Tumor necrosis factor-α receptor 1 contributes to ethanol-induced vascular reactive oxygen species generation and hypertension

Accepted Manuscript Tumor necrosis factor-α receptor 1 (TNFR1) contributes to ethanol-induced vascular reactive oxygen species generation and hypertension Janaina A. Simplicio, Natália A. Gonzaga, Marcelo A. Nakashima, Bruno S. De Martinis, Thiago Mattar Cunha, Luis F. Tirapelli, Carlos R. Tirapelli PII:

S1933-1711(17)30263-2

DOI:

10.1016/j.jash.2017.07.008

Reference:

JASH 1061

To appear in:

Journal of the American Society of Hypertension

Received Date: 12 April 2017 Revised Date:

22 June 2017

Accepted Date: 17 July 2017

Please cite this article as: Simplicio JA, Gonzaga NA, Nakashima MA, De Martinis BS, Cunha TM, Tirapelli LF, Tirapelli CR, Tumor necrosis factor-α receptor 1 (TNFR1) contributes to ethanol-induced vascular reactive oxygen species generation and hypertension, Journal of the American Society of Hypertension (2017), doi: 10.1016/j.jash.2017.07.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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TNF-α

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Chronic ethanol consumption

TNFR1 receptor

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ROS

NO bioavailability

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ACCEPTED MANUSCRIPT Tumor necrosis factor-α receptor 1 (TNFR1) contributes to ethanol-induced vascular reactive oxygen species generation and hypertension Janaina A. Simplicio1,2, Natália A. Gonzaga1,2, Marcelo A. Nakashima2, Bruno S. De Martinis3, Thiago Mattar Cunha1, Luis F. Tirapelli4, Carlos R. Tirapelli2* 1

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Programa de Pós-graduação em Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil. 2 Laboratório de Farmacologia, Escola de Enfermagem de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil. 3 Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, USP, Ribeirão Preto, São Paulo, Brazil. 4 Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil.

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*Corresponding author: Universidade de São Paulo, Escola de Enfermagem de Ribeirão Preto, Laboratório de Farmacologia, Avenida Bandeirantes 3900, CEP 14040-902, Ribeirão Preto, SP, Brazil. Tel.: +55-16-33150532; Fax: +55-16-3315-0518; E-mail: [email protected]

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ABSTRACT We evaluated the contribution of TNF-α receptor 1 (TNFR1) to ethanol-induced hypertension and vascular oxidative stress and the possible role of perivascular adipose tissue (PVAT) in such response. Male C57BL/6 wild-type (WT) or TNFR1-deficient mice (TNFR1-/-) were treated with ethanol (20% v/v) for 12 weeks. Ethanol induced an increase in blood pressure in WT and TNFR1−/– mice at 4 and 5 weeks of treatment, respectively. Treatment with ethanol increased TNF-α and interleukin (IL)-6 levels in aortas with or without PVAT (PVAT+ and PVAT-, respectively) from WT, but not TNFR1-/- mice. Ethanol increased superoxide anion (O2) generation, TBARS concentration and the activity of superoxide dismutase (SOD) and catalase in aortas (PVAT- and PVAT+) from WT, but not TNFR1−/- mice. Conversely, ethanol consumption decreased the concentration of nitrate/nitrite (NOx) in aortas (PVAT- and PVAT+) from WT, but not TNFR1-/- mice. Treatment with ethanol increased myeloperoxidase (MPO) activity in aortas (PVAT- and PVAT+) from WT, but not TNFR1-/- mice. The major finding of our study is that TNFR1 contributes to ethanol-induced hypertension and oxidative stress in the vasculature. Additionally, TNFR1 plays a role in ethanol-induced increase in pro-inflammatory cytokines and neutrophils migration. However, PVAT does not counteract or aggravate the effects induced by ethanol. Keywords: TNF-α; Ethanol, Oxidative Stress; Perivascular adipose tissue; Hypertension.

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Introduction Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that exerts its biological effects by binding to two receptors termed TNF-α receptor 1 (TNFR1) and TNF-α receptor 2 (TNFR2). These receptors are expressed in most cell types, including vascular smooth muscle and endothelial cells.1,2 Vascular wall cells are both a source and a target of TNF-α. In the vasculature, the actions of TNF-α are mainly mediated by TNFR1.3 These actions include a reduction of nitric oxide (NO) bioavailability,4,5 and an increase in the generation of reactive oxygen species (ROS), such as superoxide anion (O2-).3,5 TNF-α-induced O2- generation in blood vessels is mainly mediated by the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.4,6 In fact, NADPH oxidase is the main source of ROS in vascular smooth muscle and endothelial cells and increased expression or activity of this enzyme is related to vascular dysfunction and hypertension.7 The deleterious effects of TNF-α in the vasculature also include the expression of genes encoding pro-inflammatory cytokines such as interleukin (IL)-6.2,3 Importantly, experimental as well as clinical studies in hypertensive individuals have shown increased plasma and vascular tissue levels of TNF-α, suggesting a potential link between this cytokine and hypertension.8,9 The perivascular adipose tissue (PVAT) surrounds most blood vessels and it is mainly composed of adipocytes. Under physiological conditions, PVAT displays an anti-contractile action trough the release of a wide range of vasoactive molecules such as NO and angiotensin 17.10 The secretory profile of PVAT is altered by cardiovascular diseases, where it may exert deleterious effects in the vasculature by producing ROS and pro-inflammatory cytokines, including TNF-α.11 PVAT-derived TNF-α is increased in pathophysiological conditions where it may act paracrinally to induce vascular dysfunction.12 Consumption of high doses of ethanol is linked to cardiovascular diseases such as hypertension. Possible mechanisms underlying ethanol-induced hypertension include an increase in sympathetic nervous system activity, stimulation of the renin-angiotensinaldosterone system, an increase of intracellular Ca2+ in vascular smooth muscle, increased oxidative stress and endothelial dysfunction.13-20 Much of the research investigating the effects of ethanol on the cardiovascular system has focused in the vasculature. In this regard, increased vascular contractility or impairment of vascular relaxation is described to contribute to ethanolinduced hypertension.15,19,20 The vascular dysfunction induced by ethanol may be the result of an increase in ROS generation, which will lead to a reduction in NO bioavailability, endothelial dysfunction and increased concentration of Ca2+ in vascular smooth muscle cells.13,15-18 Altogether, these events would induce vascular dysfunction and hypertension. In fact, chronic ethanol consumption has been described to increase blood pressure and induce ROS generation in the vasculature.17,18, 21 TNF-α has been described as an important mediator of the toxic effects exerted by ethanol in distinctive tissues.22,23 In the vasculature, ethanol consumption was previously described to increase TNF-α expression.21 More recently, we provided evidence that chronic ethanol consumption increased the circulating levels of TNF-α.18 However, the role of TNF-α in ethanol-induced hypertension and vascular dysfunction and the participation of PVAT in this response remains unclear. Although plasma levels of TNF-α have been shown to correlate positively with ethanolinduced tissue injury, little is known regarding the effects of this cytokine in ethanol-induced vascular oxidative stress. We hypothesized that ethanol consumption increases the production of TNF-α, which will ultimately contribute to the vascular oxidative stress and hypertension induced by ethanol. Considering that vascular oxidative stress is a key event associated with ethanol-induced hypertension, we evaluated whether TNF-α plays a role in mediating vascular ROS generation in response to ethanol and the role of this cytokine in ethanol-induced hypertension. As PVAT has emerged as an important component of cardiovascular diseases, we examined the possible role of PVAT in the vascular effects mediated by ethanol.

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2. Material and methods Animals and grouping All experiments were conducted as per the guidelines of Conselho Nacional de Controle de Experimentação Animal (CONCEA, Brazil) after the necessary approval of the Ethics Committee on Animal Use of the University of São Paulo, Campus of Ribeirão Preto (#12.1.1654.53.9). Male C57BL/6 wild-type (WT, n=46) or TNFR1-deficient mice (TNFR1-/-, n=52), six to eight weeks old were randomly divided into the following groups: control (WT), ethanol, TNFR1-/- and TNFR1-/- ethanol. Mice from the ethanol groups were submitted to a period of adaptation of two weeks in which they received ethanol (Synth, São Paulo, Brazil) in drinking water at 5 and 10% (v/v), respectively. From the 3rd to the 12th week, animals had free access to ethanol 20% (v/v).24 At the end of the 12th week of treatment, mice were anaesthetized intraperitoneally with urethane at 1.25 g/kg (solution of 25%, 5 ml/kg) and killed by aortic exsanguination. TNFR1-deficient mice were originally obtained from the Jackson Laboratories (Bar Harbor, ME, USA). Both WT and TNFR1-deficient mice were bred and maintained under standard conditions in the animal house of the Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil. Blood and thoracic aorta with or without PVAT (PVAT+ and PVAT-, respectively) were collected for functional and biochemical experiments. PVAT was carefully removed using fine scissors.

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Determination of plasma ethanol levels Mice were anesthetized intraperitoneally with urethane 1.25 g/kg (Sigma-Aldrich, St. Louis, MO, USA) and decapitated. Blood samples were collected from the trunk and transferred to tubes containing sodium fluoride (1 mg/ml). Plasma ethanol concentration (mg/dl) was determined by gas chromatography using a Varian CP3380 gas chromatographer (Varian, CA, USA), equipped with a flame ionization detector as described by Gonzaga et al.25.

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Determination of systolic blood pressure Blood pressure was measured weekly using a tail cuff plethysmography (Plethysmograph EFF306, Insight, Ribeirão Preto, Brazil) as described previously by Carda et al.26. Systolic blood pressure is expressed in mmHg.

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Vascular reactivity experiments Mice were anesthetized with an intraperitoneal injection of urethane 1.25 g/kg (SigmaAldrich, St. Louis, MO, USA) and killed by aortic exsanguination. Aortas (PVAT- and PVAT+) were isolated, cut into rings of approximately 3–4 mm in length and transferred to organ chambers of 5 ml containing Krebs solution with the following composition (mmol/l): NaCl, 118.0; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 15.0; glucose, 5.5; CaCl2, 2.5. The rings were connected to isometric force transducers (TRI201; Panlab, Barcelona, Spain), and stretched until they reached a basal tension of 5 mN. The aortas were continuously gassed with a mixture of 95% O2/5% CO2 at 37oC. Endothelial integrity was assessed qualitatively by the degree of relaxation caused by acetylcholine (10 µmol/l) in the presence of contractile tone induced by phenylephrine (1 µmol/l). The magnitude of contraction induced by phenylephrine did not differ between the experimental groups at 1 µmol/l (Supplementary material online, Table S1). Rings were discarded if relaxation with acetylcholine was not 70% or greater. Concentration-response curves for acetylcholine (0.1 nmol/l to 10 µmol/l) were obtained in endothelium-intact aortas (PVAT- and PVAT+). Relaxation was expressed as a percentage change from phenylephrine-contracted levels. Concentration-response curves for acetylcholine were fitted using a nonlinear interactive fitting program (Graph Pad Prism 5.01; GraphPad Software Inc., San Diego, CA, USA). Agonist potency and maximal response were expressed as pD2 (-logEC50) and Emax (maximum effect elicited by the agonist), respectively.

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Detection of O2−generation in the aorta (PVAT- and PVAT+) The aortas were homogenized in phosphate buffer using a glass-to-glass homogenizer. The generation of O2- was determined using the lucigenin-derived chemiluminescence assay as

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Visualization of NO with 4,5-diaminofluorescein diacetate (DAF-2DA) In situ production of NO was visualized using the dye DAF-2DA. Aortas (PVAT- and PVAT+) were vertically embedded in Tissue-tek® and sectioned transversely (8-µm-thick slices). The slices were incubated for 30 min with DAF-2DA (10 µmol/l) and then washed three times with cold PBS (pH 7.4). Sections were examined by fluorescence microscopy (Leica Model SPE, Leica Imaging Systems Ltd., Wetzlar, Germany) using λexcitation/ λemission of 488/530 nm. The images were captured at x400.

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Determination of the concentration of thiobarbituric acid reactive substances (TBARS) in plasma and aortas (PVAT- and PVAT+) The aortas were homogenized in radioimmunoprecipitation assay buffer (RIPA) using a glass-to-glass homogenizer and then centrifuged at 1,600 x g (10 min at 4°C). Blood was collected in heparinized tubes and centrifuged at 1,000 x g (10 min, 4 °C).The concentration of TBARS in plasma (µmol/l of plasma) and aortas (µmol/g protein) was determined colorimetrically at 540 nm using a commercially available kit (#10009055, Cayman Chemical, Ann Arbor, MI, USA). Determinationof nitrate/nitrite (NOx) concentration in plasma and aortas (PVAT- and PVAT+) NOx concentration was determined in plasma (nmol/ml) and aortas (nmol/mg) using a commercially available kit (#780001, Cayman Chemical, Ann Arbor, MI, USA) as described by Carda et al.26.

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Determination of SOD, catalase and glutathione peroxidase (GPx) activities The activity of SOD was determined in aortas and plasma. The aortas (PVAT- and PVAT+) were homogenized in PBS (pH 7.4) and centrifuged at 1,000 x g (10 min at 4°C). Blood was collected using heparinized syringes and centrifuged at 1,000 x g (10 min at 4°C). SOD activity was determined using a commercially kit (#19160, Sigma-Aldrich, St. Louis, MO, USA).The results are expressed as% inhibition rate/mg protein or % inhibition rate/ml of plasma. Catalase activity in plasma and aortas was assayed by H2O2 consumption as described by Gonzaga et al.27. Results are expressed as U/min/mg protein or U/min/ml of plasma. One catalase unit (U) was defined as the amount of enzyme required to decompose 1 µmol of H2O2/min/mg protein. The aortic activity of GPx was determined using a commercially available kit (#703102, Cayman Chemical, Ann Arbor, MI, USA) as described by Marchi et al.17.The results are expressed in nmol/min/mg protein.

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Determination of reduced glutathione (GSH) in plasma and aortas (PVAT- and PVAT+) The assay used to analyze the concentration of GSH is based on the oxidation of GSH by sulfhydryl reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) to form the yellow product 5′-thio-2-nitrobenzoic (TNB). The concentration of GSH in plasma (mg/ml) and aorta (mg/mg protein) was determined spectrophotometrically at 415 nm as previously described by Gonzaga et al.27. Measurement of pro-inflammatory cytokines in plasma and aortas (PVAT- and PVAT+) Blood was collected, centrifuged at 4,000 x g (15 min at 4oC) and then stored at -80oC. The aortas were collected in tubes containing phosphate buffered saline (PBS, pH 7.4) and a protease inhibitor cocktail (#11697498001, Roche, Basel, Switzerland). Homogenization of aortas was performed using a glass-to-glass homogenizer. Then, samples were centrifuged at 10,000 x g (10 min at 4oC). Measurements of TNF-α and IL-6 were performed by enzymelinked immunosorbent assay (ELISA) following instructions of commercially available kits

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Determination of myeloperoxidase (MPO) activity in the aorta (PVAT- and PVAT+) MPO activity was determined as described by Souza et al.29 In brief, tissues were homogenized in ice-cold buffer (pH 4.7) with the following composition: 0.1 mol/l NaCl, 0.02 mol/l NaPO4, 0.015 mol/l EDTA. Then the homogenates were centrifuged at 800 x g (15 min at 4oC) and the pellet was suspended in NaPO4 buffer (0.05 mol/l, pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide and re-homogenized. Homogenate was centrifuged at 10,000 x g (15 min at 4°C) and 50 µl of the supernatant was mixed with 25 µl of tetramethylbenzidine (1.6 mmol/l) and 100 µl of H2O2 (3%). The solution was analyzed by spectrophotometry at 450 nm. The absorbance at 450 nm was used to calculate MPO activity, which is represented as absorbance (A450)/mg protein.30

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Histopathological analysis Tissue samples were fixed in 10% buffered formalin were embedded in paraffin for reparation. Sections of 5 mm were stained with hematoxylin and eosin. The sections were analyzed using a digital camera AxioCam Hrc® coupled to a binocular Zeiss® microscope (Axioskop 2 plus, Jena, Germany) and the program Axio Vision 4.6. The following morphological changes were evaluated: subendothelial edema, tunica media edema and neutrophilic infiltration. The images were captured at x1000. Statistical analysis Statistical analysis was performed using the program GraphPad® Prism 5.01 (GraphPad Software Inc., San Diego, CA, USA).Results are shown as means ± standard error of the mean (S.E.M.). Blood ethanol levels were compared by Student’s t test and the other results were analyzed by two-way analysis of variance (ANOVA) followed by Bonferroni’s comparison test. Results of statistical tests with P<0.05 were considered as significant.

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Results Body mass and plasma ethanol levels The body weights of the mice before treatment averaged 25.0 ± 0.02 g in the WT group, 25.1 ± 0.07 g in the ethanol group, 25.6 ± 0.12 g in the TNFR1-/- group and 25.7 ± 0.05 g in the TNFR1-/- ethanol group. At the end of the period of treatment, mice from the ethanol (30.4 ± 0.14 g) and TNFR1-/-ethanol groups (29.4 ± 0.20 g)showed reduced body weights, when compared to mice from the WT (32.0 ± 0.12 g) and TNFR1-/-groups (31.3 ± 0.16 g)(P<0.05; two-away ANOVA). In WT mice, plasma ethanol levels averaged 227 ± 27 mg/dl (∼49 mmol/l, n=7), while in TNFR1-/- mice plasma ethanol levels averaged 207 ± 23 mg/dl (∼45 mmol/l, n=5). The levels of ethanol in plasma were not significantly different between the groups. No ethanol was detected in mice from control groups (WT or TNFR1-/-).

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Effects of chronic ethanol consumption on systolic blood pressure Baseline values of systolic blood pressure were similar in mice from all experimental groups (Fig. 1).An increase in blood pressure was observed in WT mice treated with ethanol for 4weeks. This response increased in the following week and remained stable until the 12th week of treatment. In TNFR1-deficient mice ethanol-induced increase in blood pressure was evidenced at the 5th week of treatment. The increase in blood pressure induced by ethanol was less pronounced in TNFR1-deficient mice when compared to WT animals (Fig. 1). Effects of chronic ethanol consumption on vascular reactivity Chronic ethanol consumption did not alter the vascular relaxation induced by acetylcholine in aortas (PVAT- and PVAT+) from WT or TNFR1-deficient mice (Fig. 2A and B). The Emax and pD2 values for acetylcholine were not affected by chronic ethanol consumption (Supplementary material online, Table S2).

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ACCEPTED MANUSCRIPT Effects of chronic ethanol consumption on the vascular and systemic oxidative stress Lucigenin-derived luminescence was increased in aortas (PVAT- and PVAT+) from WT, but not TNFR1-deficient mice treated with ethanol (Fig. 3A).Similarly, chronic ethanol consumption increased the concentration of TBARS in aortas (PVAT- and PVAT+) from WT, but not TNFR1-deficient mice (Fig. 3B). Increased plasma concentration of TBARS was detected in WT mice chronically treated with ethanol, but this response was not observed in TNFR1-deficient mice (Table 1).

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Effects of chronic ethanol consumption on the vascular and systemic levels of NO Chronic ethanol consumption decreased NO generation in aortas (PVAT- and PVAT+) from WT, but not TNFR1-deficient mice (Fig. 4A). NOx concentration was decreased in aortas (PVAT- and PVAT+) from WT, but not TNFR1-deficient mice treated with ethanol (Fig. 4B). No alteration on plasma NOx concentration was observed after treatment with ethanol (Table 1).

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Effects of chronic ethanol consumption on the vascular and systemic antioxidant system Treatment with ethanol increased both SOD and catalase activity in aortas (PVAT- and PVAT+) from WT, but not TNFR1-deficient mice (Fig. 5A and B). On the other hand, chronic ethanol consumption did not alter GPx activity or the concentration of GSH in the aorta from both WT and TNFR1-deficient mice (Fig. 5C and D). Ethanol consumption did not affect SOD or catalase activity in plasma from WT or TNFR1-deficient mice. Decreased concentration of GSH was detected in plasma from WT, but not TNFR1-deficient mice (Table 1). Effects of chronic ethanol consumption on the vascular and systemic production of cytokines Increased levels of TNF-α and IL-6 were detected in aortas (PVAT- and PVAT+) from WT mice treated with ethanol. Conversely, ethanol-induced increase in TNF-α and IL-6 was not observed in aortas (PVAT- and PVAT+) from TNFR1-deficient mice (Fig. 6A and B). Increased plasma levels of IL-6 were detected in WT or TNFR1-deficient mice treated with ethanol (Table 1).

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Effects of chronic ethanol consumption on MPO activity in the mice aorta (PVAT- and PVAT+) Chronic ethanol consumption increased MPO activity in aortas (PVAT- and PVAT+) from WT, but not TNFR1-deficient mice (Fig.7).

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Histological analysis No histopathological alterations were observed in aortas from WT or TNFR1-deficient mice (Figure S1 and S2, Supplementary material online). On the other hand, edema of the subendothelial layer and tunica media was detected in aortas from ethanol and TNFR1-/- ethanol mice (Fig. 8 and Fig. S3, Supplementary material online). Neutrophilic infiltration was visualized in aortas from ethanol-treated mice (Fig. 8).

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Discussion Chronic ethanol consumption is described to increase blood pressure.16,17,19 We hypothesized that ethanol consumption increases TNF-α production and that this cytokine may contribute to vascular oxidative stress and hypertension induced by ethanol. Our results show that ethanol-induced hypertension is partially mediated by TNF-α/TNFR1. Increased plasma and vascular tissue levels of TNF-α were detected in hypertensive individuals.8 Moreover, experimental studies in rats have shown that angiotensin II infusion is associated with both increased blood pressure and TNF-α production by renal endothelial cells.9 Additionally, infliximab, a TNF-α-neutralizing antibody was found to decrease blood pressure in spontaneously hypertensive rats.31 Altogether, these studies establish a relationship between TNF-α and hypertension. The mechanisms whereby TNF-α modulates blood pressure are not fully understood. One proposal is that TNF-α would induce an inflammatory process in the vasculature and that this response will ultimately lead to vascular dysfunction and remodeling either directly or indirectly through oxidative stress.2 Another possibility is that TNF-α decreases the vascular synthesis of NO leading to reduced vasodilatation.31 Of note, the increase

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in blood pressure here described occurs in mice whose ethanol levels are in the range detected in individuals who chronically consume ethanol.32 However, it is important to note that deletion of the TNFR1 gene mitigated but did not suppress the hypertensive effect of ethanol, further suggesting that other mechanisms are contributing to ethanol-induced hypertension. TNF-α binds to TNFR1 and TNFR2 to promote its vascular effects.3 Thus, it is possible that TNFR2 also contributes to ethanol-induced hypertension. In this sense, TNFR2 could be overexpressed to take the vascular functions of the suppressed TNFR1 in gene-deficient mice. Moreover, it was recently described that catecholamines released from adrenal chromaffin cells plays a role in ethanol-induced hypertension.33 Chronic ethanol consumption did not alter the relaxation induced by acetylcholine in the aorta, which is in accordance with previous findings.20,34 The effects of ethanol on acetylcholine-induced relaxation are time-dependent and alterations in the vascular responsiveness appear not to be the cause, but rather a consequence of the increase in blood pressure induced by ethanol.19 These observations may explain the lack of effect of ethanol on acetylcholine-induced relaxation. The reduced concentrations of NOx and the decreased fluorescence of DAF-2DA detected in aortas of WT mice treated with ethanol suggest that ethanol consumption reduced the basal levels of NO. Our results also show that PVAT could not prevent the decrease in NO bioavailability in the aorta and that this response is mediated by TNFR1. Decreased NO bioavailability in the vasculature is usually the result of an increased inactivation of NO by O2-. This mechanism could be contributing to decrease NO levels in our model since increased generation of O2- was detected in aortas from WT mice treated with ethanol. The lucigenin luminescence assay is used to estimate the production of O2- by the enzyme NADPH oxidase.35 The increase in lucigenin-derived luminescence was not observed in aortas from TNFR1-deficient mice, suggesting that TNF-α/TNFR1 may activate NADPH oxidase, which will in turn increase O2- generation. This result is in accordance with the fact that TNF-α-induced O2- generation in blood vessels is mainly mediated by NADPH oxidase.4,6 Our results also show that PVAT is not able to counteract ethanol-induced ROS generation. In fact, the basal production of O2- was found to be greater in aortas with PVAT (WT and TNFR1-/mice), when compared to aortas without PVAT. This is in line with the fact that PVAT is capable of producing O2- via NADPH oxidase.10,12,36 Additionally, we found increased lipid peroxidation in aortas (PVAT- and PVAT+) from WT, but not TNFR1-deficient mice treated with ethanol. Thus, our results provide evidence that ethanol-induced O2- generation and lipoperoxidation are processes mediated by TNF-α/TNFR1. Since the levels of plasma TBARS were increased in WT mice treated with ethanol, we conclude that ethanol increases oxidative stress systemically and that TNF-α/TNFR1 plays a role in such response. The primary defense against tissue damage caused by O2- is a group of oxidoreductases collectively known as SODs. These enzymes catalyze the dismutation of O2- into O2 and H2O2. In mammals, three isoforms of SOD are described: the cytoplasmic Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/ZnSOD (SOD3).37 Our findings show that ethanol consumption increased total SOD activity and that PVAT did not counteract this response. The three isoforms of SOD play an important role in regulating vascular function under pathological conditions, where increased activity of theses enzymes can be interpreted as a compensatory mechanism to protect cells against ROS-induced damage.37 Our results showing that TNFR1 modulates the increase of SOD activity is in agreement with previous findings showing that TNF-α/TNFR1 increases the activity of SOD in order to protect cells against the deleterious effects of O2-.38-40 The enzymatic antioxidant system also includes the enzymes catalase and GPx, which are responsible for H2O2 degradation. Our results corroborate previous findings showing that ethanol consumption did not affect GPx activity in the vasculature.17 We found that ethanolinduced increase in catalase activity in aortas (PVAT- and PVAT+) is mediated by TNFα/TNFR1. However, PVAT did not modulate this response. This result is in line with previous findings showing that ethanol consumption increased catalase activity in distinctive tissues.41,42 Moreover, TNF-α-induced increase in catalase activity was previously described in cardiomyocytes.43 It is important to note that despite increasing SOD and catalase activity in the

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vasculature, ethanol did not affect the activity of these enzymes in the plasma. These findings show that ethanol exerts distinctive actions in the systemic and vascular enzymatic antioxidant systems. The antioxidant system also comprises non-enzymatic antioxidants. Glutathione is a tripeptide produced by most cells and it is the main non-enzymatic mechanism of antioxidant defense against ROS. The redox-active thiol (-SH) of cysteine becomes oxidized when glutathione reduces target molecules, such as ROS. For this reason, the concentration of GSH is used to estimate the non-enzymatic antioxidant capacity. Ethanol consumption decreased plasma GSH levels and TNF-α plays a role in such response. A likely mechanism for the reduction in GSH content may be the generation of oxidants by TNF-α, which will lead to the conversion of GSH into GSSG. However, the decrease in plasma GSH levels induced by ethanol was seemed in parallel with no change in GSH concentration in arteries from ethanoltreated rats. In our study, increased levels of GSH were detected in arteries with PVAT, suggesting a role for this tissue in protecting the vasculature from oxidative damage. This observation is in line with the fact that GSH is found in PVAT.44 The effects of ethanol on cytokines levels may differ between plasma and tissue.18,45 This seems to be the case in our study since ethanol did not change plasma TNF-α levels whereas it increased the levels of this cytokine in aortas (PVAT- and PVAT+) from WT mice. Additionally, we show that ethanol-induced increase in IL-6 levels in the mice aorta is mediated by TNFR1. This finding corroborates previous studies showing that TNF-α induces IL-6 synthesis by a mechanism that involves mitogen-activated protein kinases and NF-κB activation.46,47 Moreover, we found that TNFR1 modulates TNF-α expression in aortas from ethanol-treated mice. This is in line with the fact that TNF-α modulates its own expression.48 The levels of TNF-α and IL-6 are higher in aortas with PVAT from both control and ethanoltreated mice, when compared to arteries without PVAT, indicating that PVAT is an important source of these cytokines in the vasculature. In fact, PVAT adipocytes are less differentiated from adipocytes in other fat depots and for this reason they have a secretory profile that favors the synthesis of pro-inflammatory cytokines.10,12 Our findings also show that ethanol consumption increased plasma levels of IL-6, which is agreement with previous studies.18,49 Tissue MPO increases as a result of neutrophil degranulation and for this reason it is used as a marker of neutrophil recruitment and/or activation. In the vasculature, neutrophils play an important role in inflammation since they locally secrete cytokines and chemokines that promote cellular infiltration and tissue damage.50 Our findings show that ethanol consumption induced neutrophils recruitment to the aorta via TNFR1 activation. Additionally, we found that PVAT did not counteract or increase ethanol-induced neutrophils recruitment. In summary, the major finding of our study is that TNFR1 contributes to ethanolinduced hypertension and ROS generation in the vasculature. Additionally, we demonstrate that TNFR1 plays a role on ethanol-induced increase in pro-inflammatory cytokines and neutrophils migration. Finally, we show that PVAT does not counteract or aggravate the vascular effects induced by ethanol.

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Acknowledgements

We thank Vanessa F. Borges and Carla S. Ceron for technical assistance. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (grant number 141149/2013-2) and Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (grant number 2014/09595-0). Conflict of interest statement The authors declare that there are no conflicts of interest.

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50. Phillipson M, Kubes P. The neutrophil in vascular inflammation. Nat Med 2011;17(11):1381-90.

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Fig. 1. Effect of chronic ethanol consumption on systolic blood pressure. Systolic blood pressure was measured weekly by plethysmography in mice from WT (n=10), ethanol (n=10), TNFR1-/- (n=11) and TNFR1-/-ethanol (n=11) groups. Values are the means ± S.E.M. *Compared to WT and TNFR1-/-; #Compared to TNFR1-/- ethanol (P<0.05, two-way ANOVA followed by Bonferroni’s comparison test). Fig. 2. Vascular reactivity to acetylcholine in the mice aorta. Concentration-response curves for acetylcholine were obtained in endothelium-intact aortic rings (PVAT- and PVAT+) from WT (A) or TNFR1-/- mice (B). Values are means ± SEM of 4-7 independent preparations.

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Fig. 3. Effects of chronic ethanol consumption on vascular oxidative stress. Bar graphs represent the levels of O2- (A) and TBARS (B) in the aorta (PVAT- and PVAT+) of WT and TNFR1-/- mice. Results are shown as the means ± S.E.M. of 6-10 animals. *Compared to WT, ethanol and TNFR1-/- groups (PVAT- and PVAT+); #Compared to respective groups PVAT(P<0.05, two-way ANOVA followed by Bonferroni’s comparison test). Fig. 4. Effects of chronic ethanol consumption on NOx concentration in the mice aorta. Visualization of NO generation in aorta slices using the fluorescent dye 4,5-Diaminofluorescein diacetate (DAF-2DA, A). Bar graphs represent the concentration of NOx (B) in the mice aorta (PVAT- and PVAT+). Results are shown as the means ± S.E.M. of 5-9 animals. *Compared to WT, ethanol and TNFR1-/- groups (PVAT- and PVAT+); #Compared to ethanol group PVAT(P<0.05, two-way ANOVA followed by Bonferroni’s comparison test).

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Fig. 6. Effects of chronic ethanol consumption on vascular levels of pro-inflammatory cytokines. Bar graphs represent the concentrations of TNF-α (A) and IL-6 (B) in aortas (PVATand PVAT+). Results are shown as the means ± S.E.M. of 5-8 animals. *Compared to WT, TNFR1-/-, TNFR1-/- ethanol groups (PVAT-); #Compared to WT, TNFR1-/-, TNFR1-/- ethanol groups (PVAT-) and TNFR1-/-, TNFR1-/- ethanol groups (PVAT+) (P<0.05, two-way ANOVA followed by Bonferroni’s comparison test).

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Fig. 7. Effects of chronic ethanol consumption on myeloperoxidase (MPO) activity in the mice aorta. Bar graphs represent MPO activity in the mice aorta (PVAT- and PVAT+). Results are shown as the means ± S.E.M. of 7-10 animals. *Compared to WT, TNFR1-/-, TNFR1-/- ethanol groups (PVAT- and PVAT+) (P<0.05, two-way ANOVA followed by Bonferroni’s comparison test).

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Fig. 8. Photomicrograph of the mice aorta. (A) Presence of intraluminal neutrophils in aortas from ethanol-treated mice (→). (B) Subendothelial edema (→) and edema of the tunica media in aortas from mice of the TNFR1-/- ethanol group ( ). Lumen (L). Hematoxylin-eosin staining (x1000).

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Tale 1: Effect of chronic ethanol consumption in the plasma levels of TNFα, IL-6, TBARS, NOx, GSH, and the activities of SOD or catalase. WT Ethanol TNFR1-/TNFR1-/- ethanol TNFα (pg/ml) 2.78 ± 0.17 (9) 2.80 ± 0.10 (9) 3.08 ± 0.11 (8) 3.33 ± 0.18 (6) IL-6 (pg/ml) 2.96 ± 0.37 (7) 4.78 ± 0.80* (9) 3.37 ± 0.68 (4) 5.23 ± 0.56* (5) TBARS (nmol/ml) 16.24 ± 1.49 (7) 23.92 ± 1.71** (6) 18.05 ± 1.91 (6) 16.96 ± 2.38 (6) NOx (nmol/ml) 17.29 ± 2.97 (8) 18.40 ± 3.49 (9) 18.48 ± 2.68 (9) 18.77 ± 2.32 (10) SOD activity (%inhibition rate/ml) 93.29 ± 7.28 (8) 89.48 ± 2.76 (7) 98.43 ± 0.60 (8) 95.39 ± 1.08 (8) Catalase activity (U/min/ml) 40.83 ± 3.46 (9) 45.27 ± 7.22 (9) 43.61 ± 4.41 (9) 43.05 ± 5.41 (9) GSH (mg/ml) 3.59 ± 0.61 (9) 1.58 ± 0.10** (8) 3.42 ± 0.51 (10) 3.75 ± 0.52 (8) Number between parentheses indicates the number of animals. Values are means ± S.E.M. *Compared to WT and TNFR1-/groups; **Compared to WT, TNFR1-/- and TNFR1-/- ethanol groups (P<0.05, two-way ANOVA followed by Bonferroni’s multiple comparison test). TBARS: Thiobarbituric acid reactive substances; NOx: Nitrate/nitrite; SOD: Superoxide dismutase; GSH: reduced glutathione.

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1. TNFR1 contributes to ethanol-induced hypertension and oxidative stress in the vasculature. 2. TNFR1 plays a role in ethanol-induced increase in pro-inflammatory cytokines and neutrophils migration. 3. PVAT does not counteract or aggravate the effects induced by ethanol.

ACCEPTED MANUSCRIPT Tumor necrosis factor-α receptor 1 (TNFR1) contributes to ethanol-induced vascular reactive oxygen species generation and hypertension Janaina A. Simplicio1,2, Natália A. Gonzaga1,2, Marcelo A. Nakashima2, Bruno S. De Martinis3, Thiago Mattar Cunha1, Luis F. Tirapelli4, Carlos R. Tirapelli2* 1

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Programa de Pós-graduação em Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil. 2 Laboratório de Farmacologia, Escola de Enfermagem de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil. 3 Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, USP, Ribeirão Preto, São Paulo, Brazil. 4 Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil.

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*Corresponding author: Universidade de São Paulo, Escola de Enfermagem de Ribeirão Preto, Laboratório de Farmacologia, Avenida Bandeirantes 3900, CEP 14040-902, Ribeirão Preto, SP, Brazil. Tel.: +55-16-33150532; Fax: +55-16-3315-0518; E-mail: [email protected] Materials and Methods

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Vascular reactivity experiments Aortas (PVAT- and PVAT+) were mounted in organ chambers as detailed in the manuscript. Concentration-response curves for acetylcholine (0.1 nmol/l to 10 µmol/l) were obtained in endothelium-intact aortas (PVAT- and PVAT+) pre-contracted with phenylephrine (0.1 µmol/l). The curves were analyzed by non-linear regression and agonist potency and maximal response were expressed as pD2 (-logEC50) and Emax (maximum effect elicited by the agonist), respectively.

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Histopathological analysis The aortas were stained with hematoxylin and eosin or picrosirius red. The sections were analyzed using a digital camera AxioCam Hrc® coupled to a binocular Zeiss® microscope (Axioskop 2 plus, Jena, Germany) and the program Axio Vision 4.6. The images were captured at x100 and x400.

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The pre-contraction induced by phenylephrine at 0.1 µmol/l did not differ among groups (Table S1). Table S1: Values of pre-contraction (mN) induced by phenylephrine in aortas with (PVAT+) or without perivascular adipose tissue (PVAT-). PVAT+ PVATWT 1.92 ± 0.18 (4) 1.61 ± 0.20 (4) Ethanol

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Number between parentheses indicates the number of animals. Values are the means ± S.E.M. 1

ACCEPTED MANUSCRIPT As it can be seen in Table S2, treatment with ethanol did not affect the Emax or the pD2 values for acetylcholine in the mice aorta. Table S2: Emax (% relaxation) and pD2 values for acetylcholine in endothelium-intact aortas with (PVAT+) or without perivascular adipose tissue (PVAT-). PVATPVAT+ Emax pD2 Emax pD2 WT 94.8 ± 6.5 (4) 6.9 ± 0.8 92.4 ± 9.8 (4) 7.1 ± 0.1 7.4 ± 0.4

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98.2 ± 11.6 (4)

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No histological or collagen distribution alterations were observed in aortas from WT or TNFR1-deficient mice (Fig. S1 and S2). Additionally, treatment with ethanol did not alter collagen distribution in the aorta (Data not shown).

Fig. S1. Photomicrograph of the aorta from WT mice. Lumen (L); Tunica media (*); Tunica adventitia (→); Perivascular adipose tissue (PVAT). Hematoxylin-eosin staining (x100).

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Fig. S2. Photomicrograph of the aorta (PVAT+) from WT (A) and TNFR1-/- deficient mice (B). Tunica adventitia (A); Lumen (L). Picrosirius red (A) and hematoxylin-eosin staining (B) (x400).

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Chronic ethanol consumption induced subendothelial edema and edema of the tunica media (Fig. S3).

Fig. S3. Photomicrograph of the aorta from ethanol-treated mice (PVAT+). Tunica media edema (Bottom, →); Subendothelial edema (Top, →). Hematoxylin-eosin staining (x100; x400). 3