Deposition of energy leads to vascular remodeling

Tatiana Kozbenko^1,2, Nadine Adam, Veronica Grybas¹, Benjamin Smith¹, Dalya Alomar¹, Robyn Hocking¹, Janna Abdelaziz³, Amanda Pace³, Carole Yauk², Ruth Wilkins¹, Vinita Chauhan¹

(1) Health Canada, Ottawa, Ontario, K1A 0K9, Canada  

(2) University of Ottawa, Ottawa, Ontario K1N 6N5, Canada 

(3) Carelton University, Ottawa, Ontario K1S 5B6, Canada 

Consultants

Marjan Boerma¹, Omid Azimzadeh², Steve Blattnig³, Nobuyuki Hamada⁴

(1) University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

(2) Federal Office for Radiation Protection (BfS), Section Radiation Biology, 85764 Neuherberg, Germany

(3) NASA Langley Research Center Hampton, VA  23681, USA

(4) Biology and Environmental Chemistry Division, Sustainable System Research Laboratory, Central Research Institute of Electric Power Industry (CRIEPI), Chiba, Japan

Cardiovascular disease (CVD) is a leading health risk around the world. While there are many factors contributing to its development, ionizing radiation (IR) exposure has recently been recognized as one of such factors. High-dose exposures, as in the case of patients receiving radiotherapy, are strongly linked to radiation-induced CVD, and growing evidence suggests lower-dose scenarios such as occupational exposures carry somewhat similar risks. Additionally, as space missions are planned beyond the protection of the magnetosphere, mechanistic information about the effects of radiation on the cardiovascular system is essential for developing countermeasures protecting future space travelers. The present qualitative AOP (AOP#470) summarizes the evidence for a progression beginning with the deposition of energy, which occurs in cells during exposure to radiation, and ending with abnormal vascular remodeling. The pathway is initiated by the ionization events stemming from the deposition of energy (MIE: Event #1686). The deposition of energy causes water radiolysis resulting in the creation of reactive oxygen species (ROS) at a rate that outpaces the antioxidant defense system and results in an increase in oxidative stress (KE: Event #1392). The deposition of energy can induce DNA strand breaks (KE: Event #1635) either directly or through damage from ROS. Excessive ROS levels and resulting damage to cellular compartments like DNA in turn alter signaling pathways (KE: Event #2244), increasing levels of pro-inflammatory mediators (KE: Event #1493). Within the vascular wall, activation of certain signaling molecules can alter nitric oxide (NO) levels (KE: Event #2067). All the upstream KEs of the pathway then converge to cause endothelial dysfunction (KE: Event #2068). Modified levels of NO can alter the blood flow within the endothelium resulting in subsequent compensatory abnormal vascular remodeling (AO: Event #2069). Abnormal vascular remodeling is an important precursor for many diverse cardiovascular pathologies and serves as an important marker for CVD. Vascular remodeling can include many structural changes such as increased vessel stiffness, vessel wall thickening, and decreased capillary density. Studies informing this AOP include clinical follow up studies of radiotherapy patients, epidemiological cohort studies of atomic bomb survivors and nuclear plant workers as well as biological studies using mouse and rat models. Knowledge gaps in the evidence evaluation include inconsistencies in NO evaluation, and relatively few studies exploring chronic and low dose exposures as well as a lack of studies focusing on female biology. 

Cardiovascular disease (CVD) includes any health condition affecting the heart and blood vessels. CVD is one of the leading causes of death worldwide, accounting for tens of millions of deaths yearly and surpassed in some countries by only cancer (Bray et al., 2021; Tsao et al., 2022). This class of diseases includes congenital defects, as well as CVDs that can develop throughout life such as peripheral artery disease (PAD), atherosclerosis, coronary artery disease and myocardial infarction. While the progression to a CVD outcome can take significant time, many CVDs are often preceded by much earlier changes to vascular structure. Abnormal vascular remodeling entails various structural changes of existing vasculature arising from cell death, cell migration and changes to the endothelial cell membrane. It is important to note that changes to vascular structure are not inherently detrimental, and the cardiovascular system undergoes continuous adaptation to protect vascular health (Pries et al., 2001; Santamaría et al., 2020; Zakrzewicz et al., 2002). However, certain remodeling can also serve as an important marker and risk factor for future adverse cardiovascular events (Cohn et al., 2004; Van Varik et al., 2012). Changes to vascular structure can be triggered through various perturbations such oxidative stress, inflammation, and alterations to various cellular signaling pathways. Adverse remodeling of the vasculature encompasses structural and functional changes to vessel wall intima-media, elevated stiffness, and decreased lumen diameter which are all predictive of the development of and mortality and morbidity from CVD (Heald et al., 2006; Hodis et al., 1998; Polak et al., 2011; Zieman et al., 2005).   

The risk of CVD increases with several factors such as age. There is significant evidence suggesting that environmental factors such as radiation can contribute to the risk of development (Belzile-Dugas & Eisenberg, 2021; Boerma et al., 2016; Francula-Zaninovic & Nola, 2018; Wang et al., 2019). The deposition of energy from radiation exposure is a stochastic event and any part of the human body can be affected, with adverse effects emerging years or decades after the exposure (Boerma et al., 2016; Dörr, 2015; EPRI, 2020; Menezes et al., 2018). The effects of high-dose radiation on the cardiovascular system have been well-characterized while the effects of low-dose exposure are more contended. However, growing evidence suggests that lower doses than previously thought are linked to subsequent adverse cardiovascular outcomes (Boerma et al., 2016; EPRI, 2020; Little et al., 2021; UNSCEAR, 2008). Much of the high-dose data come from follow up studies in radiotherapy patient cohorts who are shown to be at elevated risk for adverse cardiovascular events (Zou et al., 2019). In addition to clinical exposure scenarios, epidemiological studies of occupational exposures and Japanese atomic bomb survivors have also been conducted. Cohort studies of atomic bomb survivors demonstrate various ways in which CVD risk is affected by exposure parameters, including factors such as age at exposure and estimated dose received (Ozasa et al., 2012; Preston et al., 2003; Shimizu et al., 2010; Takahashi et al., 2017). Long-term follow up of individuals exposed in the Chernobyl disaster region also identified statistically significant elevation in CVD risk (Ivanov et al., 2006; Kashcheev et al., 2017). Occupational exposure studies have also been conducted in various countries in an effort to understand the relationship between low-dose chronic exposure and cardiovascular health of nuclear workers (Azizova et al., 2018; Gillies et al., 2017; Zielinski et al., 2009). Occupational exposure studies suggest positive associations between received dose and excessive relative risk of circulatory diseases (Zielinski et al., 2009), CVD mortality (Gillies et al., 2017) and occurrence of ischemic and cerebrovascular disease involving the blood vessels (Azizova et al., 2018).   

Beyond earth, space travel presents an additional radiation exposure scenario. With future missions planned beyond low Earth orbit and the protective shield of the magnetosphere, understanding the unique challenges of space radiation is crucial for protection of travelers. In space, radiation is present in the form of high linear energy transfer (LET) particles and high mass, high energy ions (HZE) which indiscriminately impacts the whole body at a low fluence rate (Baker et al., 2011; Durante & Cucinotta, 2008; Norbury et al., 2016). While the present AOP includes an MIE focused on deposition of energy following radiation exposure, it is important to note that the space exposome contains multiple stressors to which space travelers will be exposed simultaneously. Particularly important, in the case of the cardiovascular system, is microgravity. The cardiovascular system is gravity sensitive, with the endothelial layer being responsive to changes in shear stress and blood pressure (Hughson et al., 2018; Maier et al., 2015; Versari et al., 2013). Variation to the pressure gradient throughout the body can also trigger regional adaptations to vascular structure (L. F. Zhang, 2013). While determining a mechanism for an MIE of microgravity has proven challenging, microgravity exposure has been shown to contribute to other KEs in the pathway and evidence from microgravity studies has been included in the weight of evidence (WOE). 

The present AOP (#470) is a part of a broader network that links the deposition of energy with four AOs: vascular remodeling, bone loss, learning and memory impairment, and cataract formation. The network demonstrates the interconnectivity in mechanisms of non-cancer adverse outcomes for which space travelers are at risk. The development process began with the creation of a preliminary version of the network through narrative review of key literature as well as extensive subject matter expert consultation (Chauhan et al., 2021).  

Many KEs were proposed for the preliminary network; however, attention was focused on those deemed most essential for disease progression and which had greatest biological plausibility for connectivity to the rest of the pathway. The preliminary network served as the basis for the next stage of development in which a scoping review methodology was used to collect a WOE and refine KEs and KERs. Details of the methodology are described by Kozbenko et al. 2022 and are summarized below (Kozbenko et al., 2022).   

In short, literature collection consisted of structured database searches, followed by prioritization and screening stages. Focused searches were completed for each of the KERs in the pathway using combinations of key words related to involved KEs, as well as an overarching search, collecting all references broadly related to the MIE and AO. Following the literature searches, the results were processed in two stages to determine their inclusion in the pathways WOE. Both stages determined reference relevance according to inclusion and exclusion criteria that had been outlined prior to screening in a PEOE (Population, Exposure, Outcome, and Endpoint) statement.  

In the first stage, search results were prioritized using the SWIFT Review software. This software was used to identify the references most closely related to the PEOE statement as determined by the software created tags. The most relevant references identified by SWIFT were then screened by human screeners using screening forms created in Distiller SR. Screening in Distiller was split into title and abstract, full text and data extraction levels and human screeners were instructed to include references according to the same PEOE criteria. Human screeners additionally evaluated references for their demonstration of Bradford Hill criteria. At the end of the process, collected studies were used for the WOE.  

The present vascular remodeling AOP was continually refined throughout the screening process based on evidence extracted from the literature. The final pathway includes several KEs previously existing in the AOP-Wiki, these include the deposition of energy (KE #1686), oxidative stress (KE #1392), increased DNA strand breaks (KE #1635) and increased pro-inflammatory mediators (KE #1493). Newly created KEs for this pathway include altered stress response signaling (KE #2244), altered NO levels (KE #2067), increased endothelial dysfunction (KE #2068) and occurrence of abnormal vascular remodeling (AO: KE #2069). 

Summary of evidence (KE & KER Relationships and evidence)  

The AOP is supported by high biological plausibility and moderate empirical evidence. Research, primarily from laboratory studies, has supported  dose- and temporal-concordance for each KER.  

Biological Plausibility  

Described below is the well-established understanding of the mechanisms underlying this AOP with supporting literature. More detailed examples of the empirical data can be found in the individual entries for each KER. 

It is well accepted that when energy is deposited in the cell from ionizing radiation (IR), direct damage to cellular structures can occur (Desouky et al., 2015). When traveling through a cell, IR can induce the radiolysis of water forming reactive oxygen species (ROS). Deposition of energy can also induce feedback loops of ROS production where structures and molecules damaged by ROS including the mitochondria and NADPH oxidase (NOX) further produce ROS (Mittal et al., 2014; Soloviev & Kizub, 2019). Additionally, deposited energy can directly upregulate enzymes involved in ROS and reactive nitrogen species (RNS) (collectively RONS) production (de Jager, Cockrell and Du Plessis, 2017). If reactive nitrogen species RONS production outpaces the antioxidant defense, a state of oxidative stress occurs (Fletcher et al., 2010; Slezak et al., 2017; Tahimic & Globus, 2017; Wang et al., 2019). Damage to macromolecules can occur due to oxidative stress, including strand breaks to DNA, oxidation of amino acid residues in proteins and peroxidation of lipids (Ping et al., 2020). Lipid peroxidation can induce further damage to cellular structures as a chain reaction is created by the lipid peroxidation radicals, attacking other lipids, proteins and nucleic acids (Ping et al., 2020). Consequently, oxidative stress can directly lead to multiple downstream KEs including altered signaling pathways, increased DNA strand breaks, increased pro-inflammatory mediators and altered nitric oxide (NO) levels. 

DNA strand breaks in endothelial cells can be induced either directly through energy deposition or indirectly through oxidative stress. DNA strand breaks can recruit and activate the protein kinases ataxia telangiectasia mutated (ATM) and ATM/RAD3-related (ATR) (Nagane et al., 2021). Downstream signaling pathways involved in cell death and senescence like the p53/p21 pathway can be activated by ATM/ATR. Furthermore, DNA strand breaks induced by radiation directly or through oxidative stress can cause mutations or changes in transcription of proteins in signaling pathways (Ping et al., 2020; Schmidt-Ullrich et al., 2000). Therefore, DNA strand breaks will induce death and senescence of endothelial cells through altered signaling, resulting in endothelial dysfunction. 

Oxidative stress can also induce altered signaling pathways. The effects of oxidative stress on signaling pathways occur through protein oxidation of signaling components (Ping et al., 2020; Schmidt-Ullrich et al., 2000; Valerie et al., 2007). Oxidation of cysteine and methionine residues, which are particularly sensitive to oxidation, can result in structural and functional detriments to the protein (Ping et al., 2020). RONS can influence various pathways including the Akt/PI3K/mTOR pathway, where impaired cell survival signaling can induce cellular senescence (Hassan et al., 2013; Ping et al., 2020). Additionally, inhibition of tyrosine phosphatases by ROS can increase the phosphorylation of mitogen-activated protein kinase (MAPK) pathways, resulting in various downstream effects (Schmidt-Ullrich et al., 2000; Valerie et al., 2007). A phosphorylated p53 induced by oxidative DNA damage can also activate MAPK signaling and initiates a cascade ending in apoptosis (Ashcroft et al., 1999; Gen, 2004). Through affecting cell signaling pathways, damage caused by elevated RONS affects cells beyond those that have been directly irradiated (Ramadan et al., 2021). 

Excessive RONS produced by IR disrupt cellular balance and can increase pro-inflammatory mediators (Lumniczky et al., 2021; Schaue et al., 2015). Similar to activation of the immune system by damage from a pathogen, activation by oxidative stress promotes many repair mechanisms, some of which involve rapid release of pro-inflammatory cytokines (Stanojković et al., 2020). The cytokines released vary based on tissue type and radiation parameters (Di Maggio et al., 2015), but tumor necrosis factor (TNF)-α and interleukin (IL)-1 can trigger a cytokine cascade that initiates an inflammatory response (Slezak et al., 2017; Srinivasan et al., 2017). A prolonged state of inflammation in endothelial cells can lead to endothelial dysfunction (Baran et al., 2021). 

Both oxidative stress and altered signaling can directly result in altered NO levels. NO is synthesized from L-arginine by the three nitric oxide synthase (NOS) enzymes, endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). ROS can directly reduce NO levels by reacting with NO to produce the RNS peroxynitrite (Deanfield et al., 2007). Furthermore, the cofactor of NOS enzymes, tetrahydrobiopterin (BH4), can be oxidized by RONS leading to inhibition of NOS dimerization, also called NOS uncoupling (Deanfield et al., 2007). Uncoupled NOS will produce superoxide instead of NO, leading to a positive feedback loop of ROS production and reduced NO (Förstermann, 2010; Förstermann & Münzel, 2006; Mitchell et al., 2019; Nagane et al., 2021; Soloviev & Kizub, 2019). 

Modulation of NO through altered signaling pathways occurs through changing the activity of NOS enzymes. Phosphorylation of eNOS at Ser1177 will activate the enzyme while phosphorylation at Thr495 inhibits it (Förstermann, 2010; Nagane et al., 2021). Protein kinase B (Akt), part of the phosphoinositide 3-kinase (PI3K)/Akt pathway, can activate eNOS through phosphorylation at Ser1177 to increase NO production (Karar & Maity, 2011). In contrast, activation of the RhoA/Rho kinase (ROCK) pathway will inhibit NO production by destabilizing eNOS mRNA and preventing Ser1177 phosphorylation by Akt (Yao et al., 2010). Angiotensin II (AngII), the end product of the renin-angiotensin-aldosterone system (RAAS), is involved in both downregulating Ser1177 phosphorylation to prevent NO creation (Ding et al., 2020) and activating eNOS as a corrective measure (Millatt et al., 1999). Alterations to these pathways due to IR will result in changes in NO levels. 

Each of the components of the pathway described above converge at endothelial dysfunction. Endothelial cells lining the blood vessels throughout the body are an important component for maintaining vascular homeostasis (Bonetti et al., 2003; Deanfield et al., 2007). Endothelial cells are quiescent with high levels of NO most of the time (Carmeliet & Jain, 2011). Endothelial dysfunction can occur due to prolonged activation of the endothelium, characterized by the prolonged lack of bioavailable NO, lack of endothelium-dependent vasodilation and chronic pro-thrombotic and inflammatory state (Baran et al., 2021; Bonetti et al., 2003; Deanfield et al., 2007; Krüger-Genge et al., 2019). A prolonged reduction in NO will decrease vasodilation, increase leukocyte adhesion and increase fibrous plaque formation contributing to the pro-thrombotic dysfunctional environment (Schiffrin, 2008; Senoner & Dichtl, 2019; Venkatesulu et al., 2018). Furthermore, signaling in pathways like p53/p21 or PI3K/Akt/mammalian target of rapamycin (mTOR) can induce apoptosis or premature senescence of endothelial cells as part of endothelial dysfunction due to DNA damage or oxidative stress (Borghini et al., 2013; Hughson et al., 2018; Schiffrin, 2008; Senoner & Dichtl, 2019; Soloviev & Kizub, 2019). Senescent cells have decreased levels of NO production and a pro-inflammatory secretory phenotype, which feed back to further promote endothelial dysfunction (Ungvari et al., 2013; Wang et al., 2016). 

Endothelial dysfunction subsequently leads to vascular remodeling, which encompasses multiple structural changes to the vasculature. Chronic inflammation combined with impaired healing and lack of endothelium-dependent vasodilation during endothelial dysfunction increases vulnerability to damage from non-laminar flow and maladaptive repair (Sylvester et al., 2018). As compensation, vessel walls can thicken and atherosclerotic risk can increase (Hughson et al., 2018; Slezak et al., 2017; Sylvester et al., 2018). In cases of maladaptive repair of vessels, vascular remodeling can be exhibited through an increase in fibrosis (Hsu et al., 2019). The pro-thrombotic environment with increased lymphocyte adhesion induced by endothelial cell senescence can increase the likelihood of vessel occlusion, decreasing vascular density such that the corresponding increase in vascular resistance will induce remodeling as a compensatory measure (Slezak et al., 2017). Thus, increased leukocyte adhesion during endothelial dysfunction occurs early in the development of atherosclerosis (Senoner & Dichtl, 2019). Increased arterial stiffness can also occur in response to endothelial dysfunction (Boerma et al., 2015, 2016; Patel et al., 2020), with increased collagen and smooth muscle content paired with decreased elastin and degradation of the extracellular matrix (Zieman et al., 2005). The changes to the vascular structure in response to the deposition of energy are similar to a form of accelerated age-related atherosclerosis (Boerma et al., 2016; Sylvester et al., 2018; Vernice et al., 2020). 

Temporal, Dose, and Incidence Concordance  

Evidence for time, dose, and incidence concordance in this AOP is moderate. It has been repeatedly shown using many study designs and systems that deposition of energy occurs immediately following irradiation, and downstream events occur at a later timepoint. Endpoints indicating oxidative stress have been observed within minutes following irradiation (Wortel et al., 2019). Studies show that oxidative stress, increased DNA strand breaks, increased pro-inflammatory mediators, and altered signaling may occur over a similar time period; however, alteration in signaling pathways, increased DNA strand breaks, and increased pro-inflammatory mediators can be observed following oxidative stress (Ramadan et al., 2020; Baselet et al., 2017; Sakata et al., 2015; Yang et al., 1998). Increases in NO levels occur in hours to weeks after irradiation (Azimzadeh et al., 2017; Sonveaux et al., 2003; Sakata et al., 2015). Then, from weeks to months following irradiation both endothelial dysfunction and vascular remodeling occur, though concordance between these events is difficult to determine, possibly due to inter-study differences in experimental design and markers (Yentrepalli et al., 2017; Soucy et al., 2007; Yu et al., 2011; Shen et al., 2018). 

Overall, the majority of studies demonstrate that upstream KEs occur at the same or lower doses and earlier or the same time as downstream KEs. For example, endothelial cells show a dose-dependent increase in oxidative stress to X-ray irradiation at 0.1 and 5 Gy, while 0.1 Gy induced few changes in pro-inflammatory mediators with significant increases only observed at 5 Gy (Ramadan et al., 2020). Some studies also show that the upstream and downstream KEs can be observed at the same doses of radiation. For example, X-ray irradiation of mice resulted in oxidative stress, altered signaling and reduced NO levels at both 8 and 16 Gy (Azimzadeh et al., 2015). Dose concordance is not consistent across studies, but this may be due to differences in models, timepoints, and radiation types used.  

A limited number of studies support incidence concordance. In these, the upstream KE demonstrates a greater change than the downstream KE following exposure to a stressor. For example, mice exposed to 18 Gy of X-rays showed a roughly 2-fold increases in both oxidative stress and pro-inflammatory markers. A 1.3-fold increase in markers for endothelial dysfunction was observed (Shen et al., 2018).

Uncertainties and inconsistencies  

The collection of WOE identifies several important uncertainties in the literature. These include lack of quantitative understanding, low-dose or chronic-exposure studies, data from female models and consistency in measurement of NO levels.  

The WOE contained data from a wide variety of interdisciplinary fields; consequently, experimental design was equally varied. Overall, studies did not use consistent doses, radiation types, time-points, or evaluation of endpoints. Since dose and type of radiation can affect biological responses, quantitative understanding of relationships could not be determined and was low overall. Additionally, most studies used single or select doses, with limited studies exploring relatively low doses (<0.5 Gy (EPRI, 2020)). Harmonized experiments evaluating changes to adjacent endpoints across a wide range of doses or time-points with consistency of radiation type would greatly benefit quantitative understanding for this AOP.  

Similarly, the WOE is lacking in evidence using female models. Sex is an important modulating factor in cardiovascular changes and studies suggest vascular remodeling responses of astronauts can vary by sex (Hughson et al., 2016). The consequence of the general bias in clinical research (Rios et al., 2020; Yakerson, 2019) from which the current WOE draws, is the very large knowledge gaps in mechanistic data for the female body. Filling these knowledge gaps at all levels of biological organization will be an important step in solidifying the AOP.   

Evaluation of NO levels was inconsistent between studies. According to the biological plausibility, deposition of energy and subsequent oxidative stress would lead to a decrease in NO that then contributes to impaired vascular relaxation as part of endothelial dysfunction. However, primary research concludes that NO can either increase (Abdel-Magied & Shedid, 2020; Hirakawa et al., 2002; Sakata et al., 2015; Sonveaux et al., 2003) or decrease (Baker et al., 2009; Fuji et al., 2016) following irradiation. Proxy measures used to detect NO, like NOS enzyme activities or nitrite/nitrate levels, may not directly correspond to changes in NO levels. Further standardization in NO measurement and interpretation could refine this KE to become the depletion of NO. 

The present AOP serves as a platform to promote broader collaborative efforts to understand non-cancer health risks from radiation exposures. It will be a foundational AOP of regulatory interest to researchers seeking areas of knowledge gaps to prioritize research in understanding mechanisms of CVD. The AOP is also relevant to space agencies and clinicians working to improve the guidance on health risks from long-term spaceflight and radiotherapy treatments, respectively. The WOE for this AOP may also inform parameters in biologically based risk models and can serve to develop countermeasures to protect the cardiovascular systems of future space travelers on deep space missions. The present qualitative AOP can be used to guide the design of experiments that will provide quantitative understanding for the KERs to support risk-model development and inform additional guidelines for radiation protection; additionally, the identified research gaps could help prioritize research needs for funding strategies.

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