Aop:149

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AOP Title

Oxidative Stress 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); Jennifer Park (jpark@selventa.com)

Status

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Under development: Do not distribute or cite.

This AOP page was last modified on 12/11/2016.

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Abstract

States of oxidative stress in the endothelium are associated with endothelial dysfunction (Higashi et al., 2014), which is known to promote various cardiovascular-related pathologies, including renovascular hypertension (Lerman et al., 2001; Rajagopalan et al., 1996), hypercholesterolemia (Haddad et al., 2011; Ohara et al., 1993), and type II diabetes (Su et al., 2008). Oxidative stress has an inhibitory effect on endothelial NO production, which is critical for the maintenance of healthy vascular tone, and hence is an indicator of healthy endothelial function (Santos-Parker et al., 2014). Endothelial dysfunction is characterized by impairment of endothelium-dependent vasodilation and its association with hypertension is well established in clinical and animal studies (Dharmashankar and Widlansky, 2010; Silva et al., 2012).

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 (Bhatnagar et al., 2014).

Summary of the AOP

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Molecular Initiating Event

Molecular Initiating Event Support for Essentiality
Oxidative Stress, Increase Strong

Key Events

Event Support for Essentiality
Glutathione, Oxidation Strong
eNOS, S-Glutathionylation Strong
GTPCH-1, Decrease Strong
Tetrahydrobiopterin, Decrease Strong
eNOS, Uncoupling Strong
Nitric Oxide, Depletion Strong
Vasodilation, impaired Strong
Vascular Resistance, Increase Strong
Akt/eNOS activity, Decrease Strong

Adverse Outcome

Adverse Outcome
Hypertension, N/A

Relationships Among Key Events and the Adverse Outcome

Event Description Triggers Weight of Evidence Quantitative Understanding
Oxidative Stress, Increase Directly Leads to Glutathione, Oxidation Strong Moderate
Oxidative Stress, Increase Directly Leads to GTPCH-1, Decrease Moderate Weak
Glutathione, Oxidation Directly Leads to eNOS, S-Glutathionylation Strong Moderate
eNOS, S-Glutathionylation Directly Leads to eNOS, Uncoupling Strong Moderate
GTPCH-1, Decrease Directly Leads to Tetrahydrobiopterin, Decrease Strong Strong
Tetrahydrobiopterin, Decrease Directly Leads to eNOS, Uncoupling Strong Strong
eNOS, Uncoupling Directly Leads to Nitric Oxide, Depletion Strong Strong
Nitric Oxide, Depletion Directly Leads to Vasodilation, impaired Strong Moderate
Vasodilation, impaired Directly Leads to Vascular Resistance, Increase Strong Weak
Vascular Resistance, Increase Directly Leads to Hypertension, N/A Strong Moderate
Akt/eNOS activity, Decrease Directly Leads to Nitric Oxide, Depletion Strong Strong
Oxidative Stress, Increase Directly Leads to Akt/eNOS activity, Decrease Strong Moderate

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Life Stage Applicability

Life Stage Evidence Links
all life stages

Taxonomic Applicability

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

Sex Applicability

Sex Evidence Links
Unspecific

Graphical Representation

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Overall Assessment of the AOP

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).

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 (Ismail et al., 2015; 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)

References

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