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Baroreflex sensitivity and central hemodynamics after omega-3 polyunsaturated fatty acids supplementation in an animal model of menopause
VPH-06142; No of Pages 5 Vascular Pharmacology xxx (2015) xxx–xxx
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Baroreflex sensitivity and central hemodynamics after omega-3 polyunsaturated fatty acids supplementation in an animal model of menopause Pasquale Losurdo a,⁎, Andrea Grillo a, Emiliano Panizon a, Michela Zanetti b, Moreno Bardelli a, Gianni Biolo b, Bruno Fabris a, Renzo Carretta a a b
U.C.O. Medicina Clinica, Department of Medicine, Surgery and Health Sciences, University of Trieste, Ospedale di Cattinara, Strada di Fiume 447, 34149 Trieste, Italy U.C.O. Clinica Medica Generale e Terapia Medica, Department of Medicine, Surgery and Health Sciences, University of Trieste, Ospedale di Cattinara, Strada di Fiume 447, 34149 Trieste, Italy
a r t i c l e
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Article history: Received 20 July 2014 Received in revised form 19 December 2014 Accepted 21 December 2014 Available online xxxx Keywords: Menopause Omega-3 polyunsaturated fatty acids Ovariectomized rats Baroreflex Arterial stiffness Cardiovascular disease
a b s t r a c t Objective: Baroreflex sensitivity (BRS) and central arterial function are significantly worsened after menopausal transition. This study tested the hypothesis that administration of n-3 polyunsaturated fatty acids (n-3 PUFA) might contribute to prevent these adverse changes in the vascular function of ovariectomized rats, an animal model of experimental menopause. Methods: We randomized 30 female Wistar rats, 2 months old, into 3 groups: control (CTRL), sham surgery, normal diet; ovariectomized with normal diet (OVX) and ovariectomized with n-3 PUFA supplementation by daily gavage (0.8 g/kg/d) (OVX + O3). Two months after surgery, BRS was calculated as the bradycardic response to phenylephrine-induced blood pressure increase, while large artery function was estimated by the graphical analysis of the aortic pressure wave (diastolic to systolic pressure-time integral ratio, DTI/STI). Results: Ovariectomy caused a significant decrease in BRS (CTRL: 6.23 ± 0.68 ms/mm Hg; OVX: 2.85 ± 0.75; p b 0.001). n-3 PUFA supplementation prevented part of the decline of BRS caused by surgical menopause (OVX + O3: 4.75 ± 0.53; p b 0.01 vs OVX). In animals treated with n-3 PUFA, the central arterial pressure profile did not show the changes in DTI/STI ratio seen in OVX (OVX: 3.31 ± 1.72; OVX + O3: 3.83 ± 1.52; p b 0.01). Conclusions: In an experimental model of menopause, treatment with n-3 PUFA normalized central hemodynamics and prevented the decrease in BRS, associated with the reduction of compliance of the arterial wall. These findings suggest a therapeutic benefit of n-3 PUFA supplementation in the prevention of postmenopausal vascular disease. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Cardiovascular diseases remain the leading cause of death among women worldwide [1]. Although there is an increasing concern about the effects of treatments in gender subgroups [2], recent data suggest that clinical evidences for cardiovascular care are still affected by the underrepresentation of women [3]. The gender inequality in patients enrolled in randomized clinical trials [4] and in animals involved in experimental studies [5], strongly limits the progress to a truly personalized medicine, and there is an urgent need of sex specific biomedical research to avoid this longstanding gender bias.
⁎ Corresponding author at: U.C.O. Medicina Clinica — Department of Medicine, Surgery and Health Sciences, Ospedale di Cattinara, Strada di Fiume 447, 34149 Trieste, Italy. Tel.: +39 3209313940; fax: +39 040 3994912. E-mail address: [email protected] (P. Losurdo).
Epidemiologic studies suggest that the risk for a cardiovascular event is low before menopause, but rises sharply after menopausal transition [6]. This phenomenon could be explained by considering estrogen activity, which may abrogate age-related vascular remodeling in premenopausal women [7]. The changes occurring in cardiovascular system after menopause can be closely reproduced in animal models. In female rats subjected to ovarian hormone deprivation, compared to control rats, several alterations were seen both in cardiovascular function and structure [8]. Loss of estrogen activity accelerates many processes involved in vascular aging, including smooth muscular cells proliferation [9] and endothelial dysfunction [10]. Increase in arterial stiffness [11], decline in flow-mediated dilation [12] and changes in the autonomic vascular control [13] occur during the early phases of menopause. Baroreflex sensitivity (BRS), the ability to buffer the blood pressure response to pressor or depressor stimuli, is considered an established tool for the assessment of cardiovascular autonomic function. A depressed BRS has been found in menopause both in humans and in animal models [4,9],
http://dx.doi.org/10.1016/j.vph.2014.12.005 1537-1891/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Losurdo P, et al, Baroreflex sensitivity and central hemodynamics after omega-3 polyunsaturated fatty acids supplementation in an animal model of me..., Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2014.12.005
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P. Losurdo et al. / Vascular Pharmacology xxx (2015) xxx–xxx
and is linked to increased cardiovascular mortality [14,15]. However, there are few data in literature concerning the effects of pharmacologic strategies to prevent vascular hemodynamic and autonomic changes induced by ovarian hormones deprivation. Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been widely studied for their multiple benefits on cardiovascular health, but in the clinical setting their role for cardiovascular prevention is still under discussion [16]. A number of experimental studies and clinical trials have proved their ability to improve hemodynamics by reducing arterial stiffness [17]. Previous studies have also suggested the ability of n-3 PUFA to prevent cardiac arrhythmia and to modulate autonomic nervous system [18], while effects of n-3 PUFA on BRS in humans are not univocal. In a recent study from our group [19], we demonstrated the ability of n-3 PUFA to prevent the increase of arterial stiffness in ovariectomized rats, a validated animal model of experimental menopause. The purpose of the present study was to determine the effects of n-3 PUFA supplementation on BRS and on the aortic pressure wave, as a marker of the large artery elasticity, in the same experimental setting.
2. Methods 2.1. Animals Experiments were performed on female Wistar-Kyoto rats, 2 months old, initially weighing 140 to 170 g, fed with a standard rat chow (Harlan 2018, 3.4 cal/g, macronutrients composition: crude protein 18.6%, fat 6.2%, crude fiber 3.5%. Supplied by Harlan, Italy), for at least two weeks. The animals were kept in temperature-controlled facilities on a 12-hours light/dark cycle. Animals were then randomly assigned to three experimental groups: a) control group of sham operated rats receiving normal diet (CTRL group), b) ovariectomy group receiving normal diet (OVX group), c) ovariectomy group receiving normal diet with the addition of daily gavages administration of a mixture of eicosapentaenoic acids (EPA) and docosahexaenoic acids (DHA) (OVX + O3 group). Animals were sacrificed 2 months after surgical procedure. 10 animals from each of the three experimental groups were randomly selected for the following experimental phase, while other 30 randomly selected animals were included in a different experiment (data previously published [20]). All experiments were performed according to the guidelines and protocols approved by the European Union (EU Council 86/609; D.L. 27.01.1992, no. 116) and by the Animal Research Ethics Committee of the University of Trieste, Italy.
2.3. Surgical ovariectomy Bilateral ovariectomy and sham surgery were performed via a midabdominal route under Xylazine (10 mg/kg, intraperitoneal [IP] injection) and Tiletamine (40 mg/kg, IP) anesthesia. Anesthesia was assessed by complete absence of limb retraction upon painful stimulation. The fallopian tube was legated with absorbable suture, and the ovary was removed. This model of surgical menopause was validated in a previous experiment [20], which proved a significant decrease in serum estradiol levels after surgical bilateral ovariectomy, compared with shamoperated rats. 2.4. Hemodynamic measurements After 2 months since the surgical procedure, 10 animals from each experimental (CTRL, OVX and OVX + O3) group were randomly selected for the hemodynamic measurements. The animals were anesthetized with Xylazine (5 mg/kg IP) and Tiletamine (10 mg/kg IP). Nylon cannulas were introduced into the left femoral arteries up to the aortic ostia. The anatomic locations of the tips of the cannula were checked by postmortem dissection. Cannulas were connected to low-volume blood pressure (BP) transducers (STATHAM range − 50 to + 300 mm Hg), linked to a Coulbourn analog-to-digital convertor plus computer (Coulbourn Instruments; Lablink, Allentown, Pennsylvania). The frequency response of the cannula plus pressure transducer was minimum 100 Hz with 1% FS/24 Hz. Systolic and diastolic arterial BP and BP wave from the aortic ostia were recorded. The personnel who performed hemodynamic measures were blinded to study group assignment of each animal. 2.5. Determination of baroreflex After a 15 minute wait for hemodynamic stabilization after invasive arterial cannulation, the basal pressure profile was recorded. The baroreflex sensitivity was performed by injecting a single dose of phenylephrine (100 μL of 0.01 g/mL solution) directly into the cannulated vessel. Phenylephrine, a pure alpha-agonist, was administered in order to rapidly increase the blood pressure of 15–30 mm Hg, and pressure wave recorded for at least 5 min. The pressure waves were analyzed in order to quantify changes in BP and in heart rate. The slope of the linear regression line between changes in systolic blood pressure and consequent lengthening of the RR interval is a quantitative measure of the baroreceptor control of heart rate. In the analysis, five c beats where the increase in blood pressure was followed by an increase in RR interval were considered for the quantitative measurement of BRS. BRS was calculated relating the changes in BP and in RR interval, as a mean index expressed as ms/mm Hg.
2.2. Diets 2.6. Pressure wave analysis Diets were prepared weekly and stored to prevent degradation, and food was provided and removed daily. To standardize study protocol CTR and OVX rats received an equal volume of normal saline solution by gavage per day. A commercially available pharmaceutical preparation was used for n-3 PUFA treatment (EPA and DHA mixture, 0.9 to 1.5 ratio, EPA + DHA content not inferior to 85%, Eskim, SigmaTau, Italy). The manufacturing process of the drug fulfilled the good manufacturing practice standards, and the production was optimized to improve the stability of the compound. The average content of EPA and DHA was, respectively, 450 ± 50 and 395 ± 55 mg/g. A daily dose of 0.8 g/kg/d was administered to rats in the OVX + O3 group, corresponding to a 0.65 g/kg/d of purified active compound (EPA and DHA). As mentioned in our previous experiment [20], this dose of n-3 PUFA was capable to increase omega-3 index in red cell membranes of about 50%. The study dose corresponded to a human equivalent dose of about 7 g/d. Saline solution (0.9% NaCl) was manufactured by Diaco (Italy).
We performed a graphical analysis on aortic pressure waveform to calculate the time/pressure area under the pressure curves in systole and diastole. Only recordings of 10 consecutive heartbeats with stable BP values were used for the analysis. The systolic pressure–time integral (STI) and the diastolic pressure–time integral (DTI) were calculated as the mean area under the systolic and diastolic part of the waveform (Fig. 1). The relationship between diastolic and systolic pressure wave, considered a parameter evaluating central arterial function, was defined as the ratio between DTI and STI (DTI/STI). For the graphical analysis the Adobe Photoshop (Adobe Systems) v11.0 software package was used to calculate the area under the pressure curve. 2.7. Determination of blood glucose, plasma lipids, and omega-3 index Levels of glucose, total cholesterol, triglycerides, HDL, Plasma 17βestradiol, and membrane omega-3 index were obtained as referenced
Please cite this article as: Losurdo P, et al, Baroreflex sensitivity and central hemodynamics after omega-3 polyunsaturated fatty acids supplementation in an animal model of me..., Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2014.12.005
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difference: CTRL 49 g; OVX: 84 g; p b 0.05). Body weight increase was even more pronounced in animals with n-3 PUFA supplementation, compared to other groups (body weight difference: OVX + O3: 106 g; p b 0.05 vs CTRL). As evidenced also in our previous experiments in the same experimental setup [20], ovariectomy increased plasma levels of total cholesterol (CTRL: 1.1 ± 0.1 mmol/L; OVX: 1.5 ± 0.2 mmol/L; p b 0.05) and non-HDL cholesterol (CTRL: 0.4 ± 0.1 mmol/L; OVX: 1.0 ± 0.1 mmol/L; p b 0.05), while n-3 PUFA normalized plasma lipids levels (OVX + O3: total cholesterol 1.1 ± 0.1 mmol/L; p b 0.05 vs OVX; non-HDL cholesterol 0.6 ± 0.1 mmol/L; p b 0.05 vs OVX). Relative erythrocyte membrane content of EPA and DHA (n-3 PUFA index) was significantly reduced in OVX rats compared with CTRL. Treatment with n-3 PUFA markedly enhanced omega-3 index in ovariectomized rats (Table 1). 3.2. Hemodynamics and pulse wave analysis Fig. 1. The diastolic pressure–time integral and systolic pressure–time integral ratio (DTI/ STI) in ovariectomized rats (OVX), ovariectomized rats receiving n-3 PUFA supplementation (OVX + O3), and control sham-operated rats (CTRL), after surgery and 8 weeks of supplementation with n-3 PUFA. Data reported as means. Error bars ± 1 SD. *p b 0.01 versus CTRL or OVX + O3.
[20]. These data, which refer to the 30 animals considered in our previous work, were previously discussed [20] and are shown in Table 1. 2.8. Statistical analysis All data are expressed as mean ± standard deviation. The Kolmogorov–Smirnov test for normality was initially performed for all data. One-way analysis of variance was used to assess group differences. If significant differences were observed with analysis of variance, Fisher least significant difference post-hoc analysis for multiple comparisons was performed to identify the differences among the mean values in the groups. IBM SPSS Statistics v20 was used to perform all statistical analyses. A P value of less than 0.05 was considered statistically significant. 3. Results 3.1. Effects of OVX and Omega 3 on body weight, metabolic parameters and omega-3 index
Both systolic and diastolic BP levels significantly differed in rats after ovariectomy, compared to control rats (systolic BP: CTRL 128 ± 23 mm Hg; OVX 150 ± 26 mm Hg; p b 0.05; diastolic BP: CTRL 87 ± 19 mm Hg; OVX 100 ± 13 mm Hg; p b 0.05). n-3 PUFA supplementation prevented this raise in BP, leading rats in OVX + O3 group to have BP levels similar to the CTRL group (OVX + O3: systolic BP 129 ± 22 mm Hg, p b 0.05 vs OVX; diastolic BP 88 ± 14 mm Hg, p b 0.05 vs OVX). Regarding the pulse wave morphology, the DTI/STI ratio was significantly smaller in the ovariectomized group compared to controls (CTRL: 4.17 ± 1.28, OVX: 3.31 ± 1.72, p b 0.01, Fig. 1), while in the n3 PUFA supplemented rats it was comparable to the control group (OVX + O3: 3.83 ± 1.52, p b 0.01 vs OVX, Fig. 1). Heart rate at the time of the hemodynamic evaluation was not significantly different between the 3 groups (CTRL: 285 ± 16 OVX: 277 ± 17 OVX + O3: 291 ± 10, p = ns). 3.3. Effects on baroreflex sensitivity Ovariectomized rats displayed a significantly reduced BRS respect to CTRL (CTRL: 6.23 ± 0.68 ms/mm Hg, OVX: 2.85 ± 0.75 ms/mm Hg; p b 0.001, Fig. 2). Treatment with n-3 PUFA partially prevented this fall in BRS. In OVX + O3 rats BRS was increased respect to OVX group, but slightly decreased respect to CTRL group (OVX + O3: 4.75 ± 0.53 ms/mm Hg, p b 0.001 vs OVX, p b 0.05 vs CTRL Fig. 2).
Body weight increased significantly during experimental phase in all groups (Table 1, p b 0.05 for all groups). Ovariectomy induced a higher increase in body weight respect to control animals (body weight
Table 1 Body weight, metabolic parameters and omega-3 index after surgery and 8 weeks of supplementation with n-3 PUFA. Data reported as means ± SD. Body weight increased significantly in all groups *p b 0.05 for all groups. As evidenced also in our previous experiments [20], ovariectomy increased plasma levels of total cholesterol (*p b 0.05) and non-HDL cholesterol (*p b 0.05), while n-3 PUFA normalized plasma lipids levels (*p b 0.05 vs OVX; non-HDL cholesterol *p b 0.05 vs OVX). Erythrocyte membrane content of EPA and DHA (n-3 PUFA index) was significantly [20] reduced in OVX rats compared with CTRL [16]. Treatment with n-3 PUFA markedly enhanced omega-3 index [20] in ovariectomized rats (*p b 0.05).
Initial body weight (g) Final body weight (g) EPA + DHA (%) Plasma 17β-estradiol (pmol/l) Total cholesterol (mmol/l) Triglycerides (mmol/l) HDL cholesterol (mmol/l) Non-HDL cholesterol (mmol/l)
CTRL
OVX
OVX + O3
151 ± 6.3 200 ± 8 4.70 ± 0.18 65.34 ± 23.13 1.07 ± 0.11 0.71 ± 0.08 0.63 ± 0.16 0.41 ± 0.09
146 ± 15.4 230 ± 12 4.08 ± 0.11 * 5.87 ± 1.84 * 1.50 ± 0.17 * 0.58 ± 0.08 0.61 ± 0.19 1.00 ± 0.10 *
149 ± 9.4 255 ± 16 * 6.24 ± 0.18 7.34 ± 3.30 * 1.07 ± 0.07 0.58 ± 0.07 0.49 ± 0.08 0.61 ± 0.10
Fig. 2. BRS in ovariectomized rats (OVX), ovariectomized rats receiving n-3 PUFA supplementation (OVX + O3), and control sham-operated rats (CTRL), after surgery and 8 weeks of supplementation with n-3 PUFA. Data reported as means. Error bars ± 1 SD. *p b 0.001 versus CTRL or OVX + O3. †p b 0.001 versus OVX, p b 0.05 versus CTRL.
Please cite this article as: Losurdo P, et al, Baroreflex sensitivity and central hemodynamics after omega-3 polyunsaturated fatty acids supplementation in an animal model of me..., Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2014.12.005
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4. Discussion In the present study, we found that ovariectomy induced BRS reduction in female rats compared to sham-operated controls, and n-3 PUFA supplementation after ovariectomy partially prevented BRS decrease. In parallel, ovariectomized rats developed a significant change in central hemodynamic function, by increasing the systolic component of pulse wave more than the diastolic component, thus decreasing the DTI/STI ratio. Supplementation of n-3 PUFA prevented completely these changes restoring the normal appearance of the central pressure wave. A first finding from our study was the increase in BP found in ovariectomized rats when compared to controls. Onset of hypertension after loss of ovarian hormones is a well known condition. Etiology of postmenopausal hypertension is multifactorial and involves activation of renin–angiotensin system, autonomic imbalance with increased sympathetic and decreased vagal activity, inflammation and probably other mechanisms [21]. In rats supplemented with n-3 PUFA, ovariectomy did not cause an increase in BP values. The antihypertensive effect of n-3 PUFA has been demonstrated both in clinical [22] and experimental [23] conditions, and it is mediated by an increased synthesis of the vasodilator nitric oxide, a reduced production of the vasoconstrictor thromboxanes and a reduction in arterial stiffness [24]. The finding of a reduced BRS in postmenopausal state is consistent with previous studies [25,26]. In ovariectomized female rats BRS is improved by estrogen replacement therapy and cyclic BRS changes are observed during the estrous cycle, both in humans and rats, with variations mirroring the estrogen levels [27], thus suggesting a crucial role of ovarian hormones in baroreflex regulation. The decline in baroreflex function after surgical menopause represents a harmful complication, leading to an abnormal neural regulation of the heart and the vasculature and increased blood pressure variability, which is associated with an increased incidence of cardiovascular events [28]. The observed improvement in BRS given by n-3 PUFA supplementation in our experiment represents a novel therapeutical property of these agents, and can fill a gap among cardiovascular therapies, as few drugs have shown the ability to prevent baroreflex dysfunction. A specific effect of n-3 PUFA on BRS has not been previously reported, while other effects on cardiovascular parameters involving autonomic function have been demonstrated, such as reduction in resting heart rate, in blood pressure and an increased heart rate variability [18,29], suggesting a positive role of n-3 PUFA on autonomic cardiovascular regulation. Vascular stiffness represents a major determinant in the regulation of BRS. Arterial baroreceptors are stretch receptors that are stimulated by the strain of the arterial wall when blood pressure changes. An increase in the mean arterial pressure can increase depolarization of these sensory endings. As shown by previous studies, the stiffening of the baroreceptor vascular segments (in carotid artery and aorta) limits the afferent signaling of the baroreceptor reflex, thus reducing baroreflex sensitivity [30–32]. In our previous study, in the same experimental setting, we found an improvement of arterial stiffness evaluated as pulse wave velocity, following n-3 PUFA treatment in ovariectomized rats [19]. In the present study, we report again a normalization of aortic compliance, by improvement of the diastolic/systolic balance in the central pressure pulse wave evaluated at the aortic level. The elastic reservoir function of aorta and large elastic arteries helps in damping the fluctuations in blood pressure during the cardiac cycle and assists in the maintenance of organ perfusion during diastole, and has recently been studied as the ratio between DTI and STI [33]. A surrogate of this index has been studied in humans noninvasively as subendocardial viability ratio (SEVR), which is considered also a measure of the adequacy of the subendocardial muscle diastolic perfusion in response the myocardial oxygen systolic demand [34]. DTI/STI ratio may therefore represents both an evaluation the elastic properties of the aortic wall, inversely related to arterial stiffness [35], and an indirect assessment of the correctness
of the myocardial perfusion. Considering that it has been demonstrated that aortic pressure profile reflects the carotid pressure profile [36], our results can be extended to the carotid arterial viscoelastic properties, where the main receptor for baroreflex resides. In our study, both systolic and diastolic blood pressure were increased in ovariectomized rats, while in rats treated with n-3 PUFA blood pressure was similar to controls. As expected, the DTI/STI ratio was significantly decreased in ovariectomized rats, revealing a significant change in central hemodynamics after ovariectomy and an increase in arterial stiffness. n-3 PUFA supplementation prevented the decrease in DTI/STI ratio. This improvement in arterial compliance in n-3 PUFA supplemented ovariectomized rats seems to be induced by the contribution of multiple factors, like the capacity of n-3 PUFA to affect BP and lipid profile, and the reversal of endothelial dysfunction and vascular oxidative stress induced by menopause [20]. The mechanisms responsible for the alteration in the elastic properties of the arterial wall have been usually attributed to changes in the physical structure of the vessels, considered as a passive elastic conduits. But, considered the short duration of the experimental phase, it is likely that at least part of the preserved arterial compliance in the n-3 PUFA-treated group is related to functional changes in the vascular system. n-3 PUFA are able to reverse endothelial dysfunction, through an interaction with endothelial nitric oxide synthase that leads to an increased NO bioavailability [20]. NO-mediated alteration in arterial smooth muscle tone are a main determinant of arterial function, and an increased NO production can lead to a reduced wave reflection and a delayed return [37]. In our experiment we did not observe any side effect related to n-3 PUFA administration. Since we used high doses of n-3 PUFA (0.65 g/kg/d, corresponding to an approximate human equivalent dose of 7 g/d), some limits must be observed for the extension of our results to the humans. Another concern could be raised about the weight gain seen in ovariectomized animals, which was even more pronounced after n-3 PUFA administration, probably due to an increased caloric and fat intake. Although a possible neurohormonal effect of adipose tissue could not be excluded in our experiment, previous studies demonstrated that weight gain can adversely affect baroreflex sensitivity [38,39], thus suggesting that adipose tissue is not directly involved in n-3 PUFA effects on baroreflex. Our data indicate that n-3 PUFA administration prevents BRS decrease induced by estrogen deficiency, in parallel with the prevention of some detrimental changes in central arterial hemodynamic. As BRS represents an irreplaceable mechanism in cardiovascular homeostasis, this effect could be important for prevention of hypertension and of cardiovascular autonomic dysfunction associated with menopause. In clinical studies, n-3 PUFA intake has also been implied in estrogen metabolism during menopausal transition [40], suggesting a mutual relationship that needs to be further clarified. Recent clinical trials and metanalysis did not find a significant association of n-3 PUFA administration with reduction of major cardiovascular outcomes [16]. However, all the major trials assessing cardiovascular outcomes of n-3 PUFA administration present a gender disparity in patients enrollment. The percentage of female sex in these trials ranges from 21.5% to 38.5% [41–47], reflecting the known underrepresentation of female sex in cardiovascular medicine [3]. This marked gender inequality must be considered as a gap in the available evidence for n-3 PUFA administration, particularly in postmenopausal women, which present an increased cardiovascular risk. As the mechanisms of action of n-3 PUFA in cardiovascular prevention need to be further investigated, our study provides encouraging results for the use of n-3 PUFA in postmenopausal women and supports the need for testing our experimental hypothesis in a clinical setting. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Please cite this article as: Losurdo P, et al, Baroreflex sensitivity and central hemodynamics after omega-3 polyunsaturated fatty acids supplementation in an animal model of me..., Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2014.12.005
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Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph
Baroreflex sensitivity and central hemodynamics after omega-3 polyunsaturated fatty acids supplementation in an animal model of menopause Pasquale Losurdo a,⁎, Andrea Grillo a, Emiliano Panizon a, Michela Zanetti b, Moreno Bardelli a, Gianni Biolo b, Bruno Fabris a, Renzo Carretta a a b
U.C.O. Medicina Clinica, Department of Medicine, Surgery and Health Sciences, University of Trieste, Ospedale di Cattinara, Strada di Fiume 447, 34149 Trieste, Italy U.C.O. Clinica Medica Generale e Terapia Medica, Department of Medicine, Surgery and Health Sciences, University of Trieste, Ospedale di Cattinara, Strada di Fiume 447, 34149 Trieste, Italy
a r t i c l e
i n f o
Article history: Received 20 July 2014 Received in revised form 19 December 2014 Accepted 21 December 2014 Available online xxxx Keywords: Menopause Omega-3 polyunsaturated fatty acids Ovariectomized rats Baroreflex Arterial stiffness Cardiovascular disease
a b s t r a c t Objective: Baroreflex sensitivity (BRS) and central arterial function are significantly worsened after menopausal transition. This study tested the hypothesis that administration of n-3 polyunsaturated fatty acids (n-3 PUFA) might contribute to prevent these adverse changes in the vascular function of ovariectomized rats, an animal model of experimental menopause. Methods: We randomized 30 female Wistar rats, 2 months old, into 3 groups: control (CTRL), sham surgery, normal diet; ovariectomized with normal diet (OVX) and ovariectomized with n-3 PUFA supplementation by daily gavage (0.8 g/kg/d) (OVX + O3). Two months after surgery, BRS was calculated as the bradycardic response to phenylephrine-induced blood pressure increase, while large artery function was estimated by the graphical analysis of the aortic pressure wave (diastolic to systolic pressure-time integral ratio, DTI/STI). Results: Ovariectomy caused a significant decrease in BRS (CTRL: 6.23 ± 0.68 ms/mm Hg; OVX: 2.85 ± 0.75; p b 0.001). n-3 PUFA supplementation prevented part of the decline of BRS caused by surgical menopause (OVX + O3: 4.75 ± 0.53; p b 0.01 vs OVX). In animals treated with n-3 PUFA, the central arterial pressure profile did not show the changes in DTI/STI ratio seen in OVX (OVX: 3.31 ± 1.72; OVX + O3: 3.83 ± 1.52; p b 0.01). Conclusions: In an experimental model of menopause, treatment with n-3 PUFA normalized central hemodynamics and prevented the decrease in BRS, associated with the reduction of compliance of the arterial wall. These findings suggest a therapeutic benefit of n-3 PUFA supplementation in the prevention of postmenopausal vascular disease. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Cardiovascular diseases remain the leading cause of death among women worldwide [1]. Although there is an increasing concern about the effects of treatments in gender subgroups [2], recent data suggest that clinical evidences for cardiovascular care are still affected by the underrepresentation of women [3]. The gender inequality in patients enrolled in randomized clinical trials [4] and in animals involved in experimental studies [5], strongly limits the progress to a truly personalized medicine, and there is an urgent need of sex specific biomedical research to avoid this longstanding gender bias.
⁎ Corresponding author at: U.C.O. Medicina Clinica — Department of Medicine, Surgery and Health Sciences, Ospedale di Cattinara, Strada di Fiume 447, 34149 Trieste, Italy. Tel.: +39 3209313940; fax: +39 040 3994912. E-mail address: [email protected] (P. Losurdo).
Epidemiologic studies suggest that the risk for a cardiovascular event is low before menopause, but rises sharply after menopausal transition [6]. This phenomenon could be explained by considering estrogen activity, which may abrogate age-related vascular remodeling in premenopausal women [7]. The changes occurring in cardiovascular system after menopause can be closely reproduced in animal models. In female rats subjected to ovarian hormone deprivation, compared to control rats, several alterations were seen both in cardiovascular function and structure [8]. Loss of estrogen activity accelerates many processes involved in vascular aging, including smooth muscular cells proliferation [9] and endothelial dysfunction [10]. Increase in arterial stiffness [11], decline in flow-mediated dilation [12] and changes in the autonomic vascular control [13] occur during the early phases of menopause. Baroreflex sensitivity (BRS), the ability to buffer the blood pressure response to pressor or depressor stimuli, is considered an established tool for the assessment of cardiovascular autonomic function. A depressed BRS has been found in menopause both in humans and in animal models [4,9],
http://dx.doi.org/10.1016/j.vph.2014.12.005 1537-1891/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Losurdo P, et al, Baroreflex sensitivity and central hemodynamics after omega-3 polyunsaturated fatty acids supplementation in an animal model of me..., Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2014.12.005
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and is linked to increased cardiovascular mortality [14,15]. However, there are few data in literature concerning the effects of pharmacologic strategies to prevent vascular hemodynamic and autonomic changes induced by ovarian hormones deprivation. Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been widely studied for their multiple benefits on cardiovascular health, but in the clinical setting their role for cardiovascular prevention is still under discussion [16]. A number of experimental studies and clinical trials have proved their ability to improve hemodynamics by reducing arterial stiffness [17]. Previous studies have also suggested the ability of n-3 PUFA to prevent cardiac arrhythmia and to modulate autonomic nervous system [18], while effects of n-3 PUFA on BRS in humans are not univocal. In a recent study from our group [19], we demonstrated the ability of n-3 PUFA to prevent the increase of arterial stiffness in ovariectomized rats, a validated animal model of experimental menopause. The purpose of the present study was to determine the effects of n-3 PUFA supplementation on BRS and on the aortic pressure wave, as a marker of the large artery elasticity, in the same experimental setting.
2. Methods 2.1. Animals Experiments were performed on female Wistar-Kyoto rats, 2 months old, initially weighing 140 to 170 g, fed with a standard rat chow (Harlan 2018, 3.4 cal/g, macronutrients composition: crude protein 18.6%, fat 6.2%, crude fiber 3.5%. Supplied by Harlan, Italy), for at least two weeks. The animals were kept in temperature-controlled facilities on a 12-hours light/dark cycle. Animals were then randomly assigned to three experimental groups: a) control group of sham operated rats receiving normal diet (CTRL group), b) ovariectomy group receiving normal diet (OVX group), c) ovariectomy group receiving normal diet with the addition of daily gavages administration of a mixture of eicosapentaenoic acids (EPA) and docosahexaenoic acids (DHA) (OVX + O3 group). Animals were sacrificed 2 months after surgical procedure. 10 animals from each of the three experimental groups were randomly selected for the following experimental phase, while other 30 randomly selected animals were included in a different experiment (data previously published [20]). All experiments were performed according to the guidelines and protocols approved by the European Union (EU Council 86/609; D.L. 27.01.1992, no. 116) and by the Animal Research Ethics Committee of the University of Trieste, Italy.
2.3. Surgical ovariectomy Bilateral ovariectomy and sham surgery were performed via a midabdominal route under Xylazine (10 mg/kg, intraperitoneal [IP] injection) and Tiletamine (40 mg/kg, IP) anesthesia. Anesthesia was assessed by complete absence of limb retraction upon painful stimulation. The fallopian tube was legated with absorbable suture, and the ovary was removed. This model of surgical menopause was validated in a previous experiment [20], which proved a significant decrease in serum estradiol levels after surgical bilateral ovariectomy, compared with shamoperated rats. 2.4. Hemodynamic measurements After 2 months since the surgical procedure, 10 animals from each experimental (CTRL, OVX and OVX + O3) group were randomly selected for the hemodynamic measurements. The animals were anesthetized with Xylazine (5 mg/kg IP) and Tiletamine (10 mg/kg IP). Nylon cannulas were introduced into the left femoral arteries up to the aortic ostia. The anatomic locations of the tips of the cannula were checked by postmortem dissection. Cannulas were connected to low-volume blood pressure (BP) transducers (STATHAM range − 50 to + 300 mm Hg), linked to a Coulbourn analog-to-digital convertor plus computer (Coulbourn Instruments; Lablink, Allentown, Pennsylvania). The frequency response of the cannula plus pressure transducer was minimum 100 Hz with 1% FS/24 Hz. Systolic and diastolic arterial BP and BP wave from the aortic ostia were recorded. The personnel who performed hemodynamic measures were blinded to study group assignment of each animal. 2.5. Determination of baroreflex After a 15 minute wait for hemodynamic stabilization after invasive arterial cannulation, the basal pressure profile was recorded. The baroreflex sensitivity was performed by injecting a single dose of phenylephrine (100 μL of 0.01 g/mL solution) directly into the cannulated vessel. Phenylephrine, a pure alpha-agonist, was administered in order to rapidly increase the blood pressure of 15–30 mm Hg, and pressure wave recorded for at least 5 min. The pressure waves were analyzed in order to quantify changes in BP and in heart rate. The slope of the linear regression line between changes in systolic blood pressure and consequent lengthening of the RR interval is a quantitative measure of the baroreceptor control of heart rate. In the analysis, five c beats where the increase in blood pressure was followed by an increase in RR interval were considered for the quantitative measurement of BRS. BRS was calculated relating the changes in BP and in RR interval, as a mean index expressed as ms/mm Hg.
2.2. Diets 2.6. Pressure wave analysis Diets were prepared weekly and stored to prevent degradation, and food was provided and removed daily. To standardize study protocol CTR and OVX rats received an equal volume of normal saline solution by gavage per day. A commercially available pharmaceutical preparation was used for n-3 PUFA treatment (EPA and DHA mixture, 0.9 to 1.5 ratio, EPA + DHA content not inferior to 85%, Eskim, SigmaTau, Italy). The manufacturing process of the drug fulfilled the good manufacturing practice standards, and the production was optimized to improve the stability of the compound. The average content of EPA and DHA was, respectively, 450 ± 50 and 395 ± 55 mg/g. A daily dose of 0.8 g/kg/d was administered to rats in the OVX + O3 group, corresponding to a 0.65 g/kg/d of purified active compound (EPA and DHA). As mentioned in our previous experiment [20], this dose of n-3 PUFA was capable to increase omega-3 index in red cell membranes of about 50%. The study dose corresponded to a human equivalent dose of about 7 g/d. Saline solution (0.9% NaCl) was manufactured by Diaco (Italy).
We performed a graphical analysis on aortic pressure waveform to calculate the time/pressure area under the pressure curves in systole and diastole. Only recordings of 10 consecutive heartbeats with stable BP values were used for the analysis. The systolic pressure–time integral (STI) and the diastolic pressure–time integral (DTI) were calculated as the mean area under the systolic and diastolic part of the waveform (Fig. 1). The relationship between diastolic and systolic pressure wave, considered a parameter evaluating central arterial function, was defined as the ratio between DTI and STI (DTI/STI). For the graphical analysis the Adobe Photoshop (Adobe Systems) v11.0 software package was used to calculate the area under the pressure curve. 2.7. Determination of blood glucose, plasma lipids, and omega-3 index Levels of glucose, total cholesterol, triglycerides, HDL, Plasma 17βestradiol, and membrane omega-3 index were obtained as referenced
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difference: CTRL 49 g; OVX: 84 g; p b 0.05). Body weight increase was even more pronounced in animals with n-3 PUFA supplementation, compared to other groups (body weight difference: OVX + O3: 106 g; p b 0.05 vs CTRL). As evidenced also in our previous experiments in the same experimental setup [20], ovariectomy increased plasma levels of total cholesterol (CTRL: 1.1 ± 0.1 mmol/L; OVX: 1.5 ± 0.2 mmol/L; p b 0.05) and non-HDL cholesterol (CTRL: 0.4 ± 0.1 mmol/L; OVX: 1.0 ± 0.1 mmol/L; p b 0.05), while n-3 PUFA normalized plasma lipids levels (OVX + O3: total cholesterol 1.1 ± 0.1 mmol/L; p b 0.05 vs OVX; non-HDL cholesterol 0.6 ± 0.1 mmol/L; p b 0.05 vs OVX). Relative erythrocyte membrane content of EPA and DHA (n-3 PUFA index) was significantly reduced in OVX rats compared with CTRL. Treatment with n-3 PUFA markedly enhanced omega-3 index in ovariectomized rats (Table 1). 3.2. Hemodynamics and pulse wave analysis Fig. 1. The diastolic pressure–time integral and systolic pressure–time integral ratio (DTI/ STI) in ovariectomized rats (OVX), ovariectomized rats receiving n-3 PUFA supplementation (OVX + O3), and control sham-operated rats (CTRL), after surgery and 8 weeks of supplementation with n-3 PUFA. Data reported as means. Error bars ± 1 SD. *p b 0.01 versus CTRL or OVX + O3.
[20]. These data, which refer to the 30 animals considered in our previous work, were previously discussed [20] and are shown in Table 1. 2.8. Statistical analysis All data are expressed as mean ± standard deviation. The Kolmogorov–Smirnov test for normality was initially performed for all data. One-way analysis of variance was used to assess group differences. If significant differences were observed with analysis of variance, Fisher least significant difference post-hoc analysis for multiple comparisons was performed to identify the differences among the mean values in the groups. IBM SPSS Statistics v20 was used to perform all statistical analyses. A P value of less than 0.05 was considered statistically significant. 3. Results 3.1. Effects of OVX and Omega 3 on body weight, metabolic parameters and omega-3 index
Both systolic and diastolic BP levels significantly differed in rats after ovariectomy, compared to control rats (systolic BP: CTRL 128 ± 23 mm Hg; OVX 150 ± 26 mm Hg; p b 0.05; diastolic BP: CTRL 87 ± 19 mm Hg; OVX 100 ± 13 mm Hg; p b 0.05). n-3 PUFA supplementation prevented this raise in BP, leading rats in OVX + O3 group to have BP levels similar to the CTRL group (OVX + O3: systolic BP 129 ± 22 mm Hg, p b 0.05 vs OVX; diastolic BP 88 ± 14 mm Hg, p b 0.05 vs OVX). Regarding the pulse wave morphology, the DTI/STI ratio was significantly smaller in the ovariectomized group compared to controls (CTRL: 4.17 ± 1.28, OVX: 3.31 ± 1.72, p b 0.01, Fig. 1), while in the n3 PUFA supplemented rats it was comparable to the control group (OVX + O3: 3.83 ± 1.52, p b 0.01 vs OVX, Fig. 1). Heart rate at the time of the hemodynamic evaluation was not significantly different between the 3 groups (CTRL: 285 ± 16 OVX: 277 ± 17 OVX + O3: 291 ± 10, p = ns). 3.3. Effects on baroreflex sensitivity Ovariectomized rats displayed a significantly reduced BRS respect to CTRL (CTRL: 6.23 ± 0.68 ms/mm Hg, OVX: 2.85 ± 0.75 ms/mm Hg; p b 0.001, Fig. 2). Treatment with n-3 PUFA partially prevented this fall in BRS. In OVX + O3 rats BRS was increased respect to OVX group, but slightly decreased respect to CTRL group (OVX + O3: 4.75 ± 0.53 ms/mm Hg, p b 0.001 vs OVX, p b 0.05 vs CTRL Fig. 2).
Body weight increased significantly during experimental phase in all groups (Table 1, p b 0.05 for all groups). Ovariectomy induced a higher increase in body weight respect to control animals (body weight
Table 1 Body weight, metabolic parameters and omega-3 index after surgery and 8 weeks of supplementation with n-3 PUFA. Data reported as means ± SD. Body weight increased significantly in all groups *p b 0.05 for all groups. As evidenced also in our previous experiments [20], ovariectomy increased plasma levels of total cholesterol (*p b 0.05) and non-HDL cholesterol (*p b 0.05), while n-3 PUFA normalized plasma lipids levels (*p b 0.05 vs OVX; non-HDL cholesterol *p b 0.05 vs OVX). Erythrocyte membrane content of EPA and DHA (n-3 PUFA index) was significantly [20] reduced in OVX rats compared with CTRL [16]. Treatment with n-3 PUFA markedly enhanced omega-3 index [20] in ovariectomized rats (*p b 0.05).
Initial body weight (g) Final body weight (g) EPA + DHA (%) Plasma 17β-estradiol (pmol/l) Total cholesterol (mmol/l) Triglycerides (mmol/l) HDL cholesterol (mmol/l) Non-HDL cholesterol (mmol/l)
CTRL
OVX
OVX + O3
151 ± 6.3 200 ± 8 4.70 ± 0.18 65.34 ± 23.13 1.07 ± 0.11 0.71 ± 0.08 0.63 ± 0.16 0.41 ± 0.09
146 ± 15.4 230 ± 12 4.08 ± 0.11 * 5.87 ± 1.84 * 1.50 ± 0.17 * 0.58 ± 0.08 0.61 ± 0.19 1.00 ± 0.10 *
149 ± 9.4 255 ± 16 * 6.24 ± 0.18 7.34 ± 3.30 * 1.07 ± 0.07 0.58 ± 0.07 0.49 ± 0.08 0.61 ± 0.10
Fig. 2. BRS in ovariectomized rats (OVX), ovariectomized rats receiving n-3 PUFA supplementation (OVX + O3), and control sham-operated rats (CTRL), after surgery and 8 weeks of supplementation with n-3 PUFA. Data reported as means. Error bars ± 1 SD. *p b 0.001 versus CTRL or OVX + O3. †p b 0.001 versus OVX, p b 0.05 versus CTRL.
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4. Discussion In the present study, we found that ovariectomy induced BRS reduction in female rats compared to sham-operated controls, and n-3 PUFA supplementation after ovariectomy partially prevented BRS decrease. In parallel, ovariectomized rats developed a significant change in central hemodynamic function, by increasing the systolic component of pulse wave more than the diastolic component, thus decreasing the DTI/STI ratio. Supplementation of n-3 PUFA prevented completely these changes restoring the normal appearance of the central pressure wave. A first finding from our study was the increase in BP found in ovariectomized rats when compared to controls. Onset of hypertension after loss of ovarian hormones is a well known condition. Etiology of postmenopausal hypertension is multifactorial and involves activation of renin–angiotensin system, autonomic imbalance with increased sympathetic and decreased vagal activity, inflammation and probably other mechanisms [21]. In rats supplemented with n-3 PUFA, ovariectomy did not cause an increase in BP values. The antihypertensive effect of n-3 PUFA has been demonstrated both in clinical [22] and experimental [23] conditions, and it is mediated by an increased synthesis of the vasodilator nitric oxide, a reduced production of the vasoconstrictor thromboxanes and a reduction in arterial stiffness [24]. The finding of a reduced BRS in postmenopausal state is consistent with previous studies [25,26]. In ovariectomized female rats BRS is improved by estrogen replacement therapy and cyclic BRS changes are observed during the estrous cycle, both in humans and rats, with variations mirroring the estrogen levels [27], thus suggesting a crucial role of ovarian hormones in baroreflex regulation. The decline in baroreflex function after surgical menopause represents a harmful complication, leading to an abnormal neural regulation of the heart and the vasculature and increased blood pressure variability, which is associated with an increased incidence of cardiovascular events [28]. The observed improvement in BRS given by n-3 PUFA supplementation in our experiment represents a novel therapeutical property of these agents, and can fill a gap among cardiovascular therapies, as few drugs have shown the ability to prevent baroreflex dysfunction. A specific effect of n-3 PUFA on BRS has not been previously reported, while other effects on cardiovascular parameters involving autonomic function have been demonstrated, such as reduction in resting heart rate, in blood pressure and an increased heart rate variability [18,29], suggesting a positive role of n-3 PUFA on autonomic cardiovascular regulation. Vascular stiffness represents a major determinant in the regulation of BRS. Arterial baroreceptors are stretch receptors that are stimulated by the strain of the arterial wall when blood pressure changes. An increase in the mean arterial pressure can increase depolarization of these sensory endings. As shown by previous studies, the stiffening of the baroreceptor vascular segments (in carotid artery and aorta) limits the afferent signaling of the baroreceptor reflex, thus reducing baroreflex sensitivity [30–32]. In our previous study, in the same experimental setting, we found an improvement of arterial stiffness evaluated as pulse wave velocity, following n-3 PUFA treatment in ovariectomized rats [19]. In the present study, we report again a normalization of aortic compliance, by improvement of the diastolic/systolic balance in the central pressure pulse wave evaluated at the aortic level. The elastic reservoir function of aorta and large elastic arteries helps in damping the fluctuations in blood pressure during the cardiac cycle and assists in the maintenance of organ perfusion during diastole, and has recently been studied as the ratio between DTI and STI [33]. A surrogate of this index has been studied in humans noninvasively as subendocardial viability ratio (SEVR), which is considered also a measure of the adequacy of the subendocardial muscle diastolic perfusion in response the myocardial oxygen systolic demand [34]. DTI/STI ratio may therefore represents both an evaluation the elastic properties of the aortic wall, inversely related to arterial stiffness [35], and an indirect assessment of the correctness
of the myocardial perfusion. Considering that it has been demonstrated that aortic pressure profile reflects the carotid pressure profile [36], our results can be extended to the carotid arterial viscoelastic properties, where the main receptor for baroreflex resides. In our study, both systolic and diastolic blood pressure were increased in ovariectomized rats, while in rats treated with n-3 PUFA blood pressure was similar to controls. As expected, the DTI/STI ratio was significantly decreased in ovariectomized rats, revealing a significant change in central hemodynamics after ovariectomy and an increase in arterial stiffness. n-3 PUFA supplementation prevented the decrease in DTI/STI ratio. This improvement in arterial compliance in n-3 PUFA supplemented ovariectomized rats seems to be induced by the contribution of multiple factors, like the capacity of n-3 PUFA to affect BP and lipid profile, and the reversal of endothelial dysfunction and vascular oxidative stress induced by menopause [20]. The mechanisms responsible for the alteration in the elastic properties of the arterial wall have been usually attributed to changes in the physical structure of the vessels, considered as a passive elastic conduits. But, considered the short duration of the experimental phase, it is likely that at least part of the preserved arterial compliance in the n-3 PUFA-treated group is related to functional changes in the vascular system. n-3 PUFA are able to reverse endothelial dysfunction, through an interaction with endothelial nitric oxide synthase that leads to an increased NO bioavailability [20]. NO-mediated alteration in arterial smooth muscle tone are a main determinant of arterial function, and an increased NO production can lead to a reduced wave reflection and a delayed return [37]. In our experiment we did not observe any side effect related to n-3 PUFA administration. Since we used high doses of n-3 PUFA (0.65 g/kg/d, corresponding to an approximate human equivalent dose of 7 g/d), some limits must be observed for the extension of our results to the humans. Another concern could be raised about the weight gain seen in ovariectomized animals, which was even more pronounced after n-3 PUFA administration, probably due to an increased caloric and fat intake. Although a possible neurohormonal effect of adipose tissue could not be excluded in our experiment, previous studies demonstrated that weight gain can adversely affect baroreflex sensitivity [38,39], thus suggesting that adipose tissue is not directly involved in n-3 PUFA effects on baroreflex. Our data indicate that n-3 PUFA administration prevents BRS decrease induced by estrogen deficiency, in parallel with the prevention of some detrimental changes in central arterial hemodynamic. As BRS represents an irreplaceable mechanism in cardiovascular homeostasis, this effect could be important for prevention of hypertension and of cardiovascular autonomic dysfunction associated with menopause. In clinical studies, n-3 PUFA intake has also been implied in estrogen metabolism during menopausal transition [40], suggesting a mutual relationship that needs to be further clarified. Recent clinical trials and metanalysis did not find a significant association of n-3 PUFA administration with reduction of major cardiovascular outcomes [16]. However, all the major trials assessing cardiovascular outcomes of n-3 PUFA administration present a gender disparity in patients enrollment. The percentage of female sex in these trials ranges from 21.5% to 38.5% [41–47], reflecting the known underrepresentation of female sex in cardiovascular medicine [3]. This marked gender inequality must be considered as a gap in the available evidence for n-3 PUFA administration, particularly in postmenopausal women, which present an increased cardiovascular risk. As the mechanisms of action of n-3 PUFA in cardiovascular prevention need to be further investigated, our study provides encouraging results for the use of n-3 PUFA in postmenopausal women and supports the need for testing our experimental hypothesis in a clinical setting. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Please cite this article as: Losurdo P, et al, Baroreflex sensitivity and central hemodynamics after omega-3 polyunsaturated fatty acids supplementation in an animal model of me..., Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2014.12.005
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Please cite this article as: Losurdo P, et al, Baroreflex sensitivity and central hemodynamics after omega-3 polyunsaturated fatty acids supplementation in an animal model of me..., Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2014.12.005