Aopwiki

SNAPSHOT

Created at: 2017-12-04 05:24

AOP ID and Title:


AOP 149: Peptide Oxidation Leading to Hypertension
Short Title: 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)


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 Do not cite Under Development 1.50 Included in OECD Work Plan

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 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.


Background


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.

 


Summary of the AOP


Stressors


Name Evidence
Reactive oxygen species Strong

Reactive oxygen species

Compounds or environmental conditions, which generate endothelium-localised ROS in vivo are the primary source of the MIE.  Notable examples include: 

ROS/ROS donors : (Song et al. 2008, van Gorp et al. 1999, van Gorp et al. 2002, Park et al. 2013, Montecinos et al. 2007, Schuppe et al. 1992, 

Hypoxia/ischaemia : Nozik-Grayck et al. 2014, Zhang et al. 2014, De Pascali et al. 2014, 

SIN-1 (CAS № 16142-27-1) :  Das et al. 2014  

Heavy Metals (Lead, Cadmium, Mercury) : Vaziri et al. 2001, Wolf et al. 2007

Carbonyls (including methylglyoxal, N,N′-bis(2-chloroethyl)-N-nitroso-urea), acrolein) : Morgan et al. 2014, Dhar et al. 2010, Su et al. 2013, Chen et al. 2010, Chen et al. 2011, Michaud et al. 2006, Qin et al. 2016, Zhang et al. 2011

Glucose : Zou et al. 2002, Song et al. 2007, Du et al. 2013, Du et al. 2001, Dhar et al. 2010, Su et al. 2008

Ultra-fine particulates : Du et al. 2013, Tseng et al. 2016

Cigarette smoke (known to contain carbonyls, metals and ROS) : Michaud et al. 2006, Zhang et al. 2006, Talukder et al. 2011

Molecular Initiating Event

Title Short name
Peptide Oxidation Peptide Oxidation

209: Peptide Oxidation

Short Name: Peptide Oxidation

Key Event Component

Process Object Action
oxidative stress increased

Biological Organization

Level of Biological Organization
Molecular

Cell term

Cell term
eukaryotic cell

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
rodents rodents Strong NCBI
human and other cells in culture human and other cells in culture Strong NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Strong
Sex Applicability
Sex Evidence
Unspecific Strong

The concentrations of GSH and GSSG have been shown in tissues of human and laboratory animals, including rats, mice and cows (Chen et al., 2010; Giustarini et al., 2013).


How this Key Event Works

Oxidative stress corresponds to an imbalance between the rate of oxidant production and that of their degradation. The term oxidative stress indicates the outcome of oxidative damage to biologically relevant macromolecules such as nucleic acids, proteins, lipids and carbohydrates. This occurs when oxidative stress-related molecules, generated in the extracellular environment or within the cell, exceed cellular antioxidant defenses. Major reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and superoxide anion, as well as 4-hydroxy- 2,3-nonenal (HNE) and related 4-hydroxy-2,3-alkenals (HAKs), major aldehydic end-products of lipid peroxidation, can act as potential mediators able to affect signal transduction pathways as well as the proliferative and functional response of target cells. H2O2 and superoxide anion may be also generated as molecular messengers within the cell as part of the cellular response to defined growth factors, cytokines and other mediators. The final consequence at tissue, cellular and molecular level is primarily affected by the steady state concentration of oxidative stress-related molecules. The main biological targets of free radicals are proteins, lipids and DNA.

Major consequences of reaction of ROS, HAKs and NO with biologically relevant macromolecules that can mediate pathophysiological effects:

ROS: DNA: oxidation, strand breaks, genotoxicity Proteins: oxidation, fragmentation, formation of carbonyls Lipids: lipid peroxidation and degradation

HAKs: DNA: adducts (low doses), strand breaks, genotoxicity (high doses) Proteins: adducts (Michael type reactions on Lys, Cys and His residues)

NO: DNA: oxidation, strand breaks Proteins: oxidation, nitrosation, nitration (nytrosylation of tyrosine) Lipids: lipid peroxidation and degradation

Continued oxidative stress can lead to chronic inflammation. Oxidative stress can activate a variety of transcription factors including NF-κB, AP-1, p53, HIF-1α, PPAR-γ, β-catenin/Wnt, and Nrf2. Activation of these transcription factors can lead to the expression of over 500 different genes, including those for growth factors, inflammatory cytokines and chemokines, which can activate inflammatory pathways. [1] [2] [3]

Glutathione (GSH) oxidation refers to the conversion of reduced glutathione to its oxidized form glutathione disulfide (GSSG) in the presence of oxidative species. GSH plays an important role as an anti-oxidant in regulating cellular redox homeostasis, and is mainly present in the cell as the reduced form (98%). Deficiency in GSH or a decrease in GSH/GSSG ratio results in decreased anti-oxidant function and increased susceptibility to oxidative stress, thus making it a marker of cellular redox status. An imbalance in GSH/GSSG ratio has been implicated in the onset and progression of human diseases, such as neurodegenerative diseases, cancers, pulmonary diseases and cardiovascular diseases (Ballatori et al., 2009; Kalinina et al., 2014)


How it is Measured or Detected

 

measuring oxidative stress

Agents for ROS detection are primarily fluorescence based, but recently luminescent based detections have been introduced. The biggest difficulty reported with much of the cellular ROS research has been with the lack of reporter agents specific for discrete molecules. ROS moieties by their nature are reactive with a number of different molecules; as such designing reporter agents has been difficult. With more specific chemistries, particularly for hydrogen peroxide, the specific mechanisms for regulation will be elucidated.

Reduced glutathione (GSH) is regenerated from its oxidized form (GSSH) by the action of an NADPH dependent reductase GSSH + NADPH + H+ à 2 GSH + NADP+ Due to the rapid nature of the reduction of GSSH relative to its synthesis or secretion, the ratio of GSH to GSSH is a good indicator of oxidative stress within cells. GSH and GSSH levels can be determined by HPLC, capillary electrophoresis, or biochemically in microplates. Several different assays have been designed to measure glutathione in samples. By using a luciferin derivative in conjunction with glutathione S-transferase enzyme the amount of GSH would be proportional to the luminescent signal generated when luciferase is added in a subsequent step. Total glutathione can be determined colorimetrically by reacting GSH with DTNB (Ellman’s reagent) in the presence of glutathione reductase. Glutathione reductase reduces GSSH to GSH, which then reacts with DTNB to produce a yellow colored 5-thio-2-nitrobenzoic acid (TNB), which absorbs at 412 nm.

Lipid peroxidation is one of the most widely used indicators of free radical formation, a key indicator of oxidative stress. Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid (TBA) reactive compounds such as malondialdehyde generated from the decomposition of lipid peroxidation products. While this method is controversial in that it is quite sensitive, but not necessarily specific to MDA, it remains the most widely used means to determine lipid peroxidation. This reaction, which takes place under acidic conditions at 90-100ºC, results in an adduct that can be measured colorimetrically at 532 nm or by fluorescence using a 530 nm excitation wavelength and a 550 nm emission wavelength. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes (IsoP) has been shown to be specific for lipid peroxidation. Unlike the TBA assay, measurement of IsoP appears to be specific to lipid peroxides, they are stable and are not produced by any enzymatic pathway making interpretation easier. There have been a number of commercial ELISA kits developed for IsoPs, but interfering agents in samples requires partial purification of samples prior to running the assay. The only reliable means for detection is through the use of GC/MS, which makes it expensive and limits throughput.

Superoxide detection is based on the interaction of superoxide with some other compound to create a measurable result. The reduction of ferricytochrome c to ferrocytochrome c has been used in a number of situations to assess the rate of superoxide formation. While not completely specific for superoxide this reaction can be monitored colorimetrically at 550 nm. Chemiluminescent reactions have been used for their potential increase in sensitivity over absorbance-based detection methods. The most widely used chemiluminescent substrate is Lucigenin, but this compound has a propensity for redox cycling, which has raised doubts about its use in determining quantitative rates of superoxide production. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical. These dyes are synthesized by reducing the iminium cation of the cyanine (Cy) dyes with sodium borohydride. While weakly fluorescent, upon oxidation their fluorescence intensity increases 100 fold. In addition to being fluorescent, oxidation also converts the molecule from being membrane permeable to an ionic impermeable moiety. The most characterized of these probes are Hydro-Cy3 and Hydro-Cy5.

Hydrogen peroxide (H2O2) is the most important ROS in regards to mitogenic stimulation or cell cycle regulation. There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products. The more commonly used substrates include diacetyldichloro-fluorescein, homovanillic acid, and Amplex® Red. In these examples, increasing amounts of H2O2 form increasing amounts of fluorescent product.

Nitric Oxide The free radical nitric oxide (•NO) is produced by a number of different cell types with a variety of biological functions. Regardless of the source or role, the free radical •NO has a very short half life (t½= 4 seconds), reacting with several different molecules normally present to form either nitrate (NO3-) or nitrite (NO2-) A commonly used method for the indirect determination of •NO is the determination of its composition products nitrate and nitrite colorimetrically. This reaction requires that nitrate (NO3) first be reduced to nitrite (NO2), typically by the action of nitrate reductase. Subsequent determination of nitrite by a two step process provides information on the “total” of nitrate and nitrite. In the presence of hydrogen ions nitrite forms nitrous acid, which reacts with sulfanilamide to produce a diazonium ion. This then coupled to N-(1-napthyl) ethylenediamine to form the chromophore which absorbs at 543 nm. Nitrite only determinations can then be made in a parallel assay where the samples were not reduced prior to the colorimetric assay. Actual nitrate levels are then calculated by the subtraction of nitrite levels from the total. [4]

 


References

Ballatori, N., Krance, S.M., Notenboom, S., Shi, S., Tieu, K., and Hammond, C.L. (2009). Glutathione dysregulation and the etiology and progression of human diseases. Biol. Chem. 390, 191–214.

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. (2010). S-glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 468, 1115–1118.

Giustarini, D., Dalle-Donne, I., Milzani, A., Fanti, P., and Rossi, R. (2013). Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat. Protoc. 8, 1660–1669.

Held P., 2010 Biotek, Measurement of ROS in Cells, http://www.biotek.com/assets/tech_resources/ROS%20Application%20Guide.pdf

Kalinina, E.V., Chernov, N.N., and Novichkova, M.D. (2014). Role of glutathione, glutathione transferase, and glutaredoxin in regulation of redox-dependent processes. Biochem. Biokhimii︠a︡ 79, 1562–1583.

Kamencic, H., Lyon, A., Paterson, P.G., and Juurlink, B.H. (2000). Monochlorobimane fluorometric method to measure tissue glutathione. Anal. Biochem. 286, 35–37.

Parola, M. and Robino, G. (2001). Oxidative stress-related molecules and liver fibrosis. J Hepatol. 35, 297-306

Reuter S. et al., (2010) Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med.49, 1603-1616

Sánchez-Valle V. et al., (2012) Role of oxidative stress and molecular changes in liver fibrosis: a review. Curr Med Chem. 19, 4850-4860

Tipple, T.E., and Rogers, L.K. (2012). Methods for the Determination of Plasma or Tissue Glutathione Levels. Methods Mol. Biol. Clifton NJ 889, 315–324


Key Events

Title Short name
KE1 : S-Glutathionylation, eNOS S-Glutathionylation, eNOS
KE2 : Decrease, GTPCH-1 Decrease, GTPCH-1
KE3 : Decrease, Tetrahydrobiopterin Decrease, Tetrahydrobiopterin
KE4 : Uncoupling, eNOS Uncoupling, eNOS
KE6 : Depletion, Nitric Oxide Depletion, Nitric Oxide
KE7 : Impaired, Vasodilation Impaired, Vasodilation
KE8 : Increase, Vascular Resistance Increase, Vascular Resistance
KE5 : Decrease, AKT/eNOS activity Decrease, AKT/eNOS activity

927: KE1 : S-Glutathionylation, eNOS

Short Name: S-Glutathionylation, eNOS

Key Event Component

Process Object Action
protein glutathionylation nitric oxide synthase, endothelial increased
protein glutathionylation cysteine residue increased

AOPs Including This Key Event

AOP ID and Name Event Type
149: Peptide Oxidation Leading to Hypertension KeyEvent

Stressors

Name
Reactive oxygen species

Biological Organization

Level of Biological Organization
Molecular

Cell term

Cell term
endothelial cell of vascular tree

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
Homo sapiens Homo sapiens Moderate NCBI
Bos taurus Bos taurus Moderate NCBI
Mus musculus Mus musculus Weak NCBI
Rattus norvegicus Rattus norvegicus Weak NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Strong
Sex Applicability
Sex Evidence
Unspecific Strong

S-glutathionylation of eNOS has been demonstrated in humans, cows, mice and rats (Chen et al., 2010; De Pascali et al., 2014; Du et al., 2013).


How this Key Event Works

S-glutathionylation is a redox-dependent, reversible post-translational modification that is involved in the regulation of various regulatory, structural, and metabolic proteins (Pastore and Piemonte, 2013). Under oxidative stress, S-glutathionylation targets cysteine residues of a protein and adds glutathione through thiol-disulfide exchange with oxidized glutathione (GSSG) or reaction of oxidant-induced protein thiyl radicals with reduced glutathione (Chen et al., 2010, 2011, Schuppe et al. 1992). Endothelial nitric oxide synthase (eNOS) regulates vascular function by generating nitric oxide which is involved in endothelium-dependent relaxation, and control of blood pressure and vascular tone. It has been shown that cysteine residues are important for the maintenance of normal eNOS function. Under oxidative stress, S-glutathionylation of eNOS was induced by GSSG at residue sites Cys 689 and Cys 908, resulting in a decrease in eNOS activity and an increase in superoxide generation, also known as eNOS uncoupling. Furthermore, eNOS S-glutathionylation was shown to be abundant in the vessel walls of spontaneously hypertension rats (SHRs), in contrast to non-hypertensive rats.   SHRs demonstrated impaired endothelium-dependent vasodilation, which was reversible upon administration of the reducing agent, dithiothreitol (Chen et al. 2010).  Similarly in human aortic endothial cells, exposure to ultrafine particles caused a decrease in NO production in a dose-depedent manner.  This was shown to be prevented upon over-expression of glutaredoxin-1, which inhibits eNOS S-glutathionylation (Du et al. 2013).


How it is Measured or Detected

There are four general approaches to detect protein S-glutathionylation (Pastore and Piemonte, 2013).

  1. Quantification of Total S-Glutathionylated Proteins: Use sample lysis or homogenization in non-reducing buffer containing N-ethylmaleimide to eliminate thiols, followed by protein precipitation, reduction of gluthionyl-protein adducts, and derivatization of protein thiols or free glutathione with fluorescence probes. Fluorescence can be measured by fluorometric analysis with or without prior HPLC separation. This method allows for quantification of glutathionylated proteins but cannot detect glutathione adducts on specific proteins.
  2. Labeling of Glutathione: Use 35S-cysteine radiolabeling or biotin labeling to detect glutathione adducts on S-thiolated proteins.
  3. Use of Anti-Glutathione Antibodies: Use commercially available anti-glutathione to detect glutathionylated proteins by Western blots, immunoprecipitation or immunocytolocalization. This method is useful for analysis of individual proteins like eNOS but not for large-scale detection of glutathionylated proteins.
  4. Top-Down Proteomic Approach: Use liquid chromatography-coupled mass spectrometry to identify S-glutathionylated proteins on whole protein extract from cells without using labeling or anti-glutathione antibody.

References

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. (2010). S-glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 468, 1115–1118.

Chen CA, Lin CH, Druhan LJ, Wang TY, Chen YR, Zweier JL.  Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling.  J Biol Chem. 2011 286(33):29098-107.

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.

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

Pastore, A., and Piemonte, F. (2013). Protein glutathionylation in cardiovascular diseases. Int. J. Mol. Sci. 14, 20845–20876.

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


935: KE2 : Decrease, GTPCH-1

Short Name: Decrease, GTPCH-1

Key Event Component

Process Object Action
proteasome complex disassembly GTP cyclohydrolase 1 decreased

AOPs Including This Key Event

AOP ID and Name Event Type
149: Peptide Oxidation Leading to Hypertension KeyEvent

Biological Organization

Level of Biological Organization
Cellular

Cell term

Cell term
endothelial cell of vascular tree

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
Homo sapiens Homo sapiens Strong NCBI
Bos taurus Bos taurus Strong NCBI
Mus musculus Mus musculus Strong NCBI
Rattus norvegicus Rattus norvegicus Weak NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Not Specified
Sex Applicability
Sex Evidence
Unspecific Not Specified

Several studies showed decreased GTPCH-1 activity and/or protein expression in cardiac reperfusion patients, bovine endothelial cells, a mouse model of diabetes and a rat model of hypertension (Cervantes-Pérez et al., 2012; Abdelghany et al., 2017; Jayaram et al., 2015; Zhao et al., 2013).

Furthermore, mice deficient in GTPCH-1 demonstrate decreased BH4 bioavailability, increased eNOS uncoupling, pulmonary vascular resistance and pulmonary hypertension (Belik et al. 2011, Nandi et al. 2005, Khoo et al. 2005).


How this Key Event Works

Guanosine triphosphate cyclohydrolase-1 (GTPCH-1) is the rate-limiting enzyme in the de novo biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial nitric oxide synthase (eNOS) and nitric oxide generation (Wang et al., 2008). GTPCH-1 catalyzes the rearrangement of GTP to 7-dihydroneopterin triphosphate, which is converted to BH4 through sequential actions of pyruvoyl tetrahydrobiopterin synthase and sepiapterin reductase. GTPCH-1 activity is regulated in a negative feedback by levels of BH4 which promotes binding of GTPCH-1 with its inhibitor GTPCH feedback regulatory protein (GFRP), but phosphorylation of GTPCH-1 reduces its binding to GFRP and prevents this negative feedback (Chen et al., 2011).  Loss or inactivation of GTPCH-1 results in decreased BH4 levels, which causes eNOS uncoupling.


How it is Measured or Detected

The activity of GTPCH-1 can be detected through the quantification of neopterin by high-performance liquid chromatography (HPLC) after the conversion of enzymatically formed dihydroneopterin triphosphate into neopterin by sequential iodine oxidation and dephosphorylation.


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).

Belik J, McIntyre BA, Enomoto M, Pan J, Grasemann H, Vasquez-Vivar J.  Pulmonary hypertension in the newborn GTP cyclohydrolase I-deficient mouse.  Free Radic Biol Med. 2011, 51(12):2227-33.

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.

Chen, W., Li, L., Brod, T., Saeed, O., Thabet, S., Jansen, T., Dikalov, S., Weyand, C., Goronzy, J., and Harrison, D.G. (2011). Role of increased guanosine triphosphate cyclohydrolase-1 expression and tetrahydrobiopterin levels upon T cell activation. J. Biol. Chem. 286, 13846–13851.

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.

Khoo JP, Zhao L, Alp NJ, Bendall JK, Nicoli T, Rockett K, et al. Pivotal role for endothelial tetrahydrobiopterin in pulmonary hypertension. Circulation. 2005;111:2126–33

Nandi M, Miller A, Stidwill R, Jacques TS, Lam AA, Haworth S, et al. Pulmonary hypertension in a GTP-cyclohydrolase 1-deficient mouse. Circulation. 2005;111:2086–90

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.

Zhao, Y., Wu, J., Zhu, H., Song, P., and Zou, M.-H. (2013). Peroxynitrite-dependent zinc release and inactivation of guanosine 5’-triphosphate cyclohydrolase 1 instigate its ubiquitination in diabetes. Diabetes 62, 4247–4256.


934: KE3 : Decrease, Tetrahydrobiopterin

Short Name: Decrease, Tetrahydrobiopterin

Key Event Component

Process Object Action
biosynthetic process 5,6,7,8-tetrahydrobiopterin decreased

AOPs Including This Key Event

AOP ID and Name Event Type
149: Peptide Oxidation Leading to Hypertension KeyEvent

Stressors

Name
Reactive oxygen species

Biological Organization

Level of Biological Organization
Cellular

Cell term

Cell term
endothelial cell of vascular tree

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
Homo sapiens Homo sapiens Moderate NCBI
Bos taurus Bos taurus Strong NCBI
Mus musculus Mus musculus Strong NCBI
Rattus norvegicus Rattus norvegicus Weak NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Not Specified
Sex Applicability
Sex Evidence
Unspecific Not Specified

Decreased BH4 is observed in humans (Jayaram et al., 2015), cows (Abdelghany et al., 2017; Whitsett et al., 2007, Wang et al., 2008), mice (Adlam et al., 2012; Chuaiphichai et al., 2014; Crabtree et al., 2009; Tatham et al., 2009; Wang et al., 2008) and rats (Cervantes-Pérez et al., 2012).


How this Key Event Works

Tetrahydrobiopterin (BH4) is an essential cofactor for a group of enzymes including aromatic acid hydroxylases, nitric oxide synthase (NOS) isoforms, and alkylglycerol monooxygenase (Wang et al., 2014). BH4 is synthesized from guanosine triphosphate through sequential reactions catalyzed by enzymes GTPCH-1, pyruvoyl tetrahydropterin synthase, and sepiapterin reductase (Tatham et al., 2009). During NOS catalysis, BH4 donates electrons to the ferrous-dioxygen complex in the oxygenase domain, leading to oxidation of L-arginine to N-hydroxy-Larginine and eventually conversion to citrulline and nitric oxide production (Chen et al., 2011; Crabtree et al., 2009). BH4 also stabilizes dimers of NOS isoforms, which is required for their enzymatic activity. When BH4 levels are decreased or limited, for example under oxidative stress conditions, BH4 can be oxidized to dihydrobiopterin (BH2) and then converted to biopterin. This reduction in BH4 availability results in NOS uncoupling where NOS is uncoupled from L-arginine oxidation and superoxide (or other reactive species) is produced rather than nitric oxide (Carnicer et al., 2012). Decreased BH4 have been demonstrated in a variety of vascular diseases such as hypertension, diabetes and atherosclerosis where endothelial dysfunction occurs.


How it is Measured or Detected

Levels of BH4, BH2 and biopterin levels can be determined by reverse-phase high-performance liquid chromatography (HPLC) followed by electrochemical detection (for BH4) and fluorescence detection (for BH2 and biopterin) (Howells et al., 1986).

A LC-MS/MS method has been published by Zhao et al. (2009), which was validated for detection in human, monkey, dog, rabbit, rat and mouse plasma, and used to support a successful drug approval submission.

ELISA kits for BH4 are also commercially available.

In each case, care must be taken to protect the sample from oxidation, and BH4 is highly redox sensitive.  Dithioerythritol is commonly used as a preservation agent.

 


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.

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.

Chen, W., Li, L., Brod, T., Saeed, O., Thabet, S., Jansen, T., Dikalov, S., Weyand, C., Goronzy, J., and Harrison, D.G. (2011). Role of increased guanosine triphosphate cyclohydrolase-1 expression and tetrahydrobiopterin levels upon T cell activation. J. Biol. Chem. 286, 13846–13851.

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.

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.

Howells, D.W., Smith, I., and Hyland, K. (1986). Estimation of tetrahydrobiopterin and other pterins in cerebrospinal fluid using reversed-phase high-performance liquid chromatography with electrochemical and fluorescence detection. J. Chromatogr. 381, 285–294.

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.

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.

Wang, Q., Yang, M., Xu, H., and Yu, J. (2014). Tetrahydrobiopterin improves endothelial function in cardiovascular disease: a systematic review. Evid.-Based Complement. Altern. Med. ECAM 2014, 850312.

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.

Zhao Y, Cao J, Chen YS, Zhu Y, Patrick C, Chien B, Cheng A, Foehr ED.  Detection of tetrahydrobiopterin by LC-MS/MS in plasma from multiple species.  Bioanalysis. 2009;1(5):895-903.

 

 


932: KE4 : Uncoupling, eNOS

Short Name: Uncoupling, eNOS

Key Event Component

Process Object Action
nitric oxide synthase, endothelial functional change

AOPs Including This Key Event

AOP ID and Name Event Type
149: Peptide Oxidation Leading to Hypertension KeyEvent

Biological Organization

Level of Biological Organization
Cellular

Cell term

Cell term
endothelial cell

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
Homo sapiens Homo sapiens Strong NCBI
Bos taurus Bos taurus Strong NCBI
Mus musculus Mus musculus Strong NCBI
Rattus norvegicus Rattus norvegicus Strong NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Not Specified
Sex Applicability
Sex Evidence
Unspecific Not Specified

eNOS uncoupling has been demonstrated in humans, cows, mice and rats (Chen et al., 2010; Crabtree et al., 2009; De Pascali et al., 2014; Du et al., 2013; Jayaram et al., 2015).


How this Key Event Works

Endothelial nitric oxide synthase (eNOS) is responsible for the generation of vascular nitric oxide (NO), a protective molecule that is involved in the regulation of endothelium-dependent vasodilation, vascular tone, and blood pressure (Förstermann and Münzel, 2006). To generate NO, eNOS hydroxylates L-arginine to N-hydroxy-L-arginine and then oxidizes N-hydroxy-L-arginine to L-citrulline and NO. This enzymatic process requires NADPH, Ca2+/calmondulin, flavin mononucleotide, flavin adenine dinucleotide and its cofactor tetrahydrobiopterin (BH4). Limiting BH4 levels or S-glutathionylation of eNOS can lead to eNOS uncoupling in which eNOS produces superoxide (or other reactive oxygen species) and less NO. The uncoupling of eNOS has been demonstrated to cause endothelial dysfunction, and is implicated in a number of cardiovascular diseases such as hypertension, atherosclerosis, hypercholesterolemia, and diabetes mellitus (Dumitrescu et al., 2007).


How it is Measured or Detected

The activity of eNOS can be measured indirectly through superoxide and NO production. Superoxides can be detected using several standard methods including lucigenin-enhanced chemiluminescence (Münzel et al., 2002; Tarpey et al., 1999), electron paramagnetic resonance (EPR) spin-trapping (Roubaud et al., 1997), and HPLC/fluorescence detector-based assay using dihydroethidium (Fink et al., 2004; Zhao et al., 2003). NO production can be measured through the conversion of L-arginine to L-citrulline (de Bono et al., 2007) , in situ fluorescent signal detection with fluorescent indicator DAF-2 DA (Itoh et al., 2000; Nagata et al., 1999; Qiu et al., 2001), EPR spin-trapping (Xia et al., 2000), and the determination of total nitrate and nitrite concentration (Crabtree et al., 2009; Du et al., 2013).


References

de Bono, J.P., Warrick, N., Bendall, J.K., Channon, K.M., and Alp, N.J. (2007). Radiochemical HPLC detection of arginine metabolism: Measurement of nitric oxide synthesis and arginase activity in vascular tissue. Nitric Oxide 16, 1–9.

Chen, X., Xu, J., Feng, Z., Fan, M., Han, J., and Yang, Z. (2010). Simvastatin combined with nifedipine enhances endothelial cell protection by inhibiting ROS generation and activating Akt phosphorylation. Acta Pharmacol. Sin. 31, 813–820.

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.

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.

Du, Y., Navab, M., Shen, M., Hill, J., Pakbin, P., Sioutas, C., Hsiai, T.K., and Li, R. (2013). 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.

Fink, B., Laude, K., McCann, L., Doughan, A., Harrison, D.G., and Dikalov, S. (2004). Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am. J. Physiol. Cell Physiol. 287, C895–C902.

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

Itoh, Y., Ma, F.H., Hoshi, H., Oka, M., Noda, K., Ukai, Y., Kojima, H., Nagano, T., and Toda, N. (2000). Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry. Anal. Biochem. 287, 203–209.

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.

Münzel, T., Afanas’ev, I.B., Kleschyov, A.L., and Harrison, D.G. (2002). Detection of Superoxide in Vascular Tissue. Arterioscler. Thromb. Vasc. Biol. 22, 1761–1768.

Nagata, N., Momose, K., and Ishida, Y. (1999). Inhibitory effects of catecholamines and anti-oxidants on the fluorescence reaction of 4,5-diaminofluorescein, DAF-2, a novel indicator of nitric oxide. J. Biochem. (Tokyo) 125, 658–661.

Qiu, W., Kass, D.A., Hu, Q., and Ziegelstein, R.C. (2001). Determinants of shear stress-stimulated endothelial nitric oxide production assessed in real-time by 4,5-diaminofluorescein fluorescence. Biochem. Biophys. Res. Commun. 286, 328–335.

Roubaud, V., Sankarapandi, S., Kuppusamy, P., Tordo, P., and Zweier, J.L. (1997). Quantitative measurement of superoxide generation using the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide. Anal. Biochem. 247, 404–411.

Tarpey, M.M., White, C.R., Suarez, E., Richardson, G., Radi, R., and Freeman, B.A. (1999). Chemiluminescent Detection of Oxidants in Vascular Tissue Lucigenin But Not Coelenterazine Enhances Superoxide Formation. Circ. Res. 84, 1203–1211.

Xia, Y., Cardounel, A.J., Vanin, A.F., and Zweier, J.L. (2000). Electron paramagnetic resonance spectroscopy with N-methyl-D-glucamine dithiocarbamate iron complexes distinguishes nitric oxide and nitroxyl anion in a redox-dependent manner: applications in identifying nitrogen monoxide products from nitric oxide synthase. Free Radic. Biol. Med. 29, 793–797.

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.


933: KE6 : Depletion, Nitric Oxide

Short Name: Depletion, Nitric Oxide

Key Event Component

Process Object Action
nitric oxide biosynthetic process nitric oxide decreased

AOPs Including This Key Event

AOP ID and Name Event Type
149: Peptide Oxidation Leading to Hypertension KeyEvent

Stressors

Name
Reactive oxygen species

Biological Organization

Level of Biological Organization
Cellular

Organ term

Organ term
blood

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
Homo sapiens Homo sapiens Strong NCBI
Bos taurus Bos taurus Strong NCBI
Mus musculus Mus musculus Strong NCBI
Rattus norvegicus Rattus norvegicus Strong NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Strong
Sex Applicability
Sex Evidence
Unspecific Strong

NO depletion was observed in humans, cows, mice and rats (Chen et al., 2010; Crabtree et al., 2009; De Pascali et al., 2014; Du et al., 2013; Jayaram et al., 2015).


How this Key Event Works

Nitric oxide (NO), constitutively produced by endothelial nitric oxide synthase (eNOS), is an important regulator of vascular homeostasis. Endothelial-derived NO promotes vasodilation and protects against atherogenesis through the inhibition of vascular smooth muscle cell proliferation and migration, platelet aggregation and adhesion, and leukocyte adherence. Its effects have an influence on vascular resistance, blood pressure, vascular remodeling and angiogenesis (Luo et al., 2000). Dysfunctional eNOS as a result of eNOS uncoupling leads to a decrease or loss of NO bioavailability and an elevation of superoxide production (Crabtree et al., 2009). The imbalance of NO and superoxide is associated with many disorders, such as hypertension, atherosclerosis, hypercholesterolemia, and diabetes mellitus.


How it is Measured or Detected

NO production can be measured through the conversion of L-arginine to L-citrulline (de Bono et al., 2007) , in situ fluorescent signal detection with fluorescent indicator DAF-2 DA (Itoh et al., 2000; Nagata et al., 1999; Qiu et al., 2001), EPR spin-trapping (Xia et al., 2000), and the determination of total nitrate and nitrite concentration (Crabtree et al., 2009; Du et al., 2013).


References

de Bono, J.P., Warrick, N., Bendall, J.K., Channon, K.M., and Alp, N.J. (2007). Radiochemical HPLC detection of arginine metabolism: Measurement of nitric oxide synthesis and arginase activity in vascular tissue. Nitric Oxide 16, 1–9.

Chen, X., Xu, J., Feng, Z., Fan, M., Han, J., and Yang, Z. (2010). Simvastatin combined with nifedipine enhances endothelial cell protection by inhibiting ROS generation and activating Akt phosphorylation. Acta Pharmacol. Sin. 31, 813–820.

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.

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.

Du, Y., Navab, M., Shen, M., Hill, J., Pakbin, P., Sioutas, C., Hsiai, T.K., and Li, R. (2013). 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.

Itoh, Y., Ma, F.H., Hoshi, H., Oka, M., Noda, K., Ukai, Y., Kojima, H., Nagano, T., and Toda, N. (2000). Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry. Anal. Biochem. 287, 203–209.

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.

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.

Nagata, N., Momose, K., and Ishida, Y. (1999). Inhibitory effects of catecholamines and anti-oxidants on the fluorescence reaction of 4,5-diaminofluorescein, DAF-2, a novel indicator of nitric oxide. J. Biochem. (Tokyo) 125, 658–661.

Qiu, W., Kass, D.A., Hu, Q., and Ziegelstein, R.C. (2001). Determinants of shear stress-stimulated endothelial nitric oxide production assessed in real-time by 4,5-diaminofluorescein fluorescence. Biochem. Biophys. Res. Commun. 286, 328–335.

Xia, Y., Cardounel, A.J., Vanin, A.F., and Zweier, J.L. (2000). Electron paramagnetic resonance spectroscopy with N-methyl-D-glucamine dithiocarbamate iron complexes distinguishes nitric oxide and nitroxyl anion in a redox-dependent manner: applications in identifying nitrogen monoxide products from nitric oxide synthase. Free Radic. Biol. Med. 29, 793–797.


937: KE7 : Impaired, Vasodilation

Short Name: Impaired, Vasodilation

Key Event Component

Process Object Action
vasodilation blood vessel abnormal

AOPs Including This Key Event

AOP ID and Name Event Type
149: Peptide Oxidation Leading to Hypertension KeyEvent

Biological Organization

Level of Biological Organization
Organ

Organ term

Organ term
circulatory system

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
Homo sapiens Homo sapiens Strong NCBI
Oryctolagus cuniculus Oryctolagus cuniculus Weak NCBI
Mus musculus Mus musculus Moderate NCBI
Rattus norvegicus Rattus norvegicus Moderate NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Strong
Sex Applicability
Sex Evidence
Unspecific Strong

Vasodilation has been observed in humans, rabbits, mice and rats.


How this Key Event Works

Vasodilation refers to the widening or increase in the diameter of blood vessels (e.g. large arteries, large veins, small arterioles) that is caused by the relaxation of vascular smooth muscle cells (VSMCs) within the walls of blood vessels, thus increasing blood flow and decreasing arterial blood pressure and heart rate (Siddiqui, 2011). VSMC relaxation is regulated through a number of mechanisms, including cyclic GMP-dependent hyperpolarization and relaxation via nitric oxide (NO), cAMP-dependent hyperpolarization via prostaglandins, and stimulation of potassium channels via endothelial-derived hyperpolarizing factors (Durand and Gutterman, 2013). Under oxidative stress, decreased NO bioavailability results in impaired vasodilation, which is associated with cardiovascular diseases such as hypertension (Silva et al., 2012).


How it is Measured or Detected

Endothelium-dependent vasodilation can be measured using invasive and non-invasive methods (Raitakari and Celermajer, 2000). For the invasive approach, vasodilation is measured after intra-arterial pharmacologic stimulation with substances that enhance NO release (e.g. acetylcholine, bradykinin). The non-invasive ultrasound-based method evaluates flow-mediated vasodilation (FMD) in the superficial arteries, such as brachial, radial, or femoral vessels.

Guidelines for the measurement of FMD have been published (Corretti et al. 2002).


References

Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery

Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J, Vogel R.  Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery.  Journal of the American College of Cardiology, 2002, 39 (2) 257-265

Durand, M.J., and Gutterman, D.D. (2013). Diversity in mechanisms of endothelium-dependent vasodilation in health and disease. Microcirc. N. Y. N 1994 20, 239–247.

Raitakari, O.T., and Celermajer, D.S. (2000). Flow-mediated dilatation. Br. J. Clin. Pharmacol. 50, 397–404.

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

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


951: KE8 : Increase, Vascular Resistance

Short Name: Increase, Vascular Resistance

Key Event Component

Process Object Action
increased systemic vascular resistance increased

AOPs Including This Key Event

AOP ID and Name Event Type
149: Peptide Oxidation Leading to Hypertension KeyEvent

Biological Organization

Level of Biological Organization
Organ

Organ term

Organ term
circulatory system

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
Homo sapiens Homo sapiens Strong NCBI
Rattus norvegicus Rattus norvegicus Weak NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Strong
Sex Applicability
Sex Evidence
Unspecific Strong

Increased SVR was observed in humans, pigs and rats (Siddiqui 2011, Dikalova et al. 2016, 

 


How this Key Event Works

Vascular resistance is the resistance to blood flow in the circulatory system (Siddiqui, 2011). Systemic vascular resistance (SVR), also known as total peripheral resistance, refers specifically to the resistance to blood flow offered by the peripheral circulation. Vasodilation decreases SVR while vasoconstriction or impaired vasodilation increases SVR.


How it is Measured or Detected

Vascular resistance cannot be measured by any direct means, but can be calculated using a formula: (mean arterial pressure minus mean right arterial pressure) divided by cardiac output (Siddiqui 2011).


References

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

Dikalova A, Aschner JL, Kaplowitz MR, Summar M, Fike CD.  Tetrahydrobiopterin oral therapy recouples eNOS and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs.  Am J Physiol Lung Cell Mol Physiol. 2016, 1;311(4):L743-L753.

 

 


973: KE5 : Decrease, AKT/eNOS activity

Short Name: Decrease, AKT/eNOS activity

Key Event Component

Process Object Action
catalytic activity nitric oxide synthase, endothelial decreased
catalytic activity AKT kinase decreased

AOPs Including This Key Event

AOP ID and Name Event Type
149: Peptide Oxidation Leading to Hypertension KeyEvent

Stressors

Name
Reactive oxygen species

Biological Organization

Level of Biological Organization
Cellular

Cell term

Cell term
endothelial cell of vascular tree

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
Homo sapiens Homo sapiens Strong NCBI
Bos taurus Bos taurus Strong NCBI
Rattus norvegicus Rattus norvegicus Strong NCBI
Mus musculus Mus musculus Strong NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Not Specified
Sex Applicability
Sex Evidence
Unspecific Not Specified

Decreased Akt and eNOS activity was observed in humans, cows, mice and rats following exposure to stressors.

Cigarette smoke exposure was shown to inhibit the phosphorylation of AKT and eNOS in VEGF-stimulated human umbilical vein endothelial cells (HUVECs), resulting in decreased NO levels (Michaud et al. 2006).

In rat aortic rings, exposure to methylglyoxal and high concentrations of glucose decreased endothelium-dependent relaxation.  Further experiments in rat endothelial cells and HUVECs demonstrated a reduction in eNOS phosphorylation and activity, and reduced NO levels in response to the same stressors (Dhar et al. 2010).

In bovine aortic endothelial cells, AKT and eNOS phosphorylation were decreased following exposure to the peroxynitrite source; SIN-1, with an associated reduction in NO bioavailability.  These effects were ameliorated by treatment with the ROS scavenger DMPO (Das et al. 2014).

eNOS knockout mice are routinely used as models of hypertension.  Such mice display reduced bioavailability of NO and impaired vasodilation (Huang et al. 1995).

Reduced AKT/eNOS phosphorylation was reported under conditions of hyperglycaemia (in mice) and in HUVECs following treatment with high concentrations of glucose.  Aortic rings from hyperglycaemic mice demonstrated impaired vasodilation.  Resveratrol treatment was shown to improve vasodilation and eNOS phosphorylation in wild-type mice, but not AKT knockout mice.  Transfection of HUVECs with AKT siRNA abolished resveratrol-enhanced eNOS phosphorylation and NO release (Li et al. 2017),


How this Key Event Works

Endothelial nitric oxide synthase (eNOS) is responsible for the generation of nitric oxide (NO), which is an important regulator of vascular homeostasis. The activity of eNOS can be regulated through calmodulin-mediated dimerization, tetrahydrobiopterin-mediated conversion of L-arginine to L-citrulline, protein-protein interactions with heat shock protein 90 and caveolin, S-nitrosylation, acetylation and phosphorylation (Atochin et al., 2007; Qian and Fulton, 2013). eNOS has been shown to be phosphorylated at multiple sites including tyrosine (Y), serine (Ser) and threonine (Thr) residues. Serine-threonine protein kinase AKT, a multifunctional regulator of cellular processes like glucose metabolism and proliferation, can directly phosphorylate eNOS at Ser1177/Ser1179, leading to increased eNOS enzymatic activity and subsequent NO production (Dimmeler et al., 1999; Fulton et al., 1999). Inhibition of AKT or a mutation of the AKT phosphorylation site on eNOS attenuates eNOS phosphorylation and its activity, resulting in decreased NO bioavailability and endothelial dysfunction (Dimmler et al. 1999)


How it is Measured or Detected

Western blot analysis can be performed to determine the expression levels of phosphorylated eNOS, phosphorylated Akt, total Akt and total eNOS proteins using the appropriate anti-phospho-eNOS, anti-phospho-Akt, anti-eNOS, and anti-Akt antibodies. Alternatively, eNOS activity can be measured using the conversion of L-arginine to L-citrulline assay.

ELISA kits for AKT/eNOS and phospho AKT/eNOS expression are commercially available.


References

Atochin, D.N., Wang, A., Liu, V.W.T., Critchlow, J.D., Dantas, A.P.V., Looft-Wilson, R., Murata, T., Salomone, S., Shin, H.K., Ayata, C., et al. (2007). The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo. J. Clin. Invest. 117, 1961–1967.

Das A, Gopalakrishnan B, Druhan LJ, Wang TY, De Pascali F, Rockenbauer A, Racoma I, Varadharaj S, Zweier JL, Cardounel AJ, Villamena FA.  Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelialcells via eNOS phosphorylation.  Br J Pharmacol. 2014, 171(9):2321-34. doi: 10.1111/bph.12572.

Dhar A, Dhar I, Desai KM, Wu L.  Methylglyoxal scavengers attenuate endothelial dysfunction induced by methylglyoxal and high concentrations of glucose.  Br J Pharmacol. 2010, 161(8):1843-56.

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.

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.

Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC.    Hypertension in mice lacking the gene for endothelial nitric oxide synthase.  Nature. 1995, 377(6546):239-42.

Li JY, Huang WQ, Tu RH, Zhong GQ, Luo BB, He Y.  Resveratrol rescues hyperglycemia-induced endothelial dysfunction via activation of Akt.  Acta Pharmacol Sin. 2017, 38(2):182-191.

Michaud SE, Dussault S, Groleau J, Haddad P, Rivard A.J. Cigarette smoke exposure impairs VEGF-induced endothelial cell migration: role of NO and reactive oxygen species.  Mol Cell Cardiol. 2006 Aug;41(2):275-84.

Qian, J., and Fulton, D. (2013). Post-translational regulation of endothelial nitric oxide synthase in vascular endothelium. Oxid. Physiol. 4, 347.


Adverse Outcomes

Title Short name
Hypertension Hypertension

952: Hypertension

Short Name: Hypertension

Key Event Component

Process Object Action
hypertension increased

AOPs Including This Key Event

AOP ID and Name Event Type
149: Peptide Oxidation Leading to Hypertension AdverseOutcome

Stressors

Name
Reactive oxygen species

Biological Organization

Level of Biological Organization
Individual

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
Homo sapiens Homo sapiens Strong NCBI
Mus musculus Mus musculus Strong NCBI
Rattus norvegicus Rattus norvegicus Strong NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Strong
Sex Applicability
Sex Evidence
Unspecific Strong

Animal models including mouse and rat models are routinely used to study hypertension, and have been shown to reflect human physiology relating to hypertension (Leong et al., 2015).


How this Key Event Works

Hypertension is an important cardiovascular risk factor and considered one of the leading causes of cardiovascular morbidity and mortality (Kizhakekuttu and Widlansky, 2010). It is defined as a chronic elevation in blood pressure and is characterized by elevated systemic vascular resistance due to dysregulated vasomotor function and structural remodeling (Lee and Griendling, 2008). Although many genetic and environmental factors contribute to the development to hypertension, oxidative stress appears to be the main pathway involved in its pathogenesis. Excessive reactive oxygen species (ROS) contributes to endothelial nitric oxide synthase (eNOS) uncoupling, resulting in increased superoxide production but decreased nitric oxide (NO), a critical regulator of vascular homeostasis (Silva et al., 2012). Depletion of NO leads to impaired endothelium-dependent vasodilation, thus promoting endothelial dysfunction, which is a hallmark of hypertension.


How it is Measured or Detected

Arterial blood pressure is commonly measured using a sphygmomanometer, which provides systolic and diastolic blood pressure measurements in millimeters of mercury (mmHg).

Pathological hypertension is characterised according to current guidelines; https://www.nice.org.uk/guidance/cg127/evidence

Stage 1 hypertension : Clinic blood pressure is 140/90 mmHg or higher and subsequent ambulatory blood pressure monitoring (ABPM) daytime average or home blood pressure monitoring (HBPM) average blood pressure is 135/85 mmHg or higher.

Stage 2 hypertension : Clinic blood pressure is 160/100 mmHg or higher and subsequent ABPM daytime average or HBPM average blood pressure is 150/95 mmHg or higher.

Severe hypertension : Clinic systolic blood pressure is 180 mmHg or higher or clinic diastolic blood pressure is 110 mmHg or higher.


References

Durand, M.J., and Gutterman, D.D. (2013). Diversity in mechanisms of endothelium-dependent vasodilation in health and disease. Microcirc. N. Y. N 1994 20, 239–247.

Kizhakekuttu, T.J., and Widlansky, M.E. (2010). Natural antioxidants and hypertension: promise and challenges. Cardiovasc. Ther. 28, e20–e32.

Leong, X.-F., Ng, C.-Y., Jaarin, K., Leong, X.-F., Ng, C.-Y., and Jaarin, K. (2015). Animal Models in Cardiovascular Research: Hypertension and Atherosclerosis, Animal Models in Cardiovascular Research: Hypertension and Atherosclerosis. BioMed Res. Int. BioMed Res. Int. 2015, 2015, e528757.

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


Scientific evidence supporting the linkages in the AOP

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
Peptide Oxidation directly leads to KE2 : Decrease, GTPCH-1 Moderate Weak
KE1 : S-Glutathionylation, eNOS directly leads to KE4 : Uncoupling, eNOS Strong Moderate
KE2 : Decrease, GTPCH-1 directly leads to KE3 : Decrease, Tetrahydrobiopterin Strong Strong
KE3 : Decrease, Tetrahydrobiopterin directly leads to KE4 : Uncoupling, eNOS Strong Strong
KE4 : Uncoupling, eNOS directly leads to KE6 : Depletion, Nitric Oxide Strong Strong
KE6 : Depletion, Nitric Oxide directly leads to KE7 : Impaired, Vasodilation Strong Moderate
KE7 : Impaired, Vasodilation directly leads to KE8 : Increase, Vascular Resistance Moderate Weak
KE8 : Increase, Vascular Resistance directly leads to Hypertension Moderate Weak
KE5 : Decrease, AKT/eNOS activity directly leads to KE6 : Depletion, Nitric Oxide Strong Strong
Peptide Oxidation directly leads to KE5 : Decrease, AKT/eNOS activity Strong Moderate
Peptide Oxidation directly leads to KE1 : S-Glutathionylation, eNOS Moderate Weak

Graphical Representation

Overall Assessment of the AOP


Domain of Applicability

Life Stage Applicability
Life Stage Evidence
All life stages
Taxonomic Applicability
Term Scientific Term 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
Unspecific

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

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    

 

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 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).

Quantitative Consideration

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)


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.

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).

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