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Effects of progesterone treatment on endothelium-dependent

coronary relaxation in ovariectomized rats

Tagana Rosa da Cunha, Jéssyca Aparecida Soares Giesen, Wender

Nascimento Rouver, Eduardo Damasceno Costa, Marcella Daruge
Grando, Virgínia Soares Lemos, Lusiane Maria Bendhack, Roger
Lyrio dos Santos

PII: S0024-3205(20)30138-7
DOI: https://doi.org/10.1016/j.lfs.2020.117391
Reference: LFS 117391

To appear in: Life Sciences

Received date: 6 September 2019

Revised date: 22 January 2020
Accepted date: 30 January 2020

Please cite this article as: T.R. da Cunha, J.A.S. Giesen, W.N. Rouver, et al., Effects of
progesterone treatment on endothelium-dependent coronary relaxation in ovariectomized
rats, Life Sciences(2020), https://doi.org/10.1016/j.lfs.2020.117391

This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
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version to give early visibility of the article. Please note that, during the production
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© 2020 Published by Elsevier.

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Effects of progesterone treatment on endothelium-dependent coronary

relaxation in ovariectomized rats

Tagana Rosa da Cunhaa, Jéssyca Aparecida Soares Giesena, Wender

Nascimento Rouvera, Eduardo Damasceno Costab, Marcella Daruge Grandoc,

Virgínia Soares Lemosb, Lusiane Maria Bendhackc, Roger Lyrio dos Santosa,*

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Department of Physiological Sciences, Health Sciences Center, Federal

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University of Espirito Santo, Vitoria, ES, Brazil
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Department of Physiology and Biophysics, Biological Sciences Institute,
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Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
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Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo,

Ribeirão Preto, Brazil

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Corresponding author:
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Roger Lyrio dos Santos, PhD.

Department of Physiological Sciences

Health Sciences Center Federal University of Espirito Santo

Marechal Campos avenue, 1468, 29050-755, Vitoria, ES, Brazil

Telephone +55 (27) 3335-7353 - Fax +55 (27) 3335-7330

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E-mail: rogerlyrio@hotmail.com

Key words: Progesterone, endothelium, ovariectomized rats, coronary

reactivity.

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Effects of progesterone treatment on endothelium-dependent coronary

relaxation in ovariectomized rats

ABSTRACT

Aim: Although progesterone (P4) has a beneficial effect on the cardiovascular

system, P4 actions on the coronary bed have not yet been fully elucidated. This
study evaluated the effect of progesterone treatment on endothelium-dependent
coronary vascular reactivity in Wistar rats.

Main methods: Eight-week-old adult rats were divided into Sham,

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Ovariectomized (OVX), Ovariectomized and progesterone treated (OVX P4).
The OVX P4 group received daily doses of progesterone (2 mg/kg/day).

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Vascular reactivity was assessed by a modified Langendorff technique. The
intensity of eNOS, Akt, and gp91phox protein expression was quantified by
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Western blotting. Superoxide anion (O2●-) and hydrogen peroxide (H2O2)
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production was measured by dihydroethidium and 2',7'-dichlorofluorescein,
respectively.
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Key findings: Treatment with P4 was able to prevent the reduction in baseline
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coronary perfusion pressure induced by ovariectomy. We observed that

endothelium-dependent coronary vasodilation was reduced in the OVX group
and potentiated in the OVX P4 group. Following the inhibition of the nitric oxide
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(NO) pathway, the bradykinin-induced relaxing response was potentiated in the

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OVX P4 group. With regard to the combined inhibition of NO and prostanoids

pathways, the OVX P4 group showed a greater relaxing response, similar to
what was found upon individual inhibition of NO. After the combined inhibition of
NO, prostanoids and epoxyeicosatrienoic acids’ pathways, the vasodilatory
response induced by BK was abolished in all groups.

Significance: Treatment with P4 prevented oxidative stress induced by

ovariectomy. These results suggest that progesterone has a beneficial action on
the coronary vascular bed.

Keywords: Progesterone, endothelium, ovariectomized rats, coronary

reactivity.
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INTRODUCTION

The improvement in life quality and health care that took place in the last
decade culminated in an increase in life expectancy. In developed countries,
women's life expectancy is 80 years. As women spend a third of their lives in
post-menopause [1], the climacteric period, which takes place during the
transition between the reproductive and non-reproductive periods, have gained
relevance in recent years [2]. Clinical studies have shown that female sex
hormones exert beneficial effects on cardiovascular functions [3], with women of

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reproductive age usually having lower cardiovascular risk than men of the same

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age.
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Cardiovascular disease is a major cause of death in postmenopausal women in
the Western world [4], with coronary artery disease being particularly relevant in
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this population [5]. Consequently, there is an intense interest in the
development of hormone replacement therapy (HRT) for women, with the aim of
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inhibiting the development of postmenopausal cardiovascular diseases [6,7].

Initially, HRT treatment used only estrogen. Later, however, it was proposed
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that progesterone should be added, to prevent estrogen-induced side effects

such as breast and uterine cancer [2].
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However, some clinical studies such as the Heart and Estrogen-Progestin

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Replacement Study (HERS I and HERS II) and the Women's Health Initiative
(WHI) have not confirmed the beneficial effects of hormone replacement on the
cardiovascular system of postmenopausal women [8,9]. These negative
findings regarding the effects of hormone replacement on the vascular system
may be related to the number of women studied, their advanced age and
especially the progestin used then, medroxyprogesterone acetate (MPA). It has
been shown that MPA and P4 regulate vascular function in opposite ways, due
to the fact that the latter stimulates NO production, whereas the former inhibits
its synthesis in endothelial cells [10].

Sex hormones act on the endothelium and synthesis of vasoactive molecules.

The endothelium exerts a determinant function in the control of vascular
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homeostasis, participating in the regulation of intracellular signals, permeability

and vascular tone [11]. In coronary arteries, estrogen modulates the release of
vasodilators derived from the endothelium, i.e, NO, PGI2 and EDHF [12,13].

Current knowledge on how P4 acts at the vascular system is limited compared

to the large amount of evidence available for estrogen, especially in the
coronary bed. The protective functions of progesterone in the cardiovascular
system have received little attention, although there is growing evidence that
progesterone lowers blood pressure [14] and has potent vasodilator [15] and
natriuretic effects [16]. In experimental models, progesterone has been shown

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to modulate the synthesis of endothelial vasodilators, increasing the synthesis
and action of NO and PGI2 by genomic and non-genomic mechanisms [10,17].

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Besides, progesterone induces endothelium-independent relaxation in rabbit
coronary arteries and rat aorta in vitro [17].
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Therefore, although there is ample evidence in the literature that P4 exerts a
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beneficial effect on the vascular system [10,14,15,17,18], the actions of P4
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treatment on the coronary vascular bed have not yet been fully elucidated.
Thus, this study sought to evaluate the effect of progesterone treatment on
endothelium-dependent coronary vascular reactivity in ovariectomized rats.
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MATERIAL AND METHODS

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Experimental Animals
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In this study, 8-week-old adult Wistar rats (Rattus norvegicus albinus) were
reared at the animal facility in the Centre of Health Sciences at UFES. All
procedures were approved by the Institutional Ethics Committee for Animal
Care and Use of the Federal University of Espirito Santo under protocol #
063/2017. The animals were housed in collective cages (five animals per cage)
and received food (Purina Labina) and water ad libitum. They were kept under
controlled temperature conditions (22–24°C) and humidity (40–60%), with a
12/12 h light-dark cycle. The animals were randomly divided into three groups:
Sham, ovariectomized (OVX), OVX and treated with progesterone (OVX P4).

Ovariectomy
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Ovariectomy was performed under general anesthesia with ketamine (80

mg/kg) and xylazine (12 mg/kg), i.p. The rats were subjected to a bilateral
incision in the skin followed by an incision in the muscular layer, opening the
peritoneal cavity for posterior ligation of the uterine horn and removal of the
ovaries. The tube was ligated with a suture line and the ovaries were removed.
Muscle and skin were then sutured. After the surgery the animals received an
antibiotic injection (2.5% enrofloxacin, 0,1 mL, i.m.). SHAM rats were incised
and sutured, but the ovaries were left intact [18].

Hormone replacement

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Progesterone replacement (Progesterone micronized - Atymed) was performed

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for 15 days by subcutaneous injections at the concentration of 2 mg/kg/day,
mimicking physiological concentration, as previously described [19]. Groups
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that did not receive treatment had the same volume administered, containing
only the vehicle used to dilute progesterone (sunflower oil). The animals treated
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with progesterone received the first dose on the same day of ovariectomy to
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avoid low hormonal post-surgery.

Non-invasive Assessment of Blood Pressure

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Before any measurements were taken, it was necessary to adapt the animals to
the equipment (IITC INC/Life Science, 23924 Victory Blvd, Woodland Hills, Ca
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91367-1253 USA) within 3 days prior to the day of registration. The animals
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were allowed to pace for about 10 minutes in the equipment, avoiding the
influence of stress on blood pressure. On the day of registration, the animals
were accommodated in a heated acrylic chamber at a temperature of 29 to
32°C, with a pneumatic wrist cuff coupled to the proximal region of the tail. A
sphygmomanometer was automatically inflated and deflated and the value of
systolic blood pressure (SBP) obtained through transducer signals coupled to a
computer, as previously described [20,21]. An average of no less than 3
recordings per animal was obtained, and those associated with tail movement
were discarded.

Vaginal smear
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The estrous cycle of the females was monitored by means of vaginal smears.
The vaginal secretion of each animal was removed daily between 08:00 and
09:00 a.m. and the vaginal epithelial cells examined under the optical
microscope as described by Marcondes et al. [22], to identify the cell types
present during the various phases of the estrous cycle. The experiments were
carried out during the diestrus phase of the normal estrous cycle, the longest
and best representative phase of P4 levels during the cycle.

Studies in Isolated Heart

The Langendorff perfusion method used in this study has been previously

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described in detail [12]. The hearts of animals anesthetized with ketamine (80

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mg/kg) and xylazine (12 mg/kg), i.p., were extracted and transferred to the
Langendorff apparatus (Hugo Sachs Electronics, March-Hugstetten, Germany).
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Afterwards, the aorta was cannulated and perfused with modified Krebs solution
containing 120 mM NaCl, 1.25 mM CaCl2.2H2O, 5.4 mM KCl, 2.5 mM MgSO4
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.7H2O, 2.0 mM NaH2PO4.H2O, 27.0 mM NaHCO3, 1.2 mM Na2SO4, 0.03 mM
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EDTA and 11.0 mM glucose, continuously heated at 37°C in water and

equilibrated with a mixture of 95% oxygen and 5% carbon dioxide at a
controlled pressure of 100 mmHg to yield a pH of 7.4. Coronary flow was kept
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constant at 10 mL/min so that changes in CPP would be directly related to

changes in vascular resistance. Left ventricular (LV) isovolumetric pressure was
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maintained by inserting a latex balloon into the LV, which was pressurized to
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maintain intraventricular diastolic pressure at 10 mmHg. After 40 minutes of

stabilization the baseline coronary perfusion pressure (CPP) was determined.
Vascular response to Bradykinin (BK) was evaluated by means of a dose-
response curve (0.1 - 1000 ng) for BK (Sigma, St. Louis, MO), obtained by in
bolus infusion before and after the infusion with individual or combined inhibitors
of NOS (Nω-Nitro-L-arginine methyl ester hydrochloride, L-NAME, 100 μM),
cyclooxygenase (indomethacin, 2.8 μM), or cytochrome P450 (clotrimazole,
0.75 μM).

Western Blotting
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Coronary arteries of sham, OVX and OVX P4 rats were collected, frozen in
liquid nitrogen and stored at -80 °C. Next, each coronary was homogenized in
25 μL of RIPA supplemented with protease and phosphatase inhibitors (Tris-
HCl 65.2 mmol/L, 154 mmol/L NaCl, 1% NP-40, 0.25% sodium deoxycholate,
0.8 mmol EDTA 1 mM PMSF, 10 mM sodium orthovanadate, 100 mM sodium
fluoride, 10 mM sodium pyrophosphate, and protease inhibitor). The
homogenates were centrifuged at 10,000 rpm for 10 minutes at 4°C for the
removal of debris. Protein concentration in the samples was determined by
colorimetric method (Bio Rad DC Protein assay). Aliquots of the lysate
containing 20 µg of total protein were mixed with sample buffer (1x), boiled for 5

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minutes and applied to the gel. Protein separation was done by 10%
polyacrylamide gel electrophoresis at 4°C for approximately 2 hours at 150 V.

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The molecular weights were estimated with the Prestained Protein Ladder
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(Abcam). After running the gel, the proteins were electrotransferred to
nitrocellulose membranes and blocked with 5% skim milk in TBS-T (0.13 M
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NaCl, 20 mM tris, pH 7.6, 0.1% tween 20) for 2 hours. The blocking solution
was then withdrawn and the membranes washed 36 times with TBS-T for 5
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minutes. Next, they were incubated overnight at 4°C with mouse primary
antibodies to eNOS (1:1000, Abcam, cat. ab76198) and gp91phox (1:1000,
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Abcam, cat. ab80897), and rabbit primary antibody to Akt (1:1000, Abcam, cat.
ab8805), all diluted in TBS-T with 3% BSA (bovine serum albumin). After 6
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washing steps of 5 minutes each with TBS-T, anti-mouse (1:2000) or anti-rabbit

peroxidase (1:2000) conjugated secondary antibodies diluted in 1% BSA were
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applied to the membranes for 1 hour at room temperature, what was followed
by 6 more washing steps. Chemiluminescence detection was performed using
the luminol reagent (Santa Cruz biotechnology) followed by exposure to
radiographic film. Protein quantification was done by densitometry using the
application software ImageJ.

Detection of superoxide production

After performing coronary artery incisions, the slides were incubated with the
probe dihydroetidine (DHE; Invitrogen™), according to the protocol described
by Silva et al. [23]. Afterwards, two slides per animal were protected from light
and incubated, one of them with DHE (5 μM) for 30 minutes at 37°C to
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investigate basal O2●- production, while the other was incubated with DHE +
Tiron (10 μM) for 30 minutes, thus working as a negative control for basal O2●-
production. Digital images were obtained under a 40x objective using the Zeiss
Axio Imager fluorescence microscope with Apotome module from the
ICB/UFMG Image Processing and Acquisition Center. The images were
analyzed with the Image J 1.48 software, using optical density analysis for the
fluorescence of the area of interest (ROI manager). Two incisions from each
animal were evaluated, with ten areas of interest being selected in each
analyzed section. Samples consisted of 4 animals per experimental group.

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Detection of hydrogen peroxide production

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Analysis of H2O2 production by microscopy was done indirectly through the use
of the 2'-7'-dichlorodihydrofluorescein-diacetate probe (DCF-DA; Invitrogen ™),
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according to Somberg et al. [24]. Afterwards, two slides per animal were
protected from light and incubated: the first one with DCF (10 μM) for 30
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minutes at 37°C to investigate basal production of H2O2, and the second one
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with DCF + catalase (1000 u/mL), working as a negative control for basal H2O2
production. Digital images were obtained under the 40x objective using the
Zeiss Axio Imager fluorescence microscope with the Apotome module of the
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ICB/UFMG Image Processing and Acquisition Center. The images were

analyzed with Image J software (version 1.48), using optical density analysis for
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the fluorescence of the area of interest (ROI manager). Ten areas of interest
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were selected for each section analyzed, and 2 incisions of each animal were
excluded. Samples consisted of 4 animals per experimental group.

Statistical analysis

Data analysis was performed using the statistics software Graph-Pad Prism 6.
The data were expressed as mean ± standard error of the mean (SEM).
Comparison between groups was performed by one-way ANOVA, apart from
the vasodilator response to bradykinin (BK), which was analyzed by two-way
ANOVA. In both cases, the post hoc test used was the Tukey's test, and a
significance level of p < 0.05 was adopted for this study.
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RESULTS
Effects of Progesterone on Systolic Blood Pressure (SBP)

SBP data are summarized in Figure 1. We found no differences between the

groups as to SBP: Sham (117.8 ± 5 mmHg), OVX (131 ± 11 mmHg) and OVX
P4 (121.2 ± 6 mmHg).

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Figure 1. Systolic blood pressure of SHAM (n=15), OVX (n=15) and OVX P4 (n=15) groups.
Values were expressed as mean ± SEM. Data were analyzed with one-way ANOVA followed by
Tukey's post hoc test.
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Basal CPP and Coronary Vascular Reactivity

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As expected, CPP was found to be different between the studied groups. We

observed that, although ovariectomy decreased CPP (SHAM = 86 ± 3 vs OVX =
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70 ± 3 mmHg), the treatment with P4 was able to prevent this reduction (OVX
P4 = 92 ± 6 mmHg).
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Figure 2. Baseline coronary perfusion pressure (CPP) in isolated hearts from normotensive rats
of SHAM (n=15), OVX (n=15) and OVX P4 (n=15) groups. Values were expressed as mean ±

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SEM. * p<0.05 compared to SHAM, # p<0.05 compared to OVX. Data were analyzed with one-
way ANOVA followed by Tukey's post hoc test.
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Next, we analyzed the functionality of the endothelium through the bradykinin-
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induced vasodilator response. Our data suggest that a substantial increase in
endothelium-dependent vasodilation took place in P4-treated rats (SHAM =
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14.7 ± 1.6 % vs OVX P4 = 23.9 ± 2.8 %), showing that progesterone was not
only able to prevent the deleterious effects induced by ovariectomy, but also
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potentiated BK-induced vasodilatation in this vascular bed when compared to

Sham animals (Figure 3A).
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After the nonspecific inhibition of the NOS enzyme with L-NAME (Figure 3B),
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the vasodilatory response induced by BK was potentiated in the OVX P4 group

(23.9 ± 2.8 % to 35.2 ± 2.6 %). Nevertheless, after the inhibition of the
prostanoid pathway with indomethacin (Figure 3C), the relaxing response to BK
was similar to what had been observed for the baseline curve of bradykinin
shown in Figure 3A (SHAM: 11.3 ± 1.5 %; OVX: 4.0 ± 0.7 %; OVX P4: 26.8 ±
0.4 %). These results suggest that the prostanoid pathway does not participate
in the BK-induced vasodilatation.

The combined inhibition of the NOS and COX enzymes (Figure 3D) revealed
that the progesterone-treated group showed a stronger relaxing response
(SHAM = 19.4 ± 2 %; OVX = 8.5 ± 1.1 %; OVX P4 = 33.2 ± 3 %) similar to that
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promoted by the individual inhibition of NOS, suggesting that the main pathway
involved in this response is the NO pathway. When the combined inhibition of
NOS, COX and CYP enzymes was performed, the vasodilatory response
induced by BK was abolished in all groups (Figure 3E), showing that, as
expected, the vasodilation seen in this study was endothelium-dependent.

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Figure 3. Vasodilator response to increasing concentrations of bradykinin (BK 10 -10 M).
Curves obtained before (A, n=15) and after inhibition with L-NAME (B, n=8), indomethacin (C,
n=8), L-NAME + indomethacin (D, n=8), L-NAME + indomethacin + clotrimazole (E, n=7).
Values expressed as mean ± SEM * p<0.05 to indicate differences between groups. Analyses
were performed with two-way ANOVA followed by Tukey's post hoc test.
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Analysis of Protein Expression (Western Blotting)

Protein expression analysis revealed that eNOS was decreased in the OVX
group, while Akt, which is upstream in the NO formation pathway, was found to
be increased in both OVX and OVX P4 groups. However, there was no
difference as to NADPH oxidase subunit expression (gp91phox) between
groups (Figure 4).

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Figure 4. Analysis of eNOS, Akt, and gp91phox protein expression in SHAM, OVX and OVX P4
groups. Each sample was obtained from coronary septal arteries and the analyses of different
proteins were performed on the same gels. Data were expressed as mean ± SEM; n=4 per
group. One-way ANOVA followed by Tukey's post hoc test was used. * p<0.05 compared to the
Sham group, # p<0.05 compared to OVX.

Superoxide Anion Analysis

The DHE oxidative assay revealed an intense fluorescent signal in the OVX
group when compared to Sham and OVX P4 animals, showing that the
treatment with P4 was able to prevent the increase in superoxide anion
production induced by ovariectomy (Figure 5).

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Figure 5. Influence of progesterone treatment on superoxide anion production in coronary

arteries. Panels show fluorescence intensity (red) in the coronary arteries of rats from Sham,
OVX and OVX P4 groups using dihydroethidium (DHE) staining. The bar graph shows mean
DHE fluorescence for each group (AU: arbitrary units) (n=4). Data were expressed as mean ±
SEM. * p<0.05, significant difference between compared groups. The analyses were performed
by one-way ANOVA followed by Tukey's post hoc test. Scale bar: 20 μm.

Hydrogen Peroxide Analysis

The highest fluorescent signal, which corresponds to a higher hydrogen

peroxide production, was found in the sham group, the opposite being true for
the OVX group. One can observe that treatment with P4 attenuated the

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decrease in hydrogen peroxide production induced by ovariectomy.

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Figure 6. Influence of progesterone treatment on hydrogen peroxide production in coronary

arteries. Panels show fluorescence intensity (green) in the coronary arteries of rats from the
Sham, OVX and OVX P4 groups using 2′,7′-dichlorofluorescein (DCF) staining. The bar graph
shows mean DCF fluorescence for each group (AU: arbitrary units) (n=4). Data were expressed
as mean ± SEM. * p<0.05, significant difference between compared groups. The analyses were
performed by one-way ANOVA followed by Tukey's post hoc test. Scale bar: 20 μm.

DISCUSSION

The main finding of this study was that progesterone treatment prevented the
impairment in endothelium-dependent vasodilation induced by ovariectomy in
the coronary bed. Besides, we found that the group treated with P4 had a

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higher vasodilator response, when compared to the other groups.

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Our results have also revealed the existence of an important difference in
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baseline coronary perfusion pressure (CPP) in the Sham and OVX P4 groups
compared to the OVX group (Figure 3). Treatment with P4 was able to prevent
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the reduction of baseline CPP induced by ovariectomy, thus suggesting that
female sex hormones modulate this parameter. In the studies by Moysés et al.
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[25], the baseline CPP of females was found to be higher than that of males. It
is believed that the higher CPP shown by females may be related to a beneficial
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effect, as in gonadectomized females baseline CPP was equal to that of males.

Also, a higher baseline CPP in the coronary arteries could allow for more potent
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vasodilation in situations of increased blood flow [26]. Although the mechanisms

behind this difference remain to be fully elucidated, one might suggest that, as
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estrogen acts on the renin-angiotensin system, indirectly increasing the activity

of angiotensin II, it could thus modulate the control of coronary tone [27].
Besides, it is known that estrogen can also promote the increase of calcium
levels through the activation of L-type calcium channels through a non-genomic
molecular mechanism, thus contributing to the increase in CPP [28]. It may be
that progesterone plays a similar role in modulating coronary tone, though this
particular point remains to be investigated.

It is known that the deficiency of sex hormones can cause oxidative stress,
consequently decreasing the bioavailability of vascular NO [29,30]. This
reduction in NO levels could lead to increased systemic vascular resistance
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and, therefore, increased blood pressure. However, Fabricio et al. [31] observed
an increase in SBP only by the 8th week after ovariectomy. Therefore, we
believe that the 15-day ovariectomy period adopted in this study was not
sufficiently long to change blood pressure parameters, but was effective in
promoting endothelial dysfunction.

Regarding the BK-induced response, Figure 3A shows that the P4-treated

group had a higher vasodilator response than the Sham group. This response
suggests that progesterone seems to positively modulate the synthesis of
endothelial vasodilators, as BK acts by releasing endothelial mediators. Initially,

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BK interacts with its endothelial receptor (B2), which consequently induces the
production and release of NO, PGI2, and EDH [32]. Therefore, a higher BK-

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induced vasodilator response may indicate a higher production/release of
endothelial vasodilators in the OVX P4 group.
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One possible explanation for this increased relaxation may involve the absence
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of a negative E2 modulating effect on P4, as the OVX P4 group was treated
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with P4 alone. Indeed, Miller & Vanhoutte [33], using coronary arteries from
ovariectomized dogs treated with estrogen, progesterone, or both hormones,
demonstrated that acetylcholine vasodilation was greater in the estrogen-
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treated group than in the progesterone or estrogen plus progesterone-treated

groups. Those results suggest that there may be a competitive interaction
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between the two hormones, which would explain the lower relaxation observed
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when they are both present. In fact, in our study, as the treatment included only
progesterone, the response was probably enhanced by the absence of estrogen
and its competitive – or negative – modulation effect.

Among the endothelial factors involved in the BK-induced relaxation, we

analyzed the participation of nitric oxide (NO), prostacyclin (PGI 2) and
endothelium-dependent hyperpolarization (EDH), in order to assess the degree
of importance of these mediators. For this purpose, we inhibited the synthesis of
these endothelium-derived mediators. We found that the BK-induced
vasodilatory response was potentiated in the OVX P4 group after inhibition of
the NO pathway. That clearly indicates that this pathway appears to act by
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negatively modulating relaxation in these animals, for this response is increased

upon inhibition of NO synthesis.

It is well established that ovarian steroids are capable of stimulating NO-

independent vasodilation-related enzymes, i.e., COX and CYP [10,32–35],
which could explain, at least in part, the potentiation of the response observed
in the OVX P4 group. As these animals were treated only with progesterone,
the competition that would occur if another ovarian steroid (E2 vs P4) was
present does not take place, which could account for the enhanced vasodilatory
response observed in this group [36,37]. In the OVX group, as there are no

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ovarian steroid hormones present, one would indeed expect this potentiating
effect to be prevented.

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As discussed above, we believe that NO synthesis, which occurs exclusively by
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the action of NOS, being, therefore, inhibited in the presence of L-NAME, could
prevent the potentiation of the vasodilator response observed in OVX P4
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animals. In addition, NO can impair other relaxation pathways, not only in a
direct way [38] but also indirectly, as when it reacts with O2●- – which can be
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generated as a result of NOS uncoupling [39–41] – to form ONOO- [42]. ONOO-

, which is, therefore, the result of NO oxidation, is one of the major sources of
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endothelial oxidative damage [43]. This reactive nitrogen species is capable of

impairing other relaxation pathways, i.e., prostanoid (PGI2) formation and EDH,
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by inhibiting prostacyclin synthase [44] and reducing K+ channel activity, thus

mediating EDH [45]. Therefore, in the absence of NO, ONOO- levels would
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most likely be reduced, which could result in a further potentiation of these

additional relaxation pathways in OVX P4 animals, along with that promoted by
the aforementioned progesterone’s exclusive action that takes place in the
absence of estrogen competition [36,37].

Considering that progesterone is known to modulate COX activity [10,33], the

effects of the nonspecific inhibition of the prostanoid formation pathway were
evaluated. However, we did not observe the participation of this particular
pathway in the vasodilator response to BK, which led us to investigate the
involvement of EDH in this response.
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It has been shown that epoxyeicosatrienoic acids (EETs) are the most likely
candidates to promote EDH in the coronary bed [46–48]. Therefore, to analyze
the contribution of this phenomenon to the vasodilator response to BK, we
performed the combined inhibition of NO synthesis, prostanoids, and EETs,
which resulted in the relaxation being completely abolished. These data, in
addition to confirming that EETs do act in EDH in the coronary bed, provide
evidence of the participation of EDH in BK-induced relaxation in this bed.

One of the goals of this study was to evaluate whether progesterone can
prevent a possible endothelial dysfunction promoted by ovariectomy. Therefore,

of
we sought to understand how P4 modulates the generation of reactive oxygen
species. When investigating the expression of NADPH oxidase, we found no

ro
difference in gp91phox subunit expression between groups. This goes against
the findings reported by Wassmann et al. [49], in which a 4-week treatment with
-p
P4 increased NADPH oxidase activity in mice. NADPH oxidase is a
re
transmembrane enzyme complex, being the main responsible for the production
of O2●- [50].
lP

On the other hand, treatment with progesterone was able to prevent the
reduction in eNOS expression induced by ovariectomy. In addition, the
na

treatment induced alterations in this pathway by increasing Akt expression.

These results corroborate other studies that demonstrated that progesterone
ur

increases NO synthesis in vascular endothelial cells through the non-genomic

Jo

activation of the Akt/PI3K and MAPK pathways [39,45].

Akt/PI3K expression was found to be increased also in the ovariectomized

group, which was not the case for SHAM animals. That may be the result of an
attempt to increase NO production by enhancing eNOS phosphorylation in OVX
animals, since eNOS expression is reduced in this group. Lower eNOS
expression may be associated with endothelial dysfunction, and thus Akt/PI3K
would be increased in order to indirectly minimize the effects of this dysfunction.

In addition, DHE-fluorescence was also higher in OVX animals, pointing to a

higher O2●- production. As NADPH oxidase activity was not increased in this
group, the increase in O2●- availability may be related to eNOS dysfunctionality.
 Journal Pre-proof

The reaction of O2●- with NO to form ONOO- may lead to a decrease in the
availability of tetrahydrobiopterin (BH4). BH4 is an important cofactor for NOS
functionality, and its reduction causes eNOS dissociation, which leads to a
decrease in NO formation and an increase in O2 production, contributing to
increased oxidative stress [43,51]

Besides, elevated O2●- levels may be related to a reduced expression of the

antioxidant enzyme superoxide dismutase (SOD), as H2O2 levels were found to
be reduced in OVX animals. SOD prevents the accumulation of O2●- by
catalyzing its dismutation into H2O2 in the endothelial cell [43]. In fact, a study

of
by Kang et al. [52] showed a decrease in endothelium-dependent vasodilation
due to the reduced NO production in coronary arterioles of OVX rats. This effect

ro
has been associated with a reduction in SOD expression.
-p
Although treatment with P4 did not completely prevent the reduction in H2O2
bioavailability, the OVX P4 group had higher levels of H2O2 when compared to
re
the OVX group. Indeed, progesterone treatment was able to partially prevent
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the oxidative stress.

CONCLUSION
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In conclusion, the data reported here suggest that P4 has a beneficial action on
the coronary bed and could prevent the development of the endothelial
ur

dysfunction that follows the deficiency or cessation of sex hormone production.

Thus, we consider that the role of P4 in hormone replacement therapy may go
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beyond inhibiting the side effects of E2. Our results contribute to a better
understanding of the effects of P4 treatment on the coronary vascular
homeostasis, as well as to the development of therapies to counteract the
cardiovascular abnormalities associated with a decrease in sex hormone
production.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived
as prejudicing the impartiality of the research reported.

Funding
 Journal Pre-proof

This work was supported by CAPES.

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