Abstract
The role of androgens in vascular reactivity is controversial, particularly regarding their age-related actions. The objective of this study was to conduct a temporal evaluation of the vascular reactivity of resistance arteries of young male rats, as well as to understand how male sex hormones can influence the vascular function of these animals. Endothelium-mediated relaxation was characterized in third-order mesenteric arteries of 10-, 12-, 16-, and 18w (week-old) male rats. Concentration–response curves to acetylcholine (ACh, 0.1 nmol/L–10 µmol/L) were constructed in arteries previously contracted with phenylephrine (PE, 3 µmol/L), before and after the use of nitric oxide synthase or cyclooxygenase inhibitors. PE concentration–response curves (1 nmol/L–100 μmol/L) were also built. The levels of vascular nitric oxide, superoxide anion, and hydrogen peroxide were assessed and histomorphometry analysis was performed. The 18w group had impaired endothelium-dependent relaxation. All groups showed prostanoid-independent and nitric oxide-dependent vasodilatory response, although this dependence seems to be smaller in the 18w group. The 18w group had the lowest nitric oxide and hydrogen peroxide production, in addition to the highest superoxide anion levels. Besides functional impairment, 18w animals showed morphological differences in third-order mesenteric arteries compared with the other groups. Our data show that time-dependent exposure to male sex hormones appears to play an important role in the development of vascular changes that can lead to impaired vascular reactivity in mesenteric arteries, which could be related to the onset of age-related cardiovascular changes in males.
Introduction
Cardiovascular disease is the leading cause of death worldwide and men are more susceptible to developing it when compared with premenopausal women (Benjamin et al. 2018). This is attributed to female sex hormones having a protective action on the cardiovascular system (Farhat et al. 1996, Miller & Harman 2017), whereas male sex hormones have been suggested to play a controversial and sometimes deleterious role (Pantalone et al. 2019).
Androgens are secreted mainly by the testicles (Nieschlag & Nieschlag 2019) and are crucial for the development of male characteristics during the embryonic period and puberty, as well as for sexual maturation (Perusquía & Stallone 2010, Nieschlag & Nieschlag 2019). In adulthood, androgens such as testosterone and dihydrotestosterone are essential for maintaining male reproductive functions and behavior (Heemers et al. 2006). Androgens can act on several systems, in addition to those related to reproductive tissues (Heemers et al. 2006), including the cardiovascular system (Herald et al. 2017). Since men are more susceptible to cardiovascular events, androgens have been associated with deleterious effects (Tostes et al. 2016). The effects by which male sex hormones modulate the cardiovascular system may involve the stimulation of important pathways that increase oxidative stress (Reckelhoff et al. 2000, Tostes et al. 2016). There is, however, evidence that androgens may have beneficial effects on the cardiovascular system (Rouver et al. 2015, Perusquía et al. 2017). For instance, while orchiectomy increased mean blood pressure, this parameter was preserved in testosterone-treated and intact rats, indicating that androgens have an antihypertensive effect (Perusquía et al. 2017, 2019).
Changes in blood pressure variables may be due to changes in vascular reactivity, especially in resistance arteries (Onaka et al. 1998). For this reason, to identify the effects of androgens on arterial tone modulation is crucial to discern how these hormones act on resistance vessels. Accordingly, studies have shown that the effects of androgens on a wide variety of arterial segments involve a rapid, non-genomic action. For instance, testosterone was shown to vasodilate the thoracic aorta of rats (Bucci et al. 2009) and porcine coronary arteries (Deenadayalu et al. 2012). This androgen also induced relaxation on canine basilar arteries (Ramírez-Rosas et al. 2011) and rat mesenteric microvessels (Puttabyatappa et al. 2013). We have previously shown that hormonal dysfunction impairs endothelium-dependent coronary vascular reactivity in male rats (Rouver et al. 2015).
Although androgens have beneficial actions, there is evidence of them having a deleterious role. For example, these hormones can increase the production of reactive oxygen species, potentially contributing to oxidative stress in the vasculature (Tostes et al. 2016) and consequently decreasing nitric oxide (NO) bioavailability (Zou et al. 2002). Furthermore, it is known that vascular structure (Lacolley et al. 2018) and functionality (Novella et al. 2013) are impaired with age, and these changes in vasculature can also be associated with androgens (Fu et al. 2008), which can modulate blood pressure and vascular reactivity of rats (Dalmasso et al. 2017, Perusquía et al. 2017, 2019, Isidoro et al. 2018). Given the controversial role of male sex hormones, the objective of this study was to conduct a temporal evaluation of the vascular reactivity of resistance arteries of young rats, as well as to understand how male sex hormones can influence the vascular function of these animals. Our hypothesis is that a time-dependent exposure to male sex hormones can modulate the vascular reactivity of third-order mesenteric arteries.
Materials and methods
Experimental animals
We used Wistar male rats (Rattus norvegicus) provided by the animal facility of the Health Sciences Center of the Federal University of Espirito Santo. All procedures were conducted in accordance with the recommendations of the Brazilian Guidelines for the Care and Use of Animals for Scientific and Didactic Purposes and the Guidelines for the Practice of Euthanasia and approved by the Animal Ethics Committee from the Federal University of Espirito Santo (No. #07/2016). The animals were maintained in group-housing under controlled conditions of temperature (22–24°C) and humidity (40–60%), with a 12/12 h light–darkness cycle with water and food ad libitum. The animals were grouped according to their age in weeks (w), as follows: 10w, 12w, 16w, and 18w.
Vascular reactivity
The vascular reactivity of mesenteric arteries was assessed through a resistance myograph system (620 M; Danish Myo Technology, Aarhus, Denmark). The protocols were performed as previously described by Mulvany & Halpern (1977). To avoid interference with the sustained phase of the contractile response, the rats were euthanized by decapitation without anesthesia (Hatano et al. 1989). Third-order mesenteric arteries were identified, isolated, and dissected from the adjacent tissue. Artery segments were cut into 2-mm rings and mounted between two tungsten threads (40 μm in diameter) inside chambers filled with Krebs solution containing: NaCl 119 mmol/L, KCl 4.7 mmol/L, KH2PO4 0.5 mmol/L, NaHCO3 13.4 mmol/L, MgSO4.7H2O 1.17 mmol/L, CaCl2.2H2O 2.5 mmol/L, and glucose 5.5 mmol/L, kept at 37°C and aired with carbogenic mixture (95% O2 and 5% CO2). The rings were gradually stretched until their internal diameters corresponded to a transmural pressure of 100 mmHg, after which the internal circumference (IC1) was normalized to a set fraction of the internal circumference (IC100). Thus, IC1 was calculated by multiplying IC100 by 0.9. The vessels were challenged twice with phenylephrine (PE, 3 μmol/L) in order to elicit reproducible contractile responses. A vessel fragment simultaneously monitored for PE alone during the same time frame of the acetylcholine (ACh) curve worked as a control for the protocols. Endothelial viability and integrity were assessed through the administration of ACh (10 μmol/L) in rings previously contracted by PE (3 μmol/L). The endothelium was considered viable and intact when the relaxation response observed was ≥80%. Concentration–response curves to the cumulative addition of ACh (0.1 nmol/L–10 μmol/L) were constructed following the induction of contraction with PE (3 μmol/L). The vasodilator effect of ACh was investigated in the presence of the inhibitors: Nω-nitro-l-arginine methyl ester (l-NAME, nitric oxide synthase (NOS) inhibitor, 300 μmol/L; Sigma), indomethacin (INDO, cyclooxygenase (COX ) inhibitor, 10 μmol/L; Sigma), and a combination of l-NAME (300 μmol/L) and INDO (10 μmol/L). The vessels were incubated for 30 min before ACh concentration–response curves were performed, and the percent relaxation was determined using a LabChart 8 data acquisition system (AD Instruments Pty Ltd., New South Wales, Australia). PE concentration–response curves (1 nmol/L–100 μmol/L) were also constructed.
Vascular nitric oxide (NO), superoxide anion (O2•−), and hydrogen peroxide (H2O2) levels
The levels of NO, O2•−, and H2O2 were measured by fluorescence microscopy in third-order mesenteric artery sections as previously described, with modifications (Cunha et al. 2020, Giesen et al. 2020). The third-order mesenteric artery segments of the 10w and 18w groups were soaked in a frozen cryoprotection liquid (Tissue-Tek® OCT™, Sakura®, Torrance, CA, USA) and kept at −80°C until use. Cross-sectional (10 µm) cuts were obtained using a cryostat (Leica 1850, Leica).
The production of NO was measured using the 4,5-diaminofluorescein diacetate fluorescent probe (DAF-2DA, Sigma-Aldrich, 10 µmol/L). The slides were equilibrated for 10 min in phosphate buffer (0.1 mmol/L, pH = 7.4) containing CaCl2 (0.45 mmol/L) and incubated for 30 min with DAF-2DA in a humid chamber under light at 37°C. Sections from each artery were evaluated as to basal NO production or using l-NAME (300 µmol/L).
For the measurement of H2O2, the 2', 7' dichlorodihydrofluorescein diacetate fluorescent probe (H2DCF-DA; Life Technologies; 10 µmol/L) was used. The slides were equilibrated for 10 min in a phosphate buffer and incubated with the H2DCF-DA probe for 30 min. The sections were evaluated as to basal H2O2 production or using catalase (1000 units/mL). For the measurement of O2•− production, the dihydroethidium probe (DHE; Invitrogen; 5 µmol/L) was used. Slides underwent the aforementioned equilibration and DHE incubation protocol and were evaluated as to basal O2•− production or using tiron (10 µmol/L).
All slides were washed three times at 10-min intervals. Digital images were acquired with a fluorescence microscope (Zeiss, Axio Observer Z1), equipped with a fluorescein filter and an AxioCam MRm camera under a 40× objective. Fluorescence intensity was measured using ImageJ software (version 1.46, NHI) and expressed in arbitrary units (A.U.).
Histomorphometry
Third-order mesenteric arteries were removed and fixed in PBS-formalin at room temperature. Sections of the vessels were stained with hematoxylin and eosin and examined for morphological parameters according to the literature (Ximenes et al. 2017). A histomorphometric image analysis system was used with a digital camera (Axio-Cam ERc 5S) coupled to a light microscope (Olympus AX70; Olympus). High-resolution images (2048×1536 pixels) were captured using the Carl Zeiss AxioVision Rel. 4.8 software and photomicrographs were analyzed using a 20× objective. The cross-sectional area, thickness, and lumen of mesenteric vessel walls (which included all vascular tunics/area) were calculated using ImageJ software. We used four animals from each group.
Collagen density surface
Picrosirius red-stained third-order mesenteric artery sections were used to obtain 15 mesenteric tissue photomicrographs with a 20× objective lens. All areas analyzed were randomly chosen, and those without vascular tunics were avoided. The images were converted to high-contrast black and white images to visualize stained collagen fibers and analyzed with ImageJ software. The results represent the relative amount of collagen in the total mesenteric wall area, as previously described (dos Santos et al. 2012).
Scanning electron microscopy
Third-order mesenteric arteries were collected, washed, opened longitudinally for 10 min with PBS, and fixed with 2.5% v/v glutaraldehyde. The mesenteric arteries were post-fixed in 1% v/v osmium tetroxide, 3.8% v/v potassium ferricyanide, and 2.5 mmol/L CaCl2, dehydrated in acetone, and embedded in Epon 812. Ultra-thin sections were examined under a Zeiss EM 10C microscope. For scanning electron microscopy, spheroids were dried using the critical point method, coated with a 20-nm layer of gold, and examined under a Zeiss 940 DSM microscope (dos Santos et al. 2012).
Non-invasive blood pressure assessment
Systolic blood pressure (SBP) was assessed as previously described (Rouver et al. 2015). Briefly, after a period of adaptation, the animals were placed in a heated chamber within a container and restrained with a pneumatic cuff attached to the proximal region of the tail. A sphygmomanometer was inflated and deflated automatically, and SBP was recorded using a transducer. The temperature was maintained between 29 and 32°C for 40 min, during which the animals remained in the chamber (IITC INC/Life Science, 23924 Victory Blvd, Woodland Hills, CA, USA). An average of three measurements were obtained, with a maximum difference of 10 mmHg, and measurements associated with animal movements were discarded.
Serological measurements
After euthanasia, blood samples (5 mL) were collected for the measurement of hormone levels. The samples were centrifuged (centrifuge Excelsa IV Model 280R) at 2656 g for 15 min at 4°C and the serum was collected and stored at 20°C. Testosterone and estradiol were measured by chemiluminescence microparticle immunoassay using ARCHITECT 2nd Generation Testosterone and ARCHITECT Estradiol Kits, respectively. Quantification limits were ≤0.15 nmol/L for testosterone and ≤5 pg/mL for estradiol.
Statistical analysis
Data were analyzed using the GraphPad Prism 6 (GraphPad Software). All data are expressed as mean ± s.e.m. Data normality was evaluated through the Shapiro–Wilk test. Once normality was confirmed, changes in vascular reactivity were evaluated by two-way ANOVA followed by Bonferroni post hoc test. SBP, area under the curve (AUC), Rmax, and fluorescence intensity were evaluated through one-way ANOVA followed by Tukey post hoc test or unpaired Student’s t test when appropriate. For the analysis of histomorphometry parameters (non-Gaussian data), we used the Mann–Whitney test. The significance level of P < 0.05 was adopted.
Results
Vascular reactivity
The ACh concentration–response curves built to assess the vascular reactivity of third-order mesenteric arteries revealed that the 18w group had an impaired endothelium-dependent relaxation response compared with the other groups (Fig. 1A). These results were confirmed by the AUC (10w, 238 ± 14; 12w, 232 ± 19; 16w, 215 ± 20; 18w, 153 ± 22 A.U.) and maximum relaxation response data (10w, 94 ± 2; 12w, 97 ± 2; 16w, 87 ± 3; 18w, 67 ± 8% of relaxation) (Fig. 1B and C, respectively).
Vascular reactivity of third-order mesenteric arteries from male Wistar rats. (A) Concentration–response curve to ACh (10−10– 10−5 M), (B) area under the curve (AUC), and (C) maximal relaxation response of 10-, 12-, 16-, and 18-week-old animals. Values are expressed as mean ± s.e.m. Two-way ANOVA was used followed by Bonferroni post hoc test. *P < 0.05 compared with 10w, #P < 0.05 compared with 12w, and +P < 0.05 compared with 16w. n = 7 for each group.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0147
Since only third-order mesenteric arteries from 18w animals had an impaired ACh-induced relaxation response compared with younger groups and orchiectomized groups (data not shown), we present here the protocols to identify endothelial mediators only in the 10w and 18w groups. Thus, to identify possible endothelial mediators involved in the ACh-induced relaxation response, we performed non-selective incubations with COX and NOS inhibitors (Fig. 2). The AUC data showed that the ACh-induced vasodilatory response in 10w and 18w vascular segments does not seem to involve the prostanoid (PN) pathway (such as prostaglandins and thromboxane) (Fig. 2A and 2D). On the other hand, the NO pathway (Fig. 2B and 2E) seems to play an important role in the ACh-induced vasodilation response of both 10w (238 ± 14 to 26 ± 5 A.U.) and 18w groups (153 ± 22 to 31 ± 9 A9 A.U.). In addition, the combined inhibition of COX and NOS (Fig. 2C and 2F) confirmed NO participation in this response (10w (238 ± 14 to 39 ± 12 A.U.) and 18w (153 ± 22 to 46 ± 21 A.U.)). Furthermore, as can be seen in Fig. 2H, the NO vasodilatation response seems to be diminished in the 18w group, which may have contributed to the lower AUC in this group (Fig. 2G).
Participation of endothelial mediators in the vasodilatory effects of third-order mesenteric arteries from 10w and 18w groups. (A and D) Effect of cyclooxygenase (COX) inhibition with indomethacin (INDO, 10 μM; 10w (n = 7) and 18w (n = 6)); (B and E) nitric oxide synthase (NOS) inhibition with Nω-nitro-l-arginine methyl ester (l-NAME, 300 μM; n = 7 for the 10w group and n = 6 for the 18w group); and (C and F) combined inhibition of NOS and COX with l-NAME and INDO (10w n = 7 and 18w n = 6). (G) Area under the curve (AUC) of 10w and 18w groups and (H) contribution (delta of the AUC before and after inhibition) of prostanoids (PNs), nitric oxide (NO), and PNs plus NO to the concentration–response curve to ACh (10−10 –to 10−5 M). Values are expressed as mean ± s.e.m. Two-way ANOVA was used followed by Bonferroni post hoc test. *P < 0.05 compared to their respective control group.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0147
In addition to the endothelium-mediated vasodilatory response, we evaluated the PE-induced vasoconstrictor response in third-order mesenteric arteries (Fig. 3A). We observed that 16w and 18w groups had a higher contractile response when compared to the 10w and 12w groups, which was confirmed by the AUC (16w, 40 ± 1 and 18w, 37 ± 1 A.U.; 10w, 28 ± 1 and 12w, 29 ± 1 A.U.) (Fig. 3B). The highest maximal vasoconstrictor response was that of the 18w group (19 ± 1 mN) (Fig. 3C).
Vascular reactivity of third-order mesenteric arteries of male Wistar rats. (A) Concentration–response curves to PE (10−9 to 10−4 M), (B) area under the curve (AUC), and (C) maximal contractile response in 10w (n = 6), 12w (n = 6), 16w (n = 6), and 18w (n = 6). Values are expressed as mean ± s.e.m. Two-way ANOVA was used followed by Bonferroni post hoc test. *P < 0.05 compared with 10w, #P < 0.05 compared to 12w, and +P < 0.05 compared with 16w.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0147
Fluorescence analysis for nitric oxide (NO), superoxide anion (O2•−), and hydrogen peroxide (H2O2)
Under basal conditions, the fluorescence emitted by the oxidation of DAF-2DA by NO (Fig. 4) was lower in third-order mesenteric arteries of the 18w group (1.4 ± 0.03 A.U.) than in those of the 10w group (2.2 ± 0.06 A.U.). Also, the intensity of the fluorescence emitted by the oxidation of DHE by O2•− (Fig. 5) was higher in third-order mesenteric arteries from the 18w group (2.7 ± 0.1 A.U.) compared with those of the 10w group (2.2 ± 0.04 A.U.). Moreover, the intensity of the DCF fluorescence induced by H2O2 (Fig. 6) was lower in third-order mesenteric arteries from 18w animals (2.7 ± 0.06 A.U.) than in those of the 10w group (3.4 ± 0.1 A.U.).
Analysis of the fluorescence emitted by DAF-2DA in third-order mesenteric arteries of male Wistar rats. Protocols were performed in the absence or presence of nitric oxide synthase (NOS) inhibition (Nω-nitro-l-arginine methyl ester (l-NAME), 300 μM) and the fluorescence emission was quantified. Scale bar = 20 μm. Values are expressed as mean ± s.e.m. Two-way ANOVA was used followed by Tukey’s post hoc test. *P < 0.05 compared with the same group under basal conditions and #P < 0.05 compared with the respective 10w group. n = 4 for each group. A full color version of this figure is available at https://doi.org/10.1530/JME-22-0147.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0147
Analysis of the fluorescence emitted by DHE in third-order mesenteric arteries of male Wistar rats. Protocols were performed in the absence or presence of Tiron (10 µM) as O2•− scavenger and the fluorescence emission was quantified. Scale bar = 20 μm. Values are expressed as mean ± s.e.m. Two-way ANOVA was used followed by Tukey’s post hoc test. *P < 0.05 compared with the same group under basal conditions and #P < 0.05 compared with the respective 10w group. n = 4 for each group. A full color version of this figure is available at https://doi.org/10.1530/JME-22-0147.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0147
Analysis of the fluorescence emitted by H2DCF-DA in third-order mesenteric arteries of male Wistar rats. Protocols were performed in the absence or presence of catalase (1000 units/mL), which degrades H2O2, and the fluorescence emission was quantified. Scale bar = 20 μm. Values are expressed as mean ± s.e.m. Two-way ANOVA was used followed by Tukey’s post hoc test. *P < 0.05 compared with the same group under basal conditions and #P < 0.05 compared with the respective 10w group. n = 4 for each group. A full color version of this figure is available at https://doi.org/10.1530/JME-22-0147.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0147
Morphological analysis
To conduct a morphological assessment of the third-order mesenteric arteries, we analyzed representative images of arteries from the 10w (Fig. 7A and 7A1) and 18w groups (Fig. 7B and 7B1), and Picrosirius red-stained arteries from 10w (Fig. 7C) and 18w groups (Fig. 7D). Arteries from the 18w group had increased lumen (9.55 ± 0.43 µm2) (Fig. 7E) compared with those of the 10w group (7.69 ± 0.43 µm2). The cross-sectional area was measured, and a reduction was observed in the 18w group (2.78 ± 0.07 µm2) (Fig. 7G) compared with the 10w group (1.89 ± 0.12 µm2). Moreover, the mesenteric artery wall (tunica media, Fig. 7F) was thinner in the 18w group (21.52 ± 0.83 µm) than in the 10w group (27.18 ± 1.15 µm). In addition, collagen fiber deposition (Fig. 7C) was lower in the 18w group (37.36 ± 0.62%) than in the 10w group (32.01 ± 1.24%).
Histomorphometry analysis of H&E and Picrosirius red-stained third-order mesenteric artery sections from 10- (10w) and 18-week-old (18w) groups. (A and A1) third-order mesenteric artery section from the 10w group showing normal morphology. (B and B1) third-order mesenteric artery section from the 18w group showing signs of vascular atrophy. Representative image of collagen density in (C) 10w and (D) 18w groups. (E) lumen, (F) wall thickness, (G) crosssection area, and (H) collagen density of third-order mesenteric arteries from the 10w and 18w groups. Scale bar = 50 and 20 μm. Values are expressed as mean ± s.e.m. Student’s t-tests were used. *P < 0.05 compared to 10w group. n = 4 for each group. A full color version of this figure is available at https://doi.org/10.1530/JME-22-0147.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0147
Scanning electron microscopy (SEM) images of the third-order mesenteric arteries (Fig. 8) revealed that the endothelial surface of 10w arteries had a normal squamous appearance and that the epithelial nucleus cell (arrowhead) was regular (Fig. 8A and 8A1). In the 18w group, an abnormal squamous-looking epithelium was observed, suggesting denudation of the superficial endothelial aspect (asterisk), which leads to an irregular endothelial surface with accumulation of blood cells and contracted aspect (Fig. 8B and 8B1).
Scanning electron microscopy (SEM) showing the endothelial surface in third-order mesenteric arteries of 10- (10w) and 18-week-old (18w) groups. (A and A1) Endothelial cells of third-order mesenteric arteries from the 10w group had a normal squamous appearance and a regular epithelial nucleus cell (arrowhead) (bar = 5 μm). (B and B1) Irregular endothelial surface of third-order mesenteric arteries from the 18w group, suggesting denudation of the superficial endothelium with blood cell accumulation and contracted aspect (asterisk) (Scale bar = 2 μm). n = 4 for each group. A full color version of this figure is available at https://doi.org/10.1530/JME-22-0147.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0147
Hormonal level and systolic blood pressure assessment
There were no differences in serum levels of testosterone and estrogen in the groups studied. 18w animals had higher SBP values compared with 10w ones (Table 1).
Measurement of systolic blood pressure by tail plethysmography and hormone levels
10 weeks | 12 weeks | 16 weeks | 18 weeks | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
N | Mean | s.e.m. | N | Mean | s.e.m. | N | Mean | s.e.m. | N | Mean | s.e.m. | |
Systolic blood pressure (mmHg) | 7 | 116 | 4 | 7 | 122 | 3 | 7 | 128 | 4 | 7 | 132* | 4 |
Estrogen levels (μg/dL) | 6 | 20 | 2 | 6 | 16 | 1 | 6 | 22 | 2 | 6 | 22 | 2 |
Testosterone levels (μg/dL) | 6 | 382 | 66 | 6 | 789 | 160 | 6 | 460 | 97 | 6 | 424 | 67 |
Data are expressed as mean ± s.e.m. Systolic blood pressure, estrogen, and testosterone levels in male Wistar rats from 10 to 18 weeks of age. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 compared with 10w.
Discussion
The main finding of our study was that the vascular reactivity of third-order mesenteric arteries from 18w animals was impaired when compared with 10w animals. This difference in reactivity was accompanied by functional and morphological alterations, changes in the release of factors derived from the endothelium that could, together, be involved in blood pressure changes found in the 18w group.
Our first observation was that animals from the 18w group had reduced endothelium-dependent relaxation in third-order mesenteric arteries when compared with 10w animals. These results suggest that a time-dependent exposure to male sex hormones could modulate vascular reactivity in mesenteric arteries, leading to an impairment of endothelium-dependent relaxation with age. Indeed, in gonadectomized animals, this impairment was not observed (data not shown). It is known that the damage in the cardiovascular system that takes place in the presence of male sex hormones is attenuated by orchiectomy (Oloyo et al. 2016).
Male sex hormones can modulate vascular parameters (Reckelhoff et al. 1999; Perusquía et al. 2019). Indeed, orchiectomy impaired the vascular reactivity of internal pudendal arteries of Wistar rats 4 weeks after the surgery took place (Alves-Lopes et al. 2017). In addition, the endothelium-dependent coronary vascular reactivity of normotensive male Wistar rats was impaired 15 days after orchiectomy (Rouver et al. 2015). In contrast, Ferrer et al. (Ferrer et al. 1999) found an improvement in the vasodilator response to ACh in a segment of the thoracic aorta of rats 4 months after orchiectomy. It has been demonstrated that hormonal deficiency in male rats, 4 weeks after castration, did not alter vascular reactivity in second-order mesenteric arteries (Toot et al. 2012). Similar results were reported by Oloyo et al. (Oloyo et al. 2016) while studying the internal iliac artery of rats 6 weeks after orchiectomy. All these findings underline the complexity of the roles male sex hormones play on the cardiovascular system and attest that such roles can induce a variety of responses that may differ depending on the vascular bed and the species studied.
In addition, 18w animals had an increase in the contractile response that was not observed in orchiectomized animals either (data not shown). English et al. (English et al. 2000), when studying the coronary arteries of both young (3–4 months old) and elderly (22–26 months old) rats, demonstrated that coronary vessels of elderly animals were less sensitive to the vasodilator effects of testosterone compared with those of young animals. In elderly animals, this outcome was followed by hypertrophy of vascular myocytes and thickening of the smooth muscle layer (English et al. 2000). Therefore, we verified through histomorphometry whether such morphological changes were also present in third-order mesenteric arteries of 18w animals, considering that only this group had altered vascular reactivity. The results demonstrated that 18w animals had reduced vascular smooth muscle walls when compared with 10w animals.
It is known that aging is responsible for the stiffening and thickening of the vascular wall (Lacolley et al. 2018, Mukherjee et al. 2020). In addition, there is an inverse relationship between testosterone levels and thickening of the vascular wall, so that the decline in androgen levels with age correlates with increased vascular wall in humans (Fu et al. 2008, Mukherjee et al. 2020). For laboratory rats, 14 days are equivalent to approximately 1 year of human life (Quinn 2005). Thus, an 18-year-old human would be equivalent to a 6-month-old rat (Sengupta 2013). In the present study, animals with the longest observation time were 4.5 months old (18w) and thus considered young despite the established reproductive phase (Quinn 2005, Sengupta 2013, Silva et al. 2013). Thus, we believe our vascular morphology results can be explained by age and time-dependent exposure to male sex hormones since there was no difference in the concentration of testosterone and estrogen between the groups studied. In addition to causing androgen-promoted vascular wall stiffening, age is known to lead to endothelial dysfunction (Delp et al. 2008, Moreau et al. 2020). That may involve the participation of male sex hormones (Lopes et al. 2012, Moreau et al. 2020), which would act by inducing the formation of reactive oxygen species, such as O2•−, in blood vessels (Tostes et al. 2016, Moreau et al. 2020).
Scanning electron microscopy data revealed an abnormality in the appearance of the endothelial tissue in 18w animals, suggesting a lesion in the endothelial surface of third-order mesenteric arteries that led to an irregular endothelial surface. This result shines some light on the reasons underlying the impaired endothelium-dependent relaxation in this group. In experimental preparations with an intact endothelium, a vasoconstrictor stimulus had a reduced effect compared with an endothelium-denuded preparation (Sakata et al. 1989). A possible explanation may involve the passage of calcium ions (Ca2+) through vascular smooth muscle cells to the underlying endothelial cells (Dora et al. 1997). After vasoconstrictor stimulation, the influx of Ca2+ into smooth muscle cells via myoendothelial junctions (gap junctions) reaches adjacent endothelial cells, leading to the formation of endothelial-derived factors such as NO (Dora et al. 1997). Thus, in arteries with compromised endothelium, the production of endothelium-dependent relaxation factors, which depends on Ca2+ influx into endothelial cells (Félétou et al. 2012), would not occur, so the contractile stimulation on smooth muscle would remain unopposed.
Considering that the 18w group presented an abnormal endothelial surface, impaired endothelium-dependent relaxation, and greater contractile response, our next step was to evaluate the involvement of possible endothelial mediators. Thus, ACh concentration–response curves were constructed in the presence of inhibitors of NO and PN pathways. We observed that NO-mediated relaxation in the 18w group was decreased compared with that of the 10w group, indicating that this pathway was impaired in 18w animals. This dysfunction can be due to an imbalance in the production of vasodilating and vasoconstricting agents, in which the bioavailability of relaxation factors is reduced while vasoconstricting agents have an enhanced action (Furchgott & Vanhoutte 1989, Qiao et al. 2008, Félétou et al. 2012). In this study, NO signaling seems to have been impaired, since there was no difference when COX was inhibited.
The fluorescence data revealed that the 18w group had decreased NO levels compared with the 10w group. Thus, the decreased response to ACh in 18w animals could be related to a reduction in NO bioavailability. The decline in endothelium function due to senescence with a gradual decrease in endothelial vasodilator mediators, such as NO, has already been demonstrated in experimental models that used 24-month-old rodents (Yildiz 2007, Delp et al. 2008, Yang et al. 2009). However, our results showed that the impairment in NO bioavailability can already be noticed in young, 4.5-month-old male animals.
It is well known that androgens can induce the formation of reactive oxygen species, such as O2•− (Tostes et al. 2016). For instance, testosterone stimulates the activity of the NADPH oxidase in the vascular smooth muscle of normotensive rats (Chignalia et al. 2012), which can increase the O2•− production (Rodiño-Janeiro et al. 2013) in the vascular system (Paik et al. 2014). Once formed, O2•− can act directly as a vasoconstrictor, increasing sensitivity to Ca2+, which subsequently leads to arterial constriction (Knock et al. 2009). O2•− can also modulate vascular reactivity indirectly by decreasing NO bioavailability through the formation of ONOO−, a product of the reaction between O2•− and NO (Zou et al. 2002). Our fluorescence data revealed that O2•− was present at higher levels in the 10w group. As there was no difference in hormone values between the groups, the increase in superoxide formation in 18w animals may have been due to time-dependent exposure to male sex hormones. Therefore, we believe that, at least in part, the lower endothelium-dependent relaxation response in this group may be also due to O2•− causing a decrease in NO bioavailability. In addition, our data showed that the amount of H2O2 was lower in the 18w group. Since H2O2 can be considered an endothelium-dependent vasodilating agent (Matoba et al. 2000), its reduction may have contributed to the damage found in this group.
Regarding the association between androgens and increased blood pressure in rats, studies are still inconsistent as to how these hormones act. While Perusquía et al. (Perusquía et al. 2017, 2019) have shown a progressive increase in the mean arterial pressure of Wistar rats from the fifth week of orchiectomy, Loh & Salleh (Loh & Salleh 2017) showed that normotensive rats submitted to orchiectomy had a decrease in mean blood pressure after 8 weeks. In our study, the higher SBP values found in the 18w group may be, at least in part, related to the morphological changes and impaired vascular function found in this group.
The divergences regarding blood pressure results may be related to the experimental model used in each study. Our animals were 10 weeks old at the beginning of the protocol, while Perusquía and colleagues started with older animals at 18–21 weeks old (Perusquía et al. 2017, 2019). In addition, we used Wistar rats, while Loh & Salleh used Wistar Kioto (Loh & Salleh 2017). The mechanisms by which androgens can modulate blood pressure are well established in hypertensive animal models (Reckelhoff et al. 1997, 1999, 2000). However, the role of androgens on blood pressure in normotensive animals is not yet fully elucidated.
The time-dependent exposure to male sex hormones throughout life promotes morphological and functional changes in resistance mesenteric arteries that explain the impairment in endothelial function observed in the present study. Among the alterations found (Fig. 9), the reduction of the vascular wall, the increase of the vascular lumen, the lower production of NO and H2O2, and the higher production of O2•− could explain the increased vasoconstrictor response and the impairment in vascular relaxation found in the 18w group. Finally, these changes may be related to the development of cardiovascular events in men and their complications with age progression. Thus, we believe that androgens are involved in the development of vascular changes that may participate in the onset of age-related cardiovascular diseases in males.
Schematic representation of the main data. The 18w animals showed impaired vascular relaxation and greater contractile activity in third-order mesenteric arteries compared with 10w animals. Furthermore, 18w animals showed morphological differences in third-order mesenteric arteries, such as increased lumen and thinner artery wall. The 18w group presented an irregular endothelial surface, higher fluorescence to superoxide anion (O2•−), and lower fluorescence to both nitric oxide (NO) and hydrogen peroxide (H2O2). A full color version of this figure is available at https://doi.org/10.1530/JME-22-0147.
Citation: Journal of Molecular Endocrinology 70, 1; 10.1530/JME-22-0147
Declaration of interest
There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Fundo de Apoio a Ciência e Tecnologia do Município de Vitória (FACITEC) – 02/2020; Fundação de Amparo à Pesquisa e Inovação do Espírito Santo (FAPES) – ((grants number: #572/2018 and # Nº 03/2021 TO: 486/2021. Nº SIAFEM: 2021-QZX9N); (TO: 346/2022)); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - (grants number: #307224/2021-0 and # 406097/2021-6).
Author Contribution Statement
Wender do Nascimento Rouver: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft, Writing - Review & Editing, Visualization, Project administration. Nathalie Tristão Banhos Delgado: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft, Writing - Review & Editing, Visualization. Leticia Tinoco Gonçalves: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft, Writing - Review & Editing, Visualization. Jéssyca Aparecida Soares Giesen: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft, Writing - Review & Editing, Visualization. Charles Santos da Costa: Methodology, Formal analysis, Investigation, Writing - Review & Editing, Visualization. Eduardo Merlo: Methodology, Formal analysis, Investigation, Writing - Review & Editing, Visualization. Eduardo Damasceno Costa: Methodology, Formal analysis, Investigation, Writing - Review & Editing, Visualization. Virginia Soares Lemos: Methodology, Formal analysis, Resources, Writing - Review & Editing, Visualization. Jones Bernardes Graceli: Conceptualization, Methodology, Formal analysis, Resources, Writing - Review & Editing, Visualization. Roger Lyrio dos Santos: Conceptualization, Methodology, Formal analysis, Resources, Writing - Original Draft, Writing - Review & Editing, Visualization, Supervision.
Acknowledgements
The authors would like to thank Fabricio Bragança da Silva, Juan Carlos Arapa Diaz, Tagana Rosa da Cunha, Débora Tacon da Costa, and Matheus Zucolotto Homem for the technical assistance regarding other issues with the manuscript.
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