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Relationship: 2776

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Oxidative Stress leads to Increase, Endothelial Dysfunction

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Oxidative Stress

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Increase, Endothelial Dysfunction

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Deposition of energy leads to abnormal vascular remodeling	non-adjacent	Moderate	Low	Vinita Chauhan (send email)	Open for citation & comment

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
human	Homo sapiens	Low	NCBI
mouse	Mus musculus	Moderate	NCBI
rat	Rattus norvegicus	High	NCBI
pigs	Sus scrofa	Low	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Male	High
Female	Low
Unspecific	Low

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Adult	Moderate
Not Otherwise Specified	Low

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Oxidative stress describes the imbalances in reactive oxygen and reactive nitrogen species (RONS) radical formation as well as antioxidants and reactive oxygen species (ROS) scavengers (Beckhauser et al., 2016; Elahi et al., 2009; Ray et al., 2012). Oxidative stress can lead to endothelial dysfunction. Within the cardiovascular system, every vessel is lined with a single layer of endothelial cells (Augustin et al., 1994; Fishman, 1982). This endothelial layer plays a crucial role in the regulation of vascular homeostasis through controlling various factors such as vascular permeability, vasomotion, and immune response (Baran et al., 2021; Bonetti et al., 2003; Hughson et al., 2018; Slezak et al., 2017; Sylvester et al., 2018). Of the vascular wall components, the endothelium is also the most vulnerable to damage from ROS (Soloviev & Kizub, 2018). Endothelial cells normally exist in a quiescent state characterized by high nitric oxide (NO) bioavailability (Carmeliet & Jain, 2011); however, cells can become activated as part of a normal host-defence response following tissue injury or oxidative stress (Deanfield et al., 2007; Krüger-Genge et al., 2019). Sustained activation leads to the pathological state of endothelial dysfunction, which is defined by decreased NO bioavailability, increased vessel permeability, altered vasomotion, and a pro-thrombotic and inflammatory environment (Baran et al., 2021; Bonetti et al., 2003; Deanfield et al., 2007; Schiffrin, 2008). Shifting redox balance towards oxidation is known to indirectly lead to endothelial dysfunction through various mechanisms (Hughson et al., 2018; Ramadan et al., 2020; Soloviev & Kizub, 2018). There are several ways through which imbalanced ROS can affect endothelium function, including decreasing NO bioavailability through direct scavenging, which forms the RNS peroxynitrite (ONOO-) (Hatoum et al., 2006; Li et al., 2002; Schiffrin, 2008; Soloviev & Kizub, 2018; Venkatesulu et al., 2018), as well as impeding NO production and diffusion (Hatoum et al., 2006; Li et al., 2002; Schiffrin, 2008; Soloviev & Kizub, 2018; Venkatesulu et al., 2018; Schiffrin, 2008; Soloviev & Kizub, 2018). Additionally, elevated ROS contribute to introducing a pro-inflammatory and pro-thrombotic milieu characteristic of dysfunction (Hughson et al., 2018; Schiffrin, 2008; Slezak et al., 2017; Tapio, 2016; Venkatesulu et al., 2018). It is also linked to decreased vasomotion (Schiffrin, 2008; Soloviev & Kizub, 2018; Venkatesulu et al., 2018) and finally the onset of endothelial cell apoptosis and premature senescence (Borghini et al., 2013; Hughson et al., 2018; Tapio, 2016; Wang et al., 2016).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

The strategy for collating the evidence on radiation stressors to support the relationship is described in Kozbenko et al 2022. Briefly, a scoping review methodology was used to prioritize studies based on a population, exposure, outcome, endpoint statement.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Overall weight of evidence: Moderate

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Mechanisms for oxidative stress leading to endothelial dysfunction are outlined in various reviews on the topic (Hughson et al., 2018; Nagane et al., 2021; Slezak et al., 2017; Soloviev & Kizub, 2018; Venkatesulu et al., 2018; Wang et al., 2016).

It is broadly accepted that elevated ROS can indirectly lead to endothelial dysfunction by causing an imbalance of NO, specifically the decrease in NO bioavailability, with biologically plausible mechanisms described. Firstly, ROS can react with NO directly; if quenching outpaces NO production, it will cause reduced NO bioavailability underlying endothelial dysfunction (Hatoum et al., 2006; Li et al., 2002; Soloviev & Kizub, 2018). In particular, the superoxide anion (O2•–) reacts with NO to form peroxynitrite, both reducing available NO and further accelerating NO degradation (Li et al., 2002; Soloviev & Kizub, 2018). In addition, superoxide and peroxynitrite can uncouple eNOS, which produces more ROS instead of NO (Soloviev & Kizub, 2018). Peroxynitrite can cause cellular senescence as a part of endothelial dysfunction (Nagane et al., 2021). eNOS downregulation and subsequent drop in NO levels are caused in part by increased endothelin-1 (ET-1), a vasoconstrictor with enhanced secretion during an oxidative stress state (Marasciulo, Montagnani & Potenza, 2006; Ramadan et al., 2020). ROS is also involved in perturbing NO diffusion from the endothelial cells (Soloviev & Kizub, 2018). Overall, the decreased NO bioavailability causes reduced vasodilation and endothelial dysfunction (Soloviev & Kizub, 2018).

Oxidative stress is also established to affect endothelial function through inhibition of endothelium-dependent vasodilation (Soloviev & Kizub, 2018; Venkatesulu et al., 2018). ROS in both endothelial cells and surrounding vascular smooth muscle cells (VSMCs) act as second messengers to many cellular pathways that mediate VSMC contractility and endothelial permeability and function, causing disruption to these endothelial functions (Hughson et al., 2018; Li et al., 2002; Ramadan et al., 2020; Soloviev & Kizub, 2018; Ungvari et al., 2013; Venkatesulu et al., 2018). Specifically, impaired endothelium-dependent vasomotion following radiation (Venkatesulu et al., 2018) was suggested to be due to the loss of prostaglandin F2α (PGF2α) inhibition and therefore, vasoconstriction (Li et al., 2002).

Oxidative stress is also involved in inducing the pro-thrombotic and inflammatory environment of endothelial dysfunction. In the case of radiation-induced endothelial injury, radiation type, fraction size used, and endothelial cell model used all influence the resulting downstream endpoints (Venkatesulu et al., 2018). Possible changes to the endothelial milieu include alterations of cell adhesion molecule levels, creation of pro-thrombotic environment, endothelial cell apoptosis and inflammation (Hughson et al., 2018; Nagane et al., 2021; Slezak et al., 2017; Tapio, 2016; Venkatesulu et al., 2018). When induced by oxidative stress, nuclear factor kappa B (NF-кB) can target genes involved with the upregulation of prothrombotic markers associated with endothelial dysfunction (Slezak et al., 2017). Free radicals produced by macrophages also stimulate TGF-β, accelerating the creation of a profibrotic milieu (Venkatesulu et al., 2018). ROS can also oxidize low-density lipoproteins (LDL) resulting in structural complications, as oxidized LDL accumulates in blood circulation due to decreased cell uptake (Nagane et al., 2021; Slezak et al., 2017). Furthermore, endothelial cells can undergo morphological changes following oxidative injury; cells become enlarged and form fibrin networks, showing increased levels of activated platelets and leukocytes with membrane protrusions and pseudopodial extensions, which are all indicative of an inflammatory and pro-thrombotic state (Li et al., 2002).

Furthermore, ROS can induce premature endothelial cell senescence, which in turn contributes to overall endothelial dysfunction (Hughson et al., 2018; Nagane et al., 2021; Tapio, 2016). In contrast to replicative senescence attributed to telomere dysfunction, oxidative stress is one of several injuries causing stress-induced premature senescence (Nagane et al., 2021). This is thought to occur through oxidative stress causing the induction of the p53/p21 pathway that regulates cell senescence (Borghini et al., 2013; Wang et al., 2016). Once senescent, the endothelial cells contribute to dysfunction in multiple ways. Firstly, senescence can stimulate a pro-inflammatory response and trigger apoptosis through decreased cell repair (Nagane et al., 2021; Ramadan et al., 2020). Additionally, senescent cells themselves are sources of ROS, furthering both genomic instability (causing additional senescence in neighbouring cells) and endothelial dysfunction itself (Tapio, 2016; Wang et al., 2016). Senescent cells also lack proper endothelial cell function, contributing to changing the environment to a dysfunctional one (Hughson et al., 2018; Tapio, 2016). This lack of function includes a decrease in NO production, increased monocyte adhesion, and loss of cell barrier integrity paired with increased levels of ET-1 (Hughson et al., 2018; Nagane et al., 2021; Tapio, 2016).

Finally, oxidative stress has been shown to lead to mitochondrial dysfunction and dysregulation, which is thought to play an important role in the development of endothelial dysfunction (Borghini et al., 2013; Hughson et al., 2018; Nagane et al., 2021; Slezak et al., 2017).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Empirical evidence provides a moderate level of support to this KER. Examples of this evidence are summarized here and further in attached tables. The evidence to support the relationship between oxidative stress leading to endothelial dysfunction was gathered from studies using in vitro and in vivo rat and mice models (Delp et al., 2016; Hatoum et al., 2006; Shen et al., 2018; Soucy et al., 2007; Soucy et al., 2010; Soucy et al., 2011; Ungvari et al., 2013), in vivo pig models (Li et al., 2002) and human in vitro cells (Ramadan et al., 2020). Various stressors were applied, including X-rays, hindlimb unloading (HU), heavy ions (56Fe ions), beta-rays (32P), and gamma rays with a dose range of 0.1 to 22.5 Gy. To determine the effect of oxidative stress on endothelial dysfunction, various assays and endpoints were measured including: senescence-associated β-galactosidase (SA β-gal), insulin-like growth factor-binding protein-7 (IGFBP-7) and growth differentiation factor 15 (GDF-15) as senescence markers, caspase 3/7 activity as an apoptosis marker, endothelin-1 levels for the ratio of apoptotic to normal cells, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT) as aortic oxidative damage markers, superoxide production, vascular tension, and ROS detection via xanthine oxidase (XO) activity, and fluorescent dyes, such as dihydroethidium fluorescence.

Dose Concordance

Dose concordance between the two key events is supported by numerous studies. Hatoum et al. (2006) explored the effect of 3 to 9 cumulative X-ray doses of 0.25 Gy on murine intestinal arterioles. The study found that at the dose when superoxide and hydrogen peroxide generation increased, vasodilation in response to acetylcholine (ACh) decreased (Hatoum et al., 2006). Another study using various doses on cerebral microvascular endothelial cells found a significant change in cellular peroxide and mitochondrial oxidative stress following a 4 Gy X-ray dose, concordant with SA β-gal showing the first large increase at 4 Gy (Ungvari et al., 2013).

Ramadan et al. (2020) used X-ray irradiation in multiple types of human endothelial cells to show that ROS production is significantly higher in both the 0.1 Gy and 5 Gy compared to the control. These changes were correlated to endothelial dysfunction; SA β-gal activity and endothelial apoptosis showed a response of greater magnitude following the 5 Gy dose compared to 0.1 Gy (Ramadan et al., 2020).

Exposure of mice to 18 Gy X-rays led to a ~1.8-fold and ~2.2-fold decrease in 4-HNE and 3-NT in aorta respectively, both being markers of aortic oxidative damage. Simultaneously, the 18 Gy dose caused a ~5-fold increase in aortic apoptosis, a marker of endothelial dysfunction (Shen et al., 2018). Two studies (Soucy et al., 2007; Soucy et al., 2010) showed that varying doses (0.5 and 5 Gy) of gamma radiation led to significant increases in ROS levels in rat aorta. Subsequently, endothelial function was affected with a 0.5 Gy dose resulting in a ~30% decrease in ACh-induced vasodilation response (Soucy et al., 2007), and a 5 Gy dose leading to a ~13-15% decrease in ACh-induced vasodilation (Soucy et al., 2010).

Iron ion irradiation resulted in oxidative stress and endothelial dysfunction, both occurring after 1 Gy (Soucy et al., 2011). Similarly, Delp et al. (2016) showed total body 56Fe irradiation at 1 Gy led to a ~2-fold increase in XO activity and a ~10% decrease in ACh response in mice (Delp et al., 2016). Delp et al. (2016) also explored the effects of HU on mice and found no significant changes to XO activity or ACh response following 2-week HU, while HU in combination with 1 Gy 56Fe radiation led to a ~2.2-fold increase in XO activity and the same ~10% decrease in ACh response. Li et al. (2002) showed ~3.5-fold increase in superoxide anion production and a ~25-80% decrease in vasoconstriction and vasodilation response to various vasomotive substances following 20 Gy 32P radiation in pig coronary arteries.

Time Concordance

There is some evidence of time concordance between oxidative stress and endothelial dysfunction. Ramadan et al. (2020) used human endothelial cells and showed ROS production first increased 45 minutes after 5 Gy X-ray exposure before returning to baseline levels after 2 hours. Apoptosis markers Annexin V and Caspase 3/7 were first increased after 4 hours. Cellular senescence evaluated with the SA-β-gal activity was first measured only after 7 days (Ramadan et al., 2020). ROS production and dilation response to ACh after irradiation were measured at various times as cumulative doses were given, which showed increased ROS and decreased dilation of rat intestinal microvessels both after 5 days of cumulative 0.25 Gy X-ray doses (Hatoum et al., 2006). Work using 4-HNE and 3-NT as biomarkers of oxidative stress in mice aorta following 18 Gy 6 MV X-ray at 3, 7, 14, 28, and 84 days after irradiation showed both markers to be significantly elevated starting after 3 days. Exposure also led to significantly increased apoptosis, indicating endothelial dysfunction after 3 days (Shen et al., 2018).

Incidence Concordance

There is moderate support in current literature for an incidence concordance relationship between oxidative stress and endothelial dysfunction. Three out of the 9 primary research studies used to support this AOP demonstrated an average change to endpoints of oxidative stress that was greater or equal to that of endothelial dysfunction (Soucy et al., 2011; Soucy et al., 2010; Soucy et al., 2007).

Essentiality

Essentiality in the relationship was demonstrated in multiple studies. Studies by Soucy et al. (2007, 2010, 2011) explored the relationship between radiation exposure, ROS levels and endothelial function, all focusing on the role of XO. Soucy et al. (2007) incubated aortic rings from irradiated rats in the XO inhibitor oxypurinol (Oxp) and saw this treatment results in recovery of ACh vasodilation response. Soucy et al. (2010) showed that administration of allopurinol (a superoxide scavenger) following irradiation led to significantly decreased ROS levels. Additionally, the latter two studies showed that XO inhibition by dietary administration of Oxp significantly decreased XO activity and ROS levels while simultaneously recovering ACh response (Soucy et al., 2010, 2011). Similar results were observed when treatment with manganese tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), a SOD mimetic, returned peroxide and superoxide levels and significantly improved ACh response irradiated rats (Hatoum et al., 2006). Dietary treatment with Tempol, a water-soluble SOD-mimetic likewise increased vasomotion and decreased superoxide levels (Hatoum et al., 2006).

Human bone marrow mesenchymal stem cells (hBMSCs) have also been studied for their ability to prevent radiation-induced aortic injury. Both high and low doses of hBMSCs were shown to increase catalase and HO-1 antioxidant activity, and decrease levels of the aortic oxidative damage markers 4-HNE and 3-NT. Subsequently, this treatment also significantly decreased levels of apoptosis in the aorta (Shen et al., 2018). Finally, blocking Connexin43 hemichannels using TAT-Gap19 peptide also significantly reduced oxidative stress and resultant cell senescence and death, suggesting the role of intracellular communication in mediating radiation response (Ramadan et al., 2020).

Studies have also shown that transgenic mice overexpressing superoxide dismutase (SOD) have a twofold reduction in aortic lesions following X-ray exposure compared to control (Hughson et al., 2018). Overexpression of SOD also mitigates atherosclerotic plaque formation, further outlining the relationship between oxidative stress and the pathological environment of endothelial dysfunction (Tapio, 2016).

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Work by Ramadan et al. (2020) explored the use of TAT-Gap19 to block endothelial intracellular communication in order to modulate radiation response of intercellular connexin proteins. Overall, TAT-Gap19 was shown to reduce ROS production and subsequent senescence (SA β-gal activity) and apoptosis (Annexin V and Caspase 3/7) markers. However, treatment with TAT-Gap19 led to an increase in SA β-gal in non-irradiated control at the 9-day point. Additionally, the 0.1 Gy irradiated group showed persistent SA β-gal activity at all time points studied, while the 5 Gy group demonstrated an unexpected decrease before day 14

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Modulating factor	Details	Effects on the KER	References
Drug	MnTBAP (a superoxide dismutase mimetic)	Treatment with MnTBAP after irradiation was able to reduce superoxide and peroxide levels and restore vasodilation ability	(Hatoum et al., 2006)
Drug	Tempol (a superoxide dismutase mimetic)	Treatment with tempol after irradiation was able to restore vasodilation ability	(Hatoum et al., 2006)
Drug	TAT-Gap19 (inhibitor of connexin 43 which is associated with atherogenesis and endothelial stiffness)	Treatment with TAT-Gap19 led to a decrease in ROS and SA β-gal levels after irradiation	(Ramadan et al., 2020)
Drug	hBMSCs (protect against vascular damage through antioxidant properties)	Treatment with hBMSCs after irradiation caused increased catalase and HO-1, as well as decreased oxidative damage and apoptosis	(Shen et al., 2018)
Drug	Oxp (can inhibit XO, a source of ROS)	Treatment with Oxp showed decreased XO activity and ROS production along with increased vasodilation after irradiation	(Soucy et al., 2007; Soucy et al., 2010; Soucy et al., 2011)

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

The following are a few examples of quantitative understanding of the relationship. All data that is represented is statistically significant unless otherwise indicated.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Dose/incidence concordance

Reference	Experiment Description	Result
Soucy et al. 2007	In vivo. Sprague-Dawley rats were whole-body irradiated with 137Cs gamma radiation at 50, 160 and 500 cGy. XO is a primary source of cardiac ROS and was used as a measure of oxidative stress. Vasodilation response to ACh was used to evaluate endothelial function.	At 500 cGy, XO activity was 2-fold elevated compared to control, and there was also an increase in XO quantity. Simultaneously, endothelial dysfunction was observed as a ~30 percentage point decrease in vasodilation response to ACh.
Soucy et al. 2010	In vivo. In Sprague-Dawley rats were whole-body irradiated with 137Cs gamma radiation at 5 Gy. ROS were measured using dihydroethidium fluorescence. Aortic relaxation response to ACh was also measured.	After 5 Gy, ROS increased 1.7-fold and relaxation decreased 0.7-fold.
Soucy et al. 2011	In vivo. Wistar rats were exposed to 0.5 and 1 Gy doses of 56Fe-ion radiation. ROS production rates were evaluated using dihydroethidium fluorescence. ACh-induced vasodilation responses were measured.	56Fe-ion irradiation at 1 Gy produced a 1.8-fold increase in ROS levels. At 10-5 M ACh, aorta without irradiation relaxed by 87%, while aorta with 1 Gy irradiation had significantly lower relaxation of 76%.
Li et al. 2002	In vivo. Surgically exposed coronary arteries of yucatan pigs were irradiated with 20 Gy 32P β-irradiation. Oxidative stress was evaluated through superoxide production. Endothelial function was evaluated through endothelial-dependent vasomotor response and morphological changes.	Superoxide production increased 3.5-fold between the control and 20 Gy irradiated groups. Vasodilation response to KCl dropped over 50% in the irradiated group. Morphological changes were also observed, with irradiated arteries seeing enlarged endothelial cells, formation of fibrin networks, activated platelets, leukocytes exhibiting membrane protrusions and pseudopodial extensions all indicative of an inflammatory and pro-thrombotic state of endothelial dysfunction.
Shen et al. 2018	In vivo. Male mice were irradiated with 18 Gy X-rays, oxidative stress was measured with 4-HNE and 3-NE oxidative damage markers, and endothelial dysfunction was determined through apoptosis.	4-HNE showed a maximum increase of ~1.8-fold and 3-NT showed a maximum increase of ~2.3-fold. Apoptosis levels peaked at a ~5-fold increase above control levels.
Ramadan et al. 2020	In vitro. Telomerase-immortalized human Coronary Artery and Microvascular Endothelial cells (TICAE) and Telomerase Immortalized human Microvascular Endothelial cells (TIME) were exposed to X-rays (0.1 and 5 Gy). ROS production was measured using CM-H2DCFDA combined with Incucyte live cell imaging. Endothelial dysfunction was evaluated through: Endothelial apoptosis Annexin V and Caspase 3/7 marker levels Dextran fluorescein dye uptake level by necrotic cells Cell senescence SA β-gal activity IGFBP-7 and GDF-15 senescence marker levels Endothelin-1 levels	ROS production was increased in TIME cells after 0.1 and 5 Gy dose. In both coronary and endothelial cells, apoptosis mainly occurred after 5 Gy radiation. With coronary cells demonstrating an increase in Annexin V and Caspase 3/7 markers and endothelial cells showing elevated Annexin V and membrane leakage. SA β-gal activity significantly increased for both 0.1 and 5 Gy doses. IGFBP-7 and GDF-15 levels were also elevated in both cell types; GDF-15 increasing at both 0.1 and 5 Gy doses, while IGFBP-7 only showed significant elevation at the 5 Gy dose. With the 0.1 Gy dose, there was a significant increase in SA β-gal activity of ~3-fold, while at 5 Gy the activity increased ~5-fold. Endothelin-1 was significantly elevated following 5 Gy irradiation in both cell types.
Ungvari et al. 2013	In vitro. Cerebral microvascular endothelial cells (CMVECs) from F344×BN rats were harvested and cultured. Following culture, cells were irradiated with 137Cs gamma radiation at doses between 2-8 Gy. Oxidative stress was evaluated through cellular peroxide and superoxide production. Endothelial dysfunction was evaluated through cell senescence via SA-β-gal presence, and apoptosis via caspase 3/7 maker and ratio of apoptotic:viable cells.	Oxidative stress increased in a dose-dependent manner following irradiation. Change of ROS became significant after 4 Gy at a ~1.5-fold increase and reached ~3-fold increase at the highest studied dose of 8 Gy. Mitochondrial oxidative stress also became significant after 4 Gy and increased linearly for a peak of a ~1.5-fold increase at 8 Gy. Endothelial cell senescence and apoptosis were similarly found to increase in a dose dependent manner. With ~30% of cells being SA-β-gal positive after 8 Gy irradiation, signalling premature senescence. Ratio of dead cells peaked at 10% and Caspase 3/7 peaking at a ~5.5-fold change following 18h post irradiation.
Hatoum et al. 2006	In vivo. Effect of cumulative radiation doses on rat gut microvessels was studied. Rats were exposed to 1 to 9 fractions of 250 cGy for a total dose of up to 2250 cGy. Following exposure, the animals were euthanized, and submucosal vessels isolated. Oxidative stress was measured through superoxide and peroxide levels. Endothelial function was assessed through ACh vasodilation response and NO bioavailability via DAF-FM fluorescence.	After the final cumulative dose of 2250 cGy, superoxide was ~1.6-fold elevated and peroxides were ~1.7-fold elevated compared to non-irradiated controls. ROS levels increased sharply after the second dose, immediately preceding drop in ACh vasodilation response. Max dilation dropped from 87% to 3% between pre-irradiation and post-final radiation dose. ACh response remained within control levels following fractions 1 and 2, however following fraction 3, response dropped below 30% for all remaining doses. Following all radiation doses, NO bioavailability dropped ~0.8-fold.
Delp et al. 2016	In vivo. The effects of HU and 1 Gy dose of 56Fe radiation of the gastrocnemius muscle feed arteries and coronary arteries of C57BL/6 mice was studied. Xanthine oxidase (XO) levels were used as a measure of ROS production and therefore oxidative stress. Endothelial function was evaluated through vasodilation response to ACh.	Following 2-week HU, there were no significant changes to XO levels or vasomotor response. Following total body irradiation with 1 Gy, there was a ~2-fold increase in XO activity in both gastrocnemius muscle feed and coronary arteries. Vasodilation response subsequently decreased ~10 percentage points. Combined HU and total body irradiation led to a ~2.3-fold increase in XO activity in both artery types and vasodilation response decrease of ~10 percentage points.

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Time concordance

Reference	Experiment Description	Result
Soucy et al. 2007	In vivo. Sprague-Dawley rats were whole-body irradiated with 137Cs gamma radiation at various doses. 2 weeks after irradiation, the animals were euthanized, and aortas were harvested. XO was used as a measure of oxidative stress. Dose-dependent vasodilation response to ACh was used to evaluate endothelial function.	XO activity was elevated 2-fold compared to control, and there was also an increase in XO quantity. Simultaneously, endothelial dysfunction was seen with a ~30% decrease in vasodilation response to ACh.
Soucy et al. 2010	In vitro. Sprague-Dawley rats were whole-body irradiated with 137Cs gamma radiation. 2 weeks after receiving radiation dose, the animals were euthanized and aortas were harvested. ROS were measured using dihydroethidium fluorescence. Aortic relaxation response to ACh was also measured.	ROS increased 1.7-fold and relaxation simultaneously decreased 0.7-fold.
Soucy et al. 2011	In vivo. Wistar rats were exposed to 56Fe-ion radiation. Rats were euthanized at 4 months post-irradiation and aorta was harvested. ROS production rates evaluated using dihydroethidium along the ACh-induced vasodilation response were measured.	ROS levels increased by 75% 4 months post-irradiation. ACh vasodilation response decreased by 13%.
Shen et al. 2018	In vivo. Male mice were irradiated with 18 Gy X-rays. Immunohistochemical staining assessed oxidative stress using 4-HNE and 3-NE as markers for oxidative damage. Endothelial dysfunction was determined through apoptosis.	Exposure to 18 Gy caused increased 4-HNE and 3-NT levels. 4-HNE showed a maximum ~1.8-fold increase at 14-days post radiation, and 3-NT showed a maximum ~2.3-fold increase 7 days post radiation. Apoptosis levels peaked at 7 days post-irradiation with a ~5-fold increase above control levels.
Ramadan et al. 2020	In vitro. TICAE and TIME cells were exposed to X-rays (0.1 and 5 Gy). Oxidative stress was evaluated through intracellular ROS production. Endothelial dysfunction was evaluated through: Endothelial apoptosis Annexin V and Caspase 3/7 marker levels Dextran fluorescein dye uptake level by necrotic cells Cell senescence SA β-gal activity IGFBP-7 and GDF-15 senescence marker levels Endothelin-1 levels	Highest response was observed for both doses at 45 minutes after irradiation followed by a decline at the 2- and 3-hour time points but remaining elevated above non-irradiated control levels. Endothelial cells studied produced more ROS than the coronary cells. Caspase 3/7 and annexin V increased linearly until 100h. SA β-gal activity significantly increased at 7 and 9 days. GDF-15 and IGFBP-7 were increased after 7 days. Endothelin-1 was found to be significantly elevated after 7 days.
Ungvari et al. 2013	In vitro. Cerebral microvascular endothelial cells (CMVECs) from F344×BN rats were harvested and cultured. Following culture, cells were irradiated with 137Cs gamma radiation. Oxidative stress was evaluated through cellular peroxide and superoxide production. Endothelial dysfunction was evaluated through cell senescence via SA-β-gal presence, and apoptosis via caspase 3/7 maker and ratio of apoptotic:viable cells.	Superoxide and peroxide increased 1 day but not 14 days post-irradiation ~30% of cells were SA-β-gal positive after 8 Gy irradiation, measured 7 days post-irradiation. 24 h after irradiation, 10% of cells were dead. Caspase 3/7 increased from 2 to 18 h, peaking at a ~5.5-fold change following 18 h post-irradiation but decreased at 24 h.
Hatoum et al. 2006	In vivo. Effect of cumulative radiation doses on rat gut microvessels was studied. Rats were exposed to 1 to 9 cGy in 3 fractions per week on alternate days for 3 successive weeks for a total dose of up to 2250 cGy over a total time of 19 days. Oxidative stress was measured through superoxide and peroxide levels from various fluorescent markers. Endothelial function was assessed through ACh vasodilation response.	After 19 days, superoxide was ~1.6-fold elevated and peroxides were ~1.7-fold elevated compared to non-irradiated controls. ROS levels increased at day 5, at the same time as a drop in ACh vasodilation response. Max dilation dropped from 87% to 3% between day 1 and day 19.

Known Feedforward/Feedback loops influencing this KER

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Domain of Applicability

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The evidence is derived from rat in vivo and in vitro models. Mice cell-derived studies were also available but less in-vivo evidence was available from this species. There was a low number of studies containing human or pig models to support this KER. Males have been studied more often than females. There are a few studies with unspecified lifestage of models, while the studies with a defined age typically used adult models.

References

List of the literature that was cited for this KER description. More help

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