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Relationship: 2773

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

Increased, Altered Signaling leads to Altered, Nitric Oxide Levels

Upstream event
The causing Key Event (KE) in a Key Event Relationship (KER). More help
Downstream event
The responding Key Event (KE) in a Key Event Relationship (KER). More help

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 vascular remodeling adjacent Vinita Chauhan (send email) Under development: Not open for comment. Do not cite

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 Moderate NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help
Sex Evidence
Male Low
Female Low
Unspecific Moderate

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

Nitric oxide (NO) is a diffusible molecule found in various cell types throughout the body, including the neurons, macrophages, and vascular endothelial cells (Bruckdorfer, 2005). NO produced in endothelial cells is one of the components responsible for vasodilation (Soloviev & Kizub, 2019; Wang, Boerma & Zhou, 2016). Multiple signaling pathways can regulate NO levels. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway can activate nitric oxide synthase (NOS), an enzyme that produces NO, through phosphorylation (Hemmings & Restuccia 2012; Nagane et al., 2021). The RhoA/Rho kinase (ROCK) pathway inhibits both the expression and phosphorylation of NOS (Yao et al., 2010). Furthermore, the renin-angiotensin-aldosterone system (RAAS) can both inhibit NOS to reduce vasodilation and activate NOS as a countermeasure for vasoconstriction (Millatt, Abdel-Rahman & Siragy, 1999). The extracellular signal-regulated protein kinase 5 (ERK5)/kruppel-like factor 2 (KLF2) pathway can increase transcription of endothelial NOS (eNOS), which results in increased NO levels (Paudel, Fusi & Schmidt, 2021). Lastly, the acidic sphingomyelinase/ceramide pathway can activate NADPH oxidase (NOX) production of reactive oxygen species (ROS) that react with NO, resulting in lower NO levels (Soloviev & Kizub, 2019). Alterations in these pathways will result in altered NO levels.

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

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help
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 signaling pathways and altered NO levels is well-supported by the literature. Studies have shown that altered signaling pathways lead to altered NO levels (Azimzadeh et al., 2015; Azimzadeh et al., 2017; Hasan, Radwan & Galal, 2019; Shi et al., 2012; Siamwala et al., 2010).

Without measuring NO levels directly, NOS levels can be used as a proxy to measure NO production. eNOS and inducible NOS (iNOS) are common points for assessing NO levels indirectly. Decreased NOS protein expression often corresponds to a decrease in NO. However, it is important to note that NOS levels do not perfectly correlate with NO levels. Increased NOS can also decrease NO if paired with a simultaneous increase in ROS, which, through oxidizing the enzyme’s cofactor BH4, causes NOS uncoupling (Ceriello, 2003; Forstermann, 2010; Zhang et al., 2009). Uncoupled NOS produces additional ROS that react with NO and reduce its overall abundance. Therefore, in this case, higher levels of NOS correlate to increased quantity of uncoupled NOS and a subsequent drop in NO bioavailability (Soloviev & Kizub, 2019).

Multiple signaling pathways can influence NO levels. Under normal physiological conditions, the PI3K/Akt pathway is regulated by various growth factors and other signaling molecules to modulate eNOS phosphorylation and therefore NO production (Hemmings & Restuccia, 2012). Phosphorylation of eNOS can affect its function. Phosphorylation at Ser1177 activates the enzyme, while phosphorylation at Thr495 acts in reverse and decreases enzymatic activity instead (Forstermann, 2010; Nagane et al., 2021). In endothelial cells, Thr495 is constitutively phosphorylated by kinases like protein kinase C (PKC) (Forstermann, 2010). Following Akt activation, eNOS is phosphorylated on Ser1177 to activate the enzyme and thus NO production is upregulated (Karar & Maity, 2011). Although peroxisome proliferator-activated receptor α (PPARα) can increase eNOS directly through transcription, it can also increase vascular endothelial growth factor (VEGF) (Du, Wagner & Wagner, 2020) which can activate the PI3K pathway to activate eNOS (Hicklin & Ellis, 2005). Alternatively, activation of the RhoA/ROCK pathway leads to a decrease in NO bioavailability (Yao et al., 2010). The RhoA/ROCK pathway causes eNOS mRNA destabilization and prevents Ser1177 eNOS phosphorylation by Akt (Yao et al., 2010). However, phosphorylation of RhoGDI, a regulator in the RhoA/ROCK pathway, causes increased affinity to GTP-RhoA, sequestering the active form of RhoA and preventing eNOS inhibition (Dovas & Couchman, 2005) The RAAS pathway results in the production of angiotensin II (AngII) which can cause various downstream effects on vascular homeostasis, including inducing vasoconstriction (Millatt, Abdel-Rahman & Siragy, 1999). AngII can downregulate eNOS Ser1177 phosphorylation to prevent vasodilation (Ding et al., 2020; Millatt, Abdel-Rahman & Siragy, 1999), or activate eNOS as a corrective measure (Millatt, Abdel-Rahman & Siragy, 1999). As a result, depending on the mechanism, NO levels can either increase or decrease after AngII stimulation. The ERK5/KLF2 pathway results in activation of the KLF2 transcription factor, which increases transcription of eNOS (Paudel, Fusi & Schmidt, 2021).

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
  • Due to the high reactivity of NO, it can be difficult to obtain its direct measures (Luiking, Engelen & Deutz, 2010). The inconsistencies in NO levels may be attributed to the challenges in measuring NO. 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.

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

BPF (ACE inhibitor)

BPF treatment led to decreased iNOS, angiotensin II and aldosterone following irradiation.

Hasan, Radwan & Galal, 2019

Drug

LY294002 (PI3K inhibitor)

LY294002 treatment led to reduced phosphorylation of both Akt and eNOS.

Shi et al., 2012;

Drug

Wortmanin (PI3K inhibitor)

Wortmanin decreased NO production.

Siamwala et al., 2010

Drug

Fenofibrate (PPARα activator)

Treatment with PPARα prevented the decrease in eNOS levels after irradiation.

Azimzadeh et al., 2021

Drug

Atorvastatin (HMG-CoA reductase inhibitor)

Treatment with atorvastatin increased KLF2 and eNOS levels slightly.

Sadhukhan et al., 2020 

Drug

Gamma tocotrienol (HMG-CoA reductase inhibitor)

Treatment with gamma tocotrienol prevented the radiation-induced decrease in KLF2 and eNOS levels.

Sadhukhan et al., 2020 

Drug

Geranylgeranyltransferase I inhibitor 298

Treatment with Geranylgeranyltransferase I inhibitor 298 prevented the radiation-induced decrease in KLF2 and eNOS levels.

Sadhukhan et al., 2020 

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

Dose Concordance

Reference

Experiment Description

Result

Shi et al., 2012

In vitro. Microgravity was stimulated with a clinostat using rotation at a slow speed to negate centrifugal force and simulate weightlessness. eNOS expression and Akt (signaling molecule) in HUVEC-C were measured by western blot.

After clinorotation, eNOS and p-eNOS expression increased by 5.2-fold and 5.5-fold respectively.

p-Akt increased by 2.9-fold, while there was no significant change in Akt after clinorotation in HUVEC-C.

Hasan, Radwan & Galal, 2019

In vivo. Rat heart serum were exposed to irradiation by 6 Gy Gamma rays. iNOS levels were measured by ELISA. RAAS indices, AngII and aldosterone, were also measured in serum by ELISA.

Irradiation with 6 Gy led to a 3.3-fold increase in iNOS expression, ~1.4-fold increase in AngII and aldosterone.

Azimzadeh et al., 2015

In vivo. Mice were exposed to heart X-ray irradiation at either 8 or 16 Gy. Levels of proteins in the insulin-dependent PI3K/Akt pathway with and without phosphorylation were determined along with levels of NO and eNOS. Protein levels in various signaling pathways were measured using immunoblotting, and NO was measured using an ELISA assay.

Many key proteins in each pathway showed significant changes in abundance and phosphorylation after 8 and 16 Gy irradiation. For example, phosphorylation of the insulin receptor IGFR1 decreased 0.4-fold after 8 Gy and 0.25-fold after 16 Gy. Similarly, p-Akt decreased 0.2-fold at 8 Gy and 0.1-fold at 16 Gy. p-eNOS decreased 0.6-fold after 8 Gy and 0.15-fold after 16 Gy. The ERK/MAPK pathway was found decreased 0.5-fold at 16 Gy and the p38/MAPK pathway was found increased 1.3-fold at 16 Gy. NO decreased 0.33-fold after 8 Gy and 0.25-fold after 16 Gy.

Azimzadeh et al., 2017

In vitro. HCAECs were irradiated with 0.5 Gy X-ray irradiation over 1 minute. Phosphorylated RhoGDI and eNOS levels were determined using immunoblotting, and NO levels were determined using ELISA assay.

p-RhoGDI decreased 0.7-fold, p-eNOS decreased 0.6-fold, HSP90 (positive regulator of eNOS) decreased 0.75-fold and NO decreased 0.8-fold after 0.5 Gy irradiation.

Sonveaux et al., 2003

In vitro. Following X-ray irradiation of BAECs and HUVECs, levels of eNOS, p-eNOS, Akt, and p-Akt were measured with immunoblotting at various doses (0.86 Gy/min).

Compared to control, 24h after irradiation, the ratio of p-Akt/Akt increased 1.3-fold after 2 Gy (not significant) and 5.6-fold after 6 Gy. eNOS increased 1.3-fold after 2 Gy (not significant), 2.1-fold after 4 Gy (not significant), 3-fold after 6 and 8 Gy, 4.3-fold after 10 Gy and 3.4-fold after 20 Gy. Compared to control, 24h after irradiation, p-eNOS increased 1.2-fold after 2 Gy and 1.7-fold after 6 Gy.

Azimzadeh et al., 2021

In vivo. Male C57BL/6J were irradiated with an acute dose of 16 Gy X-rays. Activation of the PI3K-Akt pathway was determined through p-PPARα (deactivated) levels from Ponceau S staining. eNOS activity and NO were measured using fluorometric assay and Griess assay, respectively. The level of proteins in MAPK pathways were determined by ELISA in heart tissue.

After 16 Gy, p-PPARα increased 1.3-fold. After 16 Gy, p-ERK increased 1.5-fold, and p-p38 increased 1.3-fold, eNOS was unchanged, p-eNOS decreased 0.8-fold and NO decreased to 65% of control levels.

Sadhukhan et al., 2020

In vitro. HUVECs were irradiated with various regimens and doses of gamma radiation. Signaling from the ERK5/KLF2 pathway was determined through KLF2 and p-ERK5 levels from Western blot. eNOS levels were determined by Western blot. Fractionated doses were separated by 24h.

p-ERK5 decreased 0.6-fold after 5 doses of 2 Gy, increased 1.5-fold after acute 10 Gy, decreased 0.5-fold after 5 doses of 2.5 Gy, and increased 1.3-fold after acute 12.5 Gy. KLF2 and eNOS showed similar responses but were often still slightly decreased after acute doses.

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

Azimzadeh.et al., 2017

In vitro. AECs were irradiated with 0.5 Gy X-ray irradiation over 1 minute. Phosphorylated RhoGDI and eNOS levels were determined using immunoblotting, and NO levels were determined using ELISA assay. Measurements were taken after 1, 7 or 14 days.

p-RhoGDI significantly decreased after 1 day, while NO only significantly decreased after 7 days. However, p-eNOS significantly decreased after 1 day.

Sonveaux et al., 2003

In vitro. Levels of eNOS, p-eNOS, Akt, and p-Akt after X-ray irradiation of BAECs and HUVECs were measured with immunoblotting at various doses (0.86 Gy/min).

p-Akt was significantly increased 24h after irradiation, while eNOS was significantly increased after 12, 24, and 48h and p-eNOS was significantly increased after 24h.

Sadhukhan et al., 2020

In vitro. HUVECs were irradiated with various regimens and doses of gamma radiation. Fractionated doses were separated by 24h. Signaling from the ERK5/KLF2 pathway was determined through KLF2 and p-ERK5 levels from Western blot. eNOS levels were determined by Western blot. Measurements were taken 4 or 24h post-irradiation.

After 4h, p-ERK5 decreased 0.6-fold after 5 doses of 2 Gy, increased 1.5-fold after acute 10 Gy, decreased 0.5-fold after 5 doses of 2.5 Gy, and increased 1.3-fold after acute 12.5 Gy. Both KLF2 and eNOS decreased similar to p-ERK5 after 4h and were slightly decreased after 24h.

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

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 evidence for the KER comes from rat and mouse models. Most evidence regarding sex and lifestage is unspecified with a low support of evidence coming from adult models.

References

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

Ancion, A. et al. (2019), “A Review of the Role of Bradykinin and Nitric Oxide in the Cardioprotective Action of Angiotensin-Converting Enzyme Inhibitors: Focus on Perindopril”, Cardiology and Therapy, Vol. 8/2, Springer, London, https://doi.org/10.1007/S40119-019-00150-W.

Azimzadeh, O. et al. (2021), “Activation of pparα by fenofibrate attenuates the effect of local heart high dose irradiation on the mouse cardiac proteome”, Biomedicines, Vol. 9/12, Multidisciplinary Digital Publishing Institute, Basel, https://doi.org/10.3390/biomedicines9121845.

Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, International Journal of Radiation Biology, Vol. 93/9, Informa, London, https://doi.org/10.1080/09553002.2017.1339332.

Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, Journal of Proteome Research, Vol. 14/2, American Chemical Society, Washington, https://doi.org/10.1021/pr501141b.

Bruckdorfer, R. (2005), “The basics about nitric oxide”, Molecular Aspects of Medicine, Vol. 26/1-2, Elsevier, Amsterdam, https://doi.org/10.1016/j.mam.2004.09.002.

Ceriello, A. (2003), “New Insights on Oxidative Stress and Diabetic Complications May Lead to a “Causal” Antioxidant Therapy”, Diabetes Care, Vol. 26/5, American Diabetes Association, Arlington County, https://doi.org/10.2337/DIACARE.26.5.1589

Ding, J. et al. (2020), “Angiotensin II Decreases Endothelial Nitric Oxide Synthase Phosphorylation via AT1R Nox/ROS/PP2A Pathway”, Frontiers in physiology, Vol.11, Frontiers Media SA, Lausanne, https://doi.org/10.3389/fphys.2020.566410.

Dovas, A. and J. R. Couchman (2005), “RhoGDI: multiple functions in the regulation of Rho family GTPase activities”, Biochemical Journal, Vol. 390, Portland Press, London, https://doi.org/10.1042/BJ20050104.

Du, S., N. Wagner and K. D. Wagner (2020), “The Emerging Role of PPAR Beta/Delta in Tumor Angiogenesis”, PPAR Research, Vol. 2020, Hindawi, London,  https://doi.org/10.1155/2020/3608315.

Förstermann, U. (2010), “Nitric oxide and oxidative stress in vascular disease”, Pflugers Archiv : European journal of physiology, Vol. 459/6, Springer, London, https://doi.org/10.1007/s00424-010-0808-2.

Hasan, H. F., R. R. Radwan and S. M. Galal (2019), “Bradykinin‐potentiating factor isolated from Leiurus quinquestriatus scorpion venom alleviates cardiomyopathy in irradiated rats via remodelling of the RAAS pathway”, Clinical and Experimental Pharmacology and Physiology, Vol. 47/2, Wiley-Blackwell, Hoboken, https://doi.org/10.1111/1440-1681.13202.

Hemmings, B. A. and D. F. Restuccia (2012), “PI3K-PKB/Akt Pathway”, Cold Spring Harbor Perspectives in Biology, Vol. 4/9, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, https://doi.org/10.1101/CSHPERSPECT.A011189.

Hicklin, D. J. and L. M. Ellis (2005), “Role of the Vascular Endothelial Growth Factor Pathway in Tumor Growth and Angiogenesis”, Journal of Clinical Oncology, Vol. 23/5, American Society of Clinical Oncology, Virginia,  https://doi.org/10.1200/JCO.2005.06.081.

Karar, J. and A. Maity (2011), “PI3K/AKT/mTOR Pathway in Angiogenesis”, Frontiers in Molecular Neuroscience, Vol. 4, Frontiers Media SA, Lausanne, https://doi.org/10.3389/FNMOL.2011.00051.

Luiking, Y. C., M. P. Engelen and N. E. Deutz (2010), “Regulation of nitric oxide production in health and disease”, Current Opinion in Clinical Nutrition and Metabolic Care, Vol. 13/1, Lippincott Williams and Wilkins Ltd., Philadelphia, https://doi.org/10.1097/MCO.0b013e328332f99d.

Millatt, L. J., E. M. Abdel-Rahman and H. M. Siragy (1999), “Angiotensin II and nitric oxide: a question of balance”, Regulatory Peptides, Vol. 81/1-3, Elsevier, Amsterdam, https://doi.org/10.1016/S0167-0115(99)00027-0.

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.  

Paudel, R., L. Fusi and M. Schmidt (2021), “The MEK5/ERK5 Pathway in Health and Disease”, International Journal of Molecular Sciences, Vol. 22/14, Multidisciplinary Digital Publishing Institute, Basel, https://doi.org/10.3390/ijms22147594.

Sadhukhan, R. et al. (2020), “Fractionated radiation suppresses Kruppel-like factor 2 pathway to a greater extent than by single exposure to the same total dose”, Scientific Reports, Vol. 10/1, Springer, London, https://doi.org/10.1038/s41598-020-64672-3.

Shi, F. et al. (2012), “Effects of Simulated Microgravity on Human Umbilical Vein Endothelial Cell Angiogenesis and Role of the PI3K-Akt-eNOS Signal Pathway”, PLoS ONE, Vol. 7/7, PLOS, San Francisco, https://doi.org/10.1371/journal.pone.0040365.

Siamwala, J. H. et al. (2010), “Simulated microgravity perturbs actin polymerization to promote nitric oxide-associated migration in human immortalized Eahy926 cells”, Protoplasma, Vol. 242/1, Springer, London, https://doi.org/10.1007/S00709-010-0114-Z.

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.

Sonveaux, P. et al. (2003), “Irradiation-induced Angiogenesis through the Up-Regulation of the Nitric Oxide Pathway: Implications for Tumor Radiotherapy”, Cancer Research, Vol. 63, American Association for Cancer Research, Philadelphia, https://aacrjournals.org/cancerres/article/63/5/1012/511021/Irradiation-induced-Angiogenesis-through-the-Up.

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

Yao, L. et al. (2010), “The role of RhoA/Rho kinase pathway in endothelial dysfunction”, Journal of Cardiovascular Disease Research, Vol. 1/4, Elsevier, Amsterdam, https://doi.org/10.4103/0975-3583.74258.

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.