High glucose causes vascular dysfunction through Akt/eNOS pathway: reciprocal modulation by juglone and resveratrol

2018 ◽  
Vol 96 (8) ◽  
pp. 757-764 ◽  
Author(s):  
M. Bilgehan Pektas ◽  
Ozge Turan ◽  
Gozde Ozturk Bingol ◽  
Esra Sumlu ◽  
Gökhan Sadi ◽  
...  
Keyword(s):  
Insulin Signaling ◽  
High Glucose ◽  
Vascular Function ◽  
Vascular Reactivity ◽  
Vascular Dysfunction ◽  
Expression Profiles ◽  
Vascular Complications ◽  
Rat Aorta ◽  
High Fructose ◽  
Fructose 2

Transient elevations in blood glucose level may lead to changes in vascular function. Herein, we investigated the effects of high-glucose or high-fructose challenge, as well as potential influence of juglone or resveratrol on vascular reactivity, Akt/eNOS, and insulin signaling effectors in rat aorta. Aortic segments of rats were incubated with high glucose (30 mmol/L) or high fructose (2 mmol/L) in the absence and presence of juglone (5 μmol/L) or resveratrol (10 μmol/L). Acute high-glucose incubation markedly decreased acetylcholine-induced relaxation, which is further inhibited by juglone, but ameliorated by resveratrol. Incubation with high glucose caused significant reduction in pAkt/total Akt and peNOS/total eNOS ratios, as well as in the expression of some genes involved in insulin signaling. Juglone produced a further impairment, whereas resveratrol resulted in an improvement on the expression profiles of these proteins and genes. Acute exposure of aortic segments to high glucose causes a reduction in acetylcholine-induced relaxation in association with suppression of Akt/eNOS pathway, as well as several genes in insulin signaling pathway. Juglone and resveratrol have opposite actions on vascular relaxation and the above signaling targets. These findings could be relevant for the treatment of hyperglycemia-induced vascular complications.

Endothelin-mediated in vivo pressor responses following TRPV1 activation

2011 ◽  
Vol 301 (3) ◽  
pp. H1135-H1142 ◽  
Author(s):  
Vahagn A. Ohanyan ◽  
Giacinta Guarini ◽  
Charles K. Thodeti ◽  
Phani K. Talasila ◽  
Priya Raman ◽  
...  
Keyword(s):  
Vascular Function ◽  
Vascular Reactivity ◽  
Transient Receptor Potential ◽  
Vascular Dysfunction ◽  
Vascular Complications ◽  
Receptor Potential ◽  
Immunoblot Analysis ◽  
Trpv1 Channels ◽  
Transient Receptor

Transient receptor potential vanilliod 1 (TRPV1) channels have recently been postulated to play a role in the vascular complications/consequences associated with diabetes despite the fact that the mechanisms through which TRPV1 regulates vascular function are not fully known. Accordingly, our goal was to define the mechanisms by which TRPV1 channels modulate vascular function and contribute to vascular dysfunction in diabetes. We subjected mice lacking TRPV1 [TRPV1(−/−)], db/ db, and control C57BLKS/J mice to in vivo infusion of the TRPV1 agonist capsaicin or the α-adrenergic agonist phenylephrine (PE) to examine the integrated circulatory actions of TRPV1. Capsaicin (1, 10, 20, and 100 μg/kg) dose dependently increased MAP in control mice (5.7 ± 1.6, 11.7 ± 2.1, 25.4 ± 3.4, and 51.6 ± 3.9%), which was attenuated in db/db mice (3.4 ± 2.1, 3.9 ± 2.1, 7.0 ± 3.3, and 17.9 ± 6.2%). TRPV1(−/−) mice exhibited no changes in MAP in response to capsaicin, suggesting the actions of this agonist are specific to TRPV1 activation. Immunoblot analysis revealed decreased aortic TRPV1 protein expression in db/db compared with control mice. Capsaicin-induced responses were recorded following inhibition of endothelin A and B receptors (ETA /ETB). Inhibition of ETA receptors abolished the capsaicin-mediated increases in MAP. Combined antagonism of ETA and ETB receptors did not further inhibit the capsaicin response. Cultured endothelial cell exposure to capsaicin increased endothelin production as shown by an endothelin ELISA assay, which was attenuated by inhibition of TRPV1 or endothelin-converting enzyme. TRPV1 channels contribute to the regulation of vascular reactivity and MAP via production of endothelin and subsequent activation of vascular ETA receptors. Impairment of TRPV1 channel function may contribute to vascular dysfunction in diabetes.

Omega-3E treatment regulates matrix metalloproteinases and prevents vascular reactivity alterations in diabetic rat aortaThis article is one of a selection of papers published in a special issue on Advances in Cardiovascular Research.

2009 ◽  
Vol 87 (12) ◽  
pp. 1063-1073 ◽  
Author(s):  
Esma N. Zeydanli ◽  
Belma Turan
Keyword(s):  
Matrix Metalloproteinases ◽  
Vascular Reactivity ◽  
Vascular System ◽  
Vascular Dysfunction ◽  
Vascular Complications ◽  
Close Correlation ◽  
Tissue Level ◽  
Rat Aorta ◽  
Diabetic Rat ◽  
Cardiovascular Research

It is known that increased generation of oxidants and (or) reduced endogenous antioxidant defense mechanisms are associated with the etiology of diabetic vascular complications. Although a close correlation exists between increased oxidative stress and the activation of matrix metalloproteinases (MMPs), little is known about the effect of hyperglycemia on the regulation and contribution of MMPs in the vascular system. Therefore, we aimed to examine whether omega-3E (50 mg/kg per day for 4 weeks), a long-chain (n-3) polyunsaturated fatty acid enriched with vitamin E, has a beneficial effect on vascular dysfunction via affecting MMPs in streptozotocin-diabetic rat aorta. Omega-3E treatment improved the diabetes-induced impairment of phenylephrine-induced contraction and isoproterenol-induced relaxation responses of aorta. It also exhibited marked protection against diabetes-induced degenerative changes in smooth muscle cell morphology. Biochemical data showed that this treatment significantly prevented important changes, such as inhibition of MMP-2 and MMP-9 activity, loss of tissue inhibitor of matrix metalloproteinase-4 (TIMP-4) protein, increase in tissue levels of thiol oxidation, endothelin-1, protein kinase C (PKC), and cAMP production, and decrease in tissue level of nitrite. These results indicated that omega-3E significantly improved impaired vascular responses and regulated the activity of MMPs via preventing oxidative injury. Overall, the data suggest that omega-3E ameliorates or prevents vascular reactivity alterations in diabetes. Such an observation provides preliminary evidence for omega-3E’s potential as a therapeutic agent for the prevention of vascular disorders in diabetes.

Abstract P217: Nox Compartmentalization, Protein Oxidation and ER Stress in Vascular Smooth Muscle Cells in Hypertension

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Livia L Camargo ◽  
Augusto C Montezano ◽  
Adam Harvey ◽  
Sofia Tsiropoulou ◽  
Katie Hood ◽  
...  
Keyword(s):  
Er Stress ◽  
Protein Oxidation ◽  
Vascular Function ◽  
Vascular Reactivity ◽  
Stress Proteins ◽  
Vascular Dysfunction ◽  
Mesenteric Arteries ◽  
Resistance Arteries ◽  
Post Translational Modification ◽  
Wistar Kyoto

In hypertension, activation of NADPH oxidases (Noxs) is associated with oxidative stress and vascular dysfunction. The exact role of each isoform in hypertension-associated vascular injury is still unclear. We investigated the compartmentalization of Noxs in VSMC from resistance arteries of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Expression of Nox1 and Nox4 was increased in SHR cells (96.6±28.7% and 48.2±21.2% vs WKY, p<0.05), as well as basal ROS levels measured by chemiluminescence (110.2±26.4% vs WKY, p<0.05) and amplex red (105.2±33.2% vs WKY, p<0.05). Phosphorylation of unfolded protein response activators, PERK and IRE1α, and expression of ER chaperone BiP were elevated in SHR cells (p<0.05 vs WKY), indicating activation of ER stress response. Immunoblotting after organelle fractionation demonstrated that Noxs are expressed in an organelle-specific manner, with Nox1, 2 and 4 present in plasma membrane, ER and nucleus, but not in mitochondria. In SHR cells, NoxA1ds (Nox1 inhibitor, 10μM) and GKT136901 (Nox1/4 inhibitor, 10μM) decreased AngII-induced ROS levels (p<0.001 vs Ctl). Additionally, mito-tempol (mitochondrial-targeted antioxidant, 50nM) and 4-PBA (ER stress inhibitor, 1mM) decreased basal ROS levels in SHR cells (p<0.05 vs Ctl). Furthermore, oxidation of the antioxidant enzymes Peroxiredoxins (Prx) was increased in SHRSP compared to WKY (2.51±0.14 vs 0.56±0.07, p<0.001). One-dimensional isoelectric focusing revealed that cytosolic Prx2 and mitochondrial Prx3 were more oxidized in SHRSP than WKY cells. Using a biotin-tagged dimedone-based probe (DCP-Bio) we identified oxidation of ER stress proteins BiP and IRE1. To investigate the effect of protein oxidation in vascular function, vascular reactivity was evaluated in isolated mesenteric arteries. Inhibition of general oxidation (DTT 1mM; Emax: 111.7±33.1) and peroxiredoxin (Conoidin A 10nM; Emax: 116.0±7.3) reduces vascular contraction in response to noradrenalin in WKY rats (Emax: 166.6±30.2; p<0.05). These findings suggest an important role for Nox1/4 in redox-dependent organelle dysfunction and post-translational modification of proteins, processes that may play an important role in vascular dysfunction in hypertension.

Abstract 230: Evaluation of Hyperglycemia-Driven Alterations on Vascular Endothelial Function

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Brian R Hoffmann ◽  
Anthony R Prisco ◽  
Jingli Wang ◽  
Tisha M Suboc ◽  
Michael E Widlansky ◽  
...  
Keyword(s):  
High Glucose ◽  
Vascular Function ◽  
Serotonin Receptor ◽  
Protein Identification ◽  
Vascular Dysfunction ◽  
Causative Factor ◽  
The United States ◽  
Flow Chamber ◽  
Protein Glycosylation ◽  
Angiotensin Ii Receptor

Type 2 diabetes mellitus (T2DM) afflicts ~25 million people in the United States and hyperglycemia is a major causative factor contributing to vascular dysfunction among this population. This study evaluated the hypothesis that hyperglycemia-induced changes in endothelial glycoproteome leads to alterations of important homeostatic signaling pathways that result in vascular dysfunction. Utilizing targeted glycoprotein enrichment coupled with tandem mass spectrometry for protein identification and quantification, the cell surface glycoproteins were assessed from rat microvascular endothelial cells (RMVECs) cultured in ‘normal’ glucose (5 mM) or ‘high’ glucose (25 mM). Altogether, 273 N-glycosylation modified proteins were identified; 65 proteins uniquely N-glycosylated and 22 proteins significantly increased in N-glycosylation (p<0.05) from ‘high’ glucose RMVECs. Additionally, 499 O-glycoproteins were identified; 78 proteins uniquely O-glycosylated and 65 proteins significantly increased in O-glycoslyation (p<0.05) from ‘high’ glucose RMVECs. The type-1A angiotensin II receptor (AT1; p=0.029), along with numerous cell adhesion molecules, had increased N-glycosylation in ‘high’ glucose RMVECs. Numerous ion channels important for vascular function and the 5-HT2A serotonin receptor (p=5.3e-28), recently shown to contribute to vascular dysfunction in T2DM, had elevated O-glycosylation in ‘high’ glucose RMVECs. In order to evaluate these targets, functional assays were developed to measure endothelial and vascular function. Immobilization assays in a parallel plate flow chamber (PPFC) indicated that ‘high’ glucose significantly increased RMVEC adhesion to gelatin substrate under laminar flow (p<0.05). In addition, incubation of RMVECs under ‘high’ glucose conditions increased the adherence of healthy platelets to RMVECs, suggesting the importance of adhesion protein glycosylation in endothelial dysfunction. Finally, studies in human subcutaneous arterioles demonstrated that acute exposure to ‘high’ glucose impaired acetylcholine-induced vasodilation. Utilizing these assays, differentially hyperglycemia-regulated targets will be functionally evaluated for potential therapeutic intervention.

scholarly journals Metformin prevented high glucose-induced endothelial reactive oxygen species via OGG1 in an AMPKα-Lin-28 dependent pathway

2020 ◽  
Author(s):  
Liangliang Tao ◽  
Xiucai Fan ◽  
Jing Sun ◽  
Zhu zhang
Keyword(s):  
High Glucose ◽  
Vascular Function ◽  
Umbilical Vein ◽  
Vascular Complications ◽  
Diabetic Patients ◽  
Type 2 Diabetic Patients ◽  
Umbilical Vein Endothelial Cells ◽  
Dependent Pathway

AbstractMetformin improved vascular function in obese type 2 diabetic patients. 8-oxoguanine glycosylase (OGG1), a main DNA glycosylase, was involved in vascular complications in diverse diseases. However, whether metformin suppressed endothelial ROS via OGG1 pathway was unclear. Human umbilical vein endothelial cells (HUVECs) were exposed to HG (high glucose) or HG with metformin. OGG1 and AMPfα levels were measured after metformin treatment while HG-caused ROS was measured by DHE prober. Diabetic mice were induced by daily intraperitoneal injections of streptozotocin (STZ). Metformin reduced Endothelial ROS caused by HG via upregulating OGG1. Additionally, OGG1 protein expression was dependent on its mRNA stability, which was reversed by genetic inhibition of AMPKα and Lin-28. The role of OGG1 on ROS stimulated by HG was partially dependent on NFKB/NOX4 pathway in HUVECs. These results suggested that metformin contacted HG-induced endothelial ROS via AMPKα/Lin-28/OGG1 pathway.

Abstract 234: High Glucose Exacerbates the Lipopolysaccharide-induced Impairment of Nitrergic Relaxation to Electrical Field Stimulation of Rat Corpus Cavernosum

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Inger S Stallmann-Jorgensen ◽  
R. Clinton Webb
Keyword(s):  
High Glucose ◽  
Vascular Function ◽  
Vascular Dysfunction ◽  
Corpus Cavernosum ◽  
Electrical Field Stimulation ◽  
Maximal Response ◽  
Field Stimulation ◽  
Electrical Field ◽  
System Risk ◽  
Relaxation Responses

Hypertension (HTN) and diabetes (DM) are comorbid conditions, each associated with inflammation. Lipopolysaccharide (LPS) is elevated in the serum of patients with DM and capable of robustly activating the immune system. Risk of erectile dysfunction (ED) is increased in both HTN and DM. Vascular relaxation is needed to regulate vascular tone and erectile function. Our previous studies showed that corpus cavernosum (CC) nitrergic relaxation to electrical field stimulation (EFS) was reduced after LPS exposure. It is not known whether LPS-induced inflammation plays a role in DM-associated ED. Our study aim was to examine vascular function of rat CC under high glucose conditions in the presence of LPS. We hypothesized that high glucose would exacerbate the LPS-induced decrease in EFS relaxation. Isolated rat CC strips incubated with control (5mM) or high glucose (HG, 25 mM) media with or without LPS (1 ug/mL) for 6 h. CC strips were mounted in a myograph and pre-contracted with phenylephrine (PE, 10 -5 M) before eliciting relaxation responses using 20-volt stimuli of 1, 2, 4, 8, and 16 Hz. Four Hz produced the half maximal response (%PE contraction) and was used to compare treatment effects. LPS-treated CC (7.13±1.53%) relaxed less than Control CC (19.1±2.67%). The decrease in relaxation was greatest in HG+LPS-treated CC (-3.58±1.43%), Fig. 1. Thus, we show that the LPS-induced reduction in relaxation is exacerbated in the HG+LPS condition, suggesting that elevated LPS and hyperglycemia act synergistically in the promotion of vascular dysfunction and ED.

Abstract MP250: Reduction In Endothelial Aryl Hydrocarbon Receptor Nuclear Translocator (arnt) Mediates Vascular Dysfunction In Mice With Type 2 Diabetes Mellitus

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Tu Nguyen ◽  
Kaichao Pan ◽  
Maura Knapp ◽  
Mei Zheng ◽  
Nikola Sladojevic ◽  
...  
Keyword(s):  
Cell Viability ◽  
High Glucose ◽  
Vascular Dysfunction ◽  
Vascular Complications ◽  
Tube Formation ◽  
Diabetic Mice ◽  
Aryl Hydrocarbon ◽  
Diabetic Vascular Complications ◽  
High Fat Chow

Background: Endothelial dysfunction, especially at the microvasculature level, is one of the most deleterious events in diabetes. ARNT is a transcription factor that functions as a master regulator of glucose homeostasis, but its role in diabetic vascular complications is poorly understood. Results and method: We found a reduction in ARNT expression in microvascular endothelial cells (MVECs) derived from type 2 diabetic mice (db/db). Thus, we generated an inducible, EC-specific ARNT-knockout mutation ( Arnt ΔEC, ERT2) to address the hypothesis that aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. We show here that loss of ARNT in the endothelium mimics diabetic phenotypes, such as impairs blood flow recovery after hindlimb ischemia, delays wound healing, and exacerbates infiltration of pro-inflammatory neutrophils after myocardial infarction. Interestedly, the degree of these impairments in the KO mice was more remarkable in diabetic animals induced with high-fat chow. In addition, the siRNA-mediated knockdown of ARNT activity reduced tube formation and cell viability measurements in HUVECs cultured under high-glucose conditions. The Arnt ΔEC, ERT2 mutation also reduced measures of cell viability while increasing the production of reactive oxygen species (ROS) in MVECs isolated from mouse skeletal muscle, and the viability of Arnt ΔEC, ERT2 MVECs under high-glucose concentrations increased when the cells were treated with a ROS inhibitor. Conclusion: Collectively, these observations suggest that declines in endothelial ARNT expression contribute to the suppressed angiogenic phenotype in diabetic mice and that the cytoprotective effect of ARNT expression in ECs is at least partially mediated by declines in ROS production. Endothelial ARNT might be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic complications.

scholarly journals Chemerin receptor blockade improves vascular function in diabetic obese mice via redox-sensitive and Akt-dependent pathways

2018 ◽  
Vol 315 (6) ◽  
pp. H1851-H1860 ◽  
Author(s):  
Karla Bianca Neves ◽  
Aurelie Nguyen Dinh Cat ◽  
Rheure Alves-Lopes ◽  
Katie Yates Harvey ◽  
Rafael Menezes da Costa ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Insulin Resistance ◽  
Insulin Signaling ◽  
Vascular Function ◽  
Glucose Transporter ◽  
Critical Role ◽  
Vascular Complications ◽  
Mesenteric Arteries ◽  
Nitric Oxide Signaling ◽  
Vascular Oxidative Stress

Chemerin and its G protein-coupled receptor [chemerin receptor 23 (ChemR23)] have been associated with endothelial dysfunction, inflammation, and insulin resistance. However, the role of chemerin on insulin signaling in the vasculature is still unknown. We aimed to determine whether chemerin reduces vascular insulin signaling and whether there is interplay between chemerin/ChemR23, insulin resistance, and vascular complications associated with type 2 diabetes (T2D). Molecular and vascular mechanisms were probed in mesenteric arteries and cultured vascular smooth muscle cells (VSMCs) from C57BL/6J, nondiabetic lean db/m, and diabetic obese db/db mice as well as in human microvascular endothelial cells (HMECs). Chemerin decreased insulin-induced vasodilatation in C57BL/6J mice, an effect prevented by CCX832 (ChemR23 antagonist) treatment. In VSMCs, chemerin, via oxidative stress- and ChemR23-dependent mechanisms, decreased insulin-induced Akt phosphorylation, glucose transporter 4 translocation to the membrane, and glucose uptake. In HMECs, chemerin decreased insulin-activated nitric oxide signaling. AMP-activated protein kinase phosphorylation was reduced by chemerin in both HMECs and VSMCs. CCX832 treatment of db/db mice decreased body weight, insulin, and glucose levels as well as vascular oxidative stress. CCX832 also partially restored vascular insulin responses in db/db and high-fat diet-fed mice. Our novel in vivo findings highlight chemerin/ChemR23 as a promising therapeutic target to limit insulin resistance and vascular complications associated with obesity-related diabetes. NEW & NOTEWORTHY Our novel findings show that the chemerin/chemerin receptor 23 axis plays a critical role in diabetes-associated vascular oxidative stress and altered insulin signaling. Targeting chemerin/chemerin receptor 23 may be an attractive strategy to improve insulin signaling and vascular function in obesity-associated diabetes.

Vascular actions of insulin with implications for endothelial dysfunction

2009 ◽  
Vol 297 (3) ◽  
pp. E568-E577 ◽  
Author(s):  
Maria Assunta Potenza ◽  
Francesco Addabbo ◽  
Monica Montagnani
Keyword(s):  
Endothelial Dysfunction ◽  
Signaling Pathways ◽  
Insulin Signaling ◽  
Intracellular Signaling ◽  
Vascular Function ◽  
Molecular Mechanisms ◽  
Dynamic Control ◽  
Vascular Complications ◽  
Short Review ◽  
Insulin Resistant

Hemodynamic actions of insulin depend largely on the hormone's ability to stimulate synthesis and release of endothelial mediators, whose balanced activity ensures dynamic control of vascular function. Nitric oxide (NO), endothelin-1 (ET-1), and reactive oxygen species (ROS) are important examples of endothelial mediators with opposing properties on vascular tone, hemostatic processes, and vascular permeability. Reduced NO bioavailability, resulting from either insufficient production or increased degradation of NO, characterizes endothelial dysfunction. In turn, endothelial dysfunction predicts vascular complications of metabolic and hemodynamic disorders. In the cardiovascular system, insulin stimulates the production and release of NO, ET-1, and ROS via activation of distinct intracellular signaling pathways. Under insulin-resistant conditions, increased insulin concentrations and/or impaired insulin-signaling pathways in the vasculature may contribute to imbalance in secretion of endothelial mediators that promote pathogenesis of vascular abnormalities. This short review describes signaling pathways involved in insulin-stimulated release of NO, ROS, and ET-1 and suggests possible molecular mechanisms by which abnormal insulin signaling may contribute to endothelial dysfunction.

scholarly journals Effect of Caffeic Acid Phenethyl Ester on Vascular Damage Caused by Consumption of High Fructose Corn Syrup in Rats

2016 ◽  
Vol 2016 ◽  
pp. 1-8
Author(s):  
Aburrahman Gun ◽  
Mehmet Kaya Ozer ◽  
Sedat Bilgic ◽  
Nevin Kocaman ◽  
Gonca Ozan
Keyword(s):  
Metabolic Syndrome ◽  
Caffeic Acid ◽  
Vascular Function ◽  
Vascular Dysfunction ◽  
Caffeic Acid Phenethyl Ester ◽  
High Fructose Corn Syrup ◽  
Corn Syrup ◽  
Beneficial Effects ◽  
Fructose Intake ◽  
High Fructose

Fructose corn syrup is cheap sweetener and prolongs the shelf life of products, but fructose intake causes hyperinsulinemia, hypertriglyceridemia, and hypertension. All of them are referred to as metabolic syndrome and they are risk factors for cardiovascular diseases. Hence, the harmful effects of increased fructose intake on health and their prevention should take greater consideration. Caffeic Acid Phenethyl Ester (CAPE) has beneficial effects on metabolic syndrome and vascular function which is important in the prevention of cardiovascular disease. However, there are no known studies about the effect of CAPE on fructose-induced vascular dysfunction. In this study, we examined the effect of CAPE on vascular dysfunction due to high fructose corn syrup (HFCS). HFCS (6 weeks, 30% fed with drinking water) caused vascular dysfunction, but treatment with CAPE (50 micromol/kg i.p. for the last two weeks) effectively restored this problem. Additionally, hypertension in HFCS-fed rats was also decreased in CAPE supplemented rats. CAPE supplements lowered HFCS consumption-induced raise in blood glucose, homocysteine, and cholesterol levels. The aorta tissue endothelial nitric oxide synthase (eNOS) production was decreased in rats given HFCS and in contrast CAPE supplementation efficiently increased its production. The presented results showed that HFCS-induced cardiovascular abnormalities could be prevented by CAPE treatment.

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