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Relationship: 2789

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

Altered, Nitric Oxide Levels leads to Increase, Endothelial Dysfunction

Upstream event

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

Altered, Nitric Oxide Levels

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	adjacent	Moderate	Low	Vinita Chauhan (send email)	Open for citation & comment	WPHA/WNT Endorsed

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
rat	Rattus norvegicus	Moderate	NCBI
rabbit	Oryctolagus cuniculus	Low	NCBI

Sex Applicability

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

Sex	Evidence
Male	Moderate
Female	Low
Unspecific	Low

Life Stage Applicability

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

Term	Evidence
Adult	Low
Not Otherwise Specified	Moderate

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

Altered nitric oxide (NO) levels can lead to endothelial dysfunction (Soloviev & Kizub, 2019). In a functional endothelium, NO is bioavailable and is involved in preventing inflammation, proliferation and thrombosis (Deanfield, Halcox & Rabelink, 2007; Kruger-Genge et al., 2019). An increase in reactive oxygen species (ROS) along with increased NO can drive cellular senescence in endothelial cells (ECs) and catalyze endothelial dysfunction (Nagane et al., 2021; Wang, Boerma & Zhou, 2016). Another driver of endothelial dysfunction is reduced vasomotion. In a functional state, the endothelium requires a balance of vasoconstrictors and vasodilators (like NO); an interruption of this balance can lead to dysfunction (Deanfield, Halcox & Rabelink, 2007; Marti et al., 2012; Nagane et al., 2021; Schulz, Gori & Münzel, 2011; Soloviev & Kizub, 2019). Decreased NO due to direct reactions with ROS or uncoupling of NOS enzymes will lead to a reduced ability of smooth muscle cells (SMCs) to relax (Soloviev & Kizub, 2019).

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

The biological plausibility surrounding the connection between altered NO levels leading to endothelial dysfunction is well-supported by literature. NO is synthesized from L-arginine and oxygen with the aid of enzymes and cofactors (Nagane et al., 2021). NO regulates ECs by binding to soluble guanylyl cyclase (sGC) to create cGMP and activate protein kinase G (PKG), leading to activation of Ca2+-dependent vasodilation and smooth muscle relaxation (Nagane et al., 2021; Soloviev & Kizub 2019). NOS isoforms, such as inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) that synthesize NO are indirect measures of NO. Lower NO reduces the ability of SMCs relaxation and dilates the blood vessel leading to an inability to control vasodilation, a component of endothelial dysfunction (Soloviev & Kizub 2019).

Increased expression of NOS enzymes can result in reduced NO levels in the case of insufficient L-arginine substrate or BH4 cofactor leading to ROS production instead of NO (Zhang et al., 2009). ROS can decrease NO bioavailability by uncoupling/downregulating eNOS or converting NO to peroxynitrite (Mitchel et al 2019; Schulz, Gori & Münzel, 2011; Soloviev & Kizub 2019; Wang, Boerma & Zhou, 2016). A further decrease in NO occurs as peroxynitrite oxidizes BH4 to BH2 and induces eNOS to produce ROS, continuing the uncoupling of more NOS enzymes (Hong et al., 2013; Soloviev & Kizub, 2019; Zhang et al., 2009). A reduction in NO bioavailability due to ROS is an important mediator of endothelial dysfunction (Schulz, Gori & Münzel, 2011).

Another component of endothelial dysfunction influenced by NO levels is cellular senescence (Nagane et al., 2021). iNOS expression increases following an increase in oxidative stress (Nathan & Xie, 1994). In the presence of oxidative stress, NO is converted to peroxynitrite, which is a reactive nitrogen species (RNS) that can modify proteins and lead to cellular senescence (Hong et al., 2013; Nagane et al., 2021; Soloviev & Kizub, 2019). Although NO can increase at first and cause cell senescence, senescent ECs show downregulation and/or uncoupling of eNOS that contributes to a decrease of NO in the endothelium (Wang, Boerma & Zhou, 2016). These changes in senescent ECs lead to endothelial dysfunction.

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

The empirical evidence to support this KER was gathered from research that utilized in vivo rat and rabbit models (Soucy et al., 2010; Soucy et al., 2011; Hong et al., 2013; Yan et al., 2020; Zhang et al., 2009), ex vivo rabbit models and in vitro HUVEC models (Hong et al., 2013). Stressors used to alter NO levels and increase endothelial dysfunction include 56Fe ions (Soucy et al., 2011), X-rays (Hong et al., 2013; Yan et al., 2020), gamma rays (Soucy et al., 2010) and altered gravity by hindlimb unweighting (HU) (Zhang et al., 2009). Irradiation dose levels ranged from 0.5 Gy to 16 Gy (Hong et al., 2013; Soucy et al., 2010; Soucy et al., 2011; Yan et al., 2020). Studies used various endpoints to measure NO levels while endothelial function was consistently determined through relaxation response to acetylcholine (ACh). Methods to measure NO included DAF-FM DA fluorescent probe (Soucy et al., 2010; Soucy et al., 2011), Griess reagent NO assay kit (Yan et al., 2020), eNOS dimer to monomer ratio (Yan et al., 2020), and NOS protein and mRNA levels (Hong et al., 2013; Zhang et al., 2009).

Dose Concordance

There is moderate evidence to demonstrate dose concordance between a decrease in nitric oxide and endothelial dysfunction. Iron-56 ion irradiation in rat aorta showed a decrease in both NO levels and endothelial relaxation at 1 Gy. However, endothelial relaxation did not significantly decrease at a lower dose of 0.5 Gy (Soucy et al., 2011). After 5 Gy gamma irradiation, NO production and endothelial relaxation both decreased 0.7-fold in rat aorta (Soucy et al., 2010). Increased NOS levels can cause further NO decreases due to uncoupling and production of more peroxynitrite, which is the result of NO reacting with ROS and can therefore, be used to determine NO levels (Hong et al., 2013; Zhang et al., 2009). In rat mesenteric arteries, NO levels decreased 0.6-fold after 4 Gy X-ray irradiation, while endothelial relaxation decreased 0.1-fold compared to non-irradiated arteries (Yan et al., 2020). X-ray irradiated human umbilical vein endothelial cells (HUVECs) showed significantly increased iNOS and peroxynitrite after 4 Gy, and X-ray irradiated rabbit carotid arteries had decreased relaxation after 8 and 16 Gy (Hong et al., 2013). Hong et al (2013) also showed that endothelial relaxation was lower after 16 Gy than 8 Gy ex vivo.

Altered gravity resulted in an increase of eNOS and increase of iNOS in carotid arteries (Zhang et al., 2009). Increases in eNOS and iNOS corresponded to a decrease in carotid artery relaxation from 64% to 33% at the same level of HU (Zhang et al., 2009). These studies showed increases in NOS isoforms and corresponding decreases in endothelial function, such as increased vasoconstriction and impaired vasodilation following altered gravity.

Time Concordance

Evidence of time concordance between altered NO levels and endothelial dysfunction is limited from the studies cited. HUVECs irradiated with 4 Gy X-rays displayed an increase in nitrotyrosine (peroxynitrite biomarker indicating reduced NO) after 6 hours post-irradiation (Hong et al., 2013). While rabbit carotid arteries in vivo and ex vivo irradiated with 8 or 16 Gy X-rays showed decreased ACh-induced endothelial relaxation only after 20 hours post-irradiation (Hong et al., 2013).

Incidence Concordance

There is limited support in current literature for an incidence concordance relationship between altered NO and endothelial dysfunction. A primary research study that supports this AOP demonstrated an average change to endpoints of altered NO that was greater or equal to that of endothelial dysfunction (Zhang et al., 2009).

Essentiality

Many studies show the essentiality of decreased NO levels in endothelial dysfunction. After stressors like irradiation, xanthine oxidase (XO) can produce cardiac ROS that can react with NO and decrease its concentration or oxidize BH4 and uncouple eNOS. Oxypurinol (Oxp), an inhibitor of XO, has been shown to reverse these effects and reduce ROS levels, restore NO levels, and increase endothelial relaxation following irradiation (Soucy et al., 2010; Soucy et al., 2011). L-nitroarginine (L-NA, general NOS inhibitor) and aminoguanidine (AG, specific iNOS inhibitor) together were able to reduce relaxation of rabbit carotid arteries, suggesting that reduced NO levels are a key cause to endothelial dysfunction (Hong et al., 2013). In addition, L-NA and AG were able to reduce iNOS and nitrotyrosine (peroxynitrite biomarker) levels after irradiation (Hong et al., 2013). Gch1 is an enzyme involved in the synthesis of BH4, a cofactor for eNOS coupling. DAHP, a Gch1 inhibitor, caused the ratio of coupled-to-uncoupled eNOS to decrease and endothelial relaxation to also decrease, showing how coupled eNOS is necessary for endothelial function (Yan et al., 2020). Angiotensin II (AngII) type 1 (AT1) receptor activation can activate NOS. HU rats treated with losartan, an AT1 receptor antagonist, show reduced NOS levels and increased endothelial relaxation (Zhang et al., 2009).

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

Directionality of NO changes cannot be compared between studies due to a variety of experimental conditions like stressor type, dose, dose rate, model and time course of the experiment.

Irradiating in vivo rabbit carotid arteries with X-rays showed that endothelial dysfunction was higher after 8 Gy than 16 Gy (Hong et al., 2013). This was inconsistent with the ex vivo model, where endothelial dysfunction was highest after 16 Gy (Hong et al., 2013). Endothelial dysfunction was shown through a relaxation response to ACh.

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	AG (selective iNOS inhibitor)	AG treatment prevented radiation-induced increase in peroxynitrite and endothelial dysfunction along with L-NA treatment.	Hong et al., 2013
Drug	L-NA (general NOS inhibitor)	L-NA treatment prevented radiation-induced increase in peroxynitrite and endothelial dysfunction	Hong et al., 2013
Drug	DAHP (Gch1 inhibitor to inhibit BH4 synthesis)	DAHP (100 mg/kg/body weight) further decreased eNOS, nitrite concentration and endothelial relaxation after irradiation.	Yan et al., 2020
Drug	Losartan (AT1 receptor antagonist)	Losartan restored the levels eNOS and iNOS expression and improved endothelial relaxation after HU.	Zhang et al., 2009
Drug	Oxp (XO inhibitor)	Oxp increased NO levels and endothelial relaxation.	Soucy et al., 2010, 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 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., 2011	In vivo. 3-4 months-old rats were irradiated with 1 Gy 56Fe ions. Endothelial NO levels from male rat aorta were measured with a DAF-FM DA fluorescent probe in response to ACh-induced relaxation. Vascular tension response to ACh was measured in male rat aorta after 0, 0.5 and 1 Gy 56Fe ion irradiation. Doses were given at 0.5 Gy/min.	Iron ion irradiation at 1 Gy produced a decrease of 0.8-fold in NO levels compared to aorta without irradiation. At 10-5 M ACh, aorta without irradiation relaxed by 88%, while aorta with 1 Gy irradiation had significantly lower relaxation of 75%. No significant changes were observed at 0.5 Gy.
Hong et al., 2013	In vivo, in vitro and ex vivo. HUVECs were irradiated with 4 Gy X-rays (2.7 Gy/min) and amounts of eNOS, iNOS and nitrotyrosine (a biomarker for peroxynitrite) were determined by western blot. Rabbit carotid arteries were irradiated with 8 or 16 Gy X-rays. In vivo arteries were irradiated at 4.1 Gy/min, and ex vivo arteries were irradiated at 3.9 Gy/min. Arteries were contracted with phenylephrine then relaxed with ACh to determine vascular responsiveness, which was measured with a computerized automated isometric transducer system.	In 4 Gy irradiated HUVECs, iNOS was increased 6.6-fold and nitrotyrosine was increased 6.4-fold. eNOS expression did not change. The responsiveness of the ex vivo carotid artery to ACh-induced relaxation was 77.4% without irradiation, 65.7% with 8 Gy and 60.1% with 16 Gy. The in vivo irradiated carotid artery also showed decreased ACh-induced relaxation, but relaxation was lowest after 8 Gy.
Zhang et al., 2009	In vivo. HU rats were exposed to altered gravity conditions as a stressor. Western blot was used to measure eNOS and iNOS protein in arteries. Endothelial dysfunction was determined by vasodilation.	Following HU, there was a 2-fold increase of eNOS in carotid arteries compared to control. A 4.3- and 3.3-fold increase in iNOS in carotid and cerebral arteries, respectively, was found in HU rats. Vasodilation was reduced by ~30% in the ACh induced relaxation of basilar arteries in HU rats. Vasoconstriction was increased in HU rats by 1.6 and 1.8-fold in the basilar artery in response to KCl (100 mmol/L) and 5-hydroxytryptamine (5-HT), respectively, and 1.2 and 1.3-fold in the carotid artery in response to KCl and phenylephrine (PE) respectively
Soucy et al., 2010	In vivo. In 4-month-old rats were irradiated with 0 or 5 Gy 137Cs gamma radiation. Altered NO levels and endothelial function were investigated through fluorescent measurements of NO and vascular tension dose responses.	After 5 Gy NO production decreased 0.7-fold. There was a 0.7-fold decrease of relaxation response to ACh after 5 Gy compared to control.
Yan et al., 2020	In vivo. Rats were irradiated with 4 Gy abdominal X-ray radiation. Nitrite and eNOS levels were measured by NO assay kit and western blot, respectively. Endothelial dysfunction was determined by changes in vasodilation.	After radiation nitrite (NO metabolite and marker) levels and the eNOS ratio decreased 0.6-fold. With increasing ACh concentration the control group dropped to ~7% constriction while the irradiated group remained at ~75% constricted.

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
Hong et al., 2013	In vivo and ex vivo. HUVECs were irradiated with 4 Gy X-rays (2.7 Gy/min) and amounts of iNOS and nitrotyrosine (a biomarker for peroxynitrite) were determined by western blot at various times over 6 hours. Rabbit carotid arteries were irradiated with 0, 8 or 16 Gy X-rays and the contraction was measured every 2 minutes for 10 minutes. In vivo arteries were irradiated at 4.1 Gy/min, and ex vivo arteries were irradiated at 3.9 Gy/min. Arteries were contracted with phenylephrine then relaxed with ACh to determine vascular responsiveness, which was measured with a computerized automated isometric transducer system.	After 4 Gy X-ray irradiation, iNOS levels in HUVECs increased consistently over 6 hours, while nitrotyrosine did not change at the 1.5- or 3-hour timepoint, but then increased at 6 hours. 20 hours after irradiation, relaxation with ACh was increased in the irradiated arteries compared to the non-irradiated arteries. This occurred in both the in vivo and ex vivo models.

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Not Identified

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

The majority of the evidence is derived from in vivo rat models. A limited number of studies were in human and rabbit models. The relationship has been more commonly shown in vivo male animals, specifically in adult male rodents.

References

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

Deanfield, J.E., J. P. Halcox and T. J. Rabelink (2007), “Endothelial function and dysfunction: Testing and clinical relevance”, Circulation, Vol. 115/10, Lippincott Williams & Wilkins, Philadelphia, https://doi.org/10.1161/CIRCULATIONAHA.106.652859.

Hong, C. W. et al. (2013), “Involvement of inducible nitric oxide synthase in radiation-induced vascular endothelial damage”, Journal of Radiation Research, Vol. 54/6, Oxford University Press, Oxford, https://doi.org/10.1093/JRR/RRT066.

Krüger-Genge, A. et al. (2019), “Vascular Endothelial Cell Biology: An Update”, International Journal of Molecular Sciences, Vol. 20/18, Multidisciplinary Digital Publishing Institute, Basel, https://doi.org/10.3390/ijms20184411.

Marti, C. N. et al. (2012), “Endothelial dysfunction, arterial stiffness, and heart failure”, Journal of the American College of Cardiology, Vol. 60/16, Elsevier, Amsterdam, https://doi.org/10.1016/J.JACC.2011.11.082.

Mitchell, A. et al. (2019), “Cardiovascular effects of space radiation: implications for future human deep space exploration”, European Journal of Preventive Cardiology, Vol. 26/16, SAGE Publishing, Thousand Oaks, https://doi.org/10.1177/2047487319831497.

Nagane, M. et al. (2021), “DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases”, Journal of Radiation Research, Vol. 62/4, Oxford University Press, Oxford, https://doi.org/10.1093/JRR/RRAB032.

Nathan, C. and Q. W. Xie (1994), “Regulation of biosynthesis of nitric oxide”, Journal of Biological Chemistry, Vol. 269/19, American Society for Biochemistry and Molecular Biology, Rockville, https://doi.org/10.1016/S0021-9258(17)36703-0 

 Schulz, E., T. Gori and T. Münzel (2011), “Oxidative stress and endothelial dysfunction in hypertension”, Hypertension Research, Vol. 34/6, Nature Portfolio, London, https://doi.org/10.1038/hr.2011.39.

Soloviev, A. I. and I.V. Kizub (2019), “Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction”, Biochemical pharmacology, Vol. 159, Elsevier, Amsterdam, https://doi.org/10.1016/j.bcp.2018.11.019.   

Soucy, K. G. et al. (2011), “HZE 56Fe-ion irradiation induces endothelial dysfunction in rat aorta: Role of xanthine oxidase”, Radiation Research, Vol. 176/4, Radiation Research Society, Bozeman, https://doi.org/10.1667/RR2598.1.

Soucy, K. G. et al. (2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, Journal of Applied Physiology, Vol. 108/5, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00946.2009.

Wang, Y., M. Boerma and D. Zhou. (2016), “Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases”, Radiation research, Vol. 186/2, Radiation Research Society, Bozeman, https://doi.org/10.1667/RR14445.1.  

Yan, T., et al. (2020), “Ionizing radiation induces BH4 deficiency by downregulating GTP-cyclohydrolase 1, a novel target for preventing and treating radiation enteritis”, Biochemical Pharmacology, Vol. 180, Elsevier, Amsterdam, https://doi.org/10.1016/J.BCP.2020.114102.

Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, Journal of Applied Physiology, Vol. 106/1, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.01278.2007.