2006) [National Council on Radiation Protection and Measurements, 2006]. Of all procedures, computed tomography (CT) scanning involves large doses of radiation (10-15 mSv in case of cardiac CT) and accounts for 75% of total doses from medical radiation [5, 31]. Although long term effects of CT scanning on the development of 7

cardiovascular disease remain unknown, data from other low dose radiation-related epidemiological studies suggest that CT scanning may increase the risk of cardiovascular disease later in life. Multiple published reports also demonstrate the relationship between low dose IR and late occurring cardiovascular disease [30, 32]. In particular, a report by the Health Protection Agency in the United Kingdom estimated that risk of circulatory disease increases above 0.5 Sv (AGIR, Circulatory Disease Risk, Report of the Independent Advisory Group on Ionising Radiation, London: Health Protection Agency). However, these results presented high heterogeneity in the risk estimation by different groups, which can at least be partially explained by differences in lifestyle among subjects. Taking all medium- and low- dose studies together, the UK Health Protection Agency estimates an excess relative risk (ERR) per Sv of 0.09 (95% CI: 0.07, 0.12) indicating a small but strong evidence against the null hypothesis (AGIR, Circulatory Disease Risk, Report of the Independent Advisory Group on Ionising Radiation, London: Health Protection Agency). Thus, these studies suggest that cardiovascular complications may arise from occupational or environmental exposure to low dose IR.

3.4. Galactic cosmic rays: Recent evidence on the effects of low IR doses on the heart and the cardiovascular system has raised concerns about risks associated with long duration space travel. Galactic Cosmic Rays (GCRs) are the main source of radiation exposure to astronauts traveling in space. GCRs primarily consist of protons, helium nuclei (Z=2), and higher atomic number particles such as iron (Z=26). The energy of these particles can be very high (1000 MeV/nucleon or more) and in many cases powerful enough to penetrate spacecraft hulls and interior materials. Because of highlinear energy transfer (LET), the physical dose needed to produce a biological effect from high-charge and -energy (HZE) particle radiation is much lower compared to γ-rays [5]. Carcinogenesis is the most common risk factors among astronauts travelling in space and exposed to high-LET space radiation [33]. However, based on a NASA report published in August 2013, heart attack is the second leading cause of death (after non-flight accidents) in astronauts who participated in the Apollo 11, 12, and 14– 8

17 Moon missions [34]. In a recent report, Cucinotta et al. estimated that the inclusion of cardiovascular and circulatory disease will increase the percentage risk of GCR exposure-induced death by about 40% compared with that from carcinogenesis alone [35]. For a 1000-day space mission to Mars it is estimated that every cell in the body will be exposed to high energy proton every 3 days and helium every 30 days [36, 37]. Additionally, at least 3.2 x 1012 cell nuclei will be exposed to iron (56Fe) ions during the course of the journey [38]. The resulting physical radiation dose incurred is estimated to be 0.4 Gy, corresponding to 1.1 Sv [38].

Previous reports showed degenerative changes in coronary arteries of mouse orbital regions exposed to a single dose of 0.1 or 0.2 Gy of

56Fe

particles [39]. This suggests

that the dose incurred during space travel is sufficient to cause cardiovascular complications. Yu et al. reported that exposure of male apolipoprotein E-deficient (ApoE−/−) mice to 2 to 5 Gy 56Fe ions can accelerate the development of atherosclerosis in irradiated portions of the aorta. Irradiated mice also showed rapid progression to advanced disease, accompanied by aortic foot lesions, thickening of the carotid artery and larger necrotic cores [40]. Furthermore, HZE-particle radiation has been shown to have detrimental effects on the rat endothelium. Soucy et al. observed increased aortic stiffness and endothelial dysfunction in rats exposed to 1 Gy of

56Fe

particle radiation

[41]. In another study, Tungjai et al. examined the effects of silicon ( 28Si) ions, another type of HZE particle found in GCR, on heart tissue. Adult male CBA/CaJ mice exposed to different doses of

28Si

ions radiation showed increased apoptotic cell death and

inflammatory responses in heart tissue up to 6 months after radiation exposure [42]. Moreover, HZE-particle radiation exposure also impairs blood vessel formation or angiogenesis [43-47]. Furthermore, Grabham et al. used an endothelial cell threedimensional culture model to show that low-LET HZE particles inhibit early stages of vasculogenesis while high-LET HZE particles affect later stages of the disease, thereby preventing migration of endothelial cells to form tubes [48]. Yan et al. performed whole body irradiation in mice with 50 cGy of 1 GeV/nucleon protons and found increased cardiac hypertrophy and fibrosis, ultimately leading to heart failure 10 months after 9

irradiation [34]. In contrast, exposure to a lower dose of

56Fe

particle (15 cGy of 1

GeV/nucleon) had a negative impact on homeostasis and cardiac function only 1 month after irradiation [34]. Interestingly, proton-irradiated mice affected by acute myocardial infarction (AMI) at different times post-irradiation did not exhibit any negative effects during post-AMI recovery. In contrast, 56Fe ion irradiation negatively affected recovery of the heart from an adverse cardiac event [34]. These observations suggest that more than one factor may contribute to RICD including radiation type, time after radiation exposure and presence or absence of additional cardiovascular complications. However,

the

calculation

of

the

relative

biological

effectiveness

(RBE)

for

cardiovascular/circulatory disease has been challenging due to the scarcity of valid experimental model systems. 3.5. Radiotherapy: RICD is a prominent and serious side effect of radiation exposure and occurs as a consequence of treating tumors in the thoracic region (e.g. lung cancer, esophageal cancer, mediastinal lymphoma and breast cancer) [49-51]. Patients, receiving high doses of radiation during radiotherapy (RT), may exhibit myocardial damage and injury to the cardiac vasculature [52]. The extent of the damage depends on both radiation dose and irradiated volume. Recent advancements in RT and the development of sophisticated techniques, especially image guided therapy, have reduced radiation exposure to the heart but despite the progress made the mean radiation doses are still high. Taylor and colleagues reported that during tangential breast RT, both the heart and the left anterior descending artery received a high average dose of radiation, 2.3 Gy and 7.6 Gy, respectively, with certain components of the heart receiving as much as 20 Gy of radiation [53]. In accordance with these findings, a vast number of reports suggest that the risk of cardiac disease is greater in patients exposed to radiation at a young age (e.g. pediatric cancer patients). In Hodgkin’s lymphoma patients cardiac radiation exposure to 1500 centigray or more increased the relative hazard of congestive heart failure by 6-fold [54]. In addition, the cumulative incidence of adverse cardiac outcomes in cancer survivors continues to increase up to 30 years after diagnosis [54]. Indeed, cardiovascular morbidity and mortality are significantly higher in long term survivors of various childhood cancers [55], 10

and survivors are 5 to 10 times more likely than their healthy siblings to experience heart disease later in life [56]. A study conducted among irradiated Hodgkin’s lymphoma pediatric patients found the patients to be at increased risk of developing cardiovascular diseases, with a 13.1 excess absolute risk per 10,000 person years for cardiovascular death [57]. Studies also showed irradiated Hodgkin’s disease patients have increased chances of coronary artery bypass grafting, coronary intervention, pacemaker implantation, pericardial surgery and heart failure, as well as higher mortality rate [58, 59]. Tukenova et al. [60] evaluated long-term risk of death as a result of cardiac diseases in childhood cancer survivors previously treated with RT; they found a high long-term risk of cardiovascular disease-related death among patients who received a radiation dose greater than 5 Gy [60]. Furthermore, they also observed a linear relationship between the average dose of radiation to the heart and the risk of mortality due to cardiac complications [60].

Based on a limited number of studies performed among adult cancer survivors, women treated for left sided breast cancer are at higher risk of developing cardiovascular disorders when compared with women treated for right sided breast cancer [61]. This result was further corroborated by another study performed in 35000 breast cancer patients (the mean dose to the whole heart was 6.3 Gy and 2.7 Gy for left and right sided tumors, respectively). While no difference in mortality rate was observed in women exposed to radiation in either left or right sides, the incidence ratio of heart disease was increased in those with left sided tumors [62]. Another independent study conducted in 2168 European women, who had received RT for breast cancer (19582001), found that the risk of cardiovascular disease (e.g. myocardial infarction, coronary revascularization and death from ischemic heart disease) increased linearly with the mean dose delivered to the heart (overall average of the mean doses to the whole heart was 4.9 Gy) [63]. The magnitude of the risk was 7.4% per Gy, with no apparent risk threshold. The risk started to increase within the first 5 years after exposure and continued for at least 20 years [63].

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In addition to cancer treatment, RT for other nonmalignant diseases also increases the chances of developing cardiovascular disorders. For example, a study conducted at the University of Chicago indicated that peptic ulcer patients treated with fractionated RT had increased risk of coronary heart disease and relative mortality at a dose of just 2.6 Gy to the heart [64]. In 2001, the Hyogo Ion Beam Medical Center in Japan became the first institution in the world to provide both proton therapy and carbon-ion therapy for cancer treatment [65]. Due to a unique pattern of energy deposition as a function of depth in tissue, hadron (proton and neutron) and heavy ion (carbon) therapy are considered superior to conventional RT. Because of high-LET, the physical dose needed to produce a biological effect from proton, helium or carbon irradiation is lower as compared to X-rays (photons). To our knowledge, no study has been conducted in human to estimate the risk of cardiovascular disease following proton, helium and carbon radiation exposure. Additional human subject trials are needed to determine the RBE of proton, helium and carbon radiation and determine their potential role in the promotion of cardiovascular disease.

4. Mechanism of radiation-induced cardiac toxicity

Radiation-induced cardiovascular disease (RICD) is a progressive disorder, which may take years to decades to manifest [52, 66]. Also, it can affect all structures of the heart, including the pericardium, the myocardium, the valves and the conduction system [49, 67]. Pathological changes after exposure to RT which contributes to the clinical outcome is well documented however, the underlying biological mechanisms promoting debilitating chronic heart problems from early asymptomatic conditions is still unclear. It is accepted that majority of the early damage appears to be from acute and chronic inflammatory changes, which leads to vascular dysfunction, cardiac remodeling and atherosclerosis. In addition, persistent or irreparable DNA lesions, dysfunctional telomeres, persistent oxidative stress also contribute to the pathogenesis of RICD. Furthermore, the development of the disease may depend on the interaction of different genetic and environmental factors as well (Figure 1 and Table 3). Understanding the 12

biological mechanism and characterizing their relative contribution to pathogenesis of RICD will be an important step in evaluation of viable target of therapeutics.

4.1. DNA damage response signaling: Growing evidences show that cellular DDR signaling may play a key role in the development of radiation-induced cardiac complications [68]. DNA damage of exposed tumor tissue, leading to decreased growth or cell death, is one of the detrimental effects of IR exploited as a cornerstone in cancer RT [69]. However, DNA breaks can also occur in cardiomyocytes of the heart tissue during RT. Furthermore, it is known that DSBs generated in genomic DNA by IR are not efficiently repaired in the heart [70, 71]. A recent report found that IR exposure increased the expression of Bax/Bcl2 which is associated with an elevated number of apoptotic cardiomyocytes [72]. p53, one of the major effectors in DDR signaling pathway, plays a critical role in cell cycle arrest and apoptosis in response to DNA damage. In order to determine the role of p53 in the protection of heart during radiation, Lee and colleagues selectively deleted p53 in mouse endothelial cells utilizing the CreloxP system. They observed that deletion of p53 resulted in cardiac ischemia, myocardial hypoxia and changes in vascular permeability. These changes ultimately resulted in heart failure characterized by myocardial necrosis, systolic dysfunction, and cardiac hypertrophy, signifying the importance of p53 in the protection of cardiomyocytes from myocardial injury [73]. Consistent with this hypothesis, a recent report showed that chronic radiation-induced DNA damage and oxidative stress result in the induction of the p53/p21 pathway, inhibiting the replication potential of primary human umbilical vein endothelial cells and leading to premature senescence [74]. Taken together these results suggest that radiation-induced DNA damage plays a crucial role in the development of cardiovascular disorders, along with the vital contribution of DDR signaling in the regulation of cardiomyocyte turnover. However, further detailed experimental evidences are required to establish a strong link between DDR signaling in RICD.

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4.2. Oxidative stress: Reactive oxygen species (ROS) are known to play important roles in vascular physiology. In addition to causing direct damage to DNA, proteins or lipids, IR also induces the formation of water radiolysis products containing ROS. Examples of products include nitric oxide (NO), superoxide (O2-), hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) [75, 76]. ROS can induce various forms of DNA damage, including oxidized bases, SSBs or DSBs, inter- and intra-strand crosslinks and DNA-protein crosslinks, which can significantly alter the structure of DNA ultimately leading to cell cycle arrest, apoptosis, mutations and many other effects, as previously reviewed [77, 78]. ROS act through different signal transduction pathways, including regulation of protein kinases and phosphatases activities [79], and gene expression alteration [80] and cardiac tissue remodeling [81]. Low concentrations of ROS are important for homeostasis maintenance, myocyte contraction and proper functioning of endothelial cells [82]. Thus, the production of ROS can be beneficial for cells under physiological conditions [81]. However higher concentrations of ROS contribute to cellular injury [83, 84] via mitochondrial dysfunction and bioenergetic decline [81]. Data also indicated that ROS-mediated oxidative stress is an important feature of cardiovascular conditions including atherosclerosis, hypertension, and congestive heart failure [85, 86]. At the cellular level, oxidative stress can induce myocardial remodeling characterized by hypertrophy, apoptosis, altered gene expression and increased matrix metalloproteinase activity [87]. Li et al. further showed that cardiomyocytes exposed to excess ROS were distinctively larger than control cardiomyocytes. Investigators treated chick embryos with 2 2’-azobis (2-amidinopropane) dihydrochloride (AAPH) to generate free radicals. AAPH-induced increased ROS (from free radicals) caused cardiomegaly in cardiomyocytes, which was attributed to hypertrophy. Excess ROS were found to inhibit Wnt signaling while inducing VEGF signaling, promoting angiogenesis and causing enlarged coronary arteries in AAPH-treated hearts [88].

Recent findings by Puente et al. indicated a much larger role of ROS in cardiomyocyte development and regeneration [9]. They showed that transition to the oxygen-rich postnatal environment after birth results in cell-cycle arrest of cardiomyocytes. They 14

exposed neonatal mice to a hyperoxic or hypoxic environment and measured the effects of different oxygen levels on cardiomyocyte cell size, heart to body weight ratio, cell cycle progression, extent of DNA damage and DDR signaling. They concluded that under physiological conditions mitochondrial-derived ROS induce oxidative DNA damage and activate DDR, resulting in cell cycle arrest in the majority of cardiomyocytes shortly after birth [9]. The activity of ROS can be counter-balanced by the production of antioxidants, including glutathione by cells to ensure quenching of excess free radicals [89]. However, glutathione and other reducing agents are also oxidized during their activities, requiring energy to be produced. The capacity of cells to maintain this balance is eventually diminished and ROS start to predominate. When more radicals are produced than quenched this can easily be detected as DNA damage, for instance in plaque-derived cells [90]. Plaques are known to have the highest rates of senescent and apoptotic cells [91]. Inhibition of ROS (by using ROS scavengers) and ROS-induced DNA damage signaling response can improve cardiac function after an injury from radiation exposure and increase the proliferation potential of cardiomyocytes [9]. These observations are in agreement with animal model data indicating that increased oxidative stress due to elevated ROS production by mitochondria is a hallmark of heart failure [87]. In light of previous reports showing ROS-induced DDR signaling as a central player in the regulation of cardiomyocyte renewal potential, Canseco et al. examined the effect of mechanical unloading on the proliferation potential of cardiomyocytes before and after implantation of left ventricular assist devices (LVADs) in advanced heart failure patients. They found that postnatal physiological increase in mechanical load leads to the build-up of mitochondrial content with a concomitant increase in ROS production and subsequent DNA damage in cardiomyocytes [92]. The same group observed that this can be reversed by LVAD implantation leading to mechanical unloading. Patients on LVAD for longer duration showed a significant decrease in DDR signaling in cardiomyocytes accompanied by a switch from hypertrophic to hyperplastic cardiomyocyte growth, with an increased number of cardiomyocytes re-entering the cell cycle [92].

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The next obvious objective is to determine how new cardiomyocytes are derived when ROS-induced DNA damage prompts them to exit the cell cycle shortly after birth. In the adult heart new cardiomyocytes are derived from pre-existing ones [93-95]. However, the identity of these parent cardiomyocytes was unknown until recently. Kimura et al. hypothesized the presence of a small number of cycling cardiomyocytes in the adult heart residing in hypoxic microenvironments and thus protected from oxidative DNA damage [96]. They used a sophisticated fate mapping technique based on the stabilization of hypoxia-responsive protein Hif-1α. They developed a transgenic mouse model expressing a fusion protein where the oxygen-dependent degradation (ODD) domain of Hif-1α is fused to the tamoxifen-inducible CreERT2 under the cardiomyocytespecific MHC promoter, thereby fate mapping hypoxic cardiomyocytes and their progeny after tamoxifen administration. Using the Hif-1α protein tracking technique they identified a small population of hypoxic cardiomyocytes that contribute to new cardiomyocyte generation in the adult heart. Hypoxic cardiomyocytes share many features with neonatal cardiomyocytes including smaller size, mononucleation and absence of oxidative DNA damage [96]. Taken together, these data suggest that ROSmediated oxidative DNA damage is a central player in cardiomyocyte development and disease.

In addition to nuclear DNA, other cellular organelles are also affected by IR [97]. Most notably, mitochondria occupy about 30% of total cardiac cell volume [98] and carry extra nuclear DNA, making them a prime target for radiation-induced DNA damage. Mitochondria are the principal generators of ROS in the cell under physiological conditions. An increasing body of evidence indicates mitochondrial ROS as important mediators of disease and redox signaling in the cardiovascular system. Radiation exposure causes excess mitochondrial ROS production leading to enhanced mitochondrial oxidative stress [86]. Changes in the regulation of proteins involved in energy metabolism and antioxidant response have also been observed in vivo after IR exposure [99]. Immediately after radiation, an instant increase in mitochondrial ROS in human cells was observed [100, 101]. However, once the acute increase in ROS 16

subsides, a delayed second wave of ROS generation has been observed, likely contributing to long term radiation-induced toxicity. Mitochondria are the source of this increased ROS production [102]. Kobashigawa et al. observed persistently high mitochondrial ROS level even 7 days after initial radiation exposure [103]. Furthermore, this delayed, persistent elevation in ROS levels resulted in elevated mitochondrial respiration, ATP production, increased mitochondrial content, ultimately causing cell cycle arrest [77]. Although some of these observations were made in cells other than cardiomyocytes, mitochondria of cardiomyocytes are likely to retain even higher levels of ROS even long after initial radiation exposure because of their relative abundance and bigger size in cardiomyocytes. Indeed, excess production of ROS from mitochondria is responsible for the inflammatory vascular reactions which have been implicated in pathogenesis of atherosclerosis [104]. Mitochondrial dysfunction and increased oxidative stress have also been heavily linked to myocardial ischemia [81].

Damaged mitochondria can also enhance ROS production by neighboring mitochondria (bystander effect) by virtue of altered Ca+2 ion signaling which results into the amplification of the effects of radiation [105]. Similar observations were made by Brady and colleagues in rodent cardiomyocytes through ROS rather than Ca+2 ions [106]. These observations are in agreement with the “ROS-induced ROS release” and “mitochondrial ROS-induced ROS” theory originally proposed by Zorov and colleagues [107, 108], indicating a mitochondrial ROS-driven positive feedback loop may be involved in these events. IR impairs the electron transport chain leading to persistent elevation in mitochondrial oxidative stress. This positive feedback loop results in enhanced depolarization and greater ROS production by non targeted bystander mitochondria, leading to further cell damage [20]. Furthermore, in addition to crosstalk between neighboring mitochondria, changes in mitochondria could be relayed to the nucleus via retrograde signaling. Different groups also reported that crosstalk between damaged mitochondria and the nucleus may also be altered following radiation damage. Mitochondrial ROS can influence the cellular response to radiation exposure and

17

regulation of cell cycle checkpoints [109, 110]. Overall, radiation exposure leads to persistent mitochondrial oxidative stress and that may play a role in RICD.

4.3. Telomere erosion: A growing body of evidence suggests progressive telomere erosion as an important marker in the pathobiology of vascular disease, coronary heart disease and premature myocardial infarction [111, 112]. Leukocytes from coronary artery disease patients have shorter telomeres when compared with healthy controls, suggesting a possible link between telomere length and cardiovascular disease risk [113, 114]. Reduced telomere length is found in aging endothelial cells of the abdominal aorta and iliac arteries [115, 116]. In post-natal mice and humans, telomerase ceases to express leading to progressive telomere shortening. A recent study by Bär et al. showed that cardiac tissue specific activation of telomerase (tert) can protect mice from heart failure following myocardial infarction [117]. Data generated from a telomerase-deficient (mTerc-/-) mouse model indicates an association between short telomeres and increased radiosensitivity [118-120]. Although human data is scarce, telomere erosion is a common event in cancer patients undergoing RT [121]. Also, analysis of telomere length from peripheral blood samples showed significant telomere shortening in Chernobyl clean-up workers both in early and in late periods (up to 20 years) after lowdose irradiation [122]. This further supports the observation that progressive telomere erosion and telomere dysfunction is not just a biomarker, rather an important player in the pathobiology of vascular disease, coronary heart disease and myocardial infarction mediated heart failure. However, whether RICD arises because of telomere attrition in cardiomyocytes remains to be seen. Also, the underlying mechanism of radiation induced telomere shortening needs to be elucidated.

It is known that ROS can trigger telomere shortening. Von Zglinicki et al. observed telomere attrition in cultured cells in response to ROS-induced mitochondrial dysfunction; this effect can be reversed when cells are grown in a hypoxic environment or in the presence of antioxidants [123]. This is supported by observations of chronic hypoxia preserving telomere length and extending the life span of cells by the promotion 18

of telomerase activity [124, 125]. Epidemiological evidence also indicates a positive correlation between short telomere length and cardiovascular disorders characterized by chronic oxidative stress and inflammation such as coronary atherosclerosis, myocardial infarction and hypertension [113, 126]. However, further studies are needed to support the hypothesis that ROS mediated increased oxidative stress promote telomere erosion, contributing to cardiovascular disease.

4.4. Alterations in RNA and protein expressions: Low-dose IR (200 mGy) has been found to alter the cytoplasmic proteome in vitro; the dose rate also appears to influence these changes. Pluder and colleagues used mass spectrometry to detect changes in the cytoplasmic proteome of a human endothelial cell line (EA.hy926) after exposure to 200 mGy of γ-irradiation at two different dose rates (20 mGy/min and 190 mGy/min), at 4h and 24h [127]. Changes in the expression of 15 proteins were found to be different in irradiated cells as compared to non-irradiated controls. Furthermore, pathway analysis showed that low-dose exposure to radiation affects the Ran and RhoA pathways and causes alterations in fatty acid metabolism and stress response in endothelial cells [127]. Another study by Barjaktarovic and colleagues found that a single 200 mGy radiation dose significantly altered the expression of 28 proteins in endothelial cells. Subsequent bioinformatics analysis indicated that “cellular assembly and organization, cellular function and maintenance and molecular transport” are the most significant radiation-responsive networks [128].

The same group also reported that radiation

exposure leads to the deregulation of several microRNAs, most notably miR21 and miR146B. Subsequent analysis also predicted that microRNA deregulation is at least partially responsible for altered protein expression after radiation exposure; this indicates the involvement of small non-coding RNAs in the cellular response to IR exposure [128]. Interestingly, microRNAs were recently found to be implicated in the regulation of radiation-induced DNA damage and induction of premature senescence [129], highlighting the growing importance of non-coding RNAs in development and disease.

19

4.5. Genetic risk factors: Recent reports have shown that genetic risk factors play an important role in the development of atherosclerosis after radiation exposure. Genetically predisposed ApoE-/- mice irradiated with a single dose of 14 Gy showed rapid onset and growth of atherosclerotic plaque formation as compared to their wild type counterparts [130]. This result suggests that exposure to high dose radiation accelerates the development of atherosclerosis in individuals who are genetically predisposed to RICD development. This finding was corroborated by an independent study conducted in ApoE-/- mice indicating the development of atherosclerosis following exposure to low dose radiation (0.025-0.5 Gy) [131]. Studies conducted in ApoE-/- mice revealed that low dose rate radiation (1 mGy/min) (early or late stage disease) slowed disease progression, while high dose rate (about 150 mGy/min) exposure during earlystage disease produced both protective and detrimental effects; data overall suggest that the dose rate also influences disease outcome [131, 132]. Interestingly, the tumor suppressor p53 gene also found to be important in the development of atherosclerosis, at least in genetically predisposed ApoE-/- mice [74]. During early disease stage, following low doses of radiation delivered at a low dose rate (1 mGy/min), a reduced function of p53 did not influence disease outcome. However, during late disease stages, a reduced p53 function had detrimental effects [74]. These observations suggest that radiation-induced atherosclerosis is a multifaceted disease depending on factors that include total dose of radiation, dose rate, genetic predisposition, p53 status and other tumor suppressors.

4.6. Endothelial dysfunction, alterations in proinflammatory molecules and platelet activities: Apart from DDR signaling and oxidative stress, IR can also cause acute damages to endothelial cells, and changes in inflammation, lipid accumulation and coagulants. These can result in endothelial dysfunction, which appears to be the principle mechanism by which radiation exposure causes vascular damage.

4.6.1. Endothelial dysfunction: Endothelial dysfunction is a well established response to cardiovascular risk factors and precedes the development of atherosclerosis. Early 20

animal and human subject studies have shown that endothelial cell damage and a decrease in capillary density are early events in radiation-induced heart tissue damage [133, 134]. Subsequent studies reported that capillary loss, characterized by focal loss of the endothelial cell marker enzyme alkaline phosphatase, may be associated with ischemic myocardial degeneration and heart failure following radiation exposure [135]. However, since alkaline phosphatase loss is also observed in other disease conditions unrelated to radiation [136], its pathological role in the development of radiation-induced atherosclerosis is unclear.

4.6.2. Alterations in proinflammatory molecules: Radiation damage results in both acute and chronic changes in cardiac tissue. Inflammation is a common event following radiation exposure which initiates tissue fibrosis [137]. Upregulation of several proinflammatory molecules is observed following endothelial cell irradiation. An increased production of proinflammatory adhesions molecules E-selectin, P-selectin (both mediate leukocyte rolling), ICAM1 (important in leukocyte arrest) and PECAM1 (involved in leukocyte transmigration) in endothelial cells has been observed both in vitro and in vivo by different groups following irradiation [138-141]. Also, NF-ĸB mediated cellular signaling appears to be involved in the inflammation process [142, 143]. Although the exact mechanism of cardiomyopathy promoted by inflammatory molecules has not been elucidated, these factors contribute to structural changes in the micro vessels of the myocardium after radiation exposure [133, 144, 145] most probably by recruiting inflammatory leukocytes like neutrophils and monocytes [50]. Furthermore, several cytokines are up-regulated after irradiation of endothelial cells. IL-8, a chemo attractant for leukocytes and known inducer of endothelial cell proliferation is upregulated in a time- and dose-dependent fashion after radiation exposure [140, 146]. IL-8-induced endothelial cell proliferation leads to radiation damage, mitotic death and apoptosis before the development of radiation-induced cardiomyopathy [147]. In addition to IL-8, other cytokines such as TGF-β1 and IL-1β have also been implicated in the advancement of atherosclerotic lesions by promoting endothelial and fibroblast proliferation, increased collagen deposition and fibrosis [148]. 21

4.6.3. Alterations in coagulation and platelet activities: In addition to inflammation mediated by upregulation of proinflammatory molecules leading to endothelial cell dysfunction, coagulation and platelet activity alterations have also been observed following radiation exposure. Multiple reports suggested increased deposition and/or release of the von Willebrand factor in endothelial cells 5 hours after total body irradiation with 4 Gy, or 16 months after heart irradiation with 15 Gy. These activities ultimately lead to increased platelet adherence and thrombus formation in capillaries and arteries [149-151]. These observations are also consistent with animal model data, indicating an inflammatory, thrombotic plaque phenotype following high dose radiation exposure. This proinflammatory environment coupled with collagen deposition and increased recruitment of leukocytes and fibroblasts results in tissue remodeling, cardiac fibrosis and atherosclerosis which is a major endpoint of RICD [130, 152-154].

5. Conclusions and future directions

Cardiovascular complications may arise from radiation exposure. Clinical studies have identified risk factors, but the actual biological mechanism driving disease pathogenesis after radiation exposure has not been clarified. The up-regulation of proinflammatory molecules, p53 status and genetic risk factors undoubtedly contribute to the pathophysiology of the disease but may not be major causes. Careful studies of inherent cellular processes regulating cardiovascular development under physiological conditions are needed. The importance of DDR signaling in cardiomyocyte development and proliferation has not been explored before. In this regard, the role of ROS-induced DDR in limiting the cardiomyocyte proliferation potential may be highly relevant. Even more important is the notion of a cycling cardiomyocyte population maintaining their proliferation potential under hypoxic conditions and probably generating new cardiomyocytes after insult or injury. Another potentially intriguing question is why adult cardiomyocytes are unable to re-enter the cell cycle on demand after injury or insult (e.g. after radiation exposure). Radiation-induced delayed cardiotoxicity may possibly 22

occur due to the ablation of this small number of cycling cardiomyocytes, though additional testing is needed to support this hypothesis. Cycling cardiomyocytes are protected from oxidative stress-induced DNA damage under physiological conditions and

maintain

their

proliferative

potential

throughout

an

individual’s

lifespan.

Cardiomyocytes may be equally or more susceptible to radiation-induced DNA damage or lose their proliferation potential when ROS levels are elevated long after radiation exposure. Thus, radiation-induced direct DNA damage combined with excess oxidative stress acutely reduces their turnover rate. Furthermore, the degree and type of DNA damage, the ability of cycling cardiomyocytes to repair DNA damage and the specific pattern of DDR activation in cardiomyocytes are unknown and need to be carefully examined. Moreover, whether the rate of cardiomyocyte turnover is also dependent on the extent of DNA damage still needs to be tested. Another important aspect requiring further elucidation is the fate of cycling cardiomyocytes upon aging. Age may contribute to cardiotoxicity following radiation exposure. Finally, permanent reduction in the rate of cardiomyocyte turnover following exposure to IR also needs to be investigated.

If the hypothesis regarding the ablation of cycling cardiomyocytes by radiation is proven, the next obvious step is to test if populations of cycling cardiomyocytes can be protected from the deleterious effects of radiation. ROS-mediated DNA damage due to irradiation is well accepted. This occurs either by the direct effect of radiation on the nucleus or secondary damage to other cellular components, including mitochondria, resulting in persistent increases of ROS production. The secondary damage mechanism is likely to mediate a more lasting effect on DNA. Similarly, irreparable DSBs can also trigger increased ROS production [155]. Thus, using ROS scavengers in the prevention and reversal of IR-induced reduction in cardiomyocyte turnover is a promising strategy. The effectiveness of these strategies in the clinical setting needs to be explored. Using nuclear or mitochondrial-targeted ROS scavengers before (prevention) or after (reversal of cardiomyocyte turnover) radiation may be an ideal approach to prevent DNA damage and cardiovascular complications due to radiation exposure.

23

6. Acknowledgements

We thank Dr. Damiana Chiavolini for the critical reading of our manuscript. This work was

supported

by the

National

Aeronautics and

Space

Association grants

NNX13AD57G and NNX15AE06G (to A.A.).

24

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Figure legends

Figure 1:

Schematics showing pathophysiology of ionizing radiation induced heart

risks. ROS- reactive oxygen species; DDR-DNA damage response signaling.

39

Table 1: Sources of environmental/occupational radiation and their estimated contribution to total average dose received in the United States. Source

Worldwide annual average dose+ (mSv)

Individual dose received (range)+ (mSv)

Percent contribution in US++ (mSv)

Natural sources of exposure Inhalation (radon & thoron)

1.26

0.2-10

37

External terrestrial radiation

0.48

0.3-1

3

Galactic cosmic radiation

0.39

0.2-1

5

Ingestion

0.29

0.3-1

5

0.6 (range 0.03-2 depending on the country

3 per individual in US

48

Occupational exposure

0.005

0-20

<0.01

Industrial exposure

0.007

Depends on location

<0.01

Consumer products

Not known

Varies between countries; 0.13 per individual in US

2

Artificial sources of exposure

Medical diagnosis (not therapy) including nuclear medicine

+ ++

Sources and Effects of Ionizing Radiation, UNSCEAR report to general assembly, 2008 National Council on Radiation Protection and Measurements, 2006 40

Table 2: Spectrum of radiation induced cardiovascular disease.

Type of cardiovascular risks

Acute pericarditis

Delayed pericarditis

Myocardial disease

Valvular disease

Radiation dose

Symptoms and pathogenesis

40 Gy

Comparatively rare but may arise due to radiation of large mediastinal tumours adjacent to the heart. Acute pericarditis is driven by inflammation and fibrin deposition. Injury to the pericardium can also lead to ischemia and fibrosis. Other symptoms include fever, tachycardia, chest pain and pericardial rub. Pericardial effusion is also seen in some patients.

40 Gy

20% of patients, especially those showing effusions in acute stages develop chronic pericarditis later in life. Pericardial effusions are often characterized by cardiomegaly, fibrous adhesions, high protein content and an increase in serum inflammatory markers. This is accompanied by increased collagen deposition and thickening of the pericardium resulting into cardiac tamponade in rare cases.

>30 Gy

Reduced myocardial compliance with varying levels of interstitial fibrosis. The myocardial fibrosis is often asymptomatic and can remain undetected for years following radiation. Abnormal motion of the wall, left ventricular hypokinesis and defective myocardial relaxation are some of the signs which can be detected by ECG before the disease becomes symptomatic. Other symptoms include dyastolic dysfunction, restrictive cardiomyopathy and small vessel ischemic disease. Even if acute myocardial damage is moderate, continued myocardial remodeling often leads to progressive myocardial dysfunction and heart failure. Clinically significant cardiomyopathy is seen in patients exposed to chemotherapy as well as very high radiation doses (>60 Gy).

>30 Gy

Radiation associated valvular disease is seen among 81% of the patients. The left sided valves; aortic and mitral valves are more commonly affected than the right sided tricuspid and pulmonary, probably because of higher pressure across left sided valves. Valvular disease is a late complication, with asymptomatic valvular thickening is detected 11.5 years after radiation and symptomatic valvular dysfunction is detected only after 16.5 years following IR exposure. Endocardial fibrosis characterized by thickening and calcification of the valvular endocardium are the early events which drives valvular dysfunction. 41

Coronary artery disease

Cardiac arrythmias

6-40 Gy

Due to high number of atherosclerotic patients often it is difficult to distinguish radiation associated coronary artery disease (CAD) patients from typical atherosclerotic CAD patients. Risk factors for CAD include anterior exposure without shielding, high radiation dose, exposure at a young age, and prior history of heart disease. Traditional risk factors like hypertension, smoking, male gender and hyperlipidemeia or diabetes mellitus also contribute to radiation induced CAD to varying degrees. The proximal coronary vessels; proximal right coronary, left main or proximal left anterior descending arteries being in the range of radiation are mostly affected.The early events are microvascular damage, inflammation and fibrosis. Symptoms often include chest pain, angina, dyspnea, heart failure and sudden death in certain patients.

Not known

Conduction system abnormalities are less well documented compared to other cardiac abnormalities following IR. Non-specific ECG abnormalities like repolarisation abnormalities are often observed upto one year following IR but generally asymptomatic and transient. More serious arrhythmias and infranodal conduction are sometimes observed as late complications (>10 years after treatment). Right bundle branch block is more commonly observed than left bundle branch block probably due to the anterior location of the right ventricle, absorbing higher radiation dose. Conduction difficulties may arise due to direct damage to structures like sinoatrial (SA) and atrioventricular (AV) nodes or because of microvascular damage leading to cardiomyocte conduction abnormailities. Complete heart block and sudden death is rarely seen.

42

Table 3: Biological mechanisms contributing to radiation induced cardiotoxicity.

Suggested Mechanism

Where

References

Results

Upregulation of proinflammatory molecules

E- selectin [138]; P-selectin [138]; ICAM-I Vascular endothelial cells [139]; PECAM-I [140, 141].

Endothelial dysfunction, capillary loss, structural changes in the blood vessels of myocardium, Atherosclerosis and tissue fibrosis.

Upregulation of cytokines and growth factors

IL-6 [140, 141]; IL-8 [146]; TGF-β1 and Vascular endothelial cells IL- β1 [148]; NF-ĸb activation [142, 143].

Inflammation, collagen deposition, proliferation, apoptosis of endothelial cells, fibrosis and atherosclerotic plaque formation.

Prothrombotic effects

Endothelial cells, capillaries and arteries of myocardium

Increased release of von Willebrand factor [149-151].

Increased platelet adherence and thrombus formation in the capillaries and arteries and atherosclerosis.

Myocardium

Myocardial fibrosis [144, 145]; Reduced capillary density and loss of capllilary endothelial cells [134, 158]; focal loss of alkaline phosphatase [135].

Endothelial cell swelling, proliferation, lymphocyte adhesion and extravasation, cardiomyopathy and heart failure.

Endothelial cells

Complex interplay of ApoE-/- genetic predisposition [130]; dose rate [131]; p53 status and early or late stage disease [111].

Atherosclerotic plaque formation.

Myocardial degeneration

Genetic and environmental factors

43

Proteome and transcriptome alterations

Radiation induced direct DNA damage and increased reactive oxygen species production

Human vascular endothelial cells

Changes in cytoplasmic protein expression involved in RAN and RhoA pathways, fatty acid metabolism and stress response pathways [127]; changes in cellular- assembly, organisation, function, maintenance and molecular transport [128]; deregulation of non coding microRNAs [128].

Accelerated atherosclerosis.

Cardiomyocytes and vascular endothelial cells

Increased Bax/Bcl2 expression [72]; enhanced p53-p21 signaling [74, 160]; telomere attrition [111, 112]; intracellular oxidative stress [9, 85, 86, 92, 96]; mitochondrial dysfunction [81, 105].

Mitotic arrest, cell cycle exit increased apoptosis and premature senescence of cardiomyocytes and vascular endothelial cells.

44