API

Aop: 149

AOP Title

?


Peptide Oxidation Leading to Hypertension

Short name:

?

Hypertension

Authors

?


British American Tobacco: Frazer Lowe (Frazer_Lowe@bat.com); Linsey Haswell; Marianna Gaca

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

Selventa: Vy Hoang (vhoang@selventa.com)

Point of Contact

?


Frazer Lowe

Contributors

?


  • Frazer Lowe

Status

?

Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite Under Development 1.50 Included in OECD Work Plan


This AOP was last modified on September 05, 2017 05:00

?

Revision dates for related pages

Page Revision Date/Time
Peptide Oxidation September 16, 2017 10:14
Oxidation, Glutathione August 17, 2017 08:39
S-Glutathionylation, eNOS September 16, 2017 10:16
Decrease, GTPCH-1 September 16, 2017 10:16
Decrease, Tetrahydrobiopterin September 16, 2017 10:16
Uncoupling, eNOS September 16, 2017 10:16
Depletion, Nitric Oxide September 16, 2017 10:16
impaired, Vasodilation September 16, 2017 10:16
Increase, Vascular Resistance September 16, 2017 10:16
Hypertension August 17, 2017 09:02
Decrease, Akt/eNOS activity September 16, 2017 10:16
Peptide Oxidation leads to Oxidation, Glutathione November 29, 2016 20:46
Peptide Oxidation leads to Decrease, GTPCH-1 November 29, 2016 20:48
Oxidation, Glutathione leads to S-Glutathionylation, eNOS November 29, 2016 20:46
S-Glutathionylation, eNOS leads to Uncoupling, eNOS November 29, 2016 20:46
Decrease, GTPCH-1 leads to Decrease, Tetrahydrobiopterin November 29, 2016 20:46
Decrease, Tetrahydrobiopterin leads to Uncoupling, eNOS November 29, 2016 20:46
Uncoupling, eNOS leads to Depletion, Nitric Oxide November 29, 2016 20:46
Depletion, Nitric Oxide leads to impaired, Vasodilation November 29, 2016 20:46
impaired, Vasodilation leads to Increase, Vascular Resistance November 29, 2016 20:48
Increase, Vascular Resistance leads to Hypertension November 29, 2016 20:48
Decrease, Akt/eNOS activity leads to Depletion, Nitric Oxide November 29, 2016 20:48
Peptide Oxidation leads to Decrease, Akt/eNOS activity November 29, 2016 20:48
Reactive oxygen species August 15, 2017 10:43

Abstract

?


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 relatively 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 in regards 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.


Background (optional)

?


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


Summary of the AOP

?



Stressors

?

Name Evidence Term
Reactive oxygen species Strong

Molecular Initiating Event

?

Title Short name
Peptide Oxidation Peptide Oxidation

Key Events

?

Title Short name
Oxidation, Glutathione Oxidation, Glutathione
S-Glutathionylation, eNOS S-Glutathionylation, eNOS
Decrease, GTPCH-1 Decrease, GTPCH-1
Decrease, Tetrahydrobiopterin Decrease, Tetrahydrobiopterin
Uncoupling, eNOS Uncoupling, eNOS
Depletion, Nitric Oxide Depletion, Nitric Oxide
impaired, Vasodilation impaired, Vasodilation
Increase, Vascular Resistance Increase, Vascular Resistance
Decrease, Akt/eNOS activity Decrease, Akt/eNOS activity

Adverse Outcome

?

Title Short name
Hypertension Hypertension

Relationships Between Two Key Events (Including MIEs and AOs)

?

Title Directness Evidence Quantitative Understanding
Peptide Oxidation leads to Oxidation, Glutathione Directly leads to Strong Moderate
Peptide Oxidation leads to Decrease, GTPCH-1 Directly leads to Moderate Weak
Oxidation, Glutathione leads to S-Glutathionylation, eNOS Directly leads to Strong Moderate
S-Glutathionylation, eNOS leads to Uncoupling, eNOS Directly leads to Strong Moderate
Decrease, GTPCH-1 leads to Decrease, Tetrahydrobiopterin Directly leads to Strong Strong
Decrease, Tetrahydrobiopterin leads to Uncoupling, eNOS Directly leads to Strong Strong
Uncoupling, eNOS leads to Depletion, Nitric Oxide Directly leads to Strong Strong
Depletion, Nitric Oxide leads to impaired, Vasodilation Directly leads to Strong Moderate
impaired, Vasodilation leads to Increase, Vascular Resistance Directly leads to Strong Weak
Increase, Vascular Resistance leads to Hypertension Directly leads to Strong Moderate
Decrease, Akt/eNOS activity leads to Depletion, Nitric Oxide Directly leads to Strong Strong
Peptide Oxidation leads to Decrease, Akt/eNOS activity Directly leads to Strong Moderate

Network View

?

 

Life Stage Applicability

?

Life stage Evidence
All life stages

Taxonomic Applicability

?

Term Scientific Term Evidence Link
human Homo sapiens Strong NCBI
mouse Mus musculus Moderate NCBI
rat Rattus norvegicus Moderate NCBI
cow Bos taurus Moderate NCBI

Sex Applicability

?

Sex Evidence
Unspecific

Graphical Representation

?

Click to download graphical representation template

W1siziisijiwmtcvmdgvmtcvnjjyzdeydxhkzv9bt1bfmtq5x0pqry5qcgcixsxbinailcj0ahvtyiisijuwmhg1mdaixv0?sha=741e0da29129b48d

Overall Assessment of the AOP

?



This section addresses the relevant domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and weight of evidence for the overall hypothesised AOP (i.e., including the MIE, KEs and AO) as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). It draws upon the evidence assembled for each KER as one of several components which contribute to relative confidence in supporting information for the entire hypothesised pathway. An important component in assessing confidence in supporting information as a basis to consider regulatory application of AOPs beyond that described in Section 6 is the essentiality of each of the key events as a component of the entire pathway. This is normally investigated in specifically-designed stop/reversibility studies or knockout models (i.e., those where a key event can be blocked or prevented). Assessment of the overall AOP also contributes to the identification of KEs for which confidence in the quantitative relationship with the AO is greatest (i.e., to facilitate determining the most sensitive predictor of the AO).

Instructions

To edit the “Overall Assessment of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Overall Assessment of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page.  The new text should appear under the “Overall Assessment of the AOP” section on the AOP page.

Domain of Applicability

?

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 not sex-dependent or associated with a certain life stage. It is well-documented and well-studied in humans, cows and rodents.


Essentiality of the Key Events

?

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 shouldn't 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) AKT kinase phosphorylates eNOS in humans 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 cells by 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 in literature as having a deterimental effect on vasodilation, 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

High 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 total peripheral resistance (TPR) or systemic vascular resistance (SVR) 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    

 


Weight of Evidence Summary

?

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 It is well-established that tetrahydrobiopterin (BH4) is highly susceptible to oxidation by reactive oxygen species, leading to dysfunction eNOS function (Lee and Griendling, 2008). Several studies demonstrated that GTPCH-1, the rate-limiting enzyme for BH4 synthesis, is also 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 deletion of GTPCH-1 led to the deficiency 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, leading to decreased NO and increased superoxide 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 demonstrated a response-response relationship between reduced glutathione (GSH) and eNOS S-glutathionylation in human and bovine aortic endothelial cells, respectively (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).

Quantitative Considerations

?

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 a few stressor. 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)

?


At their discretion, the developer may include in this section discussion of the 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. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale. Detailing such considerations can aid the process of transforming narrative descriptions of AOPs into practical tools. In this context, it is necessarily beneficial to involve members of the regulatory risk assessment community on the development and assessment team. The Network view which is generated based on assessment of weight of evidence/degree of confidence in the hypothesized AOP taking into account the elements described in Section 7 provides a useful summary of relevant information as a basis to consider appropriate application in a regulatory context. Consideration of application needs then, to take into consideration the following rank ordered qualitative elements: Confidence in biological plausibility for each of the KERs Confidence in essentiality of the KEs Empirical support for each of the KERs and overall AOP The extent of weight of evidence/confidence in both these qualitative elements and that of the quantitative understanding for each of the KERs (e.g., is the MIE known, is quantitative understanding restricted to early or late key events) is also critical in determining appropriate application. For example, if the confidence and quantitative understanding of each KER in a hypothesised AOP are low and or low/moderate and the evidence for essentiality of KEs weak (Section 7), it might be considered as appropriate only for applications with less potential for impact (e.g., prioritisation, category formation for testing) versus those that have immediate implications potentially for risk management (e.g., in depth assessment). If confidence in quantitative understanding of late key events is high, this might be sufficient for an in depth assessment. The analysis supporting the Network view is also essential in identifying critical data gaps based on envisaged regulatory application.

Instructions

To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page.


References

?


AbdelGhany, T., Ismail, R., Elmahdy, M., Mansoor F, Zweier J, Lowe, F., and Zweier, JL. (2017). Cigarette Smoke Constituents Cause Endothelial Nitric Oxide Synthase Dysfunction and Uncoupling due to Depletion of Tetrahydrobiopterin with Degradation of GTP Cyclohydrolase.  Nitric Oxide (Under review).

Adlam, D., Herring, N., Douglas, G., De Bono, J.P., Li, D., Danson, E.J., Tatham, A., Lu, C.-J., Jennings, K.A., Cragg, S.J., et al. (2012). Regulation of β-adrenergic control of heart rate by GTP-cyclohydrolase 1 (GCH1) and tetrahydrobiopterin. Cardiovasc. Res. 93, 694–701.

Alp, N.J., Mussa, S., Khoo, J., Cai, S., Guzik, T., Jefferson, A., Goh, N., Rockett, K.A., and Channon, K.M. (2003). Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression. J. Clin. Invest. 112, 725–735.

Antoniades, C., Cunnington, C., Antonopoulos, A., Neville, M., Margaritis, M., Demosthenous, M., Bendall, J., Hale, A., Cerrato, R., Tousoulis, D., et al. (2011). Induction of vascular GTP-cyclohydrolase I and endogenous tetrahydrobiopterin synthesis protect against inflammation-induced endothelial dysfunction in human atherosclerosis. Circulation 124, 1860–1870.

Bendall JK, Douglas G, McNeill E et al.  Tetrahydrobiopterin in cardiovascular health and disease.  Antioxid Redox Signal. (2014) 20(18):3040-77.

Berg, T., and Jensen, J. (2011). Simultaneous parasympathetic and sympathetic activation reveals altered autonomic control of heart rate, vascular tension, and epinephrine release in anesthetized hypertensive rats. Front. Neurol. 2, 71.

Bhatnagar, A., Whitsel, L.P., Ribisl, K.M., Bullen, C., Chaloupka, F., Piano, M.R., Robertson, R.M., McAuley, T., Goff, D., Benowitz, N., et al. (2014). Electronic cigarettes: a policy statement from the American Heart Association. Circulation 130, 1418–1436.

Brandes, R.P. (2014). Endothelial dysfunction and hypertension. Hypertension 64, 924–928.

Brett, S.E., Cockcroft, J.R., Mant, T.G., Ritter, J.M., and Chowienczyk, P.J. (1998). Haemodynamic effects of inhibition of nitric oxide synthase and of L-arginine at rest and during exercise. J. Hypertens. 16, 429–435.

Carnicer, R., Hale, A.B., Suffredini, S., Liu, X., Reilly, S., Zhang, M.H., Surdo, N.C., Bendall, J.K., Crabtree, M.J., Lim, G.B.S., et al. (2012). Cardiomyocyte GTP cyclohydrolase 1 and tetrahydrobiopterin increase NOS1 activity and accelerate myocardial relaxation. Circ. Res. 111, 718–727.

Cervantes-Pérez, L.G., Ibarra-Lara, M. de la L., Escalante, B., Del Valle-Mondragón, L., Vargas-Robles, H., Pérez-Severiano, F., Pastelín, G., and Sánchez-Mendoza, M.A. (2012). Endothelial nitric oxide synthase impairment is restored by clofibrate treatment in an animal model of hypertension. Eur. J. Pharmacol. 685, 108–115.

Chan, S.S., Tse, M.M., Chan, C.P., Tai, M.C., Graham, C.A., and Rainer, T.H. (2016). Haemodynamic changes in emergency department patients with poorly controlled hypertension. Hong Kong Med. J. Xianggang Yi Xue Za Zhi Hong Kong Acad. Med. 22, 116–123.

Chen, C.-A., Wang, T.-Y., Varadharaj, S., Reyes, L.A., Hemann, C., Talukder, M.A.H., Chen, Y.-R., Druhan, L.J., and Zweier, J.L. (2010a). S-glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 468, 1115–1118.

Chen, C.-A., Lin, C.-H., Druhan, L.J., Wang, T.-Y., Chen, Y.-R., and Zweier, J.L. (2011). Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling. J. Biol. Chem. 286, 29098–29107.

Chen CA, De Pascali F, Basye A et al. Redox modulation of endothelial nitric oxide synthase by glutaredoxin-1 through reversible oxidative post-translational modification.  (2013)  Biochemistry. 52(38):6712-23

Choi, Y.-J., Yoon, Y., Lee, K.-Y., Hien, T.T., Kang, K.W., Kim, K.-C., Lee, J., Lee, M.-Y., Lee, S.M., Kang, D.-H., et al. (2014). Uric acid induces endothelial dysfunction by vascular insulin resistance associated with the impairment of nitric oxide synthesis. FASEB J. 28, 3197–3204.

Choi, Y.-J., Yoon, Y., Lee, K.-Y., Kang, Y.-P., Lim, D.K., Kwon, S.W., Kang, K.-W., Lee, S.-M., and Lee, B.-H. (2015). Orotic Acid Induces Hypertension Associated with Impaired Endothelial Nitric Oxide Synthesis. Toxicol. Sci. 144, 307–317.

Chuaiphichai, S., McNeill, E., Douglas, G., Crabtree, M.J., Bendall, J.K., Hale, A.B., Alp, N.J., and Channon, K.M. (2014). Cell-autonomous role of endothelial GTP cyclohydrolase 1 and tetrahydrobiopterin in blood pressure regulation. Hypertension 64, 530–540.

Crabtree, M.J., Tatham, A.L., Al-Wakeel, Y., Warrick, N., Hale, A.B., Cai, S., Channon, K.M., and Alp, N.J. (2009). Quantitative regulation of intracellular endothelial nitric-oxide synthase (eNOS) coupling by both tetrahydrobiopterin-eNOS stoichiometry and biopterin redox status: insights from cells with tet-regulated GTP cyclohydrolase I expression. J. Biol. Chem. 284, 1136–1144.

Das, A., Gopalakrishnan, B., Druhan, L.J., Wang, T.-Y., De Pascali, F., Rockenbauer, A., Racoma, I., Varadharaj, S., Zweier, J.L., Cardounel, A.J., et al. (2014). Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelial cells via eNOS phosphorylation. Br. J. Pharmacol. 171, 2321–2334.

De Pascali, F., Hemann, C., Samons, K., Chen, C.-A., and Zweier, J.L. (2014). Hypoxia and reoxygenation induce endothelial nitric oxide synthase uncoupling in endothelial cells through tetrahydrobiopterin depletion and S-glutathionylation. Biochemistry (Mosc.) 53, 3679–3688.

Dessy, C., Moniotte, S., Ghisdal, P., Havaux, X., Noirhomme, P., and Balligand, J.L. (2004). Endothelial beta3-adrenoceptors mediate vasorelaxation of human coronary microarteries through nitric oxide and endothelium-dependent hyperpolarization. Circulation 110, 948–954.

Dhar, A., Dhar, I., Desai, K.M., and Wu, L. (2010). Methylglyoxal scavengers attenuate endothelial dysfunction induced by methylglyoxal and high concentrations of glucose. Br. J. Pharmacol. 161, 1843–1856.

Dharmashankar, K., and Widlansky, M.E. (2010). Vascular endothelial function and hypertension: insights and directions. Curr. Hypertens. Rep. 12, 448–455.

Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., and Zeiher, A.M. (1999). Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601–605.

Du, J., Fan, L.M., Mai, A., and Li, J.-M. (2013). Crucial roles of Nox2-derived oxidative stress in deteriorating the function of insulin receptors and endothelium in dietary obesity of middle-aged mice. Br. J. Pharmacol. 170, 1064–1077.

Du, X.L., Edelstein, D., Dimmeler, S., Ju, Q., Sui, C., and Brownlee, M. (2001). Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J. Clin. Invest. 108, 1341–1348.

Du, Y., Navab, M., Shen, M., Hill, J., Pakbin, P., Sioutas, C., Hsiai, T.K., and Li, R. (2013b). Ambient ultrafine particles reduce endothelial nitric oxide production via S-glutathionylation of eNOS. Biochem. Biophys. Res. Commun. 436, 462–466.

Dumitrescu, C., Biondi, R., Xia, Y., Cardounel, A.J., Druhan, L.J., Ambrosio, G., and Zweier, J.L. (2007). Myocardial ischemia results in tetrahydrobiopterin (BH4) oxidation with impaired endothelial function ameliorated by BH4. Proc. Natl. Acad. Sci. U. S. A. 104, 15081–15086.

Durand MJ, Gutterman DD.  Diversity in mechanisms of endothelium-dependent vasodilation in health and disease.  Microcirculation. (2013) 20(3):239-47

Eugene, A.R. (2016). The influences of nitric oxide, epinephrine, and dopamine on vascular tone: dose-response modeling and simulations. Hosp. Chron. Nosokomeiaka Chron. 11, 1–8.

Förstermann, U., and Münzel, T. (2006). Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 113, 1708–1714.

Foëx, P., and Sear, J.W. (2004). Hypertension: pathophysiology and treatment. Contin. Educ. Anaesth. Crit. Care Pain 4, 71–75.

Fulton, D., Gratton, J.P., McCabe, T.J., Fontana, J., Fujio, Y., Walsh, K., Franke, T.F., Papapetropoulos, A., and Sessa, W.C. (1999). Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399, 597–601.

Galougahi, K.K., Liu, C.-C., Gentile, C., Kok, C., Nunez, A., Garcia, A., Fry, N.A.S., Davies, M.J., Hawkins, C.L., Rasmussen, H.H., et al. (2014). Glutathionylation Mediates Angiotensin II–Induced eNOS Uncoupling, Amplifying NADPH Oxidase-Dependent Endothelial Dysfunction. J. Am. Heart Assoc. 3, e000731.

van Gorp, R.M.A., Heeneman, S., Broers, J.L.V., Bronnenberg, N.M.H.J., van Dam-Mieras, M.C.E., and Heemskerk, J.W.M. (2002). Glutathione oxidation in calcium- and p38 MAPK-dependent membrane blebbing of endothelial cells. Biochim. Biophys. Acta 1591, 129–138.

van Gorp, R.M., Broers, J.L., Reutelingsperger, C.P., Bronnenberg, N.M., Hornstra, G., van Dam-Mieras, M.C., and Heemskerk, J.W. (1999). Peroxide-induced membrane blebbing in endothelial cells associated with glutathione oxidation but not apoptosis. Am. J. Physiol. 277, C20–C28.

Haddad, P., Dussault, S., Groleau, J., Turgeon, J., Maingrette, F., and Rivard, A. (2011). Nox2-derived reactive oxygen species contribute to hypercholesterolemia-induced inhibition of neovascularization: effects on endothelial progenitor cells and mature endothelial cells. Atherosclerosis 217, 340–349.

Higashi, Y., Maruhashi, T., Noma, K., and Kihara, Y. (2014). Oxidative stress and endothelial dysfunction: clinical evidence and therapeutic implications. Trends Cardiovasc. Med. 24, 165–169.

Huang PL.  Mouse models of nitric oxide synthase deficiency. (2000) J Am Soc Nephrol. 11 Suppl 16:S120-3.

Huang PL, Huang Z, Mashimo H et al. Hypertension in mice lacking the gene for endothelial nitric oxide synthase.  (1995) Nature. 377(6546):239-42.

Jayaram, R., Goodfellow, N., Zhang, M.H., Reilly, S., Crabtree, M., De Silva, R., Sayeed, R., and Casadei, B. (2015). Molecular mechanisms of myocardial nitroso-redox imbalance during on-pump cardiac surgery. Lancet Lond. Engl. 385 Suppl 1, S49.

Landmesser, U., Dikalov, S., Price, S.R., McCann, L., Fukai, T., Holland, S.M., Mitch, W.E., and Harrison, D.G. (2003). Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J. Clin. Invest. 111, 1201–1209.

Lee, M.Y., and Griendling, K.K. (2008). Redox signaling, vascular function, and hypertension. Antioxid. Redox Signal. 10, 1045–1059.

Lerman, L.O., Nath, K.A., Rodriguez-Porcel, M., Krier, J.D., Schwartz, R.S., Napoli, C., and Romero, J.C. (2001). Increased oxidative stress in experimental renovascular hypertension. Hypertension 37, 541–546.

Li, J., Zhou, Z., Jiang, D.-J., Li, D., Tan, B., Liu, H., and Li, Y.-J. (2007). Reduction of NO- and EDHF-mediated vasodilatation in hypertension: role of asymmetric dimethylarginine. Clin. Exp. Hypertens. N. Y. N 1993 29, 489–501.

Lowe FJ., Luettich K, Talikka M, Hoang V, Haswell LE., Hoeng J, and Gaca MD. Development of an Adverse Outcome Pathway for the Onset of Hypertension by Oxidative Stress-Mediated Perturbation of Endothelial Nitric Oxide Bioavailability.  Applied In Vitro Toxicology. (2017), 3(1): 131-148.  

Luo, Z., Fujio, Y., Kureishi, Y., Rudic, R.D., Daumerie, G., Fulton, D., Sessa, W.C., and Walsh, K. (2000). Acute modulation of endothelial Akt/PKB activity alters nitric oxide–dependent vasomotor activity in vivo. J. Clin. Invest. 106, 493–499.

Mayet, J., and Hughes, A. (2003). Cardiac and vascular pathophysiology in hypertension. Heart Br. Card. Soc. 89, 1104–1109.

McVeigh, G.E., Allen, P.B., Morgan, D.R., Hanratty, C.G., and Silke, B. (2001). Nitric oxide modulation of blood vessel tone identified by arterial waveform analysis. Clin. Sci. Lond. Engl. 1979 100, 387–393.

Michaud, S.E., Dussault, S., Groleau, J., Haddad, P., and Rivard, A. (2006). Cigarette smoke exposure impairs VEGF-induced endothelial cell migration: role of NO and reactive oxygen species. J. Mol. Cell. Cardiol. 41, 275–284.

Mitchell BM , Dorrance AM, Webb RC. GTP cyclohydrolase 1 inhibition attenuates vasodilation and increases blood pressure in rats.  (2003) Am J Physiol Heart Circ Physiol. 285(5):H2165-70.

Montecinos, V., Guzmán, P., Barra, V., Villagrán, M., Muñoz-Montesino, C., Sotomayor, K., Escobar, E., Godoy, A., Mardones, L., Sotomayor, P., et al. (2007). Vitamin C is an essential antioxidant that enhances survival of oxidatively stressed human vascular endothelial cells in the presence of a vast molar excess of glutathione. J. Biol. Chem. 282, 15506–15515.

Nozik-Grayck E, Woods C, Taylor JM et al. Selective depletion of vascular EC-SOD augments chronic hypoxic pulmonary hypertension.  Am J Physiol Lung Cell Mol Physiol. (2014) 307(11):L868-76. 

Ohara, Y., Peterson, T.E., and Harrison, D.G. (1993). Hypercholesterolemia increases endothelial superoxide anion production. J. Clin. Invest. 91, 2546–2551.

Ozaki, M., Kawashima, S., Yamashita, T., Hirase, T., Namiki, M., Inoue, N., Hirata, K., Yasui, H., Sakurai, H., Yoshida, Y., et al. (2002). Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice. J. Clin. Invest. 110, 331–340.

Park, W.H. (2013). The effects of exogenous H2O2 on cell death, reactive oxygen species and glutathione levels in calf pulmonary artery and human umbilical vein endothelial cells. Int. J. Mol. Med. 31, 471–476.

Paulis, L., Zicha, J., Kunes, J., Hojna, S., Behuliak, M., Celec, P., Kojsova, S., Pechanova, O., and Simko, F. (2008). Regression of L-NAME-induced hypertension: the role of nitric oxide and endothelium-derived constricting factor. Hypertens. Res. Off. J. Jpn. Soc. Hypertens. 31, 793–803.

Rajagopalan, S., Kurz, S., Münzel, T., Tarpey, M., Freeman, B.A., Griendling, K.K., and Harrison, D.G. (1996). Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. J. Clin. Invest. 97, 1916–1923.

Santos-Parker, J.R., LaRocca, T.J., and Seals, D.R. (2014). Aerobic exercise and other healthy lifestyle factors that influence vascular aging. Adv. Physiol. Educ. 38, 296–307.

Schuppe, I., Moldéus, P., and Cotgreave, I.A. (1992). Protein-specific S-thiolation in human endothelial cells during oxidative stress. Biochem. Pharmacol. 44, 1757–1764.

Scotland RS, Madhani M, Chauhan S et al.  Investigation of vascular responses in endothelial nitric oxide synthase/cyclooxygenase-1 double-knockout mice: key role for endothelium-derived hyperpolarizing factor in the regulation of blood pressure in vivo.  (2005)  Circulation. 111(6):796-803.

Sélley, E., Kun, S., Szijártó, I.A., Laczy, B., Kovács, T., Fülöp, F., Wittmann, I., and Molnár, G.A. (2014). Exenatide induces aortic vasodilation increasing hydrogen sulphide, carbon monoxide and nitric oxide production. Cardiovasc. Diabetol. 13, 69.

Shinozaki, K., Nishio, Y., Okamura, T., Yoshida, Y., Maegawa, H., Kojima, H., Masada, M., Toda, N., Kikkawa, R., and Kashiwagi, A. (2000). Oral administration of tetrahydrobiopterin prevents endothelial dysfunction and vascular oxidative stress in the aortas of insulin-resistant rats. Circ. Res. 87, 566–573.

Siddiqui, A. (2011). Effects of Vasodilation and Arterial Resistance on Cardiac Output. J. Clin. Exp. Cardiol. 02.

Sindler, A.L., Devan, A.E., Fleenor, B.S., and Seals, D.R. (2014). Inorganic nitrite supplementation for healthy arterial aging. J. Appl. Physiol. Bethesda Md 1985 116, 463–477.

Silva, B.R., Pernomian, L., and Bendhack, L.M. (2012). Contribution of oxidative stress to endothelial dysfunction in hypertension. Front. Physiol. 3, 441.

Song, P., Wu, Y., Xu, J., Xie, Z., Dong, Y., Zhang, M., and Zou, M.-H. (2007). Reactive nitrogen species induced by hyperglycemia suppresses Akt signaling and triggers apoptosis by upregulating phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in an LKB1-dependent manner. Circulation 116, 1585–1595.

Song, P., Xie, Z., Wu, Y., Xu, J., Dong, Y., and Zou, M.-H. (2008). Protein kinase Czeta-dependent LKB1 serine 428 phosphorylation increases LKB1 nucleus export and apoptosis in endothelial cells. J. Biol. Chem. 283, 12446–12455.

Stamler, J.S., Loh, E., Roddy, M.A., Currie, K.E., and Creager, M.A. (1994). Nitric oxide regulates basal systemic and pulmonary vascular resistance in healthy humans. Circulation 89, 2035–2040.

Su, Y., Liu, X.-M., Sun, Y.-M., Jin, H.-B., Fu, R., Wang, Y.-Y., Wu, Y., and Luan, Y. (2008). The relationship between endothelial dysfunction and oxidative stress in diabetes and prediabetes. Int. J. Clin. Pract. 62, 877–882.

Su, Y., Qadri, S.M., Wu, L., and Liu, L. (2013). Methylglyoxal modulates endothelial nitric oxide synthase-associated functions in EA.hy926 endothelial cells. Cardiovasc. Diabetol. 12, 134.

Talukder MA, Johnson WM, Varadharaj S et al.  Chronic cigarette smoking causes hypertension, increased oxidative stress, impaired NO bioavailability, endothelial dysfunction, and cardiac remodeling in mice.  (2011) Am J Physiol Heart Circ Physiol. 300(1):H388-96.

Tatham, A.L., Crabtree, M.J., Warrick, N., Cai, S., Alp, N.J., and Channon, K.M. (2009). GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression. J. Biol. Chem. 284, 13660–13668.

Tiefenbacher CP, Chilian WM, Mitchell M et al. Restoration of endothelium-dependent vasodilation after reperfusion injury by tetrahydrobiopterin. (1996) Circulation. 94:1423–1429.

Uruno, A., Sugawara, A., Kanatsuka, H., Kagechika, H., Saito, A., Sato, K., Kudo, M., Takeuchi, K., and Ito, S. (2005). Upregulation of nitric oxide production in vascular endothelial cells by all-trans retinoic acid through the phosphoinositide 3-kinase/Akt pathway. Circulation 112, 727–736.

Vásquez-Vivar J, Martásek P, Whitsett J et al. The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study.  (2002) Biochem J. 362(Pt 3):733-9.

Wang, S., Xu, J., Song, P., Wu, Y., Zhang, J., Chul Choi, H., and Zou, M.-H. (2008). Acute inhibition of guanosine triphosphate cyclohydrolase 1 uncouples endothelial nitric oxide synthase and elevates blood pressure. Hypertension 52, 484–490.

Whitsett, J., Picklo, M.J., and Vasquez-Vivar, J. (2007). 4-Hydroxy-2-nonenal increases superoxide anion radical in endothelial cells via stimulated GTP cyclohydrolase proteasomal degradation. Arterioscler. Thromb. Vasc. Biol. 27, 2340–2347.

Wilkinson, I.B., MacCallum, H., Cockcroft, J.R., and Webb, D.J. (2002). Inhibition of basal nitric oxide synthesis increases aortic augmentation index and pulse wave velocity in vivo. Br. J. Clin. Pharmacol. 53, 189–192.

Zhang, W., Han, Y., Meng, G., Bai, W., Xie, L., Lu, H., Shao, Y., Wei, L., Pan, S., Zhou, S., et al. (2014). Direct renin inhibition with aliskiren protects against myocardial ischemia/reperfusion injury by activating nitric oxide synthase signaling in spontaneously hypertensive rats. J. Am. Heart Assoc. 3, e000606.

Zhao, H., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vásquez-Vivar, J., and Kalyanaraman, B. (2003). Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic. Biol. Med. 34, 1359–1368.

Zou, M.-H., Hou, X.-Y., Shi, C.-M., Nagata, D., Walsh, K., and Cohen, R.A. (2002). Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase. J. Biol. Chem. 277, 32552–32557.

Zweier JL, Chen CA, Druhan LJ.  S-glutathionylation reshapes our understanding of endothelial nitric oxide synthase uncoupling and nitric oxide/reactive oxygen species-mediated signaling.  (2011) Antioxid Redox Signal. 14(10):1769-75.