Beneficial effects of nebivolol and hydrochlorothiazide combination in spontaneously hypertensive rats

Abstract

Objective

This study examined the combined effect of nebivolol (NEB) and hydrochlorothiazide (HCTZ) on cardiovascular function in spontaneously hypertensive rats (SHR).

Methods

SHR normotensive male rats were randomly assigned to five groups (n = 8 per group): (i) SHR control group; (ii) NEB 2 mg/kg per day group; (iii) HCTZ 10 mg/kg per day group; (iv) NEB 2 mg/kg per day + HCTZ 10 mg/kg per day group; (v) Eight age-matched Wistar-Kyoto normotensive male rats served as the control group. All groups were treated orally for 8 weeks.

Results

The combination of NEB + HCTZ synergistically reduced systolic blood pressure and heart rate compared with either monotherapy. HCTZ increased water intake, which is a sign of diuresis. NEB reduced plasma angiotensin II concentration, which was increased in SHR and after HCTZ treatment. NEB + HCTZ increased plasma nitric oxide (NO) concentration and NO synthase activity, which were both reduced in SHR. NEB + HCTZ normalized femoral arterial vasorelaxation induced by acetylcholine, which was impaired in SHR.

Conclusions

The combination of NEB + HCTZ provided a number of beneficial and additive effects due to the synergistic characteristics of both drugs, in an experimental rat model of hypertension.

Keywords

Beta-blocker diuretic hydrochlorothiazide hypertension nebivolol spontaneously hypertensive rats

Introduction

Hypertension is a cardiovascular risk factor in which sustained high blood pressure (BP) affects endothelial function and artery remodelling. The treatment of hypertension is based on several classes of antihypertensive drugs including β-blockers, angiotensin-converting enzyme inhibitors, calcium channel blockers, sartans and diuretics. Although pharmacological therapy plays a primary role in hypertension management, drugs alone are not effective for every patient.¹

The use of antihypertensive agent monotherapy is usually only indicated for patients with mild-to-moderate hypertension, whereas combination therapy is used in patients with severe hypertension, or in those who do not respond to initial monotherapy.² The increasing average age of the population in the Western world and the concomitant increase in the prevalence of associated pathologies (such as atherosclerosis) have reinforced the need for multidrug treatment, in order to combine the benefits of one class of antihypertensive agent with those of another.^2,3

The β-blockers have been used in the treatment of hypertension for the last four decades. Apart from lowering BP, they have antianginal and antiarrhythmic actions that effectively reduce rates of cardiovascular disease and mortality.³ Thiazide diuretics have been available for a longer time period than β-blockers, but remain some one of the most effective therapies for treating hypertension.⁴ Thiazide diuretics are believed to act by blocking renal sodium absorption via the NaCl cotransporter.⁵ Using a combination of a β-blocker and a thiazide is a well-established antihypertensive treatment regimen,^6–8 given that these agents exert a more marked antihypertensive effect when used together and that, in combination, the metabolic side-effects of the diuretic are reduced.⁹ Nebivolol (NEB), a third-generation β-blocker, has the highest β-1 adrenoceptor selectivity compared with other third-generation β-blockers and is devoid of intrinsic sympathomimetic activity.^10,11 Along with peripheral vasodilatation and release of nitric oxide (NO)-inducing benefits (such as antioxidant activity and reversal of endothelial dysfunction), NEB facilitates better protection from cardiovascular events, compared with classic β-blockers.^12,13

The present study investigated the effect of NEB and hydrochlorothiazide (HCTZ), alone and in combination, on cardiovascular function in spontaneously hypertensive rats (SHR), in order to determine the potential synergistic antihypertensive effects of both drugs in this animal model.

Materials and methods

Animals

The study was conducted in the Department of Pharmacology, Shanxi Medical University, Taiyuan, Shanxi Province, China. Animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication 86–23, revised 1986).¹⁴ The study was reviewed and approved by Shanxi Medical University Ethics Committee. Thirty-two male SHR rats aged 3 months (body weight, range 250–300 g) and eight age-matched Wistar-Kyoto (WKY) normotensive rats (body weight, range 250–300 g) were obtained from the Animal Centre of the Institute of Science and Technology, Shanghai, China. All rats were housed in a standard experimental animal laboratory for 2 weeks prior to the start of the experiments. Rats were housed in 2–3 animals per cage; temperature of their housing was maintained at 22–24℃ with a 12-h light–dark cycle and free access to water.

Experimental design

Rats were randomly divided into five groups: (i) SHR control group (n = 8): SHR rats treated with vehicle (distilled water) once daily; (ii) NEB 2 mg/kg per day group (n = 8, SHR rats); (iii) HCTZ 10 mg/kg per day group (n = 8, SHR rats); (iv) NEB 2 mg/kg + HCTZ 10 mg/kg per day group (n = 8 SHR rats); (v) WKY control group (n = 8): WKY rats treated with vehicle (distilled water) once daily. NEB (a racemic mixture of equal parts of [+]-SRRR- and RSSS-nebivolol) was obtained from the Menarini Group (Florence, Italy). HCTZ was purchased from Sigma-Aldrich (St Louis, MO, USA).

Oral therapy continued for 8 weeks. Tail systolic blood pressure (SBP), heart rate (HR) and water intake were measured at 0, 1, 2, 4 and 8 weeks after the beginning of therapy. Therapeutic doses were chosen on the basis of our previous experience with NEB monotherapy in SHR,¹⁵ taking into account the ratio of NEB/HCTZ used in human hypertension research.¹⁶ At the end of this study, animals were anaesthetized with 3% sodium pentobarbital via intraperitoneal injection (30 mg/kg). Blood was drawn by abdominal aortic puncture, then the aorta and femoral artery were removed rapidly and placed into physiological saline solution (144 mM NaCl, 5.8 mM KCl, 1.2 mM MgCl₂, 2.5 mM CaCl₂, 11.0 mM glucose, 5.0 mM HEPES; pH 7.4) at 4℃, continuously oxygenated with 95% O₂ and 5% CO₂. Blood samples were collected in polypropylene tubes containing 50 µl of heparin (50 000 IU) and, after centrifugation at 3000 g at 4℃ for 10 min, each plasma sample was stored at −70℃ until analysis for NO, NO synthase (NOS) activity and hydroxyl radicals.

Measurement of SBP, HR and water intake

Systolic blood pressure (SBP) was measured indirectly in a conscious and slightly restrained rat using the tail cuff method. HR was calculated from the physiological tracings obtained during BP measurement. For these measurements, rats were conditioned to the restraint and the warming chamber for 10–20 min/day for ≥ 3 days before measurement. SBP and HR measurements were performed between 07.00 h and 12.00 h by the same investigator. The warming chamber (model 306 warming chamber; IITC Life Science, Woodland Hills, CA, USA) was kept at 31–33℃. There were three sensors in the warming chamber that measured BP; as one rat was tested, another rat was being warmed in advance. After 5–10 min of stabilization in the chamber, a typical run involved 10 repetitions of the automated inflation–deflation cycle. The mean of three readings within a 5–10-mmHg range was taken as the SBP. Mean daily water consumption per rat was calculated by dividing the total daily water consumption by the number of animals present in the cage.

Measurement of NO and NOS activity

The level of NO was measured indirectly as the total content of nitrite and nitrate. Briefly, nitrate was converted to nitrite with Aspergillus nitrate reductase (A012 NO kit; Nanjing Jianchen Bioengineering Institute, Nanjing, Jiangsu Province, China), and the total nitrate was measured with Griess reagent, as previously described.¹⁷ Absorbance was determined at 540 nm , using the UV6100 spectrophotometer (Mapada Instruments, Shanghai, China). NOS activity was determined using a commercially available kit (A014 NOS kit; Nanjing Jianchen Bioengineering Institute). The absorbance was determined at 530 nm using the UV6100 spectrophotometer.

Measurement of hydroxyl radicals

Hydroxyl radicals were measured as an index of reactive oxygen species. Hydroxyl radicals are very reactive and can be generated through the Fenton reaction. Hydroxyl radical concentrations were determined using a commercially available kit (A018 hydroxyl radical kit; Nanjing Jianchen Bioengineering Institute). In brief, plasma (200 µl) was added to substrate application liquid and allowed to react for 1 min at 37℃. Then, 2 ml of colour agent was added and the mixture was allowed to react for 20 min at room temperature. The absorbance of the hydroxyl radicals was determined at 570 nm using the UV6100 spectrophotometer (Mapada Instruments).

Measurement of plasma angiotensin II and renin activity

Enzyme inhibitors (containing 0.30 mol/l Na₂-edetic acid, 0.32 mol/l dimercaprol and 0.34 mol/l oxine sulphate) were added to plasma samples (200 µl for angiotensin II [Ang II]; 100 µl for plasma renin activity [PRA]). The samples were centrifuged at 3000 g for 5 min at 4℃. The supernatant was used for measuring Ang II and PRA using radioimmunoassay kits (Ang II kit [D02PZA], PRA kit [D01PZA]; Beijing North Institute of Biological Technology, Beijing, China).

Arterial relaxation

Femoral artery rings (2 mm in length) were mounted in separate 5- ml tissue baths using 40-mm steel wire in a small-vessel myograph (Model 610M; Danish Myo Technology, Aarhus, Denmark) for tension recordings. After an initial equilibration time of 30 min, the arteries were stretched stepwise to characterize the relationship between the vessel circumference and the passive wall tension. The vessel size was normalized by defining L₁₀₀ as the internal circumference that the vessel would have when relaxed and exposed to a transmural pressure of 100 mmHg. The vessels were maintained at 0.9 × L₁₀₀ to let the development of maximal active force. Vessels were contracted with 60 mmol/l KCl and then a cumulative concentration response curve to acetylcholine (ACh; 10⁻⁹–10⁻⁵ mol/l) was produced.

Histology

Thoracic aortas were isolated and cleaned. The aortas were fixed in 10% formalin for 24 h, then dehydrated in a graded series of ethanol solutions and finally rinsed with distilled water for 10 min. Next, the aortas were fixed in 4% paraformaldehyde and paraffin-wax embedded. Serial sections (5-µm thick) were cut and stained with haematoxylin and eosin. The geometry of the arteries (i.e. the media thickness [M, µm], luminal radius [L, µm] and media–to–luminal radius [M/L]) was evaluated using a microscope (Olympus AX-70 microscope; Olympus Corporation, Tokyo, Japan), with a video camera (Olympus DP71 CCD camera; Olympus) connected to an image-analysis processor (Nachet 1500; Chengdu Thai Union Technology, Chengdu, China) and a microcomputer (Macintosh II; Apple, Cupertino, CA, USA).

Statistical analyses

All data were expressed as mean ± SEM. All statistical analyses were performed using the SPSS® software package, version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Calculations for significant differences between the different groups were performed with a one-way analysis of variance (ANOVA) followed by the Bonferroni test; ANOVA for repeated measures was used as appropriate. A P-value < 0.05 was considered statistically significant.

Results

Effect on SBP, HR and water intake

Over the course of the 8-week treatment period, the mean (± SEM) SBP was higher in the SHR control group than in age-matched WKY rats at every timepoint (values at week 8: 194.5 ± 7.2 mmHg versus 129.4 ± 2.3 mmHg, respectively; P < 0.05; Figure 1). Compared with the SHR control group, treatment with 2 mg/kg per day NEB alone had no effect on the mean SBP (mean ± SEM value at week 8: 191.5 ± 6.6 mmHg), whereas 10 mg/kg per day HCTZ alone significantly reduced the mean (± SEM) SBP to 186.2 ± 6.1 mmHg at week 8 (P < 0.05). The combination of NEB and HCTZ induced a significant reduction in the mean SBP (mean ± SEM value at week 8: 171.0 ± 6.9 mmHg; P < 0.05), especially after the first week of treatment. At the end of the 8-week treatment period, NEB + HCTZ reduced the mean SBP by 23.5 mmHg compared with the SHR control group (P < 0.05).

Figure 1.

Time course of the mean systolic blood pressure (SBP) in Wistar-Kyoto (WKY) normotensive rats, control spontaneous hypertensive rats (SHR) and SHR treated with 2 mg/kg per day nebivolol (NEB), 10 mg/kg per day hydrochlorothiazide (HCTZ) or the combination of 2 mg/kg per day NEB and 10 mg/kg per day HCTZ for 8 weeks. Results are expressed as mean ± SEM. n = 8 per treatment group. ^*P < 0.05 versus control SHR group; ^#P < 0.05 versus the SHR group that received NEB + HCTZ.

The mean (± SEM) HR was higher in the SHR control group than in the age-matched WKY rats over the 8-week treatment period (values at week 8: 445.6 ± 20.4 versus 321.9 ± 50.8 beats per min, respectively; P < 0.05; Figure 2). The mean HR was not affected by HCTZ treatment alone but was significantly reduced after NEB and NEB + HCTZ treatment (P < 0.05 compared with SHR control group at weeks 2, 4 and 8; Figure 2). When SHR were treated with NEB alone, the mean HR was initially reduced but then gradually increased after 1 week’s treatment, although it remained significantly lower than that in the SHR control group at all timepoints (P < 0.05); the mean HR was reduced by NEB + HCTZ treatment and continued to fall significantly over the treatment period (P < 0.05 compared with the SHR control at weeks 2, 4 and 8).

Figure 2.

Time course of the mean heart rate in Wistar-Kyoto (WKY) normotensive rats, control spontaneous hypertensive rats (SHR) and SHR treated with 2 mg/kg per day nebivolol (NEB), 10 mg/kg per day hydrochlorothiazide (HCTZ) or the combination of 2 mg/kg per day NEB and 10 mg/kg per day HCTZ for 8 weeks. Results are expressed as mean ± SEM. n = 8 per treatment group. ^*P < 0.05 versus SHR control group; ^#P < 0.05 versus the SHR group that received NEB + HCTZ.

There was no difference in the mean water intake among the WKY, SHR control and NEB groups, but the mean water intake increased significantly in all rats treated with HCTZ (P < 0.05), especially after 1 week’s treatment (P < 0.05 versus SHR control group; Figure 3).

Figure 3.

Time course of the mean drinking water consumption in Wistar-Kyoto (WKY) normotensive rats, control spontaneous hypertensive rats (SHR) and SHR treated with 2 mg/kg per day nebivolol (NEB), 10 mg/kg per day hydrochlorothiazide (HCTZ) or the combination of 2 mg/kg per day NEB and 10 mg/kg per day HCTZ for 8 weeks. Results are expressed as mean ± SEM. n = 8 per treatment group. ^*P < 0.05 versus SHR control group; ^#P <0.05 versus the SHR group that received NEB + HCTZ.

Effect on plasma Ang II and PRA activity

The mean plasma Ang II concentration and PRA activity were significantly higher in the SHR control group compared with the WKY group after 8 weeks’ treatment (P < 0.05 for both comparisons; Table 1). Treatment with NEB alone significantly decreased the mean plasma Ang II concentration compared with that observed in the SHR control group after 8 weeks’ treatment (P < 0.05), but had no significant effect on the mean PRA activity. Treatment with HCTZ alone for 8 weeks significantly increased the mean plasma Ang II concentration compared with the SHR control group (P < 0.05), but had no significant effect on the mean PRA activity. Treatment with NEB + HCTZ significantly decreased both the mean plasma Ang II concentration and PRA activity compared with the SHR control group after 8 weeks’ treatment (P < 0.05 for both comparisons).

Table 1.

Plasma levels of angiotensin II (Ang II), plasma renin activity (PRA), nitric oxide (NO), NO synthase (NOS) activity and hydroxyl radicals in Wistar-Kyoto (WKY) normotensive rats, control spontaneous hypertensive rats (SHR) and SHR treated with nebivolol (NEB) 2 mg/kg per day, hydrochlorothiazide (HCTZ) 10 mg/kg per day or the combination of NEB 2 mg/kg per day and HCTZ 10 mg/kg per day for 8 weeks.

Study group	Ang II pg/ml	PRA ng/ml per h	NO concentration µmol/l	NOS activity U/ml	Hydroxyl radicals U/ml
SHR control	933.6 ± 42.5	7.1 ± 0.6	9.6 ± 1.5	25.0 ± 1.7	1909.5 ± 29.9
WKY control	326.3 ± 21.2^a	4.5 ± 0.8^a	34.0 ± 3.6^a	31.2 ± 1.3^a	1882.3 ± 31.5
NEB	692.1 ± 49.9^a,b	9.4 ± 0.7^b	45.2 ± 4.4^a	26.4 ± 1.9	1904.7 ± 30.4^b
HCTZ	1097.5 ± 55.9^a,b	10.7 ± 0.9^b	33.1 ± 3.9^a	25.2 ± 1.9	1909.8 ± 61.7^b
NEB + HCTZ	485.1 ± 10.6^a	4.7 ± 0.6^a	41.9 ± 3.8^a	30.3 ± 2.4^a	1769.9 ± 88.7^a

Results presented as mean ± SEM. n = 5–8 per treatment group.

^aP < 0.05 versus SHR control; ^bP < 0.05 versus NEB + HCTZ; one-way analysis of variance (ANOVA) followed by the Bonferroni test and ANOVA for repeated measures, as appropriate.

Effect on plasma NO and NOS activity

The mean plasma NO concentration and NOS activity were significantly lower in the SHR control group compared with the WKY rat group (P < 0.05 for both comparisons; Table 1). All three treatment regimens significantly increased the mean plasma NO concentrations compared with the SHR control group after 8 weeks’ treatment (P < 0.05 for all comparisons). In terms of the mean plasma NOS activity, only the combination of NEB + HCTZ resulted in a significant increase compared with the SHR control group after 8 weeks’ treatment (P < 0.05).

Effect on plasma hydroxyl radical concentrations

There was no significant difference in the mean plasma hydroxyl radical concentrations between the SHR control group and the WKY rats (Table 1). Mean plasma hydroxyl radical concentrations were not significantly changed when SHR were treated with either NEB or HCTZ alone, but were significantly reduced following 8 weeks’ treatment with the combination of NEB + HCTZ, compared with the SHR control group (P < 0.05), the NEB alone group (P < 0.05) and the HCTZ alone group (P < 0.05).

Effect on femoral artery relaxation response to ACh

The extent of relaxation in response to ACh (10⁻⁹–10⁻⁵ mol/l) of femoral artery rings, which had been precontracted with 60 mmol/l KCl, is shown in Figure 4. An impaired response to ACh was observed in the SHR control group compared with the WKY rat group (P < 0.05 for all ACh concentrations). HCTZ alone had no significant effect on relaxation compared with the SHR control group, but treatment with NEB alone or NEB + HCTZ normalized ACh-induced relaxation, compared with the SHR control group, at the majority of ACh concentrations (P < 0.05).

Figure 4.

Percentage relaxation in response to acetylcholine (ACh; 10⁻⁹–10⁻⁵ mol/l) in rat femoral artery rings precontracted with 60 mmol/l KCl taken from Wistar-Kyoto (WKY) normotensive rats, control spontaneous hypertensive rats (SHR) and SHR treated with 2 mg/kg per day nebivolol (NEB), 10 mg/kg per day hydrochlorothiazide (HCTZ) or the combination of 2 mg/kg per day NEB and 10 mg/kg per day HCTZ for 8 weeks. Results were expressed as mean ± SEM. n = 8 per treatment group. ^*P < 0.05 versus control SHR group; ^#P < 0.05 versus the SHR group that received NEB + HCTZ.

Effect on aorta morphometry

Representative photomicrographs of aortas taken from rats in the five treatment groups are shown in Figure 5. The SHR control group demonstrated the typical arterial wall hypertrophy that is associated with hypertension in the rat model. As shown in Table 2, the mean media thickness and M/L ratio of the aorta were significantly increased in the SHR control group compared with the WKY rat group (P < 0.05 for both comparisons). Treatment with NEB alone for 8 weeks had no significant effect on the mean media thickness and M/L compared with the SHR control group, but HCTZ alone and NEB + HCTZ significantly reduced the M/L compared with the SHR control group (P < 0.05 for both comparisons). There were no significant changes in the luminal radius in response to 8 weeks’ treatment with HCTZ alone or NEB + HCTZ compared with the SHR control group, but treatment with NEB alone significantly reduced the luminal radius compared with the SHR control group and the NEB + HCTZ group (P < 0.05, both comparisons).

Figure 5.

Representative photomicrographs of aortas taken from control spontaneous hypertensive rats (SHR) (A); Wistar-Kyoto (WKY) normotensive rats (B); SHR treated with 2 mg/kg per day nebivolol (NEB) (C), 10 mg/kg per day hydrochlorothiazide (HCTZ) (D) or 2 mg/kg per day NEB + 10 mg/kg per day HCTZ (E) for 8 weeks. Hypertension was associated with arterial wall hypertrophy (see SHR control group [A] compared with WKY normotensive rats [B]), which was reduced by treating the SHR with the combination of NEB + HCTZ (E).

Table 2.

Values of the media thickness (M), luminal radius (L) and M/L ratio of aortas taken from Wistar-Kyoto (WKY) normotensive rats, control spontaneous hypertensive rats (SHR) and SHR treated with nebivolol (NEB) 2 mg/kg per day, hydrochlorothiazide (HCTZ) 10 mg/kg per day or the combination of NEB 2 mg/kg per day and HCTZ 10 mg/kg per day for 8 weeks.

Study group-	Media thickness µm	Luminal radius Μm	M/L ratio %
SHR control	93.4 ± 15.0	1394.1 ± 124.7	6.7 ± 1.1
WKY control	70.9 ± 11.6^a	1335.8 ± 113.3	5.5 ± 0.9^a
NEB	76.6 ± 25.7	1179.0 ± 100.0^a,b	6.5 ± 2.0^b
HCTZ	63.3 ± 8.7^a	1347.9 ± 105.4	4.7 ± 0.4^a
NEB + HCTZ	65.8 ± 13.1^a	1371.4 ± 57.8	4.8 ± 1.1^a

Results presented as mean ± SEM. n = 5–8 per treatment group.

^aP < 0.05 versus SHR control; ^bP < 0.05 versus NEB + HCTZ; one-way analysis of variance (ANOVA) followed by the Bonferroni test and ANOVA for repeated measures as appropriate.

Discussion

The main findings of the present study were: (i) that treatment of SHR with 2 mg/kg per day NEB for 8 weeks significantly reduced HR and the mean plasma Ang II concentration, significantly increased the mean plasma NO concentration and improved endothelium-dependent vasodilatation; (ii) that there was pronounced synergy between 2 mg/kg per day NEB and 10 mg/kg per day HCTZ in countering hypertension in the SHR animal model, as demonstrated by improved endothelium-dependent arterial relaxation, together with reductions in the mean plasma Ang II concentration, PRA activity, and arterial wall hypertrophy.

The increasing mean values of SBP observed in the SHR control group paralleled the increases observed in the mean HR over the 8-week treatment period. NEB administered at a relatively low oral dose (2 mg/kg per day), which was able to improve experimental hypertension in other studies,^18–20 did not affect the mean SBP when administered alone, but instead exerted a bradycardic effect. Our current results demonstrated that NEB treatment was associated with a significant reduction in HR, which was independent of its BP-lowering effect. It should be noted that these rats were severely affected by hypertension, as indicated by their basal mean SBP value of ∼180 mmHg, which was higher than the basal mean value of 130 mmHg observed in WKY normotensive rats. A significant bradycardic effect was also observed with the combination of NEB + HCTZ. NEB is one of the most selective β-1 adrenoceptor blockers.^11,12 A reduction in HR contributes to a reduction in cardiac workload. As a thiazide diuretic, HCTZ exerts its antihypertensive effects by inhibiting Na⁺/Cl⁻ reabsorption from the distal convoluted tubules in the kidney.²¹ By reducing osmotic pressure in this way, HCTZ reduces the reabsorption of water in the distal convoluted tubules and thereby reduces plasma volume and cardiac output.²¹ The combined effect of these actions is to reduce SBP. Our current study demonstrated that the diuretic effect (seen indirectly as an increased water intake) of HCTZ treatment (alone or in combination with NEB) peaked after 1 week’s treatment, then reduced during weeks 2–8, although it always remained at a higher level compared with that seen in the other three groups. There was no significant difference between HCTZ monotherapy and the combination of NEB + HCTZ with respect to the effect on water intake, showing that the diuretic effect could be ascribed only to HCTZ.

Increased BP induces long-lasting changes in the arterial vascular wall,²² which may result from the interplay of several mechanisms.²³ In our current study, from the histological perspective, hypertrophy of the aortic wall was found in the SHR control group, which was in agreement with most of the previous studies.^24–26 After 8 weeks’ treatment with 2 mg/kg per day NEB in the present study, there were negligible antihypertensive effects and these did not prevent arterial media thickening. In our previous study, NEB administered at 8 mg/kg per day ameliorated the aortic hypertrophy observed in the SHR model.²⁷ Our current findings suggest that 2 mg/kg per day NEB, when administered alone, is unable to improve the hypertrophic changes that are typically present in the aorta of the SHR model. Our work published here demonstrated that treatment of SHR with HCTZ alone or NEB + HCTZ was associated with morphological normalization of the aortic vascular wall. These findings confirm earlier research, which showed that HCTZ resulted in beneficial vascular effects when given in combination with a β-blocker in SHR,²⁸ and suggest that HCTZ may affect hypertension-induced functional and morphological vascular alterations.

Nebivolol, a third-generation vasodilator β-blocker, has the ability to release NO from endothelial cells and to induce vasodilatation.^29,30 In SHR, the NO pathway is subjected to the mechanism of eNOS uncoupling, leading to endothelial dysfunction.¹⁹ NEB might counteract this mechanism by reducing peripheral resistance (as shown in other studies^30,31) and reducing the levels of asymmetric dimethylarginine that are increased during hypertension.¹⁵ Mason et al.¹⁹ demonstrated that the preferential formation of highly reactive oxidative products (such as peroxynitrites) and the reduced bioavailability of NO, which is evident in endothelial cells of SHR, is restored by NEB treatment. In this context, it is interesting to note that HCTZ has also been shown to possess antioxidant properties in a model of renovascular hypertension,³² therefore it can act in synergy with NEB in the eNOS uncoupling. According to our previous study,¹⁵ a lower availability of plasma NO and lower levels of eNOS activity were found in both plasma and aorta samples from SHR. In the present study, both NEB and HCTZ administered alone were able to increase the mean plasma NO concentration significantly, compared with control SHR, NEB + HCTZ did not provide greater bioavailability of plasma NO, and neither NEB nor HCTZ administered alone could increase the plasma NOS activity. These findings for plasma NOS activity were probably due to the lower dose of NEB used, compared with the greater effect observed with a dose that was four-times higher in a similar study undertaken with the β-blocker.¹⁵ The distinctive effect of the third-generation β-blocker NEB on the NO pathway was clearly different to that of the classic β-blocker, atenolol, despite both drugs having an almost similar reduction in BP.¹⁵ It is worth noting that in our current study, only the combination of NEB + HCTZ was able to restore the mean plasma NOS activity to levels similar to those of the normotensive WKY animals, which was probably due to the effective antihypertensive effects exerted by both drugs when administered in combination. In our current study, NEB restored the functional availability of NO, resulting in a better vasodilatating capability and influencing peripheral resistance.

This study also examined the effects of the combination of NEB + HCTZ on plasma Ang II concentration and PRA. HCTZ induces activation of the renin–angiotensin–aldosterone system mainly via sodium and water depletion, leading to increases in plasma renin concentration and activity, and therefore to enhanced Ang II production, which limits its antihypertensive effects.³³ Both the SHR control group and the SHR treated with HCTZ alone showed a marked increase in both markers, compared with those seen in normotensive WKY rats. Increases in Ang II concentration and PRA were normalized after the concomitant administration of NEB with HCTZ, which reduced both markers down to levels similar to those observed in normotensive WKY rats. It is known that β-1 receptor blockade decreases renin expression and secretion.³⁴ In addition, upregulation of the renin–angiotensin system (induced by treatment with angiotensin-converting enzyme inhibitors, diuretics or both) can be prevented by concomitant administration of β-blockers.³⁵ NEB has been shown to normalize renin levels that were increased by an 8% salt diet in SHR;³⁶ and to reduce Ang II concentrations in both plasma and the left ventriculum, in SHR.²⁷

In our current study, the combination of NEB + HCTZ effectively restored the endogenous vasodilative capability of the femoral artery endothelium in response to Ach, which had been markedly impaired in the SHR control group; the relaxation was reduced by 50% compared with the normotensive WKY rats. Treatment with NEB alone (or NEB + HCTZ) resulted in significant vasodilatory recovery in response to ACh, with values returning to the control normotensive WKY values in the NEB + HCTZ group. This was consistent with the findings of Guerrero et al,³¹ who reported that 8 weeks’ treatment with NEB, at a dosage of 8 mg/kg daily, gradually enhanced relaxation of the aorta in response to ACh. It has been speculated that impaired endothelium-dependent relaxation induced by ACh in SHR may result from decreased NO synthesis.³⁷ NEB, which activates the L-arginine–NO pathway,³⁰ can improve endothelial function in hypertensive patients, as shown in forearm vessels³⁸ or in the venous circulation.³⁹ Our current data indicate that, NEB, used at a low dose (2 mg/kg per day), can improve vascular endothelium-dependent relaxation. The protective effect observed in this current study was driven by NEB, because when HCTZ was administered alone it was completely inactive in restoring the ACh-induced relaxation of femoral artery rings.

In conclusion, the present study demonstrates that the combination of NEB + HCTZ provides a number of beneficial and additive effects due to the synergistic characteristics of both drugs, in an experimental rat model of hypertension.

Footnotes

Acknowledgements

The authors wish to thank Mrs Lisa Mylander, an employee of the Menarini Group (Florence, Italy), for the English revision.

Funding

The study was supported by a grant from the Menarini Group (Florence, Italy).

Declaration of conflicting interest

Stefano Evangelista is an employee of Menarini Ricerche SpA, which is part of the Menarini Group (Florence, Italy), owner of the commercial rights to nebivolol.

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