Edited by: Carlos Rosales, Universidad Nacional Autónoma de México, Mexico
Reviewed by: Owen Woodman, RMIT University, Australia; Gautham Yepuri, University of Fribourg, Switzerland
*Correspondence: Agustina Cano-Martínez
Verónica Guarner-Lans
This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Different human-like cardiomyopathies associated to β-adrenergic stimulation are experimentally modeled in animals through variations in dose, route, and duration of administration of different cardiotoxic drugs. However, associated changes in the vasculature and their relation to systemic inflammation, and the influence of cardiovascular diseases risk factors (gender and age) upon them are seldom analyzed. Here we studied the effect of age and gender on the vasoreactivity of aortas from mice subjected to
β-adrenoceptors (β-AR) are essential regulators of the cardiovascular homeostasis. They are located in the heart, but also in vascular smooth muscle cells, where they mediate vasodilating effects of endogenous catecholamines (Chruscinski et al.,
β-adrenergic stimulation also increases circulating inflammatory cells (Mills et al.,
Experimentally, different pathological cardiac phenotypes result from the stimulation with ISO depending on the dose, route or duration of the administration. These phenotypes resemble different human-like cardiomyopathies including myocardial infarction (Hohimer et al.,
Furthermore, factors such as gender and age influence the incidence of cardiovascular diseases. Aging increases 10-fold the risk of cardiovascular morbidity between ages 50 and 80 (Ghebre et al.,
Here we studied the differences in
All procedures followed the guidelines established by the Federal Regulation for Experimentation and Animal Care (SAGARPA, NOM-062-ZOO-1999, México) and the experimental protocol was registered and approved by our institution; protocol INCICH-10-695. Young (2 months old) and old (18 months old) male and female Balb/c mice were used. Animals were kept with food and water
Aortas from 4 mice of each group were obtained and carefully cleaned from connective and adipose tissue, taking care not to damage the endothelium. 1 or 2 segments from every aorta were used totaling 6–8 vascular reactivity assays. Tension measurements were made as previously described (Rubio-Ruiz et al.,
Plasma circulating levels of IL-1α (RRA00, R&D Systems), IL-4 (R4000, R&D Systems), and VCAM-1 (E-EL-R1061, Elabscience) were determined by enzyme-linked immunosorbent assay (ELISA) following the manufacturer's instructions using 10 μl of serum from all experimental animal groups (
Statistical analysis of vascular reactivity assays, pEC50 and Emax were performed by two-way ANOVA, followed by Student-Newman-Keuls or Dunn tests, using the Sigma Stat program (Jandel Scientific). When comparing control values between gender and age, Student's
The contraction induced by NE was slightly stronger in aortas from males than from females of 2 months of age (0.21 ± 0.02 g vs. 0.18 ± 0.03 g, respectively) but the difference was not statistically significant. In aged mice, contraction significantly decreased in male aortas, while it remained constant in females (old males: 0.06 ± 6.8*10−3g vs. old females: 0.17 ± 0.01 g, respectively).
Aortic rings exhibited a concentration-dependent vasorelaxation in response to Ach. The maximal relaxation was similar in aortas from male and female mice of 2 months of age (Emax = 76.7 ± 0.9% vs. 71.6 ± 0.4%, respectively) (Table
Vehicle | 6.24 ± 0.03 | 76.7 ± 0.9 | ND | ND |
ISO 5 μg/g/d | 6.13 ± 0.13 | 49.7 ± 2.7 |
6.01 ± 0.02 | 42.5 ± 0.4 |
ISO 40 μg/g/d | 6.45 ± 0.23 | 31.3 ± 2.9 |
5.47 ± 0.07 | 40.4 ± 1.7 |
ISO 80 μg/g/d | 5.94 ± 0.16 | 39.4 ± 1.9 |
6.02 ± 0.05 | 50.9 ± 1.3 |
ISO 160 μg/g/d | 5.88 ± 0.21 | 34.3 ± 3.5 |
ND | ND |
Vehicle | 6.02 ± 0.01 | 71.6 ± 0.4 | 6.29 ± 0.10 | 50.0 ± 2.2 |
ISO 5 μg/g/d | 5.54 ± 0.10 |
50.4 ± 2.8 |
6.13 ± 0.20 |
33.4 ± 2.8 |
ISO 40 μg/g/d | 6.47 ± 0.15 | 49.9 ± 3.0 |
6.23 ± 0.13 |
40.1 ± 2.6 |
ISO 80 μg/g/d | 6.18 ± 0.12 | 49.8 ± 2.6 |
5.94 ± 0.07 | 28.0 ± 1.2 |
ISO 160 μg/g/d | 5.82 ± 0.16 | 48.2 ± 3.9 |
6.02 ± 0.12 | 20.6 ± 1.2 |
Ach-induced relaxation in NE-precontracted aortic rings from 2 months-old (continuous line) and 18 months-old (discontinuous line) male
NE-induced contraction in 2 months-old (solid bars) and 18 months-old (open bars) male
As shown in Figures
Effect of ISO-treatment on vascular relaxation in aortic rings from 2 months-old male mice
Changes in plasma concentrations of circulating VCAM-1, IL-1α, and IL-4 are shown in Figure
Plasma concentrations of circulating inflammatory mediators VCAM-1
Changes in ventricular weight (VW) to body weight (BW) ratio (VW/BW) were quantified to corroborate that the doses used induced heart damage. Therefore, the observed changes in vasoreactivity can be related to cardiac damage. Table
Body weight and ventricular weight/body weight ratio from young (2 months) and old (18 months) male and female mice treated with isoproterenol.
Vehicle | 26.32 ± 0.65 | 26.65 ± 0.55 | 4.7 ± 0.06 | 31.06 ± 1.15 |
30.86 ± 0.71 |
5.0 ± 0.07 |
ISO 5 μg/g/d | 26.06 ± 0.74 | 26.53 ± 0.66 | 5.2 ± 0.09 |
30.77 ± 0.98 |
30.00 ± 0.70 |
6.0 ± 0.13 |
ISO 40 μg/g/d | 26.07 ± 0.48 | 25.90 ± 0.67 | 5.8 ± 0.06 |
34.09 ± 1.23 |
33.36 ± 1.02 |
5.7 ± 0.07 |
ISO 80 μg/g/d | 26.79 ± 0.65 | 26.60 ± 0.72 | 5.8 ± 0.09 |
32.15 ± 0.91 |
30.35 ± 0.93 |
5.8 ± 0.07 |
ISO 160 μg/g/d | 27.42 ± 0.89 | 26.92 ± 1.03 | 5.9 ± 0.10 |
32.25 ± 0.74 |
30.47 ± 0.67 |
6.0 ± 0.12 |
Vehicle | 21.94 ± 0.44 |
21.95 ± 0.52 |
4.4 ± 0.08 | 27.79 ± 0.57 |
26.93 ± 0.66 |
4.1 ± 0.12 |
ISO 5 μg/g/d | 21.91 ± 0.73 |
22.38 ± 0.85 |
5.5 ± 0.07 |
27.58 ± 0.49 |
27.30 ± 0.48 |
4.7 ± 0.08 |
ISO 40 μg/g/d | 20.83 ± 0.90 |
21.61 ± 1.00 |
5.6 ± 0.11 |
27.69 ± 1.08 |
25.36 ± 0.82 |
5.1 ± 0.09 |
ISO 80 μg/g/d | 20.97 ± 1.00 |
21.62 ± 0.88 |
5.7 ± 0.11 |
28.54 ± 1.01 |
26.59 ± 1.14 |
5.3 ± 0.14 |
ISO 160 μg/g/d | 21.44 ± 0.56 |
21.21 ± 0.72 |
6.1 ± 0.07 |
28.70 ± 1.11 |
26.52 ± 0.88 |
5.4 ± 0.08 |
Overstimulation of the β-adrenergic system with daily doses of ISO causes heart damage inducing inflammation of the tissue and having consequences on the vasculature. In this paper, we studied the effects of repeated β-adrenergic overstimulation with different doses of ISO on vascular reactivity.
We found that repeated β-adrenergic stimulation with different doses of ISO modifies vascular reactivity (VR) in an age-, gender-, and dose-dependent pattern being young males the most susceptible to vascular alterations; whereas old mice tended to keep their homeostasis, but with different strategies according to the gender. Even though changes in vascular tone may be associated with systemic inflammation induced by the repeated β-adrenergic stimulation, in our study, we found no changes in the circulating levels of the inflammatory mediators IL-1α and IL-4 and only VCAM-1 seemed to be associated with vascular alterations in old females stimulated with the highest ISO dose. However, we cannot rule out that other biochemical and cellular markers of inflammation and local inflammation markers could be modified.
Gender and age modulate the incidence and progression of vascular and heart-related diseases. In the present study, we found no differences in VR between vehicle-treated young males and females. This response is in agreement with previous reports (Júnior and Cordellini,
Vasoconstriction usually declines with age due to decreases in cell density, aortic compliance and aortic contractility and increases in aortic diameter, the thickness of the medial layer of the aortic wall and collagen volume fraction (Wheeler et al.,
Gender differences in contraction during aging may be related to sex hormones. Estrogens improve endothelial function (Usselman et al.,
Endothelium-dependent relaxation mediated by Ach has been reported to fall during rat maturation (Soltis and Newman,
Besides cardiovascular modifications, aging also influences the inflammatory system. In the present study, circulating IL-1α and IL-4 were not different among individuals from different gender and age, but young vehicle-treated males had higher levels of circulating VCAM-1. At the vascular level, previous studies indicate that circulating pro-inflammatory factors may activate endothelial cells to promote an atherogenic phenotype, which may result in endothelial dysfunction, ventricular dysfunction and heart failure (Marchais et al.,
In the present work, the repeated stimulation of the β-AR system with different doses of ISO that caused heart damage also triggered different responses in VR and inflammation according to the gender and age of the organisms.
We found that, in response to β-adrenergic stimulation, young male aortas showed a bell-shaped contraction curve with increases in 3 of 4 doses of ISO and this response was decreased with age. In young female aortas, only the highest dose of isoproterenol produced a significant increase in contraction. In old females, a decrease in contraction was registered only with the highest dose of ISO. Gender differences in the susceptibility to β-adrenergic stimulation have been previously reported, being males more susceptible (Page et al.,
The β2-AR/Giα signaling pathway (Davel et al.,
In old males, a reduced aortic response is expected since the ability of β-AR to respond to the stimulation by catecholamines is declined. In females, the selective blockage of β2-AR increases the contraction to NE (Al-Gburi et al.,
In our study, ISO-administration was accompanied by a decrease in relaxation in young males and females at all doses. However, we did not find significant changes in the
In aortas from old female mice, the highest doses of ISO significantly decreased the maximum response to Ach. Surprisingly, the treatment with ISO at 5, 40, and 80 μg/g/d induced vasorelaxation in aortas from old male mice. We do not know the exact reason for this effect, but we hypothesize that ISO could be mediating the vasorelaxant effect by the stimulation of β2-AR that activate the ATP-sensitive potassium channels in vascular smooth muscle (Fauaz et al.,
The combination of the changes in relaxation and contraction that we observed suggests that young females are more resistant than young males to vascular responses triggered by β-adrenergic stimulation. This is reflected by the decreased relaxation with all the doses without changes in contraction with the exception of the 160 dose in females, while in males, relaxation decreased and contraction increased. This last combination is more likely to lead to the occlusion of the vessels. Opposite to young mice, old mice treated with ISO tended to keep the homeostasis of the vascular function with most of the doses of ISO, but probably through different mechanisms. For instance, in our study, contraction and relaxation increased with the majority of the doses in old males; while in old females there were no changes in vasorelaxation and vasoconstriction when they were treated with different doses of isoproterenol. These results highlight the relevance of including males and females young and old when trying to test new treatments.
Even if inflammation and changes in the vascular function have been associated with several conditions that involve the β-adrenergic overactivation (Zeiher et al.,
In conclusion, our results showed that repeated β-adrenergic stimulation with different doses of catecholamines (ISO) modifies VR in an age-, gender- and dose-dependent manner being young males more susceptible to damage. Old mice tend to keep their homeostasis, but with different strategies according to the gender. These alterations in VR accompany the heart damage which was evidenced by the increases in VW/BW. A vicious cycle could be established with a tendency to threaten life unless interrupted. Although inflammation and vascular tone are associated with β-adrenergic stimulation, this association might not necessarily involve the participation of the inflammatory cytokines IL-1α and IL-4. Only circulating VCAM-1 levels showed changes in old females stimulated with the highest ISO dose. Therefore, other cellular and humoral participants of the inflammatory response should also be assayed in future studies. Importantly, these results highlight the relevance of considering the gender and age of organisms in studies aiming to determine mechanisms that lead to the incidence of diseases.
AC-M and BN-L: designed the study, performed the treatments, obtained the heart and aortic tissues and plasma samples, drafted the interpreted results and wrote and reviewed the manuscript; MR-R: performed the assay of vascular reactivity and drafted the interpreted results; IP-T: performed the assay to determine the inflammatory and nitrite profile; VG-L: was responsible reviewing the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Florencio Hernández for his help in the animal care.