Early Estrogen Replacement Therapy Attenuates Cardiac Dysfunction Caused by Aging and Ovariectomy in Female Wistar Rats.

BACKGROUND: Cardiovascular diseases (CVDs) are the leading cause of women's mortality, linked to aging and reduced estrogen during menopause. Estrogen replacement therapy (ERT) is suggested for CVDs prevention. Yet, its timing initiation remains contentious. Thus, we aimed to evaluate the effect of early and late estrogen therapy on cardiac function and lipid metabolism in ovariectomized old female Wistar rats. METHODS: Fifty randomized female Wistar rats were included in 5 groups (n = 10, 18 months old): (1) Sham, (2) 10 weeks post ovariectomy (Ovx-10 w), (3) 10 weeks post Ovx + early estrogen replacement therapy (Ovx 10 w-early ERT), (4) 20 weeks post Ovx (Ovx-20 w) and (5) Ovx 20 w-late ERT. Three days (early ERT) or 10 weeks (late ERT) after surgery 17-ß estradiol was given (5 µg/kg/day), and 10 weeks after the start of ERT, we assessed cardiac function by echocardiography, electrocardiography, and cardiac catheterization. Estradiol, cholesterol, triglyceride (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels were determined. Cardiac histology was performed with Masson's staining. RESULTS: Ovariectomy (Ovx) increases left ventricle internal systolic diameter (0.4 vs 0.3 cm, *p = 0.020) and decreases shortening fraction (40 vs 54 %, *p = 0.030) regardless of therapy. ERT prevents the increase in left ventricle mass after 10 weeks post-Ovx and the ejection fractionreduction after 20 weeks. Lower P wave amplitudes (18.8 vs 24.2 ms, *p = 0.013) were found in the Ovx-20 w group. A longer duration of the QRS complex after 20 weeks post-Ovx with and without ERT was found (32.5 and 32.1 vs 28.3 ms, *p = 0.003; *p = 0.007). Diastolic blood pressure was higher 20 weeks post-Ovx (86 vs 76 mmHg, *p = 0.047), regardless of ERT. The left ventricle (LV) -dP/dt was decreased in Ovx groups without ERT (-750 vs -1320 mmHg, *p = 0.034). An increase in LV collagen deposition was found in the Ovx 10 w group vs Sham (9.58 vs 4.54 %, *p = 0.028). Early ERT avoids the increase in body weight, cholesterol and LDL caused by Ovx. CONCLUSIONS: Ovariectomy causes time-dependent alterations in lipid metabolism, morphology, electrical activity, and heart contractile function. Early but not late ERT prevents some of these effects.

Timing Matters: Effects of Early and Late Estrogen Replacement Therapy on Glucose Metabolism and Vascular Reactivity in Ovariectomized Aged Wistar Rats.

Cardiovascular disease incidence increases after menopause due to the loss of estrogen cardioprotective effects. However, there are conflicting data regarding the timing of estrogen therapy (ERT) and its effect on vascular dysfunction associated with impaired glucose metabolism. The aim of this work was to evaluate the effect of early and late ERT on blood glucose/insulin balance and vascular reactivity in aged ovariectomized Wistar rats. Eighteen-month-old female Wistar rats were randomized as follows: (1) sham, (2) 10-week postovariectomy (10 w), (3) 10 w postovariectomy+early estradiol therapy (10 w-early E2), (4) 20-week postovariectomy (20 w), and (5) 20-week postovariectomy+late estradiol therapy (20 w-late E2). Early E2 was administered 3 days after ovariectomy and late therapy after 10 weeks, in both groups. 17ß-Estradiol (E2) was administered daily for 10 weeks (5 µg/kg/day). Concentration-response curves to angiotensin II, KCl, and acetylcholine (ACh) were performed. Heart rate (HR), diastolic and systolic blood pressure (DBP and SBP), glucose, insulin, HOMA-IR, and nitric oxide (NO) levels were determined. Higher glucose levels were found in all groups compared to the sham group, except the 20 w-late E2 group. Insulin was increased in all ovariectomized groups compared to sham. The HOMA-IR index showed insulin resistance in all ovariectomized groups, except for the 10 w-early E2 group. The 10 w-early E2 group increased NO levels vs. the 10 w group. After 10 w postovariectomy, the vascular response to KCl and Ach increases, despite early E2 administration. Early and late E2 treatment decreased vascular reactivity to Ang II. At 20-week postovariectomy, DBP increased, even with E2 administration, while SBP and HR remained unchanged. The effects of E2 therapy on blood glucose/insulin balance and vascular reactivity depend on the timing of therapy. Early ERT may provide some protective effects on insulin resistance and vascular function, whereas late ERT may not have the same benefits.

Time-Dependent Effects of Individual and Combined Treatments With Nebivolol, Lisinopril, and Valsartan on Blood Pressure and Vascular Reactivity to Angiotensin II and Norepinephrine.

Clinical guidelines suggest the combination of 2 drugs as a strategy to treat hypertension. However, some antihypertensive combinations have been shown to be ineffective. Therefore, it is necessary to determine whether differences exist between the results of monotherapy and combination therapy by temporal monitoring of the responses to angiotensin II and norepinephrine, which are vasoconstrictors involved in the development of hypertension. Thus, the purpose of this work was to determine the vascular reactivity to angiotensin II and norepinephrine in spontaneously hypertensive rat (SHR) aortic rings after treatment with valsartan, lisinopril, nebivolol, nebivolol-lisinopril, and nebivolol-valsartan for different periods of time. In this study, male SHR and Wistar Kyoto normotensive (WKY) rats were divided into 7 groups treated for 1, 2, and 4 weeks: (1) WKY + vehicle, (2) SHR + vehicle; (3) SHR + nebivolol; (4) SHR + lisinopril; (5) SHR + valsartan; (6) SHR + nebivolol-lisinopril; and (7) SHR + nebivolol-valsartan. Blood pressure was measured by the tail-cuff method, and vascular reactivity was determined from the concentration-response curve to angiotensin II and norepinephrine in aortic rings. The results showed that the combined and individual treatments reduced mean blood pressure at all times evaluated. All treatments decreased vascular reactivity to angiotensin II; however, in the case of lisinopril and nebivolol-lisinopril, the effect observed was significant up to 2 weeks. All treatments decreased the reactivity to norepinephrine up to week 4. These results show a time-dependent difference in vascular reactivity between the pharmacological treatments, with nebivolol-valsartan and nebivolol-lisinopril being both effective combinations. Additionally, the results suggest crosstalk between the renin-angiotensin and sympathetic nervous systems to reduce blood pressure and to improve treatment efficacy.

Differences in the expression of the renin angiotensin system and the kallikrein-kinin system during the course of myocardial infarction in male and female Wistar rats.

BACKGROUND: There is some evidence that components of the renin-angiotensin system and kallikrein-kinin system are not similarly regulated in both sexes. The aim of this work was to analyze the expression of angiotensin-converting enzyme, angiotensin-converting enzyme 2, angiotensin 1 receptor, angiotensin 2 receptor, beta-1 receptor, and beta-2 receptor during the evolution of myocardial infarction. METHODS: Thirty-six male and 36 female Wistar rats were used. Myocardial infarction was induced. Six groups of both sexes were formed, (n=6): (a) sham; (b) 48 h myocardial infarction; (c) one week myocardial infarction; (d) two weeks myocardial infarction; (e) three weeks myocardial infarction and (f) four weeks myocardial infarction. The expression was evaluated by real-time polymerase chain reaction on the penumbra of left ventricle. RESULTS: The mRNA expression of most biomarkers was lower in females than in males. During acute infarction, an increase of all protein expression was found in female and at two weeks while in the male only biomarker changes occurred at three weeks. In addition, in male biomarkers mRNA expression decreased during chronic infarction while in females it did not. CONCLUSIONS: The renin-angiotensin system and kallikrein-kinin system biomarkers expression occurs at earlier times in the female than in the male rat. In addition, during chronic myocardial infarction these biomarkers remained unchanged in females while in males they decreased.

Combined Antihypertensive Therapies That Increase Expression of Cardioprotective Biomarkers Associated With the Renin-Angiotensin and Kallikrein-Kinin Systems.

Antihypertensive pharmacological treatments focus on the use of angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists, and beta-blockers as single and combined treatments. The effect of single treatments on the mRNA expression of some components of the renin-angiotensin system has been studied, but not the effect of combined treatments. This study determined the expression of the AT1, AT2, B1, and B2 receptors and of the enzymes ACE and ACE2 in hypertensive rats treated with captopril-propranolol or losartan-propranolol. Methods: The mRNA expression of the receptors and enzymes was determined by reverse transcription-quantitative polymerase chain reaction in the aorta of spontaneously hypertensive rats under different treatments. Results: Rats under combined treatments showed a decrease in the expression of AT1 and ACE, and an increase in the expression of the B1 receptor (captopril + propranolol group: 0.43 ± 0.046, 2.243 ± 0.269, 3.356 ± 0.418; Group: losartan + propranolol: 0.727 ± 0.071, 0.852 ± 0.102, 1.277 ± 0.131 compared to the spontaneously hypertensive group: 1 ± 0.212, 1 ± 0.192, 1 ± 0.214). This decrease in the expression of ACE and AT1 suggests a reduction in the expression of Ang II that could be related to a lower response to this vasoconstrictor. An increase in the expression of B1 would improve vasodilation, which would be a beneficial effect of combined therapies for hypertension.

Sex Differences in Vascular Reactivity to Angiotensin II During the Evolution of Myocardial Infarction.

The influence of sex on the vascular response during the progression of myocardial infarction (MI) has not been extensively studied. In this work, we analyzed the differences of the vasoconstriction induced by angiotensin II (Ang II) in the absence and presence of valsartan (200 nM) on the aortic rings of male and female Wistar rats at 2, 4, 24, and 48 hours and 1, 2, 3, and 4 weeks after induction of MI. In the aortic rings of males, an increase was observed in the contractile response that lasts up to 4 weeks; on females, this effect is diminished since 48 hours until reaching sham group values at 2 weeks of coronary occlusion. The incubation of valsartan generated greater reduction on vasoconstriction in males than females. In relation to the determination of infarct areas, we found them between 30% and 40% in all experimental groups. In addition, the index of hypertrophy was determined and no significant changes were observed in female rats, while in males we reported an increase at 2, 3, and 4 weeks. In conclusion, we found differences in vascular reactivity due to sex, as well as on the response of Ang II via AT1 during the evolution of MI.

Combination of ß Adrenergic Receptor Block and Renin-Angiotensin System Inhibition Diminished the Angiotensin II-Induced Vasoconstriction and Increased Bradykinin-Induced Vasodilation in Hypertension.

In hypertension, the combination therapy is frequently used to obtain a better therapeutic effect and reduce adverse effects. One effective combination is with inhibitors and ß-blockers of renin-angiotensin system. Although the mechanisms of action of each drug are already known, the antihypertensive mechanism is more complex and therefore the combined treatment mechanism is unclear. Specifically, the effect of the treatments of angiotensin-converting enzyme inhibitor or AT1 receptor antagonist with ß-blocker on the angiotensin II and bradykinin reactivity has not been studied. For this reason, we evaluated the interaction between propranolol and captopril or losartan on vascular reactivity to bradykinin and angiotensin II in spontaneously hypertensive rat. We constructed concentration-response curves to angiotensin II and bradykinin after treatment of SHR with propranolol-captopril or propranolol-losartan by using rat aortic rings. While losartan or captopril with propranolol potentiated bradykinin-induced vasodilation effect, the propranolol-losartan interaction decreased the angiotensin II-induced vasoconstriction. In addition, the combinations did not reduce the heart rate significantly. These results suggest that the combined therapy decreased blood pressure to normotensive values and showed less effect for angiotensin II and greater effect for bradykinin than monotherapy which could contribute in the antihypertensive effect.

Antihypertensive Properties of a Novel Morphologic Derivative (4-tert-buthyl-2,6-bis(thiomorpholine-4-ilmethyl)phenol).

We evaluated the antihypertensive properties of 4-tert-buthyl-2,6-bis(thiomorpholine-4-ilmethyl)phenol (TBTIF). Spontaneously hypertensive rats were treated with TBTIF or captopril (both at 1 mg·kg⁻¹·d⁻¹ intramuscularly for 4 days), and their blood pressure (BP) was assessed. In some experiments, concentration response curves to angiotensin I or angiotensin II were generated in rat aortic rings and in the absence or presence of Ang-(1-7), N(G)-monomethyl L-arginine, or both; additionally, the angiotensin-converting enzyme (ACE) and ACE2 mRNA levels were quantified in the aortic rings using reverse transcription-polymerase chain reaction. TBTIF diminished BP and reduced angiotensin I- or angiotensin II-induced vasoconstriction. The presence of Ang-(1-7) induced a greater reduction in vasoconstriction, and this effect was reversed by L-N(G)-monomethyl arginine. Moreover, TBTIF decreased the mRNA of ACE and increased the mRNA of ACE2. In conclusion, TBTIF diminished rat BP through nitric oxide-dependent and nitric oxide-independent mechanisms. In contrast to captopril, TBTIF exhibits better antihypertensive properties through mechanisms that involve ACE2.