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AOP: 149


A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the AO. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE.  More help

Peptide Oxidation Leading to Hypertension

Short name
A name that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
The current version of the Developer's Handbook will be automatically populated into the Handbook Version field when a new AOP page is created.Authors have the option to switch to a newer (but not older) Handbook version any time thereafter. More help
Handbook Version v1.0

Graphical Representation

A graphical representation of the AOP.This graphic should list all KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs. More help
Click to download graphical representation template Explore AOP in a Third Party Tool


The names and affiliations of the individual(s)/organisation(s) that created/developed the AOP. More help

British American Tobacco: Frazer Lowe (; Linsey Haswell; Marianna Gaca

Philip Morris International: Karsta Luettich; Marja Talikka ; Julia Hoeng

Selventa: Vy Hoang (

Point of Contact

The user responsible for managing the AOP entry in the AOP-KB and controlling write access to the page by defining the contributors as described in the next section.   More help
Frazer Lowe   (email point of contact)


Users with write access to the AOP page.  Entries in this field are controlled by the Point of Contact. More help
  • Frazer Lowe


This field is used to identify coaches who supported the development of the AOP.Each coach selected must be a registered author. More help

OECD Information Table

Provides users with information concerning how actively the AOP page is being developed and whether it is part of the OECD Workplan and has been reviewed and/or endorsed. OECD Project: Assigned upon acceptance onto OECD workplan. This project ID is managed and updated (if needed) by the OECD. OECD Status: For AOPs included on the OECD workplan, ‘OECD status’ tracks the level of review/endorsement of the AOP . This designation is managed and updated by the OECD. Journal-format Article: The OECD is developing co-operation with Scientific Journals for the review and publication of AOPs, via the signature of a Memorandum of Understanding. When the scientific review of an AOP is conducted by these Journals, the journal review panel will review the content of the Wiki. In addition, the Journal may ask the AOP authors to develop a separate manuscript (i.e. Journal Format Article) using a format determined by the Journal for Journal publication. In that case, the journal review panel will be required to review both the Wiki content and the Journal Format Article. The Journal will publish the AOP reviewed through the Journal Format Article. OECD iLibrary published version: OECD iLibrary is the online library of the OECD. The version of the AOP that is published there has been endorsed by the OECD. The purpose of publication on iLibrary is to provide a stable version over time, i.e. the version which has been reviewed and revised based on the outcome of the review. AOPs are viewed as living documents and may continue to evolve on the AOP-Wiki after their OECD endorsement and publication.   More help
OECD Project # OECD Status Reviewer's Reports Journal-format Article OECD iLibrary Published Version
1.50 Under Development
This AOP was last modified on April 29, 2023 16:02

Revision dates for related pages

Page Revision Date/Time
Peptide Oxidation November 13, 2017 10:22
KE1 : S-Glutathionylation, eNOS November 20, 2017 05:26
KE2 : Decrease, GTPCH-1 November 09, 2017 06:42
KE3 : Decrease, Tetrahydrobiopterin November 09, 2017 06:42
KE4 : Uncoupling, eNOS November 09, 2017 06:43
KE6 : Depletion, Nitric Oxide November 09, 2017 06:43
KE7 : Impaired, Vasodilation November 09, 2017 06:44
KE8 : Increase, Vascular Resistance November 09, 2017 06:44
Hypertension September 27, 2017 12:46
KE5 : Decrease, AKT/eNOS activity November 09, 2017 06:43
Peptide Oxidation leads to Decrease, GTPCH-1 September 28, 2017 08:07
S-Glutathionylation, eNOS leads to Uncoupling, eNOS October 12, 2017 06:04
Decrease, GTPCH-1 leads to Decrease, Tetrahydrobiopterin October 12, 2017 06:33
Decrease, Tetrahydrobiopterin leads to Uncoupling, eNOS October 12, 2017 06:47
Uncoupling, eNOS leads to Depletion, Nitric Oxide October 12, 2017 07:58
Depletion, Nitric Oxide leads to Impaired, Vasodilation October 12, 2017 09:54
Impaired, Vasodilation leads to Increase, Vascular Resistance October 13, 2017 10:09
Increase, Vascular Resistance leads to Hypertension October 13, 2017 10:53
Decrease, AKT/eNOS activity leads to Depletion, Nitric Oxide October 16, 2017 06:33
Peptide Oxidation leads to Decrease, AKT/eNOS activity October 16, 2017 06:52
Peptide Oxidation leads to S-Glutathionylation, eNOS November 20, 2017 06:00
Reactive oxygen species August 15, 2017 10:43


A concise and informative summation of the AOP under development that can stand-alone from the AOP page. The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance. More help

Hypertension is a cardiovascular risk factor that has a profound influence on cardiovascular morbidity and mortality.  While good progress has been made in terms of identifying and managing this risk factor for patient care, methods to assess the potential of chemical compounds to induce hypertension, or to assess the efficacy of consumer products (e.g. e-cigarettes, tobacco heating products) targeted at reducing disease burden remain largely limited to epidemiological associations and in vivo studies. 

Here we present the supporting information on an AOP describing how vascular endothelial peptide oxidation leads to hypertension via perturbation of endothelial nitric oxide (NO) bioavailability.  The molecular initiating event is oxidation of amino acid (AA) residues on critical peptides of the NO pathway, notably protein kinase B (AKT), guanosine triphosphate cyclohydrolase-1 (GTPCH-1), endothelial nitric oxide synthase (eNOS), and also the cellular ROS scavenger; glutathione.  Oxidation of the enzymic components of the pathway lead to reduced expression of the phosphorylated proteins, and protein loss via proteasomal degradation. Oxidation of reduced glutathione to GSSG promotes bonding of GSSG to critical AA residues on eNOS, and the reduced expression of GTPCH-1 reduces bioavailability of tetrahydrobiopterin (BH4), both of which lead to uncoupling of eNOS (reduced NO production, increased superoxide production).  The combination of these molecular events lead to reduced bioavailabilty of NO, which in turn reduces the potential for vasodilation and shifts the balance of vascular tone towards vasoconstriction.  Repeated perturbation of this pathway via chronic exposure to toxicants, ultimately increases vascular resistance and contributes towards the development of hypertension.

The evidence supporting the AOP is strong from the molecular level up to impaired vasodilation, however there is a knowledge gap concerning the magnitude of contribution of this mechanism towards the development of hypertension, given the complexity of the disorder.  Further AOPs are likely required to characterise the contribution of competing/promoting mechanisms which alter vascular tone towards chronic vasoconstriction.

With respect to the regulatory context, the AOP is of relevance to the risk assessment of airborne pollutants and other chronically inhaled toxicants which affect vascular endothelial cells. Additionally, acute and chronic exposure to aerosols generated by alternative tobacco products and nicotine delivery devices are poorly understood with respect to their impact on cardiovascular disease risk. Reliable in vitro models and human clinical biomarkers would help to inform regulatory decision-making with respect to understanding the potential cardiovascular health impacts of these novel products.

AOP Development Strategy


Used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development.The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. More help

The motivation for the AOP development, a deeper explaination of the underlying biology, and KE/KER assessments can be found in Lowe et al. 2017.

Originally, "oxidative stress" was proposed as the MIE for this AOP.  Upon discussion with EAGMST, it was suggested to use a term that is more representative of the mechanism of action, hence "peptide oxidation" is proposed instead.  All references to "oxidative stress" within this wiki are made within the context that localised conditions conducive to oxidative damage within the vascular endothelium would trigger the revised MIE, and are limited to the peptide targets named within the AOP; namely GSH, GTPCH1, AKT and eNOS (although other redox sensitive peptides are undoubtedly affected also).

The mechanisms of potentially deleterious peptide oxidation by ROS/RNS are discussed by Berlett and Stadtman (1997) in the context of health effects.  References to "oxidative stress" within this wiki are made with this context in mind.


Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components.  Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). More help

Summary of the AOP

This section is for information that describes the overall AOP.The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help


Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help
Key Events (KE)
A measurable event within a specific biological level of organisation. More help
Adverse Outcomes (AO)
An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help
Type Event ID Title Short name
MIE 209 Peptide Oxidation Peptide Oxidation
KE 927 KE1 : S-Glutathionylation, eNOS S-Glutathionylation, eNOS
KE 935 KE2 : Decrease, GTPCH-1 Decrease, GTPCH-1
KE 934 KE3 : Decrease, Tetrahydrobiopterin Decrease, Tetrahydrobiopterin
KE 932 KE4 : Uncoupling, eNOS Uncoupling, eNOS
KE 933 KE6 : Depletion, Nitric Oxide Depletion, Nitric Oxide
KE 937 KE7 : Impaired, Vasodilation Impaired, Vasodilation
KE 951 KE8 : Increase, Vascular Resistance Increase, Vascular Resistance
KE 973 KE5 : Decrease, AKT/eNOS activity Decrease, AKT/eNOS activity
AO 952 Hypertension Hypertension

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarizes all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP.Each table entry acts as a link to the individual KER description page. More help

Network View

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (WoE) summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Life Stage Applicability

The life stage for which the AOP is known to be applicable. More help
Life stage Evidence
All life stages

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available. More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI
mouse Mus musculus Moderate NCBI
rat Rattus norvegicus Moderate NCBI
cow Bos taurus Moderate NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help
Sex Evidence

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). More help

Domain of Applicability

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. More help

Life Stage Applicability, Taxonomic Applicability, Sex Applicability Elaborate on the domains of applicability listed in the summary section above. Specifically, provide the literature supporting, or excluding, certain domains.

This proposed AOP is applicable to males and females, however a single study did suggest that KE responses may vary with sex (Scotland et al. 2005). 

The AOP is relevant for all life stages, as vascular oxidative stress (a common cause of the MIE) can occur at all life stages.  However, the AO via this mechanism is likely limited to adults, due to a loss of vaso-protective mechanisms with age (a major risk factor) and cardiovascular re-modelling over time.  Hypertension is known to occur in adolescents and children, however the condition can be due to a variety of causes, such as renal disease and cardiovascular complications in early life.  For example, persistant pulmonary hypertension is a consequence of failed pulmonary vascular transition at birth and leads to pulmonary hypertension with shunting of deoxygenated blood across the ductus arteriosus and foramen ovale, resulting in severe hypoxemia.  The condition is improved by administration of nitric oxide by inhalation (Lai et al. 2017).  While the initial cause of the hypertension is not vascular oxidative stress, ROS-mediated perturbations of the vascular endothelium, which arise as a result of the hypoxic conditions, contribute to the development of hypertension, and may have health implications for the neonate later in life (de Wijs-Meijler et al. 2017).  Therefore, the physiology described in the AOP is relevant to the condition, but not strictly causative.

The KEs in this AOP are well-documented and well-studied in humans, cows and rodents.  Similar outcomes are observed across these species following chemical exposure and other phenomena which cause vascular oxidative stress (e.g. hypoxia, ischaemia/reperfusion).

The specific amino-acid residues involved in post-translational modification of eNOS do differ across species, however the functional effect is similar.  For example, human eNOS is phosphorylated by AKT at serine residue 1177 (Reviewed by Heiss et al. 2014), whereas bovine eNOS is phosphoryated by AKT at serine 1179 (Chen et al. 2017).

Essentiality of the Key Events

The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently, evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence. The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs. More help

Molecular Initiating Event Summary, Key Event Summary Provide an overall assessment of the essentiality for the key events in the AOP. Support calls for individual key events can be included in the molecular initiating event, key event, and adverse outcome tables above.

The essentiality for this AOP is strong since there is direct evidence from multiple experimental studies showing that downstream key events can be prevented or inhibited if an upstream key event is blocked. An increase in intracellular glutathione, rather than the oxidized form, prevented S-glutathionylation (De Pascali et al., 2014), and inhibiting S-glutathionylation attenuated ultrafine particle-induced reduction in NO production (Du et al., 2013). In addition, increasing GTPCH-1, BH4 and Akt/eNOS activity increased eNOS activity and NO production but decreased superoxide generation (Fulton et al., 1999; Alp et al., 2003; Carnicer et al., 2012; Antoniades et al., 2011; Chen et al., 2011; De Pascali et al., 2014; Landmessar et al., 2003; Shinozaki et al., 2000; Ozaki et al., 2002). Infusion of NO donor sodium nitroprusside reduced vascular resistance, while sodium nitrite increased vasodilation (Eugene, 2016; Sindlier et al., 2014).

The essentiality of increased vascular resistance to hypertension is moderate due to the involvement of the heart in regulating blood pressure.  While increased vascular resistance is a hallmark of essential hypertension, Mayet and Hughes (2003) discussed the roles of the heart and vascular resistance and concluded that both are of importance, and should not be viewed in isolation.  Their conclusion is drawn from observations in younger adults with elevated blood pressure, who have a normal vascular resistance, but higher cardiac index.  Over time, such adults often go on to change to a phenotype with a lower cardiac index and high vascular resistance and a diagnosis of chronic hypertension.  Such changes are likely due to vascular remodelling processes with advancing age, however the specifics of this process are not well understood.

Support for Essentiality of KEs KE Description Defining Question High Moderate Low
Are downstream KEs and/or the AO prevented if an upstream KE is blocked? Direct evidence from experimental studies illustrating essentiality for at least one of the important KEs. Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE. No or contradictory experimental evidence of the essentiality of any of the KEs.
Peptide Oxidation (MIE) Generation of localised ROS and other oxidative species in the vascular endothelium leads to oxidation of amino acid residues of peptides.      
S-glutathionylation of eNOS (KE1) The oxidation of GSH into GSSG, elevates levels of GSSG, which then covalently bind to critical serine residues on the eNOS enzyme  High Protein S-Glutathionylation can only occur following covalent binding of GSSG.  GSSG commonly binds to protein thiol residues, which can affect protein function (Bendall et al 2014). S-glutathionylation of eNOS is observed under conditions of oxidative stress, at cysteine residues 689 and 908 leading to eNOS uncoupling (Chen et al. 2010).  Application of Glutaredoxin-1 or dithiothreitol removes S-glutathionylation and restores eNOS function (Chen et al. 2013, Jayaram et al. 2015)  Essentiality rating is high due to direct experimental evidence showing removal of S-glutathionylation of eNOS restores NO production.
Loss of GTPCH-1 (KE2) Oxidative damage to GTPCH-1 has been shown to lead to reduced expression/activity of the enzyme High

Numerous studies have demonstrated that deletion of GTPCH-1 led to the deficiency of BH4 in bovine and murine endothelial cells (Tatham et al. 2009, Crabtree et al. 2009, Adlam et al. 2012) and in knockout mice (Chen et al. 2011).  Gene silencing and GTPCH-1 inhibition lead to elevated blood pressure in experimental animals (Wang et al. 2008, Mitchell et al. 2003).  Essentiality rating is high due to sunstantial experimental evidence of GTPCH-1 blocking on downstream KEs.

Decreased BH4 (KE3) Oxidation of BH4 itself to BH2, and/or reduced bioavailability of BH4 by a reduction in de novo synthesis by GTPCH-1. High

Depletion of BH4 has been extensively reported to uncouple eNOS, decrease NO bioavailability and increase superoxide production.  Application of exogenous BH4 has been reported to reverse this phenomenon (De Pascali et al. 2014, Zweier et al. 2011, Dumitrescu et al. 2007, Tiefenbacher et al. 1996, Vásquez-Vivar et al. 2002)   Essentiality rating is high due to extensive direct experimental evidence.

eNOS Uncoupling (KE4) eNOS uncoupling is characterised by a loss of NO production and superoxide production as eNOS dimerisation is lost High eNOS uncoupling leads to reduction in NO bioavailability.  Reversal of eNOS uncoupling is achieved by supplementation with BH4 or removal of eNOS S-glutathionylation (as described earlier), which restores NO production (Du et al. 2013b, Nozik-Grayck et al. 2014, Talukder et al. 2011, Su et al. 2013).  Essentiality rating is high due to direct experimental evidence.
Loss of AKT/eNOS (KE5) In humans, AKT phosphorylates eNOS at serine residue 1177, leading to eNOS activation and NO production.  Loss of AKT/eNOS function depletes NO bioavailability. High

eNOS knockout mice are routinely used as models of hypertension (Huang 2000).  Aortic rings from eNOS knockout mice do not relax following application of acetylcholine, but do relax upon application of NO donor sodium nitroprusside (Huang et al. 1995).  Many animal studies demonstrated that inhibition of NO via eNOS inhibitors impaired endothelium-dependent vasodilation (Li et al. 2007, Paulis et al. 2008, Luo et al. 2000, Sélley et al. 2014).  Inhibition of Akt or mutant eNOS attenuated eNOS phosphorylation in human and bovine cells, resulting in decreased NO bioavailability (Michaud et al. 2006, Dhar et al. 2010, Dimmeler et al. 1999, Fulton et al. 1999, Das et al. 2014, Uruno et al. 2005).  Essentiality rating is high due to extensive direct experimental evidence.

NO Depletion (KE6) Nitric oxide is a potent vasodilator radical released by endothelial (cell) eNOS.  NO signalling leads to potassium ion efflux and hyperpolarization of vascular smooth muscle cells and blood vessel relaxation.  Reduction in NO bioavailability blunts this response Moderate

Depletion of NO bioavailability by pharmacological blockade of AKT and eNOS is widely reported to shift vascular tone towards a more vasoconstrictive phenotype, leading to hypertension (Li et al. 2007, Paulis et al. 2008, Scotland et al. 2005, Luo et al. 2000, Sélley et al. 2014, Haynes et al. 1993).  However gender differences in experimental animals were reported to influence vasodilation by different mechanisms (Scotland et al. 2005).  Furthermore, compensatory mechanisms affecting vascular tone are evident when bioavailability of NO is decreased (Brandes 2014, Durand et al. 2013).   Essentiality rating is moderate due to extensive evidence from pharmacological blocking studies in humans and animals which show impairment of vasodilation and elevated BP.  However, compensatory effects of other biological mechanisms on downstream KEs are evident as are gender differences in experimental animals.

Impaired vasodilation (KE7)

Vasodilation is mediated by vascular smooth muscle cells and characterized by potassium ion efflux and hyperpolarization of the tissue in response to signalling from the nervous system and chemical mediators released by the vascular endothelium

Moderate It is well accepted that vasodilation and systemic vascular resistance (SVR) are negatively correlated.  Blood flow volume is increased when blood vessels dilate due to decreased vascular resistance (Siddiqui 2011). When vasodilation is impaired as a result of NO depletion or blockade of potassium channels, vascular stiffness and SVR increase (Berg et al. 2011, Brett et al. 1998, Dessey et al. 2004, Li et al. 2007, McVeigh et al. 2001, Paulis et al. 2008, Wilkinson et al. 2002).  Essentiality rating is high due to extensive direct experimental evidence.
Increased vascular resistance (KE8) Increases in vascular tone as a result of impaired vasodilation.  Commonly referred to as systemic vascular resistance (SVR) or total peripheral resistance (TPR). Moderate It is well established that increased SVR (TPR), increased vascular stiffness and increased vascular reactivity contribute to hypertension (Brandes 2014, Foëx and Sear 2004, Mayet and Hughes 2003). In patients with hypertension, SVR was elevated in approximately 66% of enrolled patients (Chan et al. 2016).  However, due to the critical role of cardiac output in blood pressure regulation (Brandes 2014, Mayet and Hughes 2003), and other compensatory mechanisms described earlier, the essentiality for the AO is moderate.
Hypertension (AO) Chronic elevation of systolic and/or diastolic blood pressure in systemic circulation or localised organs    

Evidence Assessment

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. More help

Summary Table Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages.

Quantitative measurements with dose and time response data from published studies cited below can be found here: File:Hypertension Empirical Support Concordance Table.pdf.

Support for Biological Plausibility of KERs Defining Question High (Strong) Moderate Low (Weak)
Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge? Extensive understanding of the KER based on previous documentation and broad acceptance. KER is plausible based on analogy to,accepted biological relationships, but scientific understanding is incomplete. Empirical support for association between KEs, but the structural or functional relationship between them is not understood.
Oxidative Stress, Increase Directly Leads to Glutathione, Oxidation: Strong Multiple studies demonstrated that oxidative stress leads to the oxidation of glutathione (GSH) in the vascular endothelium. Exposure to a number of oxidants, including tert-butyl hydroperoxide, hydrogen peroxide, diamide, methylglyoxal, glucose, ischemia, and ultrafine particles caused a decrease in levels of GSH, which is indicative of its oxidation in human, bovine, and rat endothelial cells (De Pascali et al., 2014; Dhar et al., 2010; Du et al., 2013b; Montecinos et al., 2007; Park, 2013; Schuppe et al., 1992; van Gorp et al., 1999, 2002).
Oxidative Stress, Increase Directly Leads to AKT/eNOS activity, Decrease Strong Multiple experimental studies reported decreased Akt and eNOS phosphorylation/activity following oxidative stress as a consequence of exposure to peroxynitrite, high glucose, methylglyoxal, high fat, cigarette smoke extract (CSE) and ischemia in humans, bovine, mouse and rat endothelial cells (Das et al., 2014; Dhar et al., 2010; Du et al., 2001; Du et al., 2013; Michaud et al., 2006; Song et al., 2007, 2008; Su et al., 2013; Zhang et al., 2014; Zou et al., 2002).
AKT/eNOS activity, Decrease Directly Leads to Nitric Oxide, Depletion Strong Several studies demonstrated that Akt can directly phosphorylate eNOS, leading to increased eNOS enzymatic activity and subsequent NO production (Dimmeler et al., 1999; Fulton et al., 1999). Inhibition of Akt or mutant eNOS attenuated eNOS phosphorylation in human and bovine cells, resulting in decreased NO bioavailability (Das et al., 2014; Dhar et al. 2010; Dimmeler et al., 1999; Fulton et al., 1999; Michaud et al., 2006; Uruno et al., 2005).
Oxidative Stress, Increase Directly Leads to GTPCH-1, Decrease Moderate Several studies demonstrated that GTPCH-1, the rate-limiting enzyme for BH4 synthesis, is affected by oxidative stress. GTPCH-1 expression or activity was inhibited by peroxynitrite and CSE (Abdelghany et al., 2017; Zhao et al., 2003). Cardiac reperfusion patients who experienced oxidative stress had reduced GTPCH-1 activity (Jayaram et al., 2015).
GTPCH-1, Decrease Directly Leads to Tetrahydrobiopterin, Decrease Strong Many studies demonstrated that GTPCH-1 deletion led to reduced bioavailability of BH4 in bovine and murine endothelial cells (Adlam et al., 2012; Chen et al., 2011; Chuaiphichai et al., 2014; Crabtree et al., 2009; Tatham et al., 2009; Wang et al., 2008). Several studies also showed that overexpression of GTPCH-1 in human and mouse endothelium increased BH4 levels and eNOS activity (Alp et al., 2003; Antoniades et al., 2011; Carnicer et al., 2012).
Tetrahydrobiopterin, Decrease Directly Leads to eNOS, Uncoupling Strong The depletion of BH4 leading to eNOS uncoupling is well-studied. Several studies showed reduced levels of BH4 induced eNOS uncoupling by reducing eNOS activity, decreasing NO and increasing superoxide levels in bovine, murine, and rat endothelium (Chuaiphichai et al., 2014; Crabtree et al., 2009; De Pascali et al., 2014; Dumitrescu et al., 2007; Whitsett et al., 2007). Many studies demonstrated that BH4 treatment improved endothelial function by reducing eNOS-mediated superoxide generation and increasing NO formation in human, bovine, mouse, and rat endothelium (Chen et al., 2011; De Pascali et al., 2014; Landmesser et al., 2003; Ozaki et al., 2002; Shinozaki et al., 2000; Wang et al., 2014).
Glutathione, Oxidation Directly Leads to eNOS, S-Glutathionylation Strong Glutathione oxidation as determined by increased oxidized GSSG or decreased GSH levels caused S-glutathionylation of eNOS in bovine and human aortic endothelial cells, and in hypertensive rats and mice (Chen et al., 2010; De Pascali et al., 2014; Du et al., 2013).
eNOS, S-Glutathionylation Directly Leads to eNOS, Uncoupling Strong In vitro experiments showed that S-glutathionylation of eNOS significantly decreased NO activity and greatly increased superoxide generation (Chen et al., 2010). These results were observed in bovine and human aortic endothelial cells as well as in vessels of spontaneously hypertensive rats and cardiac reperfusion patients (De Pascali et al., 2014; Du et al., 2013; Jayaram et al., 2015).
eNOS, Uncoupling Directly Leads to Nitric Oxide, Depletion Strong It is well-established that uncoupling of eNOS causes eNOS to switch from producing NO to generating superoxides (Förstermann and Münzel, 2006). Studies reporting eNOS uncoupling as a result of BH4 depletion or S-glutathionylation measured levels of NO and superoxide which are indicative of eNOS uncoupling (Chen et al. 2010; De Pascali et al., 2014, Du et al., 2013; Whitsett et al., 2007).
Nitric Oxide, Depletion Directly Leads to Vasodilation, impaired Strong Vasodilation is caused by the relaxation of vascular smooth muscle cells within the walls of blood vessels, and is regulated through a number of mechanisms, including cyclic GMP-dependent hyperpolarization and relaxation via NO. Thus, alterations to NO levels have an influence on vasodilation (Silva et al., 2012). Many animal studies demonstrated that inhibition of NO via eNOS inhibitors impaired endothelium-dependent vasodilation (Li et al., 2007; Luo et al., 2000; Paulis et al., 2008; Sélley et al., 2014).
Vasodilation, impaired Directly Leads to Vascular resistance, Increase Strong It is well-accepted that vasodilation and systemic vascular resistance (SVR) are negatively correlated; blood flow is increased when blood vessels dilate due to decreased vascular resistance (Siddiqui, 2011). When vasodilation is impaired as a result of NO depletion or changes in potassium channels, vascular tone and SVR increase (Berg and Jensen, 2011; Brett et al., 1998; Dessy et al., 2004; Li et al., 2007; McVeigh et al., 2001; Paulis et al., 2008; Wilkinson et al., 2002).
Vascular resistance, Increase Directly Leads to Hypertension, N/A Strong It is well-established that increased systemic vascular resistance (SVR), increased vascular stiffness and increased vascular reactivity contribute to hypertension (Brandes, 2014; Foëx and Sear, 2004; Mayet and Hughes, 2003). In patients with hypertension, SVR was elevated in about 66% of enrolled patients (Chan et al., 2016).
Empirical Support for KERs Defining Question High (Strong) Moderate Low (Weak)
Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown? Does KEup occur at lower doses, earlier time points, and higher in incidence than KEdown ? Inconsistencies? Multiple studies showing dependent change in both events following exposure to a wide range of specific stressors. No or few critical data gaps or conflicting data. Demonstrated dependent change in both events following exposure to a small number of stressors. Some inconsistencies with expected pattern that can be explained by various factors. Limited or no studies reporting dependent change in both events following exposure to a specific stressor; and/or significant inconsistencies in empirical support across taxa and species
Oxidative Stress, Increase Directly Leads to Glutathione, Oxidation: Moderate Many studies demonstrated a dose-dependent relationship between known inducers of oxidative stress (tert-butyl hydroperoxide, hydrogen peroxide, methylglyoxal, high glucose, and ultrafine particles) and reduced GSH levels in human and rat studies (Dhar et al., 2010; Du et al., 2013; Montecinos et al., 2007; Park et al., 2013; van Gorp et al., 1999).
Oxidative Stress, Increase Directly Leads to AKT/eNOS activity, Decrease Moderate eNOS activity and reactive oxygen species (ROS) were modulated in the opposite manner by several stressors (methylglyoxal, high glucose, SIN-1, hydrogen peroxide) in human and bovine endothelial cells, resulting in increased ROS and decreased eNOS activity (Das et al., 2014; Dhar et al., 2010; Chen et al., 2010).
AKT/eNOS activity, Decrease Directly Leads to Nitric Oxide, Depletion Strong Various stress inducers (ischemia, peroxynitrite, SIN-1, insulin plus orotic acidura, etc.) showed that a decrease in AKT and/or eNOS activity led to increased eNOS uncoupling and decreased NO (Choi et al., 2014, 2015; Das et al., 2014; Dhar et al., 2010; Dumitrescu et al., 2007 Uruno et al., 2005).
Oxidative Stress, Increase Directly Leads to GTPCH-1, Decrease Weak One study in a rat model of aortic coarctation-associated hypertension provides evidence that there is a interdependence between oxidative stress and GTPCH-1 with increased ROS and decreased GTPCH-1 expression following a perturbation, but there is no dose-response or temporal data (Cervantes-Pérez et al., 2012).
GTPCH-1, Decrease Directly Leads to Tetrahydrobiopterin, Decrease Strong Exposure to a wide range of perturbations (e.g. CSE, 4-hydroxy-2-nonenal, cytokines) led to a decrease in both GTPCH-1 activity/expression and BH4 levels in human, cows and rats (Antoniades et al., 2011; Cervantes-Pérez et al., 2012; Chen et al., 2011; Ismail et al., 2015; Jayaram et al., 2015; Whitsett et al., 2007).
Tetrahydrobiopterin, Decrease Directly Leads to eNOS, Uncoupling Strong Multiple studies demonstrated strong dependency between BH4 and eNOS uncoupling; decreased BH4 along with decreased eNOS activity, decreased NO production or increased superoxide generation were observed after various perturbations (Cervantes-Pérez et al., 2012; De Pascali et al., 2014; Dumitrescu et al., 2007; Jayaram et al., 2015; Whitsett et al., 2007; Wang et al., 2008).
Glutathione, Oxidation Directly Leads to eNOS, S-Glutathionylation Moderate Treatment with GSSG induced a dose-dependent increase in human eNOS S-glutathionylation in vitro whereas 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment also resulted in increased eNOS S-glutathionylation in a dose-dependent manner in bovine aortic endothelial cells (Chen et al., 2010). Exposure to ultrafine particles or hypoxia/reoxygenation in human or bovine aortic endothelial cells respectively, demonstrated a response-response relationship between reduced glutathione (GSH) and eNOS S-glutathionylation  (Du et al., 2013; De Pascali et al., 2014).
eNOS, S-Glutathionylation Directly Leads to eNOS, Uncoupling Moderate Treatment with BCNU resulted in increased eNOS S-glutathionylation, increased superoxide generation and decreased NO production in a dose-dependent manner in bovine aortic endothelial cells (Chen et al., 2010). Exposure to hypoxia/reoxygenation and treatment with angiotensin II demonstrated a response-response relationship between eNOS S-glutathionylation and superoxide generation in human and bovine endothelial cells (De Pascali et al., 2014; Galougahi et al., 2014).
eNOS, Uncoupling Directly Leads to Nitric Oxide, Depletion Strong Multiple experiments demonstrated that eNOS uncoupling results in increased superoxide formation and decreased NO production (Chen et al., 2010; De Pascali et al., 2014; Dumitrescu et al., 2007; Wang et al., 2008; Whitsett et al., 2007; Zou et al., 2002).
Nitric Oxide, Depletion Directly Leads to Vasodilation, impaired Moderate Treatment with eNOS inhibitors and two other stressors, BCNU and diaminohydroxypyrimidine (DAHP), caused a decrease in both NO production and vasodilation (Chen et al., 2010; Paulis et al, 2008; Wang et al., 2008).
Vasodilation, impaired Directly Leads to Vascular resistance, Increase Weak No direct evidence was found for this KER, but there is indirect support. Treatment with eNOS inhibitor L-NG-monomethyl arginine citrate (L-NMMA) caused an increase in SVR and a reduction in NO (Stamler et al., 1994), while L-NG-nitroarginine methyl ester (L-NAME) decreased NO-dependent relaxation and increased blood pressure (Paulis et al, 2008). Infusion of NO donor sodium nitroprusside led to dose-dependent reductions in SVR (Eugene, 2016).
Vascular resistance, Increase Directly Leads to Hypertension, N/A Moderate Several human studies showed a dose-dependent change in SVR and hypertension following treatment with eNOS inhibitors (Brett et al., 1998; Haynes et al., 1993; McVeigh et al., 2001; Stamler et al., 1994; Wilkinson et al., 2002).

Known Modulating Factors

Modulating factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors.  More help

Summary Table Provide an overall discussion of the quantitative information available for this AOP. Support calls for the individual relationships can be included in the Key Event Relationship table above.

Overall, there is a good amount of quantitative data available for this AOP as demonstrated by the weight of evidence tables, which shows that empirical support ranges from weak to strong. Four key event relationships (KERs) have strong quantitative support: decreased AKT/eNOS activity => NO depletion, decreased GTPCH-1 => decreased BH4, decreased BH4 => eNOS uncoupling and eNOS uncoupling => NO depletion. The relationships between these key events are well described in the literature as GTPCH-1 is the rate-limiting enzyme for BH4 synthesis (Wang et al., 2008), BH4 is an essential cofactor for eNOS and function (Wang et al., 2014), and the uncoupling of eNOS causes it to generate superoxide instead of NO (Carnicer et al., 2012). As they are functionally interconnected, many studies measure these key events together; thus providing strong support for their dependency.

Two key events have limited quantitative support (oxidative stress => decreased GTPCH-1, impaired vasodilation => increased vascular resistance). Generally, the oxidation of BH4 rather than decreased GTPCH-1 is measured when cells are under oxidative stress, and ROS are assumed to be increased so no quantitative measures are taken. For vasodilation and vascular resistance, there appears to be a correlative relationship, where increased vasodilation would mean decreased vascular resistance and vice versa, so studies do not measure both key events. Several studies showed that treatment with eNOS inhibitors led to increased vascular resistance, suggesting impaired vasodilation (Li et al., 2007; McVeigh et al., 2001; Paulis et al., 2008; Wilkinson et al., 2002).

The other KERs have moderate quantitative support, meaning there were studies showing a dependent change in both key events following treatment with stressors. Several studies measured decreased NO and vasodilation following perturbations to eNOS inhibitors, BCNU and DAHP (Chen et al., 2010; Paulis et al, 2008; Wang et al., 2008).

In general, experiments with both dose-dependent and temporal response data following a stressor are not readily available for all KERs as most measurements are generally taken at one time point after a perturbation, or the measurements are for one key event, not both. However, an exhaustive literature search was not performed. An ideal experiment would be to treat cells with three to four stressors at increasing concentrations and measure the key events at different time intervals; thus providing a greater understanding of the temporal and dose-dependent responses between the key events.

Considerations for Potential Applications of the AOP (optional)

Addressess potential applications of an AOP to support regulatory decision-making.This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. More help

One of the most widely reported hypertension risk factors is tobacco smoking.  While smoking cessation remains the best way to reduce the harmful effects of tobacco smoking, tobacco harm reduction is being considered by some regulators (e.g. US Food and Drug Administration; FDA) as a complementary strategy to reduce smoking-related disease burden.  The US FDA has published guidance on assessing a “modified risk tobacco product” (MRTP), either through demonstration of reduced toxicant exposure or reduction in health risks (FDA 2012). By gaining an understanding of how, and to what extent tobacco smoke initiates biological mechanisms of hypertension, such knowledge could be utilised as a baseline for comparison purposes in toxicological assessments of the risk reduction potential of e-cigarettes. 

Given the wide variety of data requirements to support such risk assessments e.g. human exposure studies, behavioural studies, efficacy studies, in vitro studies, clinical biomarker studies, pharmacokinetic studies, quality of life studies etc., a data integration framework is required to organise these data types into a comprehensive story that (i) characterises the toxicological problem, (ii) demonstrates the likely outcome of an intervention, and (iii) can be utilised to monitor the performance of any intervention over time.  Adverse outcome pathways offer scientists and regulators a way to inform future Integrated Approaches to Testing and Assessment (IATA) for the risk assessment/harm reduction potential of electronic cigarettes and heated tobacco products.

Other applications could include informing risk assessments for long term exposure to airborne pollution, in the context of helping to set acceptable exposure limits in urban environments for specific pollutants e.g. diesel exhuast particles.  Furthermore, health supplements which purport to benefit the cardiovascular system in the context of hypertension risk, could also be assessed for efficacy.


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

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