Contribution of oxidative stress and prostanoids in endothelial dysfunction induced by chronic fluoxetine treatment.

VPH-06220; No of Pages 14 Vascular Pharmacology xxx (2015) xxx–xxx

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Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph

Contribution of oxidative stress and prostanoids in endothelial dysfunction induced by chronic fluoxetine treatment Janaina A. Simplicio a,e, Leonardo B. Resstel b, Daniela P.C. Tirapelli c, Pedro D'Orléans-Juste d, Carlos R. Tirapelli e,⁎ a

Programa de Pós-Graduação em Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, São Paulo, Brazil Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, São Paulo, Brazil Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, São Paulo, Brazil d Département de Pharmacologie, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Québec, Canada e Departamento de Enfermagem Psiquiátrica e Ciências Humanas, Escola de Enfermagem de Ribeirão Preto, Laboratório de Farmacologia, USP, Ribeirão Preto, São Paulo, Brazil b c

a r t i c l e

i n f o

Article history: Received 28 January 2015 Received in revised form 26 June 2015 Accepted 29 June 2015 Available online xxxx Keywords: Fluoxetine Endothelial dysfunction Oxidative stress Vascular reactivity Prostanoids

a b s t r a c t Objectives: The effects of chronic fluoxetine treatment were investigated on blood pressure and on vascular reactivity in the isolated rat aorta. Methods and results: Male Wistar rats were treated with fluoxetine (10 mg/kg/day) for 21 days. Fluoxetine increased systolic blood pressure. Chronic, but not acute, fluoxetine treatment increased the contractile response induced by phenylephrine, serotonin (5-HT) and KCl in endothelium-intact rat aortas. L-NAME and ODQ did not alter the contraction induced by phenylephrine and 5-HT in aortic rings from fluoxetine-treated rats. Tiron, SC-560 and AH6809 reversed the increase in the contractile response to phenylephrine and 5-HT in aortas from fluoxetine-treated rats. Fluoxetine treatment increased superoxide anion generation (O− 2 ) and the expression of cyclooxygenase (COX)-1 in the rat aorta. Reduced expression of nNOS, but not eNOS or iNOS was observed in animals treated with fluoxetine. Fluoxetine treatment increased prostaglandin (PG)F2α levels but did not affect thromboxane (TX)B2 levels in the rat aorta. Reduced hydrogen peroxide (H2O2) levels and increased catalase (CAT) activity were observed after treatment. Conclusions: The major new finding of our study is that chronic fluoxetine treatment induces endothelial dysfunction, which alters vascular responsiveness by a mechanism that involves increased oxidative stress and the generation of a COX-derived vasoconstrictor prostanoid (PGF2α). Moreover, our results evidenced a relation between the period of treatment with fluoxetine and the magnitude in the increment of blood pressure. Finally, our findings raise the possibility that fluoxetine treatment increases the risk for vascular injury, a response that could predisposes to cardiovascular diseases. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Cardiovascular mortality in patients taking psychotropic drugs is high. Tricyclic antidepressants (TCAs), among others, are associated with increased risk of cardiac arrhythmias and death [1]. Fluoxetine belongs to a class of new generation antidepressants which are collectively know as selective serotonin (5-HT) reuptake inhibitors (SSRIs). Those drugs are considered to be free from the cardiotoxic effects of TCAs. However, mounting evidences suggest that SSRIs induce cardiovascular dysfunction such as arrhythmias, electrocardiogram abnormalities and rest bradycardia [2–4]. Fluoxetine displayed potent inhibitory

⁎ Corresponding author at: Universidade de São Paulo, Escola de Enfermagem de Ribeirão Preto, Laboratório de Farmacologia, Avenida Bandeirantes 3900, CEP 14040-902 Ribeirão Preto, São Paulo, Brazil. E-mail address: [email protected] (C.R. Tirapelli).

properties on Na+, Ca2+ and K+ channels in cardiac tissue in vitro [5]. More recently, treatment with fluoxetine for 21 days was showed to induce mild hypertension and enhanced baroreflex responses associated with bradycardia [6]. The cardiac effects of fluoxetine are well characterized, but information regarding the effects of SSRIs on the vasculature is limited. Ungvari et al. [7] showed that fluoxetine induced endotheliumindependent relaxation of isolated rat cerebral arteries. Moreover, fluoxetine inhibited the contraction induced by 5-HT, noradrenaline and Bay K 8644, a voltage-dependent Ca2+ channel opener, further suggesting that fluoxetine blocks Ca2 + channels in the vascular smooth muscle. Similar results were observed in arterioles from rat skeletal muscle where fluoxetine reduced intracellular Ca2+ concentration [8]. Although in vitro studies have shown that fluoxetine affects vascular reactivity to vasoconstrictor agents, there is no evidence on the effect of chronic fluoxetine treatment on vascular responsiveness to vasoactive agents.

http://dx.doi.org/10.1016/j.vph.2015.06.015 1537-1891/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: J.A. Simplicio, et al., Contribution of oxidative stress and prostanoids in endothelial dysfunction induced by chronic fluoxetine treatment, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.015

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J.A. Simplicio et al. / Vascular Pharmacology xxx (2015) xxx–xxx

The vascular endothelium is important in the maintenance of the vascular tone since it is responsible for the production of endothelial-derived mediators involved in the contraction and relaxation of the vasculature [9]. Endothelial dysfunction results in impaired endothelium-mediated vasodilatation, increased vascular reactivity and is associated with several pathologies in the cardiovascular system, including hypertension [10]. Decreased nitric oxide (NO) bioavailability together with increased reactive oxygen species (ROS) generation contributes to the molecular events underlying endothelial dysfunction [9]. Interestingly, fluoxetine was described to reduce NO release by synovial and striatal cells [11,12]. Moreover, fluoxetine was also shown to reduce the expression of the enzyme NO synthase (NOS) in the rat hippocampus [13]. More recently, Göçmez et al. [14] suggested that chronic fluoxetine treatment impairs the synthesis or availability of NO in the corpus cavernosum. Another interesting observation is that fluoxetine induced ROS generation in human hepatocytes [15]. However, whether fluoxetine treatment increases ROS generation and reduces NO bioavailability in the vasculature remains elusive.

Since fluoxetine reduces NO bioavailability and increases ROS generation in different tissues, we hypothesized that fluoxetine treatment would induce endothelial dysfunction. Although the acute effect of fluoxetine in the vasculature in vitro was previously described, to the best of our knowledge, no studies have evaluated the effect of chronic fluoxetine treatment in the vasculature. In the present study, we investigated the effect of fluoxetine in the responsiveness of the isolated rat aorta and the mechanisms underlying such effect. 2. Methods 2.1. Experimental design Male Wistar rats were housed under standard laboratory conditions with free access to food and water. The housing conditions and experimental protocols were approved by the Animal Ethics Committee of the University of São Paulo — Campus of Ribeirão Preto (#11.1.1593.53.9) and were performed in accordance with the Brazilian animal protection

Fig. 1. Effect of chronic fluoxetine treatment on systolic blood pressure and aortic reactivity to phenylephrine and 5-HT. Systolic arterial pressure was evaluated by plethysmograph in 10 animals of each group (A). Concentration–response curves for phenylephrine (B) and 5-HT (C) were determined in endothelium-intact (Endo+) and endothelium-denuded (Endo−) rat aortic rings. Values are means ± SEM of 5 to 8 independent preparations. *Compared to control group; #compared to chronic fluoxetine group on the 7th and 11th days;**compared to chronic fluoxetine on the 16th day (p b 0.05, two-way ANOVA followed by Newman–Keuls multiple comparison test); **compared to control Endo+ (p b 0.05, ANOVA followed by Newman–Keuls multiple comparison test).



laws and institutional guidelines. The rats, initially weighing 230–260 g (50–60 days old), were randomly divided into two groups: Chronic vehicle: daily intraperitoneal (i.p.) injections of vehicle (saline + 0.2% Tween-80, 1 ml/kg) for 21 days; Chronic fluoxetine: daily i.p. injections of fluoxetine (10 mg/kg) for 21 days [6,16,17]. For blood pressure experiments, a group of rats was treated with fluoxetine for the 1st to the 16th day and then with vehicle from the 17th to the 21st day. For vascular reactivity experiments, groups treated acutely with fluoxetine or vehicle were studied: Acute vehicle: a single injection of vehicle (saline + 0.2% Tween-80, 1 ml/kg, i.p.); Acute fluoxetine: a single injection of fluoxetine (10 mg/kg, i.p.) [6,16,17]. Animals submitted to acute treatment were left undisturbed, except for cleaning the cages, in the animal care unit for the same period as animals submitted to chronic treatments. 2.2. Blood pressure measurements The systolic blood pressure was measured every 3 days in conscious rats using a noninvasive tail-cuff plethysmography (Plethysmograph EFF-306, Insight, Ribeirão Preto, Brazil). The rats were maintained for 5–10 min in a warm chamber. Three consecutive recordings (∼2 min apart) were performed and results are expressed as the mean of the three recordings. Tail-cuff recordings were obtained 30–40 min after fluoxetine injection. Systolic blood pressure is expressed as mm Hg. 2.3. Vascular reactivity experiments The rats were anesthetized intraperitoneally with urethane 25% (1.25 g/kg, Sigma-Aldrich, St. Louis, MO, USA) and killed by aortic exsanguination. The thoracic aorta was quickly removed, cleaned of adherent connective tissues, cut into rings (5–6 mm in length) and placed in 5-ml organ chambers as previously described [18]. In some rings, the endothelium was removed mechanically by gently rolling the lumen of the vessel on a thin wire. Endothelial integrity was qualitatively assessed by measuring the degree of relaxation induced by acetylcholine (1 μM) in the presence of contractile tone induced by phenylephrine (0.1 μM). For studies of endothelium-intact vessels, the ring was discarded if the relaxation induced by acetylcholine was less than 50%. For studies of endothelium-denuded vessels, the rings were discarded if there was any degree of relaxation. Cumulative concentration–response curves for phenylephrine (0.0001–10 μM) or 5-HT (0.001–100 μM) were determined on endothelium-intact and endothelium-denuded rat aortic rings. In another set of experiments, the rings were stimulated with KCl (90 mM). Contractions were expressed as changes in the displacement (grams) from baseline since no differences on tissue mass among the groups were observed (data not shown). The aortic rings were pre-contracted with phenylephrine (0.1 μM) and when the contraction reached a plateau, acetylcholine (0.0001–10 μM), carbachol (0.0003–30 μM) or SNP (0.0001–0.3 μM) were added cumulatively. The magnitude of contraction induced by phenylephrine did not differ among the experimental groups at 0.1 μM. Relaxation is expressed as a percentage change from phenylephrine-contracted levels. The agonist concentration–response curves were fitted using a nonlinear interactive fitting program (Graph Pad Prism 3.0; GraphPad Software Inc., San Diego, CA, USA). Agonist potencies and maximal responses were expressed as pD2 (negative logarithm of the molar concentration of agonist producing 50% of the maximal response) and Emax (maximum effect elicited by the agonist), respectively. 2.4. Vascular reactivity protocols Cumulative concentration–response curves for phenylephrine or 5HT were obtained in endothelium-intact aortic rings from control and 21-days fluoxetine-treated rats after incubation for 30 min with the following drugs: NG-nitro-L-arginine methyl ester (L-NAME, non-

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selective nitric oxide synthase [NOS] inhibitor, 100 μM), 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, selective guanylyl cyclase inhibitor, 1 μM), indomethacin (non-selective cyclooxygenase [COX] inhibitor, 10 μM), SC-560 (selective COX-1 inhibitor, 1 μM), SC-236 (selective COX-2 inhibitor, 10 μM), AH6809 (antagonist of prostaglandin F2α [PGF2α] receptors, 10 μM), SQ29548 (antagonist of PGH2/thromboxane A2 [TXA2] receptors, 3 μM) or tiron (superoxide anion [O− 2 ] scavenger, 1 mM). To determine the contribution of basal or spontaneous production and release of NO, concentration–response curves for L-NAME (0.1–300 μM) were obtained on endothelium-intact aortic rings that were pre-contracted submaximally with phenylephrine 0.01 μM (that is, to induce 10–30% maximal response) [18]. These experiments were performed in aortic rings from control and 21-days fluoxetine-treated rats. In another set of experiments, endothelium-intact rings were pre-contracted sub-maximally with phenylephrine (0.1 μM) and Larginine (0.1 nM–1 mM) was added cumulatively. 2.5. Determination of thromboxane B2 (TXB2) and prostaglandin F2α (PGF2α) in the rat aorta Frozen aortas were homogenized in EIA buffer and centrifuged (2000 ×g, 15 min, 4 °C). The samples (50 μl) were deproteinized by precipitation using 50 μl of absolute ethanol kept at 4 °C. The supernatant was centrifuged at 4000 ×g (10 min, 25 °C). TXB2, the stable metabolite of TXA2, and PGF2α, were measured using commercially available kits (#519031 and #516011, respectively; Cayman Chemical, Ann Arbor, MI, USA). Results were normalized for protein concentration and are expressed as pg/mg protein. Protein concentrations in all experiments were determined by the Lowry protein assay (Bio-Rad Laboratories, Hercules, CA, USA). 2.6. Detection of aortic O− 2 by lucigenin enhanced chemiluminescence The lucigenin-derived chemiluminescence assay was used to determine O− 2 levels in aortic homogenates as previously described [19–21]. Luminescence was measured in an Orion II Luminometer (Berthold detection systems, Pforzheim, Germany). Superoxide anion generation is expressed as relative light unit (RLU)/mg protein. 2.7. Determination of plasma and aortic thiobarbituric acid reactive substances (TBARS) The aortas were isolated and frozen in liquid nitrogen. The tissues were homogenized in RIPA buffer containing protease inhibitor and centrifuged (1600 ×g, 10 min, 4 °C). Blood was collected from the aorta in heparinized syringes and centrifuged at 1000 ×g (10 min, 4 °C). TBARS was determined colorimetrically at 540 nm following instructions of a commercially available kit (#10009055, Cayman Table 1 Effect of chronic treatment with fluoxetine on the Emax (grams) and pD2 values for phenylephrine and 5-HT in rat aortic rings with intact (Endo+) or denuded endothelium (Endo−). Emax

pD2

Endo+

Endo−

Endo+

Endo−

Phenylephrine Control Fluoxetine

1.2 ± 0.1 (7) 1.8 ± 0.1 (7)⁎

1.6 ± 0.2 (6)⁎ 1.9 ± 0.2 (7)⁎

7.4 ± 0.4 7.1 ± 0.1

8.0 ± 0.2⁎ 7.5 ± 0.2

5-HT Control Fluoxetine

1.2 ± 0.1 (5) 1.7 ± 0.1 (8)⁎

1.5 ± 0.1 (5)⁎ 1.6 ± 0.1 (6)⁎

5.4 ± 0.13 5.8 ± 0.14

5.9 ± 0.10 6.0 ± 0.12

Number within parentheses indicates the number of independent preparations. Values are means ± S.E.M. ⁎ Compared to respective control group Endo+ (p b 0.05, ANOVA followed by Newman– Keuls multiple comparison test).


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Chemical, Ann Arbor, MI, USA). Results are expressed as μmol/g protein or μmol/l of plasma.

2.8. Measurement of plasma and aortic nitrate/nitrite (NOx) Plasma and aortic NOx were measured using a commercially available kit (#780001, Cayman Chemical, Ann Arbor, MI, USA) as described previously [22]. Results are expressed as μmol/g protein or μmol/l of plasma.

2.9. Detection of aortic H2O2 concentration Amplex red (#A22188, Invitrogen, Carlsbad, CA, USA) was used to measure aortic H2O2 concentration. The aorta was homogenized in Krebs solution [(mM): NaCl 130; KCl 4.7; KH2PO4 1.18; MgSO4 1.17; NaHCO3 14.9; Glucose 5.5; CaCl2 1.6; pH: 7.4] with a glass-to-glass homogenizer. The homogenates were centrifuged at 10,000 rpm (10 min, 4 °C). Fifty microliters of Amplex Red reagent (10-acetyl3,7-dihydroxyphenoxazine) and horseradish peroxidase type II (0.1 unit/ml) were added to the samples (50 μl). Samples were then

Fig. 2. Effect of L-NAME, ODQ and tiron on phenylephrine and 5-HT-induced contraction in endothelium-intact aortas from control and fluoxetine-treated animals. Concentration– response curves for phenylephrine and 5-HT were determined in endothelium-intact rat aortic rings in the absence or after incubation for 30 min with L-NAME (100 μM), ODQ (1 μM) or tiron (1 mM). Values are means ± SEM of 5 to 8 independent preparations. *Compared to control group; **compared to fluoxetine group (p b 0.05, ANOVA followed by Newman–Keuls multiple comparison test).



incubated (30 min) in the dark at room temperature (25 °C) in 96-well microplates. 10-Acetyl-3,7-dihydroxyphenoxazine is a colorless, nonfluorescent reagent that reacts with H2O2 to produce resorufin, a redfluorescent compound that was analyzed using an excitation wavelength of 571 nm and an emission wavelength of 585 nm. Aortic H2O2 concentrations were calculated on the basis of H2O2 standard curve. Results are expressed as nmol/mg protein. 2.10. Determination of superoxide dismutase (SOD) and catalase (CAT) activity in the rat aorta The aortas were homogenized in 200 μl PBS buffer (pH 7.4) and centrifuged at 10,000 ×g (10 min, 4 °C). The supernatant was analyzed for SOD activity using a commercially available kit (#19160, SigmaAldrich, St. Louis, MO, USA). The results were normalized for protein concentration and SOD activity is expressed as inhibition rate %/mg protein. Aortic CAT activity was assayed by H2O2 consumption [23]. The aorta was homogenized in phosphate buffer with a glass-to-glass homogenizer as previously described [21]. Reaction buffer was added to quartz cuvettes containing 20 μl of the supernatant. The absorbance was read for 1 min at 240 nm. One CAT unit (U) was defined as the amount of enzyme required to decompose 1 μmol of H2O2/min.

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done with skim milk (7%) in Tris-buffered saline solution (Tris −10 mM, NaCl-150 mM, Tween 20) for 1 h at room temperature. Membranes were incubated (overnight at 4 °C) with the following primary antibodies: COX-1 (diluted 1:1000, sc-166573, Santa Cruz Biotechnology, Dallas, Texas, USA), COX-2 (diluted 1:1000, 4842S Cell Signaling, Danvers, Massachusetts, USA), nNOS (diluted 1:500, 4234S, Cell Signaling), iNOS (diluted 1:500, sc-650, Santa Cruz Biotechnology) or eNOS (diluted 1:500, N3893, Sigma-Aldrich, St. Louis, MO, USA). Signals were revealed with chemiluminescence after incubation with secondary antibodies for 90 min at room temperature. Signals were visualized using a ChemiDoc™ XRS+ (Bio-Rad, USA) and quantified by densitometry. Beta-actin (diluted 1:5000, sc-4778, Santa Cruz Biotechnology) was used as an internal control. 2.15. Statistical analysis Data are presented as means ± standard error of the mean (SEM). Statistically significant differences were calculated by Student's t-test, two-way or one-way ANOVA followed by Newman–Keuls multiple comparison test and p b 0.05 was considered as statistically significant. 3. Results 3.1. Body weight

2.11. Determination of reduced glutathione (GSH) in the rat aorta Aortic GSH was evaluated through its interaction with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) as previously described [21]. Results are expressed as μg GSH/mg protein. 2.12. Measurement of acetylcholinesterase (AChE) activity on plasma and aorta Plasma and aortic AChE activity was measured fluorometrically using a commercially available kit (#A12217, Invitrogen, Carlsbad, CA, USA). Results are expressed as Um/mg protein or Um/ml of plasma.

Before treatment, rats showed mean body masses of 257 ± 4 g (control group) and 245 ± 4 g (fluoxetine). After treatment for 21 days, no variation in body mass was observed in animals between the two experimental groups: 407 ± 7 g (control n = 40) and 395 ± 6 g (fluoxetine n = 40). 3.2. Effect of chronic treatment with fluoxetine on blood pressure Baseline values of systolic blood pressure were similar in control (119 ± 1 mm Hg, n = 10) and fluoxetine-treated animals (120 ± 1 mm Hg, n = 10). Significant increased blood pressure was observed in 7-days fluoxetine-treated animals. This response increased in day

2.13. Quantitative real-time polymerase chain reaction (qRT-PCR) Trizol® Reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total cellular RNA. After extraction, RNA was reverse-transcribed to single-stranded cDNA using a High Capacity Kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer's protocol. For the quantitative analysis of the genes of interest (eNOS [Rn 02132634_s1], nNOS [Rn 00583793_m1], iNOS [Rn 00561646_m1], COX-1 [Rn 01483828_m1] and COX-2 [Rn 00566881_m1]) the commercially available TaqMan Assay-on-Demand System was used. Reverse transcription was performed using 1 μg total RNA for each sample in 20 μl of the total reaction mixture. The cDNA obtained was diluted 1:10, and 4.5 μl was used for each 10 μl of the RQ-PCR mixture using the TaqMan Master Mix (Applied Biosystems). All reactions were carried out in duplicate and analyzed with the 7500 Sequence Detection System apparatus (Applied Biosystems). Data were analyzed using the ABI-7500 SDS software. The total RNA absorbed was normalized on the basis of the Ct value for the GAPDH gene (Rn 01775763_m1). The variation in expression among samples was calculated by the 2−ΔΔCt method, with the mean delta Ct value for a group of 6 samples from control rats used for calibration. 2.14. Western immunoblotting Samples were prepared as previously described [20]. Forty micrograms of protein was separated by electrophoresis on a 10% polyacrylamide gel (1.5 h at 150 V) in mini-gel apparatus (Mini Protean III, BioRad, Hercules, CA, USA). Proteins were transferred onto a nitrocellulose membrane (1.5 h at 100 V). Blockage of nonspecific binding sites was

Table 2 Effect of several inhibitors on the Emax (grams) and pD2 values for phenylephrine and 5-HT in endothelium-intact rat aortic rings. Control

Phenylephrine None (100 μM) ODQ (1 μM) Tiron (1 mM) Indomethacin (10 μM) SC-560 (1 μM) SC-236 (10 μM) AH6809 (10 μM) SQ29548 (3 μM) L-NAME

5-HT None L-NAME

ODQ Tiron Indomethacin SC-560 SC-236 AH6809 SQ29548

Fluoxetine

Emax

pD2

Emax

pD2

1.2 ± 0.1 (7) 1.9 ± 0.2 (5)⁎ 1.8 ± 0.4 (4)⁎

7.4 ± 0.4 7.9 ± 0.5

1.8 ± 0.1 (7)⁎ 1.7 ± 0.2 (5)⁎ 2.0 ± 0.2 (4)⁎ 1.1 ± 0.1 (4)⁎⁎ 1.4 ± 0.1 (4)⁎⁎ 1.3 ± 0.1 (6)⁎⁎ 1.6 ± 0.1 (8)⁎ 1.1 ± 0.1 (6)⁎⁎ 1.6 ± 0.1 (6)⁎

7.1 ± 0.1 7.6 ± 0.1

1.4 ± 0.1 (5) 1.4 ± 0.1 (5) 1.3 ± 0.1 (7) 1.4 ± 0.1 (6) 1.2 ± 0.1 (6) 1.2 ± 0.1 (6)

1.2 ± 0.1 (5) 1.6 ± 0.1 (8)⁎ 1.8 ± 0.1 (4)⁎ 1.0 ± 0.1 (5) 1.2 ± 0.1 (7) 1.2 ± 0.2 (5) 1.2 ± 0.2 (5) 1.3 ± 0.1 (6) 1.1 ± 0.1 (7)

7.3 ± 0.1 7.1 ± 0.2 6.9 ± 0.2 6.9 ± 0.1 6.9 ± 0.1 6.9 ± 0.1 6.9 ± 0.1

5.4 ± 0.1 5.6 ± 0.1 5.6 ± 0.1 5.5 ± 0.1 5.6 ± 0.2 6.9 ± 0.4 6.3 ± 0.1 5.7 ± 0.1 5.8 ± 0.2

1.7 ± 0.1 (8)⁎ 1.7 ± 0.2 (6)⁎ 1.8 ± 0.3 (4)⁎ 1.3 ± 0.2 (6)⁎⁎ 1.1 ± 0.2 (6)⁎⁎ 1.3 ± 0.1 (5)⁎⁎ 1.5 ± 0.1 (7)⁎ 1.2 ± 0.1 (5)⁎⁎ 1.5 ± 0.1 (6)⁎

7.4 ± 0.1 7.0 ± 0.2 6.8 ± 0.1 7.2 ± 0.1 7.1 ± 0.2 6.9 ± 0.1 7.2 ± 0.1

5.8 ± 0.2 5.9 ± 0.2 6.5 ± 0.2 6.3 ± 0.1 5.7 ± 0.1 6.9 ± 0.1 6.9 ± 0.1 6.4 ± 0.1 5.9 ± 0.1

Number within parentheses indicates the number of independent preparations. Values are means ± S.E.M. ⁎ Compared to respective control group without inhibitor. ⁎⁎ Compared to fluoxetine group without inhibitor (p b 0.05, ANOVA followed by Newman–Keuls multiple comparison test).


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15 and remained stable until the 21st day of treatment (Fig. 1A). A significant decrease in blood pressure was observed 1 day after interruption in fluoxetine treatment. Blood pressure values returned to control levels within 2 days after interruption in fluoxetine treatment (Fig. 1A). 3.3. Effect of acute treatment with fluoxetine on the aortic reactivity to phenylephrine, 5-HT, acetylcholine and SNP Acute treatment with fluoxetine did not alter the contraction induced by phenylephrine (Emax: 1.4 ± 0.1 g, n = 7; pD2: 7.2 ± 0.1) in endothelium-intact rings, when compared to control group (Emax: 1.3 ± 0.1, n = 6; pD2: 7.0 ± 0.2). In endothelium-denuded rings, the Emax or pD2 values for phenylephrine did not significantly differ between control (Emax: 1.4 ± 0.1, n = 7; pD2:7.5 ± 0.3) and fluoxetinetreated rats (Emax: 1.5 ± 0.1, n = 7; pD2:7.8 ± 0.2). Similarly, the contraction induced by 5-HT was not altered after acute treatment with fluoxetine in either endothelium-intact (Emax: 1.2 ± 0.1 g, n = 7; pD2: 6.2 ± 0.2) or -denuded aortic rings (Emax: 1.4 ± 0.1 g, n = 7; pD2:6.7 ± 0.1), when compared to control group with intact (Emax: 1.4 ± 0.3 g, n = 6; pD2:5.5 ± 0.2) or denuded endothelium (Emax: 1.4 ± 0.2 g, n = 7; pD2: 6.3 ± 0.1). Acute treatment with fluoxetine did not alter the relaxation induced by acetylcholine (Emax: 106.2 ± 5.2%, n = 5; pD2: 7.4 ± 0.1), when compared to control group (Emax: 99.1 ± 3.7%, n = 8; pD2: 7.2 ± 0.1). Similarly, SNP-induced relaxation of endothelium-denuded rings was not different between control (Emax: 115.3 ± 4.2%, n = 7; pD2: 7.8 ± 0.1) and fluoxetine-treated rats (Emax: 114.7 ± 4.5, n = 7; pD2: 8.2 ± 0.1). Finally, KCl-induced contraction of endothelium-intact (Endo +) or endothelium-denuded (Endo −) rings was not different between control (Endo +: 1.1 ± 0.1 g, n = 6; Endo−: 1.4 ± 0.1 g, n = 5) and fluoxetine-treated tissues (Endo+: 1.0 ± 0.1 g, n = 8; Endo−: 1.2 ± 0.1 g, n = 7). 3.4. Effect of chronic treatment with fluoxetine on the aortic reactivity to phenylephrine, 5-HT, acetylcholine and SNP Chronic treatment with fluoxetine enhanced the maximal contraction (Emax) induced by phenylephrine and 5-HT in endotheliumintact, but not endothelium-denuded aortic rings (Fig. 1B and C). However, no differences were observed in pD2 values between the experimental groups (Table 1). It was also noticeable that mechanical removal of the endothelium significantly increased Emax values for phenylephrine and 5-HT in arteries from the control rats (Table 1). Chronic treatment with fluoxetine did not alter the relaxation induced by acetylcholine (Emax: 94.6 ± 6.4%, n = 6; pD2: 7.2 ± 0.2), when compared to control group (Emax: 101.7 ± 10.3%, n = 5; pD2: 7.0 ± 0.2). Fluoxetine did not alter the endothelium-dependent relaxation induced by carbachol (Emax: 109.1 ± 8.1%, n = 7; pD2: 7.3 ± 0.2), when compared to control group (Emax: 107.5 ± 6.9%, n = 6; pD2: 7.4 ± 0.3). Similarly, SNP-induced relaxation of endothelium-denuded rings was not different between control (Emax: 121.0 ± 4.6, n = 6; pD2: 7.9 ± 0.1) and fluoxetine-treated rats (Emax: 121.4 ± 2.5, n = 5; pD2: 8.1 ± 0.2). Additionally, KCl-induced contraction of endothelium-denuded rings was not different between control (1.2 ± 0.1 g, n = 5) and chronically fluoxetine-treated aortas (1.3 ± 0.1 g, n = 6). Conversely, the contraction induced by KCl in endothelium-intact rings was increased after chronic treatment with fluoxetine (1.4 ± 0.1 g, n = 7), when compared to the control group (1.0 ± 0.1 g, n = 5) (p b 0.05; ANOVA). Pre-incubation with L-NAME significantly increased phenylephrineand 5-HT-induced contraction in endothelium-intact rings of control rats. Conversely, L-NAME did not alter the contraction induced by both agonists in tissues from the fluoxetine-treated rats (Fig. 2, Table 2).

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In the presence of indomethacin, the Emax values for phenylephrine or 5-HT in rings from fluoxetine-treated rats were reduced to values similar to the ones observed in control aortas. No changes in pD2 values were detected (Table 2). Similar results were found with SC-560, tiron and AH6809. On the other hand, neither SC-236 nor SQ29548, altered the contraction induced by phenylephrine or 5-HT in arteries from both control and fluoxetine-treated rats (Fig. 3, Table 2). L-NAME, a non-selective NOS inhibitor, induced a concentrationdependent increase in the tension of endothelium-intact aortic rings that were submaximally pre-contracted with phenylephrine. L-NAMEinduced increase in tension was not different between control (Emax: 1.1 ± 0.2 g, n = 5; pD2: 4.8 ± 0.04) and chronically fluoxetinetreated rats (Emax: 1.2 ± 0.2 g, n = 6; pD2: 4.6 ± 0.08). When the endothelium was removed, L-NAME failed to increase the tension in aortic rings from both control and fluoxetine-treated rats (data not shown). The relaxation induced by L-arginine in endothelium-intact rings did not significantly differ between the aortas from control (Emax: 90.0 ± 8.5%, n = 5; pD2: 5.9 ± 0.3) and fluoxetine-treated rats (Emax: 93.1 ± 5.3%, n = 7; pD2: 5.8 ± 0.2).

3.5. Effect of fluoxetine treatment on TXB2 and PGF2α levels in the rat aorta Chronic treatment with fluoxetine increased aortic PGF2α levels, when compared to control group. On the other hand, no differences on TXB2 levels were found between arteries from control and fluoxetine-treated rats (Fig. 4A and B). 3.6. Effect of fluoxetine treatment on O− 2 generation, TBARS levels and nitrate/nitrite (NOx) levels Fig. 5A shows that lucigenin-derived luminescence was significantly higher in aortas from fluoxetine-treated rats. On the other hand, chronic treatment with fluoxetine did not alter plasma or aortic TBARS (Fig. 5B and C). Chronic treatment with fluoxetine did not alter plasma NOx (Fig. 5D). Similarly, no difference on basal NOx levels was found between arteries from control and fluoxetine-treated rats (Fig. 5E). 3.7. Effect of fluoxetine treatment on the protein expression and mRNA levels of NOS and COX isoforms in the rat aorta Protein expression of COX-1, but not COX-2, was increased in aortas from fluoxetine-treated rats (Fig. 6). As shown in Fig. 7, chronic treatment with fluoxetine reduced nNOS expression but did not alter the expression of eNOS or iNOS in the rat aorta. Treatment with fluoxetine did not alter the mRNA levels of eNOS, nNOS, iNOS, COX-1 or COX-2 (Figs. 6 and 7). 3.8. Effect of fluoxetine treatment on H2O2 and GSH levels and SOD and CAT activities in the rat aorta Chronic treatment with fluoxetine reduced aortic H2O2 levels (Fig. 8A). Increased CAT activity was evidenced in aortas from fluoxetine-treated animals (Fig. 8B). On the other hand, no alteration on SOD activity as well as on GSH levels was observed after treatment with fluoxetine (Fig. 8C and D). 3.9. Effect of fluoxetine treatment on plasma and aortic AChE activity Chronic treatment with fluoxetine did not change plasma AChE activity (112.1 ± 9.8 mU/ml, n = 10), when compared to control group

Fig. 3. Effect of SC-560, SC-236, AH6809 and SQ29548 on phenylephrine and 5-HT-induced contraction in endothelium-intact aortas form control and fluoxetine-treated animals. Concentration–response curves for phenylephrine and 5-HT were determined in endothelium-intact rat aortic rings in the absence or after incubation for 30 min with SC-560 (1 μM), SC-236 (10 μM), AH6809 (10 μM) and SQ29548 (3 μM). Values are means ± SEM of 5 to 8 independent preparations. *Compared to other groups (p b 0.05, ANOVA followed by Newman– Keuls multiple comparison test).


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(115.3 ± 6.4 mU/ml, n = 10). Similarly, no differences on AChE activity were found between aortas from control (497.8 ± 57 mU/mg, n = 8) and fluoxetine-treated rats (527.4 ± 48 mU/mg, n = 9). 4. Discussion

Fig. 4. Effect of chronic fluoxetine treatment on PGF2α and TXB2 levels in the rat aorta. Basal levels of PGF2α (A) and TXB2, a stable metabolite of TXA2 (B) were measured in the rat aorta by ELISA. Results are presented as means ± SEM of 6 to 8 independent preparations. *Compared to control group (p b 0.05, Student's t test).

Our findings showing that chronic treatment with fluoxetine increased systolic blood pressure are in accordance with previous experimental and clinical studies [4,6,24]. Importantly, we first demonstrated a time-course for blood pressure increase associated with fluoxetine treatment. Significant blood pressure increase concomitant with fluoxetine was already observed after 7 days of treatment and up until day 15 at which time a sustained plateau is reached. Thus, results presented here evidenced a relation between the period of treatment with fluoxetine and the magnitude of blood pressure increase. Since blood pressure was already elevated in 7-day treated rats, our results support the concept that the hypertensive state associated with fluoxetine can be already apparent in early stages of the treatment. However, our data does not rule out the possibility that fluoxetine continues to display a timedependent effect at periods of treatment longer than that employed in the present study. Importantly, fluoxetine-induced hypertension is transient since blood pressure values returned to control levels within 2 days after interruption in fluoxetine treatment. This result raises the possibility that the increase in blood pressure is dependent on a direct effect of fluoxetine on the vasculature and seems not to involve morphological changes, such as arterial remodeling. Our data also shows that chronic, but not acute fluoxetine treatment induced an enhanced responsiveness to phenylephrine, 5-HT and KCl in endothelium-intact aortas. In contrast, after endothelial removal, the hyper-reactivity to these agonists was not observed, further suggesting that chronic fluoxetine treatment induces endothelial dysfunction. Our results also suggest that the hyper-reactivity to the vasoactive agents induced by fluoxetine treatment was the result of a nonspecific increase in the reactivity of the rat aorta, because the contractile response of these arteries to all vasoactive agents tested was affected by fluoxetine treatment. Our functional findings differ from those reported on rat brain arterioles where fluoxetine was found to induce NO-mediated vascular relaxation [25]. In that study, the cortex was superfused with fluoxetine (1–500 μM) and the diameter of pial vessels was monitored. Thus, the type of blood vessel studied as well as the method of fluoxetine administration could explain the discrepancy between our findings and those described by Ofek [25]. Endothelial vasoconstrictor prostanoids derived from the arachidonic acid–COX pathway modulate vascular contraction in the rat aorta [26]. The maximal contraction induced by phenylephrine and 5-HT was significantly diminished by indomethacin and SC-560 in endothelium-intact aortic rings from fluoxetine-treated rats, suggesting that the enhanced responsiveness of fluoxetine-treated aortas to these agonists was due to an increased release of endothelial vasoconstrictor prostanoids derived from COX-1. Additionally, we found increased COX-1 expression in the aorta after treatment with fluoxetine, which is consistent with our functional findings. Nevertheless, the mechanisms by which fluoxetine up-regulates COX-1 expression and whether it involves direct or indirect pathways are not yet clear. One possible explanation may involve the enhancement in vascular oxidative stress, which has been described to increase vascular COX-1 expression [27]. However, it is possible to conclude that fluoxetine increased COX-1 expression at a post-translational level since mRNA levels of COX-1 were not altered by the treatment. The enhanced PGF2α generation is consistent with the functional effect of the antagonist AH6809 on 5-HT or phenylephrine-induced contraction and suggests that PGF2α accounts for the enhanced reactivity to these vasoactive agents after treatment with fluoxetine. Taken together, our findings provide the first experimental evidence of a relationship between chronic fluoxetine consumption, up-regulation of COX-1 and increased PGF2α generation in the vasculature.



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Fig. 5. Effect of chronic fluoxetine treatment in systemic and vascular oxidative stress and NOx levels. Bar graphs represent O− 2 levels in aortic tissue (A) and TBARS concentrations in the plasma (B) or aorta (C). Basal plasma (D) and aortic (E) nitrate/nitrite (NOx) were measured colorimetrically. Results are presented as means ± SEM of 6 to 8 independent preparations. *Compared to control group (p b 0.05, Student's t test).


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Fig. 6. Effect of chronic fluoxetine treatment on the expression and mRNA levels of COX-1 and COX-2 in the rat aorta. Top panels, representative immunoblots for COX-1 and COX-2 protein expression. Bottom panels, corresponding bar graphs showing densitometric data for protein expression (left) or mRNA levels (right) of COX-1 (A) and COX-2 (B). Results are presented as means ± SEM of 4 to 8 independent preparations. *Compared to control group (p b 0.05, Student's t test).

NO displays an important vasodilator action and reduced NO bioavailability is associated with endothelial dysfunction as well as increased vascular reactivity [9]. The increase in tension induced by a NOS inhibitor reflects the amount of constitutively available NO, which modulates contractile responses of the vascular smooth muscle [28]. Our results show that the additional endothelium-dependent increase of the phenylephrine-induced contraction produced by L-NAME as well as the relaxation induced by L-arginine did not differ between groups, suggesting that fluoxetine treatment did not alter the basal production/release of NO. Additionally, in the present study, relaxation induced by SNP revealed no differences in the sensitivity to NO in the smooth muscle cells of the aortas from fluoxetine-treated rats. Finally, basal nitrate/nitrite levels did not differ between the groups. These results overall indicate that fluoxetine treatment did not alter the basal production/release of NO. Incubation of control aortas with L-NAME and ODQ significantly enhanced the maximal contraction induced by phenylephrine and 5-HT,

indicating an inhibitory role for the NO–cGMP pathway in the modulation of the contractile response of the aorta to these agonists. The lack of effect of L-NAME and ODQ in the contractile response to phenylephrine and 5-HT in aortas from fluoxetine-treated rats suggests that the enhanced responsiveness of fluoxetine-treated aortas to these agonists was due to an impairment of the endothelial NO–cGMP pathway. Vasoconstrictor agents, such as phenylephrine can modulate the production of vascular NO [29]. Thus, the increased responsiveness of aortas from fluoxetine-treated rats to phenylephrine and 5-HT could be a result of an impaired modulation of NO on the contractile response induced by these agonists. Surprisingly, fluoxetine treatment did not influence the endothelium-dependent relaxation induced by acetylcholine, despite the increase in vascular contractility. This result suggests that fluoxetine mediates a dysfunctional vasoconstriction rather than vasodilatation. A possible explanation for this response is that differential regulation of endothelial NO release may depend on the vasoactive agonist producing endothelium-dependent relaxation (such as acetylcholine), or



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Fig. 7. Effect of chronic fluoxetine treatment on the expression and mRNA levels of eNOS, nNOS and iNOS in the rat aorta. Top panels, representative immunoblots for eNOS, nNOS and iNOS protein expression. Bottom panels, corresponding bar graphs showing densitometric data for protein expression (left) or mRNA levels (right) of eNOS (A), nNOS (B) and iNOS (C). Results are presented as means ± SEM of 4 to 8 independent preparations. *Compared to control group (p b 0.05, Student's t test).


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Fig. 8. Effect of chronic fluoxetine treatment on H2O2 levels (A), CAT (B) and SOD (C) activities and GSH (D) levels in the rat aorta. Results are presented as means ± SEM of 6 to 8 independent preparations. *Compared to control group (p b 0.05, Student's t test).

contraction (such as phenylephrine) [30]. The lack of effect of fluoxetine on the relaxation induced by acetylcholine could also be explained by changes in the rate of acetylcholine hydrolysis, due to an altered AChE activity [25], or by an alteration in the expression and/or responsiveness of muscarinic receptors. However, this seems not to be the case in our study since no difference on plasma or aortic AChE activity was observed after treatment with fluoxetine. Moreover, fluoxetine treatment did not alter the relaxation induced by carbachol, a muscarinic agonist which is not easily metabolized by AChE. Both, eNOS and nNOS are constitutively expressed in the vasculature and contribute to NO generation and reduced expression of NOS isoforms in the vasculature is associated with altered vascular functionality [31–33]. For example, increased contractility in response to phenylephrine is described when eNOS is down-regulated in the vasculature [34,35]. Similarly, decreased vascular expression of nNOS is associated with a hyper-reactivity to phenylephrine [36]. Therefore, alterations in the expression of NOS isoforms might explain the increase in phenylephrine and 5-HT-induced contractions in fluoxetine-treated rats. Our findings ruled out a possible role for eNOS in such response since fluoxetine treatment did not alter eNOS expression or mRNA levels in the aorta. In the vasculature, although eNOS is the primary

source of NO under physiological conditions, NO derived from nNOS may also play an important role under various pathological conditions [37]. Thus, the post-translational down regulation of nNOS in aortas from fluoxetine-treated animals here described could contribute to the hyper-reactivity to phenylephrine and 5-HT. The induction of iNOS within the vascular wall would promote oxidative stress through formation of peroxynitrite from the reaction of iNOS-derived NO with O− 2 [38]. For this reason, iNOS induction may be noxious and might ultimately lead to vascular injury. The lack of effect of fluoxetine treatment on iNOS expression suggests that this is not a mechanisms by which fluoxetine induces endothelial dysfunction. The lucigenin-derived chemiluminescence assay used in the present study is based in the enzymatic action of the enzyme NAD(P)H oxidase [39]. Thus, the increased lucigenin-derived chemiluminescence here described suggests that the enzyme NAD(P)H oxidase is an important source of O− 2 generation by fluoxetine in the vasculature. Our functional studies demonstrated that O− 2 scavenging reduced the hyper-reactivity to phenylephrine and 5-HT induced by fluoxetine in endothelium-intact rings. These observations support the concept that O− 2 contributes to fluoxetine-induced endothelium-dependent increase in vascular contraction. This finding corroborates previous studies describing that O− 2



modulates the action of vasoconstrictor agents in pathological conditions such as hypertension and diabetes [40,41]. In addition, O− 2 may also act through activation of vasoconstrictor prostanoids by COX, which are involved in the contraction of vascular smooth muscle cells [40,42]. In the present study, we suggest that O− 2 may act through contractile prostanoids to modulate vasoconstriction since we observed no reduction in basal NO bioavailability. Fluoxetine did not alter plasma or aortic TBARS levels indicating that, despite the increase in vascular O− 2 generation, no alteration in lipid peroxidation was evidenced. Moreover, fluoxetine did not alter GSH levels in the aorta, indicating that the treatment did not diminished cellular antioxidant capacity. O− 2 can act either as an oxidizing agent or as a vascular signaling molecule. This highly unstable molecule is reduced by SOD to H2O2 [43], which is implicated in the regulation of signaling pathways that lead to vascular relaxation [44–46]. Fluoxetine did not alter SOD activity but decreased aortic H2O2 levels. H2O2 is tightly regulated by intracellular and extracellular enzymes, including CAT, which converts H2O2 into water and O2. In this line, the decrease in H2O2 here described could be the result of the increased CAT activity induced by fluoxetine treatment. Both O− 2 and H2O2 act as signaling molecules, but it is mainly H2O2 that is considered a signaling molecule because of its relative stability and sub-cellular localization [47]. Since H2O2 displays vasodilator actions [44,45] and increases phenylephrine-induced contraction at low concentrations [48], the reduction of H2O2 here observed could contribute to the hyper-reactivity to phenylephrine and 5-HT induced by fluoxetine. The present study offers new insights into the cardiotoxic effects of fluoxetine. Major new finding of our study is that chronic fluoxetine treatment induces endothelial dysfunction, which alters vascular responsiveness by a mechanism that involves increased oxidative stress and the generation of a COX-derived vasoconstrictor prostanoid. Moreover, results presented here evidenced a relation between the period of treatment with fluoxetine and the magnitude in the increment of blood pressure. Finally, our findings raise the possibility that fluoxetine treatment increases the risk for vascular injury, a response that could predisposes to cardiovascular diseases. Conflict of interest None declared. Acknowledgments We acknowledge L.H.A. de Camargo for technical support. This work was supported by funds from Fundação de Amparo à Pesquisa do Estado de São Paulo — FAPESP (#2010/05815-4 and #2013/00808-8) and NAPDIN (#11.1.21625.01.0). J.A.S. is supported by a master fellowship from CAPES. References [1] M. Bauer, P.C. Whybrow, J. Angst, M. Versiani, H.J. Möller, World Federation of Societies of Biological Psychiatry (WFSBP) Guidelines for Biological Treatment of Unipolar Depressive Disorders, part 1: acute and continuation treatment of major depressive disorder, World J. Biol. Psychiatry 3 (1) (2002) 5–43. [2] S.A. Spier, M.A. Frontera, Unexpected deaths in depressed medical in patients treated with fluoxetine, J. Clin. Psychiatry 52 (1991) 377–382. [3] T. Ravina, M.L. Suarez, J. Mendez-Castrillon, Fluoxetine-induced QTU interval prolongation, T wave alternans and syncope, Int. J. Cardiol. 65 (1998) 311–313. [4] S.P. Roose, A.H. Glassman, E. Attia, S. Woodring, E.G. Giardina, J.T. Bigger, Cardiovascular effects of fluoxetine in depressed patients with heart disease, Am. J. Psychiatry 155 (1998) 660–665. [5] P. Pacher, V. Kecskemeti, Cardiovascular side effects of new antidepressants and antipsychotics: new drugs, old concerns? Curr. Pharm. Des. 10 (2004) 2463–2475. [6] C.C. Crestani, R.F. Tavares, F.S. Guimarães, F.M. Correa, S.R. Joca, L.B. Resstel, Chronic fluoxetine treatment alters cardiovascular functions in unanesthetized rats, Eur. J. Pharmacol. 670 (2–3) (2011) 527–533. [7] Z. Ungvari, P. Pacher, V. Kecskemeti, A. Koller, Fluoxetine dilates isolated small cerebral arteries of rats and attenuates constrictions to serotonin, norepinephrine, and a voltage-dependent Ca(2+) channel opener, Stroke 30 (1999) 949–954.

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Omega 3 Polyunsaturated Fatty Acids Improve Endothelial Dysfunction in Chronic Renal Failure: Role of eNOS Activation and of Oxidative Stress.

The role of Nrf2 in oxidative stress-induced endothelial injuries.

Endothelial SHIP2 Suppresses Nox2 NADPH Oxidase-Dependent Vascular Oxidative Stress, Endothelial Dysfunction and Systemic Insulin Resistance.

eNOS‑Thr495 in endothelial cells.

Assessment of Mitochondrial Dysfunction and Monoamine Oxidase Contribution to Oxidative Stress in Human Diabetic Hearts.

Ranolazine protects from doxorubicin-induced oxidative stress and cardiac dysfunction.

Contribution of redox-dependent activation of endothelial Nlrp3 inflammasomes to hyperglycemia-induced endothelial dysfunction.