Gonadal function protects against organ culture-induced vascular damage. Involvement of prostanoids

Journal Pre-proof Gonadal function protects against organ culture-induced vascular damage. Involvement of prostanoids ´ Diva M. Villalpando, Juan Gomez Rivas, Daniel Flynn, Ferm´ın R. de Bethencourt, Mercedes Ferrer

PII:

S1098-8823(19)30157-1

DOI:

https://doi.org/10.1016/j.prostaglandins.2019.106406

Reference:

PRO 106406

To appear in:

Prostaglandins and Other Lipid Mediators

Received Date:

9 July 2018

Revised Date:

25 November 2019

Accepted Date:

23 December 2019

Please cite this article as: Villalpando DM, Rivas JG, Flynn D, de Bethencourt FR, Ferrer M, Gonadal function protects against organ culture-induced vascular damage. Involvement of prostanoids, Prostaglandins and Other Lipid Mediators (2020), doi: https://doi.org/10.1016/j.prostaglandins.2019.106406

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Gonadal function protects against organ culture-induced vascular damage. Involvement of prostanoids.

by Diva M. Villalpandoa, Juan Gómez Rivasb,c, Daniel Flynna, Fermín R. de Bethencourtb,c, Mercedes Ferrera,c,*. a

Departamento de Fisiología, Facultad de Medicina, UAM, Spain; bServicio de Urología,

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Hospital Universitario La Paz, Madrid; cInstituto de Investigación Hospital Universitario

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La Paz (IdiPAZ) Madrid, Spain

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Short title: Vascular damage, prostanoids and sex hormones

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*Corresponding author: Mercedes Ferrer, PhD. Departamento de Fisiología, Facultad de Medicina, UAM. C/Arzobispo Morcillo, 4, 28029 Madrid, Spain.

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e-mail address: [email protected]

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Highlights:

 The study analyzes the impact of androgenic deprivation on vascular vulnerability against organ culture-induced vascular damage.  Organ culture-induced vascular damage importantly increases the prostanoids release in arteries from both intact and orchidectomized rats.  The study shows a greater effect of endogenous TXA2 on the ACh-induced vasodilator response in cultured arteries from orchidectomized rats than in those with intact gonadal function.  Organ culture preserves the structural integrity of vessels.  The study shows the protective action of endogenous male sex hormones against organ culture-induced vascular damage in rat aorta. 1

Abstract

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Androgen deprivation induces vascular dysfunction in which altered release and action

of prostanoids has been extensively studied. On the other hand, the vascular organ-

culture system has been reported as a valid model for phenotypic changes that occur in

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several cardiovascular pathologies. Since there are no studies analyzing the impact of

androgenic loss on vascular vulnerability during induced vascular damage, the objective

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of this study was to analyze the possible preventive role of male sex hormones on the

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organ culture-induced vascular damage in rat aorta. The link to possible changes in gross structure was also analyzed. For this purpose, fresh and 20 h-cultured aortic

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arterial segments from intact and orchidectomized rats were used to analyze: (i) the release and vasomotor effect of the thromboxane A2 (TXA2), prostaglandin (PG) E2,

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PGF2α and PGI2; (ii) the vasodilator response induced by acetylcholine (ACh) as well as the involvement of prostanoids, in particular TXA2, in the ACh-induced response; (iii)

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the effect of activation of thromboxane/prostaglandin (TP) receptors on the AChinduced response; and (iv) the vascular structure. The results showed that organ culture: i) increased production of prostanoids; ii) increased prostanoids-induced vasomotor responses; iii) decreased ACh-induced relaxation after incubation with indomethacin, a blocker of cyclooxygenases; iv) increased the ACh-induced relaxation after incubation with the TXA2 synthase inhibitor, furegrelate, more in arteries from orchidectomized rats than in those of intact rats; v) diminished ACh-induced relaxation after U-46619 2

incubation only in arteries from orchidectomized rats; and vi) preserved the integrity of the different vascular layers. These results showed the protective role of male sex hormones against the induced vascular damage, since a decreased deleterious effect of prostanoids, in particular that of TXA2, was observed in arteries from rats with intact gonadal function. Key Words: gonadal function; androgens; organ culture; prostanoids; TXA2; vascular

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structure; rat aorta

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1.

Introduction Epidemiological studies have demonstrated a correlation between a higher

incidence of cardiovascular diseases and decreased levels of sex hormones [1-3]. The protective effects of male sex hormones on vascular function are due to its action on the synthesis/function of the different factors controlling vascular tone. For instance, we have reported that the loss of gonadal function in male rats alters the production of prostanoids [4-6], reactive oxygen species (ROS) [7, 8] and nitric oxide [9]. These

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modifications match with a thickening of the media muscular layer of mesenteric arteries from orchidectomized rats, in which the constitutive activation of epidermal

growth factor receptor (EGFR) seems to be involved [9]. According to these findings, it

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was also shown that testosterone diminished the activation of the EGFR and the ERK1/2 and Akt pathways in cultured vascular smooth muscle cells, therefore

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regulating the cell proliferation rate in the media layer of mesenteric arteries [9]. All

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these data demonstrate the ability of androgens to modulate vascular function and structure.

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It is well known that prostanoids, synthesized from arachidonic acid metabolism through the cyclooxygenase (COX) pathway, are key players in regulating vascular

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function [10, 11]. Prostanoids derived from COX-2 are increased when vascular dysfunction exist, exacerbating their vascular action that include increased

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vasoconstrictor, proliferative and pro-oxidant actions [6, 12, 13]. It is widely recognized that thromboxane A2 (TXA2) is one of the most important mediators that contributes to vascular dysfunction [14] due to its autocrine and paracrine actions through TXA2prostaglandins (TP) receptors activation [15, 16]. Additionally, overproduction of prostanoids other than TXA2, such as prostaglandin (PG) E2, PGF2α or PGI2, can be involved in vascular dysfunction by activating TP receptors [17-19].

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Apart from the numerous animal models to study cardiovascular pathologies, the vascular organ-cultured system has been reported as a valid model for the phenotypic changes that occur in endothelial [20] and in vascular smooth muscle [21, 22] cells during the development of cardiovascular diseases. Thus, modifications in the contribution of endothelial factors [20, 23], in the expression of different cell membrane receptors [24, 25] and in cell signaling pathways [26, 27], common features of several vascular diseases, have been described by using the organ culture technique.

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Additionally, an updated publication described the advantages of keeping cells on their own microenvironment (i.e., within the corresponding tissue) to study different cell modifications such as inflammatory or tumor diseases [28].

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It is remarkable to note that an important number of studies have analyzed the effects of sex hormones on vascular function, as well as the prostanoids involvement. However,

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there are no studies analyzing the impact of androgenic loss on vascular vulnerability in

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response to vascular damage applied to isolated vessels. Taken together these data and our previous results on protective effect of androgens on vascular function [4-9], we hypothesized that the arteries from orchidectomized rats could be more susceptible to

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organ culture-induced vascular damage by modulating the effect of prostanoids, in particular that of TXA2. Therefore, the objective of the present study was to analyze

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vascular vulnerability of aortic segments from intact and orchidectomized rats in

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response to the organ culture-induced vascular damage. Changes in gross structure of vessels were also analyzed. For this purpose, fresh and cultured aortic segments, from intact or orchidectomized rats, were used to evaluate: (i) the release and vasomotor effect of the prostanoids TXA2, PGE2, PGF2α and PGI2, (ii) the vasodilator response induced by acetylcholine (ACh) as well as the involvement of prostanoids, in particular that of TXA2, in the ACh-induced response, (iii) the effect of activation of TP receptors

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with the TXA2 analog, U-46619, on the ACh-induced response; and (iv) the morphological integrity of the aortic segments. 2.

Methods

2.1. Animal housing and protocols Male Sprague-Dawley rats (6 months old) were divided into two groups: intact and orchidectomized rats. All animals were housed in the Animal Facility of the

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Universidad Autónoma de Madrid (Registration number EX-021U) according to directives 609/86 CEE and R.D. 233/88 of the Ministerio de Agricultura, Pesca y Alimentación of Spain (Ref PROEX 202/16), exposed to constant temperature, 12 h

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dark/light cycle, standard feeding with rat chow and water ad libitum. Male sex

hormone deprivation was surgically induced by bilateral orchidectomy at 4.5 months of

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age under anesthesia by isoflurane inhalation. Rats were treated with 0.30 mg/Kg SC meloxicam (Metacam 5 mg/ml, Boehringer-Ingelheim) immediately after surgery and

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with 50 mg/Kg ibuprofen, orally administered for four days. The observation of seminal vesicles atrophy confirmed successful surgery. Systolic blood pressure was indirectly

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measured in awake intact and orchidectomized animals by the tail-cuff method (Letica, Digital Pressure Meter, LE5000, Barcelona, Spain) before and after orchidectomy. Six

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weeks after surgery the animals were sacrificed by CO₂ inhalation. The thoracic aorta

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was carefully dissected out, cleaned of connective tissue and placed in Krebs-Henseleit solution (KHS) at 4°C. The composition of KHS is as follows (mM): NaCl 115, CaCl₂ 2.5, KCl 4.6, KH₂PO₄ 1.2, MgSO₄·7H₂O 1.2, NaHCO₃ 25, glucose 11.1, Na EDTA 0.03. The investigation is compliant with the Guide for the Care and Use of Laboratory Animals published by the USA National Institute of Health (NIH publication number 85.23 revised 1985, Bethesda, MD, USA).

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2.2. Induction of vascular damage Aortic segments (4 mm in length) from intact and orchidectomized rats were placed in individual wells of a 12-well plate with 3 mL of serum-free Dulbecco’s modified Eagle’s medium (DMEM) and incubated 20 h at 37 oC in humidified 5% CO2 and air, as previously described [20]. The composition of the DMEM was L-glutamine 100 units/mL penicillin G and 100 µg/mL streptomycin (Sigma-Aldrich). When the aortic segments were to be exposed to a certain drugs, this drug would be diluted directly into

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the culture medium before the aortic segment was immersed. After organ culture, the medium was kept at -80°C to analyze prostanoid release. Fresh (non-incubated) and cultured (incubated) aortic segments were used for vascular reactivity experiments.

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2.3. Analysis of Prostanoid Release

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The release of TXA2, PGI2, PGF2α and PGE2 was measured using the stable metabolites

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TXB2, 6-ketoPGF1α, 13,14-dihydro-15-keto PGF2α and PGE2 respectively using the respective enzyme immunoassay kits (Cayman Chemical). DMEM culture medium used for organ culture of aortic segments from intact and orchidectomized rats was

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collected and frozen at -80 °C until the assay was performed. The different assays were carried out according to the manufacturer’s instructions. Results were expressed as a

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fraction of total protein concentration in the sample (pg of prostanoid/mL). Protein

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content was determined by using the bicinchoninic acid (BCA) method. 2.4. Vascular Reactivity The method used for isometric tension recording has been previously described [4-7, 29]. Briefly, two parallel stainless steel pins were introduced through the lumen of each vascular segment: one fixed to the bath wall and the other fixed to a force transducer

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(Grass FT03C, Grass Instruments Company, Quincy, MA, USA) which was connected to a model 7D Grass Polygraph. The vascular segments were suspended in an organ bath containing 5 ml KHS at 37°C continuously bubbled with a 95% O₂-5% CO₂ mixture (pH 7.4). Aortic segments were subject to a tension of 1g which was readjusted every 15 min during a 90 min equilibration period before drug administration. After this, the vessels were exposed to 75 mM KCl to test their functional integrity. After a washout period, the presence of functional endothelium was confirmed by the ability of

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10 µM acetylcholine (ACh) to relax segments of aorta precontracted with 0.1 µM noradrenaline (NA). To assess possible differences in the vasomotor responses induced

by TXA2, PGI2, PGF2α, or PGE2 in arteries from the four groups, concentration-

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response curves to the TXA2 mimetic hydroxy-11a, 9 a-(epoxymethano)prosta-5,13-

dienoic acid (U-46619, 0.1 nM - 1 µM), exogenous PGI2 (0.1 nM - 1 µM), PGF2α (1 nM

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- 1 µM), or PGE2 (1 nM - 10 µM) were performed.

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To analyze if the organ culture-induced vascular damage alters the endothelialdependent relaxation in aorta from intact or orchidectomized rats, cumulative concentration-response curves to ACh (0.1 nM-10 µM) were performed in NA (0.1 µM)

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precontracted aortic rings from each experimental group. To investigate the participation of prostanoids derived from COX-1 and COX-2 or TXA2 in the ACh-

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induced response after vascular damage, some aortic segments were respectively

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incubated overnight with the nonselective inhibitor of COX-1/2, indomethacin (10 M), or with the inhibitor of TXA2 synthesis, furegrelate (1 M). To analyze the effect of TP receptor activation on the ACh-induced response, as well as its possible modulation by gonadal function, aortic segments from intact or orchidectomized rats were incubated overnight with the stable TXA2 mimetic, U-46619 (1 nM).

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2.5. Confocal microscopy To study whether organ culture could influence the general vascular structure, aortic segments at the same anatomical location and from the four experimental conditions were cryoprotected with 30 % w/v sucrose in PBS and stored at -80°C until processing. Aortic ring sections were cut with a blade. The sections were stained with the nuclear dye 4’,6-diamidino-2-phenylindole (DAPI) at 10 µg/mL for 30 min at room temperature (RT) in darkness and were washed three times in PBS (20 min, RT). Two rings were

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mounted on a glass slide equipped with a small well. Glycerol was applied to each tissue section and coverslipped.

Images were obtained with a 10x air and 40x oil immersion objectives using a LEICA

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(TCS ST2 DM IRE2) laser scanning confocal microscope (488 nm excitation and 500-

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560 nm emission wavelengths) to detect the elastic lamella because elastin autofluoresces at this wavelength, and DAPI (405 nm excitation and 410-475 nm

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emission) was also used to locate cells.

Medial thickness was quantified by measuring the distance between the internal and

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external elastic lamina (3 measurements per image obtained with a 40x objective) using the ImageJ Analysis Software. The number of smooth muscle cells (SMCs) was

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determined measuring the nuclei in three equal areas in all aortic rings sections using the ImageJ Analysis Software. Three measurements were made per image in two aortic

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rings per experimental condition from 3 intact and orchidectomized rats. To detect the presence of the endothelial specific marker CD31, cryoprotected (30 % w/v sucrose in PBS) arteries from the four experimental conditions were embedded in optimum cutting temperature compound, OCT Tissue Tek, and 10 µm cryostat sections were place on a glass slide. The sections were washed in PBS-0.1 % Tween-20 (PBST) for 15 min and blocked with 5% BSA in PBST for 45 min. Incubation with the primary

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antibody (monoclonal anti-CD31 from mouse) was conducted at 1:100 dilution for 2 h at RT, followed by three 10-min rinses in PBST. The following steps were performed in the dark. Sections were incubated with the secondary antibody, a goat anti-mouse IgG – coupled to Alexa Fluor-647, for 1 h at RT, followed by three 10-min washes in PBS. Glycerol was applied to each tissue section and coverslipped. Images were obtained using a 40x oil immersion objectives using a LEICA (TCS ST2 DM IRE2) laser scanning confocal microscope (633 nm excitation and 650-720 nm emission

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wavelengths) to detect the specific antibody CD31. Elastin autofluorescence was detected using 488 nm excitation and 500-560 nm emission wavelengths.

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2.6. Drugs

Drugs used were: ACh chloride, L-NA hydrochloride, indomethacin, furegrelate, PGI2,

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PGF2α and DAPI (Sigma-Aldrich; Spain); CD31 (Abcam; Netherlands); Alexa Fluor467 anti-mouse IG (Jackson ImmunoResearch; USA); PGE2 (Cayman Chemical) and

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U-46619 (Calbiochem). Stock solutions (10 mmol/L) of drugs were prepared in distilled water, except for U-46619 and PGE2 which were dissolved in ethanol and administered

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from a prepared stock in such a way that the maximal ethanol concentration in the medium was < 0.001% (vol/vol). All these solutions were stored at -20°C and

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appropriate dilutions were made in KHS on the day of the experiment.

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2.7. Statistical analysis

Results are given as mean ± SEM (Standard Error of the Mean). The vasomotor responses elicited by U-46619, PGE2, PGF2α and PGI2 were expressed in mg. The difference of the area under curve (dAUC) for U-46619, PGE2, PGF2α and PGI2 in culture versus fresh arteries was also calculated. The maximum response (Emax values) and the negative logarithm of the prostanoids concentration producing 50% of 10

maximum response (pEC50) were calculated by a nonlinear regression analysis of each individual concentration-response curve using Graph Pad Prism Software (San Diego, CA). The relaxation induced by ACh was expressed as a percentage of the initial contraction elicited by NA. The values of Emax and pEC50 for the ACh-induced relaxation (in all experimental conditions) were also calculated. The dAUC for AChinduced response in the presence of indomethacin, furegrelate or U-46619 was also included. Statistical analysis was performed by comparing the curves obtained in

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cultured vessels from intact or orchidectomized rats with the obtained in the corresponding fresh vessels by means of two-way analysis of variance (ANOVA). The effect of different inhibitors/substances was also analyzed by ANOVA. For prostanoid

performed using unpaired Student's t test.

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release experiments, blood pressure, Emax and pEC50 the statistical analysis was

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To compare medial thickness and number of SMCs among groups, in the confocal

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microscopy experiments, a one way ANOVA followed by Dunn´s multiple comparisons test was used.

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Results

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In all analysis, statistical significance was considered at p < 0.05.

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3.1. Blood pressure

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Orchidectomy did not modify the systolic blood pressure (BP). The BP in intact rats was of 149.6 + 3 mmHg and of 148.2 + 2 mmHg six weeks later; n = 9. The BP in the orchidectomized group was of: 150.2 + 2 mmHg before and of 146.5 + 5 mmHg after surgery; n = 9 (p > 0.05). 3.2. Release of prostanoids

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Orchidectomy increased the release of all analyzed prostanoids, as previously reported for TXA2 [6, 9]. The release of TXA2, PGI2 and PGF2α was increased up to similar extent after organ culture in arteries from intact or orchidectomized rats. Organ culture induced a greater increase of PGE2 release in arteries from orchidectomized rats than in those from intact rats (Fig. 1). 3.3. Vascular reactivity

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The contractile response elicited by 75 mM KCl was increased after organ culture in arteries from intact or orchidectomized rats (fresh intact: 1679 + 137.8 mg; cultured intact: 2340 + 144.4 mg; p < 0.01; fresh orchidectomized: 1867 + 202.5 mg; cultured

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orchidectomized: 2633 + 120.5 mg; p < 0.01; n = 12-16).

The non-hydrolizable mimetic of TXA2, U-46619 induced a contractile response in

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fresh aortic segments from intact rats that was not modified after organ culture. However, when the vessels came from orchidectomized rats, organ culture increased the

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U-46619-induced response (Fig. 2; Table 1). Exogenous PGE2 induced a contractile response in fresh vessels that was increased in aorta from orchidectomized rats (Table

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1), which agrees with previous results [6]. Organ culture increased the PGE2-induced contraction more in aorta from orchidectomized rats than in those from intact rats (Fig.

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2; Table 1). Regarding prostacyclin and PGF2α, the vasodilator responses induced by

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exogenous PGI2 and by PGF2α in fresh vessels were increased after organ culture in similar extent in arteries from intact or orchidectomized rats (Fig. 2). The values of Emax and the pEC50 (Table 1) and those of the difference of the area under the curve (dAUC) (Fig. 2) point out these results. The vasodilator response induced by ACh (0.1 nM - 10 µM) was similar in aortic segments from intact or orchidectomized rats (p > 0.05). Organ culture decreased the

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ACh-induced response in segments from intact rats, while it did not significantly modify in arteries from orchidectomized rats (Fig. 3; Table 2). To investigate the contribution of prostanoids on the ACh-induced response after organ culture, vessels were incubated in the presence of indomethacin, the inhibitor of prostanoids synthesis. As indicated in Fig. 4, indomethacin decreased the ACh-induced response in arteries from intact or orchidectomized rats, but in a greater extent in aortas from orchidectomized rats particularly in response to low concentrations of ACh. These

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results suggest that organ culture induced greater participation of vasoconstrictor prostanoids in arteries from orchidectomized rats than in those from intact animals. Likewise, the incubation with furegrelate, the inhibitor of the synthesis of TXA2,

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increased the ACh-induced relaxation after organ culture more in aortic segments from

orchidectomized than in those of intact rats (Fig. 4; Table 3), which reinforces the

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possible results observed in the presence of indomethacin.

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The incubation during organ culture with the stable analogue of TXA2, U-46619 (1 nM), decreased the ACh-induced response only in arteries from orchidectomized rats

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(Fig. 5; Table 3). 3.4. Vascular structure

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Conventional fluorescent staining was performed to assess the possible effect of organ

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culture system on vascular structural integrity, and revealed the absence of gross structural changes. The endothelium integrity seems to be preserved in the cultured arteries from intact or orchidectomized rats since DAPI nuclei staining was also observed on the luminal side, adjacent to the internal elastic lamina (Fig. 6A). Medial thickness (the media-to-lumen width) was not modified by organ culture in arteries from intact or orchidectomized rats (Fig. 6A, 6B). The number of SMCs per

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section was similar in fresh or cultured arteries from intact or orchidectomized rats (Fig. 6A, 6C). Furthermore, CD31 immunofluorescent labelling experiments showed similar staining of the endothelium layer in arteries from the four experimental conditions, which confirms the presence of endothelium in both fresh and cultured arteries. Immunoflurescence was not detected when the incubation with the primary antibody

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was omitted as expected (Fig. 7).

Discussion

The present work describes for the first time the protective effect of endogenous

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androgens against organ culture-induced vascular damage, in which the TXA2 action is involved. Clinical studies have shown a correlation between hypotestosteronemia and

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incidence of cardiovascular diseases and mortality risk [1-3]. Likewise, numerous experimental studies have also reported cardiovascular protective effects of androgens

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[30, 31]. Thus, experiments from our research group have shown an increased prostanoid release in vessels from orchidectomized rats [4-6, 29, 32]. Likewise, the

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production of ROS, specifically that of superoxide anion, was also increased by orchidectomy [7, 8]. Alterations in these factors may also account for the development

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of vascular dysfunction [33].

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Different research has described organ culture of blood vessels as a valid technique to investigate the alterations that occur in endothelial and smooth muscle cells during the development of cardiovascular diseases [20-22]. In addition, the involvement of prostanoids in different models of vascular damage has been widely demonstrated [34], and previous experiments have shown that loss of gonadal function increases the production of prostanoids derived from COX-2 in male [4, 6] and female [10, 35] rats,

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creating a pro-inflammatory environment. Based on this information, the production of the prostanoids TXA2, PGE2, PGF2α and PGI2 and their vasomotor response were firstly analyzed. The results showed that the induction of vascular damage (by organ culture technique) strongly increased the production of the four prostanoids analyzed in arteries from both intact and orchidectomized rats, accordingly to the involvement of prostanoids in different models of vascular inflammation as already demonstrated [34]. Thus, the prostanoids release was increased more than 10 folds respect to fresh vessels

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(unpublished results), probably due to the accumulative release through the incubation period. However, the increase in the production of TXA2, PGF2α and PGI2 induced by organ culture was not influenced by the loss of gonadal function, while that of PGE2

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was greater in arteries from orchidectomized than in those of intact rats. On the

contrary, in arteries that were not subjected to vascular damage, orchidectomy

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significantly increased the release of these prostanoids, as extensively described in

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different arteries in previous publications [4-6, 9]. These results indicate that organ culture is a potent stimulus to activate the production of prostanoids, whose accumulation probably masks the regulation exerted by endogenous sex hormones on

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such production, that our research group have already reported [4, 6, 10, 35]. Furthermore, this pro-inflammatory condition is also accompanied by a pro-oxidant

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environment whose study is in progress.

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Once the release of prostanoids was analyzed, we proceeded to investigate whether its vasomotor action after induced vascular damage was influenced by endogenous sex hormones. Our results showed that the contraction induced by the TXA2 mimetic, U46619, was increased after vascular damage only in arteries from orchidectomized rats. Taken into account that this synthetic agonist is very potent and probably its contractile effect may do not reflect the real in vivo role of TXA2, we investigated the functional

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role of TXA2 on the ACh-induced vasodilator response by inhibiting its synthesis, as will be explained later. Regarding the contraction induced by PGE2, it was increased in arteries after vascular damage, being the increase greater in arteries from orchidectomized rats than in those from intact ones. Vascular damage increased the PGF2α-induce response in similar extent in arteries from intact or orchidectomized rats. These results indicate that vascular damage affects the agonist-induced contraction differently, reflecting the distinct signaling pathways activated by the different

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contractile agents used. In this regard, it is known that activation of TP receptors induces protein kinase C (PKC) activation [14], and that orchidectomy increases PKC

activation [36], suggesting that this event could be responsible to the increased TXA2-

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induced contraction, which was only observed in arteries from orchidectomized rats. Since the regulatory effects of PKC on the release of different cellular mediators such as

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NO [36] and/or on cellular signaling pathways activated after membrane receptors

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stimulation, such as serotonin [25] EGF or TP receptors [9] have been extensively described, future studies to explore these possibilities in our experimental model would be of great interest. Additionally, it is important to note that organ culture can induce

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both downregulation [20, 21] and upregulation [22, 25] of contractile responses, indicating that the effects are dependent on both the contractile agent and also on the

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vessel used. In this sense, it is worth noting that KCl-induced response was increased by

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organ culture up to similar extent, in arteries from both intact or orchidectomized rats, suggesting that induced-vascular damage could increase the activity of voltage operated calcium channels promoting extracellular calcium entry [37]. Confocal microscopy analysis revealed the absence of changes in vascular structure induced by organ culture, suggesting that the increased responses to contractile agents are probably due to changes in signaling pathways. Similar results were found in intrapulmonary arteries after four

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days of culture in which contractile responses to KCl and serotonin were increased without morphological alteration of the vascular wall [38]. Taken together these results support the hypothesis that in the increased TXA2- and PGE2-induced contraction different cell-signaling pathways can be altered, as previously commented. Regarding PGI2-induced relaxation, our results showed that organ culture importantly increased this vasodilator response in arteries from both intact or orchidectomized rats, which agrees with the hyperpolarizing action of PGI2 [39] and the increased participation of

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hyperpolarizing-mediated vasodilation after vascular damage was induced [20]. The fact that indomethacin decreased the ACh-induced response, together the great vasodilator effect of PGI2, points out the important contribution of vasodilator prostanoids and/or

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other vasodilator mediators. Thus, the greater reduction to low ACh concentrations

observed in aortas from orchidectomized rats suggests the participation of vasomotor

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factors other than prostanoids. Indeed, the behavior of the arteries in response to the

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vascular damage depends on the physio- or pathological condition of the animal. For instance, it is important to note that the ACh-response is diminished by vascular damage when the artery comes from intact animals, but it still maintains the same vasodilator

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sigmoid profile. In contrast, when the artery comes from orchidectomized animals, the decrease in the ACh-induced response is not statistically different, but a significant

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change in the ACh-curve profile is observed, reflecting an important alteration in the

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ability of the artery to respond to this particular vasodilator stimulus. Furthermore, the analysis of the nuclear DAPI-staining indicated the presence of endothelial cells, in fresh and cultured arteries, since labelled cells were observed adjacent to the internal elastic lamina, as previously reported [38]. Moreover, the CD31 staining also indicated that the endothelium was preserved in cultured arteries. It is interesting to mention that apart from the luminal localization of CD31, it can be also observed in the adventitia

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layer of some preparations, which may reflect the presence of mesenchymal stem cell (CD31+) as already reported [40]. In addition, it has also been described that in arteries after four days of organ culture the muscarinic receptor activation-induced relaxation was abolished, while the relaxation induced by the calcium ionophore A23187 were preserved [38], indicating the presence of viable endothelium and NO release. All these results reinforce the hypothesis about the involvement of additional factors whose vascular effects could have also been modified. In this regard, studies are in progress to

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evaluate this possibility. Beyond the global participation of prostanoids in the ACh-induced response, what

is undoubtedly important are the results on TXA2. As discussed above, the release of

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TXA2 was enhanced by the induction of vascular damage in arteries from both intact and orchidectomized animals, while its vasoconstrictor effect was only increased in

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aortas from orchidectomized rats. Based on these results, and taking into account that

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the participation of this prostanoid in endothelial dysfunction associated to cardiovascular risk factors has been well stablished [14, 41, 42], it was worth analyzing the involvement of endogenous TXA2 in the ACh-induced response in arteries in which

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vascular damage has been induced. The inhibition of TXA2 synthesis, with furegrelate, along the induction of vascular damage increased the ACh-induced response in aortic

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segments from intact and orchidectomized rats. However, the increase was much greater

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in arteries from orchidectomized than in those from intact rats. These results indicate the TXA2 involvement when vascular damage was induced, and show the greatest functional role in arteries from orchidectomized rats, which is in line with the increased vasoconstrictor response to the TXA2 mimetic, U-46619, observed in these arteries. In addition to the involvement of TXA2 in different cardiovascular diseases, it has been reported that the prostanoid production other than TXA2, such as PGI2 which is

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increased in hypertension [18] and atherosclerosis [43], PGE2 [44] and PGF2α [45] are able to activate TP receptors. However, based on the results obtained with furegrelate, indicating the functional role of TXA2 on the ACh-induced response, we analyzed the effect of activating TP receptors with the TXA2 mimetic, U-46619, during the induction of vascular damage. The results showed that U-46619 did not modify the ACh-induced response in arteries from intact rats while it decreased that obtained in arteries from orchidectomized rats, indicating that the vascular response depends again on the animal

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hormonal status and also reinforcing the results obtained on ACh-induced response in the presence of furegrelate.

Although alterations of specific intracellular signaling-pathways have not been

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investigated in this work, the results are of physiological importance since they showed

that arteries exposed to endogenous androgens are protected against the organ-culture-

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induced vascular damage. These results are in line with previous results from our

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research group [4-9; 29, 32] but contrast with some animal experimental models of cardiovascular diseases, such as different models of hypertension. In this regard, contradictory results have been described, since some authors have observed that

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orchidectomy attenuated the impaired endothelial function [46] and the increase in the BP [47] induced by the high-salt diet in rats, but these effects were reversed by the

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administration of testosterone. On the contrary, we have found that orchidectomy

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increased BP in normotensive Wistar and Wistar-Kyoto rats, and that testosterone replacement therapy prevented the orchidectomy-induced hypertension [48]. However, there are rat strains that are more resistant to the development of hypertension, as occurs in orchidectomized Sparague-Dawley rats that became hypertensive only in aging (22 months old) while remain normotensive during adulthood [9]. These results also indicate that experimental approaches with animal models are closer to physiological

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conditions than the organ culture technique, but are also more complex, and alterations in various systems other than the vascular one could condition vascular responses. However, the advantage of the organ culture technique lies in inducing vascular damage directly into the vascular wall without involving other regulatory systems or modifying general vascular structure, which allows to obtain results that complement those obtained with other models, as many studies have already validated. In summary, these results show that the induction of vascular damage produces a

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significant increase in the production and vascular effect of prostanoids. Likewise, the results show the protective role of male sex hormones against vascular damage, since a

decreased deleterious effect of prostanoids, particularly that of TXA2, was observed in

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be linked to vascular structure modifications.

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arteries from rats with intact gonadal function. These functional alterations seem not to

Funding

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This study was supported by grants from the Fondo de Investigaciones Sanitarias (PI11/00406 and PI19/01282), Comunidad de Madrid (S2013/ABI-2783, “INSPIR-

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CM”) and Fondo Europeo de Desarrollo Regional to M. Ferrer.

Declaration of interest

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The authors report no conflicts of interest.

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Acknowledgements

The authors thank Mr. David Muñoz and technical staff of the Animal Facility at the Facultad de Medicina for the care of animals, Ms. Soledad Montalbán for assistance in processing the immunohistochemistry samples and Ms. Laura Mañas and Dolores Morales for technical assistance with confocal microscopy.

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[47] Oloyo AK, Sofola OA, Yakubu MA (2016) Orchidectomy attenuates high-salt diet-induced increases in blood pressure, renovascular resistance, and hind limb vascular dysfunction: role of testosterone. Clin Exp Pharmacol Physiol 43: 82533. [48] Perusquía M, Herrera N, Ferrer M, Stallone JN (2017) Antihypertensive effects of androgens in conscious, spontaneously hypertensive rats. J Steroid Biochem Mol

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Figure Legends Fig. 1. Effect of orchidectomy on prostanoid release in cultured aorta. Release of thromboxane A2 (TXA2), prostaglandin (PG) E2, PGF2α and PGI2 in cultured aortic segments from intact and orchidectomized rats. Results are represented as mean ± SEM. Number of animals used is indicated in parenthesis. *p <0.05 compared to arterial

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segments from intact rats.

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Fig. 2. Effect of 20 h organ culture on prostanoids-induced vasomotor responses in aorta from intact and orchidectomized rats. Concentration response-curves to the non hydrolizable mimetic of TXA2, U-46619, prostaglandin (PG) E2, PGF2α and PGI2 in fresh and cultured aortic segments from intact and orchidectomized rats. Results (mean

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± SEM) are represented in mg. Number of animals is indicated in parenthesis. The statistical significances are indicated in the corresponding graphs. The effect of organ

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culture on the dAUC for each prostanoids is also included.

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Fig. 3. Effect of 20 h organ culture on the vasodilator action to Acetylcholine in aorta from intact and orchidectomized rats. Concentration response-curves to the Acetylcholine (ACh) in fresh and cultured aortic segments from intact and orchidectomized rats. Results (mean ± SEM) are represented as percentage of the inhibition of the contraction elicited by 0.1 µM noradrenaline. Number of animals is indicated in parenthesis. The statistical significance is indicated in the corresponding

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graph. The effect of organ culture on the dAUC for ACh is also included.

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Fig. 4. Orchidectomy modulates the involvement of prostanoids and of thromboxane A2 in the Acetylcholine-induced vasodilation in cultured aorta. Effect of indomethacin (Indom., 10 µM) and furegrelate (Fureg., 1 µM) on the concentration response-curves to the Acetylcholine (ACh) in cultured aortic segments from intact and orchidectomized

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rats. Results (mean ± SEM) are represented as percentage of the inhibition of the contraction elicited by 0.1 µM noradrenaline. Number of animals is indicated in parenthesis. The statistical significances are indicated in the corresponding graphs. The effect of organ culture on the dAUC for ACh, in all experimental conditions, is also

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included.

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Fig. 5. Orchidectomy modulates the Acetylcholine-induced vasodilation after thromboxane/prostanoids receptor activation in cultured aorta. Effect of the non hydrolyzable mimetic of TXA2, U-46619 (1 nM) on the concentration response-curves to the Acetylcholine (ACh) in cultured aortic segments from intact and orchidectomized

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rats. Results (mean ± SEM) are represented as percentage of the inhibition of the contraction elicited by 0.1 µM noradrenaline. Number of animals is indicated in parenthesis. The statistical significances are indicated in the corresponding graphs. The

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effect of organ culture on the dAUC for ACh is also included.

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Fig. 6. Effect of 20 h organ culture on vascular structure of aorta from intact and orchidectomized rats. Representative confocal images of fresh and cultured aortic wall of intact and orchidectomized rats obtained with a 40x objective. Autofluorescence of elastin is showed in green and DAPI-nuclei staining in blue. Images were representative of two aortic rings per experimental condition obtained from 3 intact rats and 3 orchidectomized rats. The intima layer is indicated with arrows; L: lumen (A). Quantification of medial thickness (B) and of number of smooth muscle cells (SMCs)

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(C) in fresh and cultured aortic segments from intact and orchidectomized rats. Results (mean ± SEM) are represented as the distance between the internal and external elastic lamina (B, in µm) and as the number of nuclei per area (C). The number of animals used

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was of 3 intact and 3 orchidectomized rats.

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Fig. 7. Effect of 20 h organ culture on CD31 detection in aorta from intact and orchidectomized rats. In each aortic section, CD31 detection is shown in red and elastin autofluorescence in green. Images are representative of multiple aortic sections per

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experimental condition obtained from 2 intact rats and 2 orchidectomized rats. The

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intima layer is indicated with arrows; L: lumen.

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f

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Table 1. Maximal vasomotor response (Emax) and the negative logarithm of effective concentration 50 (pEC50) of prostanoids in fresh and cultured aorta from intact and orchidectomized (Cx) rats.

4026 + 345

349 + 75

PGF2α

202 + 61

na l

Fresh

e-

PGE2

Pr

Intact rats

U-46619

pr

Emax

pEC50

PGI2

U-46619

PGE2

PGF2α

PGI2

187 + 15

6.64 + 1.03

< 5.61 + 0.10

< 7.10 + 0.13

< 7.08 + 0.3

5384 + 438*

1033 + 192*

868 + 99**

539 + 60*

7.24 + 0.10

< 5.60 + 0.09

< 7.02 + 0.10

< 7.83 + 0.18

Cx rats

3879 + 274

842 + 80#

338 + 53

279 + 62

7.53 + 0.07

< 5.74 + 0.03

< 7.16 + 0.07

< 7.32 + 0.16

1771 + 135**

821 + 135**

605 + 51**

7.50 + 0.06

< 5.81 + 0.05

< 7.28 + 0.15

< 8.16 + 0.18*

Fresh Cultured

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Cultured

6621 + 522**

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na l

Pr

e-

pr

fresh arteries; # p < 0.05 compared to fresh arteries from intact rats.

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Values (mean + SEM) are expressed in mg for Emax and in -log EC50 for pEC50. Statistical significances: * p < 0.05, **p < 0.001 compared to

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Table 2. Maximal vasodilator response (Emax) and the negative logarithm of effective concentration 50 (pEC50) of Acetylcholine in fresh and cultured aorta from intact and orchidectomized (Cx) rats. Emax

Intact rats

pEC50

86.70 + 2.54

7.237 + 0.10

Cultured

74.40 + 3.87**

6.67 + 0.17**

Cx rats

84.43 + 3.38

7.43 + 0.23

65.49 + 6.98*

7.28 + 0.26

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Fresh

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Cultured

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Fresh

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Values (mean + SEM) for Emax are expressed as percentage of the inhibition of the contraction elicited by 0.1 noradrenaline, and in -log EC50 for pEC50. Statistical

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significances: * p < 0.05, **p < 0.005 compared to fresh arteries

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Table 3. Effect of 10 µM indomethacin (Indom), 1 µM furegrealte (Fureg.) or 1 nM U46619 on the maximal vasodiator response (Emax) and the negative logarithm of effective concentration 50 (pEC50) of Acetylcholine in cultured aorta from intact and orchidectomized (Cx) rats. Emax

pEC50

Intact rats

Cx rats

Intact rats

Cultured

74.40 + 3.87

65.49 + 6.98

6.67 + 0.17

7.28 + 0.26

+Indom.

42.57 + 7.67**

44.14 + 7.76*

6.84 + 0.41

6.94 + 0.33

+Fureg.

89.17 + 4.79*

99.0 + 1.0**#

7.37 + 0.18**

8.92

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+U-46619

56.40 + 12.7

64.0 + 7.02

Cx rats

7.14 + 0.18

+

0.40**## 6.87 + 0.26

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Values (mean + SEM) for Emax are expressed as percentage of the inhibition of the contraction elicited by 0.1 noradrenaline, and in -log EC50 for pEC50. Statistical

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significances: * p < 0.05, **p < 0.005 compared to cultured arteries in absence of drugs;

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# p < 0.05; # p < 0.005 compared to cultured arteries from intact rats.

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