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Blood pressure variability, impaired autonomic function and vascular senescence in aged spontaneously hypertensive rats are ameliorated by angiotensin blockade
Atherosclerosis 236 (2014) 101e107
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Blood pressure variability, impaired autonomic function and vascular senescence in aged spontaneously hypertensive rats are ameliorated by angiotensin blockade Daisuke Sueta a, Nobutaka Koibuchi a, Yu Hasegawa a, Kensuke Toyama a, Ken Uekawa a, Tetsuji Katayama a, MingJie Ma a, Takashi Nakagawa a, Hidefumi Waki b, Masanobu Maeda b, Hisao Ogawa c, Shokei Kim-Mitsuyama a, * a
Department of Pharmacology and Molecular Therapeutics, Kumamoto University Graduate School of Medical Sciences, 1-1-1 Honjo, Kumamoto 860-8556, Japan b Department of Physiology, Wakayama Medical University School of Medicine, Wakayama, Japan c Department of Cardiovascular Medicine, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 January 2014 Received in revised form 29 May 2014 Accepted 17 June 2014 Available online 26 June 2014
Objective: Elderly hypertensive patients are characterized by blood pressure (BP) variability, impaired autonomic function, and vascular endothelial dysfunction and stiffness. However, the mechanisms causing these conditions are unclear. The present study examined the effect of angiotensin receptor blockers (ARBs) on aged spontaneously hypertensive rats (SHR). Methods: We surgically implanted telemetry devices in SHR and WKY at the age of 15 weeks (Young) and 80 weeks (Aged). Aged SHR were orally administered either olmesartan or valsartan once daily at 19:00 h (at the beginning of the dark period (active phase)) for 4 weeks to examine the effects on BP variability, impaired autonomic function, and vascular senescence. Results: Aging and hypertension in SHR additively caused the following: increased low frequency (LF) power of systolic BP, a decreased spontaneous baroreceptor reflex gain (sBRG), increased BP variability, increased urinary norepinephrine excretion, increased vascular senescence-related beta-galactosidase positive cells and oxidative stress. Treatment with olmesartan or valsartan significantly ameliorated these changes in aged SHR. However, olmesartan ameliorated these changes in aged SHR better than valsartan. The reductions in BP caused by olmesartan in aged SHR were sustained longer than reductions by valsartan. This result indicates longer-lasting inhibition of the AT1 receptor by olmesartan than by valsartan. Conclusion: ARBs ameliorated autonomic dysfunction, BP variability, and vascular senescence in aged SHR. Olmesartan ameliorated the aging-related disorders better than valsartan and was associated with longer-lasting AT1 receptor inhibition by olmesartan. Thus, the magnitude of improvement of these aging-related abnormalities differs for ARBs. © 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Circadian blood pressure Blood pressure variability Vascular senescence Angiotensin Oxidative stress
1. Introduction Most elderly hypertensive patients display blood pressure (BP) circadian rhythm abnormality, BP variability, impaired autonomic function, and vascular senescence such as vascular endothelial dysfunction, vascular remodeling, vascular stiffness, or enhanced oxidative stress [1e4]. Importantly, abnormal BP circadian rhythm
* Corresponding author. Tel./fax: þ81 96 373 5082. E-mail address: [email protected] (S. Kim-Mitsuyama). http://dx.doi.org/10.1016/j.atherosclerosis.2014.06.016 0021-9150/© 2014 Elsevier Ireland Ltd. All rights reserved.
[5,6], BP variability [7e12], impaired autonomic function [13e16], and vascular senescence-related abnormality [17e19] are associated with increased risk of cerebrovascular or cardiovascular events. However, the mechanism underlying these abnormalities in elderly patients has not been elucidated. Several clinical findings [9,20e22] demonstrated that the effects on these abnormalities in elderly hypertensive patients differ for different classes of antihypertensive drugs. There is substantial evidence [18,23e27] that angiotensin II is involved in not only the elevation of BP but also the activation of sympathetic nerve activity, vascular senescence, and vascular
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endothelial dysfunction. We previously examined the effects of an angiotensin receptor blocker (ARB) on impaired autonomic function in a rat model of metabolic syndrome. We found that ARBs significantly reduce sympathetic activity and ameliorate the impairment of baroreceptor reflex function [27]. Furthermore, angiotensin II is involved in cardiac remodeling aggravated by combination of BP variability with hypertension [28]. However, there is little information regarding the effects of ARBs on aged hypertensive rats with respect to autonomic function, BP variability, and vascular senescence. Eight ARBs are clinically available and ARBs differ in chemical structure, physicochemical, pharmacological and pharmacokinetic properties [29,30]. ARBs vary in their binding affinity to AT1 receptor [29,30]. Olmesartan is shown to have higher affinity to AT1 receptor than valsartan [29,30]. However, it remains to be determined whether different affinity to AT1 receptor can affect the effects of ARB on aged hypertensive rats. To address this issue, we examined the comparative effects of olmesartan and valsartan on aged spontaneously hypertensive rats (SHR). We analyzed their effects on autonomic function, BP variability, and vascular senescence caused by aging. Our data indicate that
these ARBs improve aging-related abnormalities in SHR and suggest that the magnitude of these beneficial effects differs for each ARB. 2. Materials and methods Supplemental Materials Supplemental appendixes.
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3. Results 3.1. Effects of aging on circadian rhythms of BP, HR, locomotor activity, autonomic function, and vascular function in WKY As shown in Supplemental Fig. 2(A), there was a significant difference in 24-h-averaged systolic BP between 85-week-old and 15-week-old WKY (126 ± 1 vs 118 ± 1 mmHg). The HR in 85-weekold WKY was significantly lower during both dark and light periods than in 15-week-old WKY (Supplemental Fig. 2(B)). The locomotor activity during the dark period in 85-week-old WKY was less than that observed in 15-week-old WKY (Supplemental Fig. 2(C)).
Fig. 1. Circadian rhythms of systolic BP (A), HR (B), and locomotor activity (C) in SHR revealed by 24-h recording with telemetry. In (A), the left panel shows hourly recordings of systolic BP over 24 h, and the right panel indicates 24-h-averaged systolic BP. In (B), the left panel shows hourly recordings of HR over 24 h, and the right panel indicates 24-haveraged HR. In (C), the left panel shows hourly recordings of locomotor activity over 24 h, and the right panel indicates 24-h-averaged locomotor activity. Values are expressed as the means ± SEM (n ¼ 5 in each group). Statistical analysis was performed by 1-factor analysis of variance (ANOVA) followed by post hoc Fisher's protected least significant difference test. Abbreviations used: Young, 15-week-old SHR; aged, 85-week-old SHR; Veh vehicle-treated 85-week-old SHR; Ol, olmesartan-treated 85-week-old SHR; Va, valsartan-treated 85-week-old SHR.
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As shown in Supplemental Fig. 3(A), LF of systolic BP in 85week-old WKY was significantly greater during both dark and light periods than in 15-week-old WKY. sBRG in 85-week-old WKY was significantly smaller during both dark and light periods than in 15-week-old WKY (Supplemental Fig. 3(B)). Supplemental Fig. 3(C) shows the CV of systolic BP in 85-weekold WKY were greater than in 15-week-old WKY. The 24-h urinary norepinephrine excretions were greater in 85week-old WKY than in 15-week-old WKY (Supplemental Fig. 3(D)). As shown in Supplemental Table 1, left ventricular weight corrected for tibia length in 85-week-old WKY was greater than 15-week-old WKY (P < 0.01). As shown in Supplemental Fig. 4(A), vascular endotheliumdependent relaxation by acetylcholine was significantly impaired in 85-week-old WKY compared with 15-week-old WKY (P < 0.01). Supplemental Fig. 4(B) demonstrates that pretreatment with LNAME almost completely abolished acetylcholine-induced vascular relaxation in both groups of WKY. There was no significant difference in vascular endothelium-independent relaxation with sodium nitroprusside between groups (Supplemental Fig. 4(C)). The 85-week old WKY displayed greater aortic SA b-galactosidase positive cell numbers (P < 0.01), greater vascular superoxide levels (P < 0.01) and greater vascular p22phox levels (P < 0.01) than 15-week-old WKY (Supplemental Fig. 4(D)e(F), respectively).
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3.2. Effect of olmesartan and valsartan on BP, autonomic function, and BP variability, in aged SHR As shown in Fig. 1(A), systolic BP over 24 h was higher in 85-week-old SHR than in 15-week-old SHR (172 ± 1 vs 154 ± 1 mmHg; P < 0.01). The once-daily oral dosing of olmesartan and valsartan significantly decreased BP in 85-week-old SHR. There was no significant difference in systolic BP between olmesartan and valsartan groups until 5 h after oral administration of each drug (during 5-h dark period). However, systolic BP after the 5-h dark period was lower in the olmesartan group than in the valsartan group (P < 0.01). As shown in Fig. 1(B), the HR in 85-week-old SHR was higher than the HR in 15-week-old SHR over 24 h (P < 0.01). The HR in the olmesartan group was significantly lower than in the valsartan group over 24 h (P < 0.01). Fig. 1(C) indicates there was no significant difference in locomotor activity over 24 h between the olmesartan and valsartan groups. Furthermore, no significant difference was noted in food intake, water intake, or urine volume between animals treated with the two drugs (data not shown). As shown in Fig. 2(A), LF of systolic BP over 24 h in 85-week-old SHR was higher than in 15-week-old SHR (P < 0.01). Once-daily oral dosing of olmesartan and valsartan significantly decreased LF of systolic BP
Fig. 2. Circadian rhythms of low-frequency (LF) power of systolic BP (A) and spontaneous baroreceptor reflex gain (sBRG) (B), systolic BP variability (C), and urinary norepinephrine excretion (D) in 15-week-old and 85-week-old SHR. In (A), the left panel indicates hourly recordings of LF of systolic BP over 24 h, and the right panel indicates 24-h-averaged LF of systolic BP. In (B), the left panel indicates hourly recordings of sBRG over 24 h, and the right panel indicates 24-h-averaged sBRG. In (C), left and right panels indicate CV-systolic BP at 0:00 h and 12:00 h, respectively. (D) Indicates 24-h-averaged urinary norepinephrine excretions. Values are the means ± SEM (n ¼ 5 in each group). Abbreviations used are the same as in Fig. 1. Statistical analysis was performed by one-factor analysis of variance (ANOVA) followed by post hoc Fisher's protected least significant difference test ((A)e(D)).
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in 85-week-old SHR. There was no significant difference in LF of systolic BP over 24 h between the olmesartan and valsartan groups. As shown in Fig. 2(B), sBRG over 24 h was lower in the 85-weekold SHR than in 15-week-old SHR (P < 0.01). The treatment with olmesartan and valsartan significantly increased sBRG in 85-weekold SHR. The sBRG over 24 h was higher for the olmesartan group than the valsartan group (P < 0.01). 3.3. Effect of olmesartan and valsartan on systolic BP variability in aging SHR As shown in Fig. 2(C), CV of systolic BP measured at 0:00 h or 12:00 h were greater in 85-week-old SHR than those in 15-weekold SHR. Olmesartan decreased CV of systolic BP in aged SHR more than valsartan. 3.4. Effect of olmesartan and valsartan on urinary norepinephrine in aged SHR Eighty five-week-old SHR exhibited greater 24-h-urinary norepinephrine excretion than 15-week-old SHR (Fig. 2(D)). Both olmesartan and valsartan significantly reduced 24-h-urinary norepinephrine excretions in 85-week-old SHR. However, olmesartan treatment led to a greater reduction of norepinephrine excretions than valsartan. 3.5. Effect of olmesartan and valsartan on vascular endothelial function, cardiac weight, and aortic thickness in aged SHR As shown in Fig. 3(A), vascular endothelium-dependent relaxation by acetylcholine was significantly impaired in aged SHR compared to
young SHR (P < 0.01). Olmesartan ameliorated the impairment of acetylcholine-induced vascular relaxation in 85-week-old SHR more than valsartan. As shown in Fig. 3(B), pretreatment with L-NAME almost completely abolished acetylcholine-induced vascular relaxation in all groups of rats. The data in Fig. 3(C) show there was no significant difference in sodium nitroprusside-induced endotheliumindependent vascular relaxation. As shown in Supplemental Table 2, the left ventricular weight of 85-week-old SHR was significantly greater than that of 15-weekold SHR (P < 0.01). The LV weight in olmesartan-treated SHR was significantly lower than in valsartan-treated SHR (P < 0.05). Olmesartan tended to reduce aortic thickness compared to vehicle or valsartan, although the difference did not reach statistical significance. 3.6. Effects of olmesartan and valsartan on vascular senescence, superoxide, and AT1 receptor in aged SHR As shown in Fig. 4, aortic SA b-galactosidase positive cell numbers in 85-week-old SHR were greater than those in 15-weekold SHR. Although once-daily oral dosing of olmesartan and valsartan significantly decreased aortic SA b-galactosidase positive cell numbers in 85-week-old SHR, aortic SA b-galactosidase positive cell numbers in the olmesartan group were significantly less than in the valsartan group (P < 0.05). As shown in Fig. 5, 85-week-old SHR showed greater aortic superoxide levels (P < 0.01) and greater NADPH oxidase subunit p22phox levels (P < 0.01) than 15-week-old SHR. Olmesartan and valsartan significantly attenuated aortic superoxide (P < 0.01) and p22phox (P < 0.01) levels of aged SHR. However, olmesartan caused a more significant reduction of aortic superoxide levels and p22phox
Fig. 3. Vascular endothelium-dependent relaxation with acetylcholine without L-NAME pretreatment (A) and with L-NAME pretreatment (B), and endothelium-independent relaxation with sodium nitroprusside (C) in SHR. Acetylcholine-induced vascular relaxation was examined without pretreatment with L-NAME (A), and with pretreatment with L-NAME (100 mmol/l) (B). Abbreviations used are the same as in Fig. 1. Values are the means ± SEM (n ¼ 7). Statistical analysis was performed by one-factor analysis of variance (ANOVA) with repeated measurements followed by post hoc Fisher's protected least significant difference test (A). Acetylcholine-induced vascular relaxation without pretreatment with L-NAME was significantly influenced by acetylcholine (P < 0.01) and age (P < 0.01). Acetylcholine-induced vascular relaxation with pretreatment with L-NAME was significantly influenced by acetylcholine (P < 0.01) but not influenced by age (P ¼ 0.0756). Sodium nitroprusside-induced vascular relaxation was significantly influenced by acetylcholine (P < 0.01) but not influenced by age (P ¼ 0.2146).
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Fig. 4. Effect of olmesartan and valsartan on senescence-associated beta-galactosidase (SA b-gal) positive cell numbers in the aortas in aging SHR. Upper panels indicate representative photomicrographs of aortic sections stained with senescence-associated b-galactosidase (SA b-gal). Values are the means ± SEM (n ¼ 7). Statistical analysis was performed by 1-factor analysis of variance (ANOVA) followed by post hoc Fisher's protected least significant difference test. Abbreviations used: Young, 15-week-old SHR; Aged, 85-week-old SHR; Veh, vehicle-treated 85-week-old SHR; Ol, olmesartan-treated 85-week-old SHR; Va, valsartan-treated 85-week-old SHR.
levels in 85-week-old SHR than valsartan. Aortic extracellular-SOD, copperezinc-SOD, and manganese-SOD levels in 85-week-old SHR were not significantly changed by olmesartan or valsartan (data not shown). Vascular AT1 receptor was significantly greater in 85-week-old SHR than 15-week-old SHR (P < 0.01) (Fig. 5(D)). 4. Discussion The major findings of this work are the following: (1) aging led to increased BP variability, enhancement of sympathetic activity, impairment of autonomic function, and the increase in vascular senescence and oxidative stress; (2) hypertension exacerbated these changes caused by aging; (3) ARBs ameliorated these changes in aged SHR; (4) the magnitude of improvements of these agingrelated changes may differ across ARBs. It is well recognized that elderly hypertensive patients are generally characterized by the abnormality of BP circadian rhythm, BP variability, the impairment of autonomic function, and vascular endothelial dysfunction and stiffness. However, it is unclear whether aging or high BP contributes more to such changes in elderly hypertensive patients. To address this issue, we compared BP circadian rhythm, BP variability, autonomic function, and vascular function in young and old WKY and SHR. The comparison between young and old normotensive WKY showed that the impairment of
autonomic function, BP variability, vascular endothelial dysfunction, vascular senescence and oxidative stress were significantly enhanced in old WKY compared with young WKY. These results demonstrate that aging itself causes the impairment of autonomic function, BP variability, vascular endothelial dysfunction, and vascular senescence. The comparison between young and old SHR indicated that the abnormalities caused by aging were more pronounced in SHR than in WKY. Therefore, aging and hypertension seem to additively enhance the impairment of autonomic function, BP variability, and vascular senescence. Aged SHR are regarded as a useful animal model of elderly hypertensive patients. There is no convincing evidence supporting an optimal hypertensive therapy for elderly patients. Thus, it is unclear which antihypertensive drug is optimal for the treatment of elderly patients. Accumulating experimental evidence [18,24,25,27] indicates that angiotensin II is involved in the activation of the sympathetic nervous system [31] and the pathogenesis of vascular senescence. Therefore, we investigated the effects of two ARBs, olmesartan and valsartan, on aged SHR to determine the potential benefit of angiotensin receptor inhibition in improvement of aging-associated disorder. We found that treatment with olmesartan or valsartan significantly reduced HR, and ameliorated sympathetic activity as shown by the significant reduction of LF of systolic BP and urinary norepinephrine excretion, the most popular marker of sympathetic activity. Additionally, the treatments also improved baroreceptor
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Fig. 5. Effects of olmesartan and valsartan on vascular superoxide levels (A), NADPH oxidase subunit p22phox (C) and AT1 receptor (D) of aged SHR. Upper panels in (A) indicate representative photomicrographs of aortic sections stained with dihydroethidium. (B) indicate representative photographs of western blot. Statistical analysis was performed by 1factor analysis of variance (ANOVA) followed by post hoc Fisher's protected least significant difference test. Abbreviations used are the same as in Fig. 4. DHE, dihydroethidium. Values are the means ± SEM (n ¼ 7).
reflex function, as shown by the significant increase in sBRG. These findings suggest that ARB may improve aging-related autonomic dysfunction. Furthermore, both ARBs ameliorated BP variability in aged SHR, as shown by the reduction of CV of systolic BP. Taken together with the findings that the enhancement of sympathetic activity contributes to the pathogenesis of BP variability, our results suggest that the amelioration of BP variability [32] by ARBs may be attributable to the suppression of sympathetic activity. Vascular senescence plays a causative role in vascular diseases such as vascular endothelial dysfunction, vascular remodeling, and atherosclerosis [18,19]. Of note, vascular AT1 receptor levels in aged SHR was significantly higher than in young SHR, supporting the notion that vascular renin-angiotensin system may be enhanced in aged SHR. Previous in vitro and in vivo studies indicate that angiotensin II, through activation of AT1 receptor, increases vascular oxidative stress by increasing p22phox, a key subunit of NADPH oxidase [33e35]. However, there is no report comparing the effects of different ARBs on vascular oxidative stress in aged SHR. In the present study, both olmesartan and valsartan significantly ameliorated the impairment of vascular endothelial function in aged SHR and reduced vascular superoxide levels and NADPH oxidase subunit p22phox in aged SHR. Thus, the amelioration of vascular oxidative stress by these ARBs appears to be at least in part mediated by the reduction of p22phox. Moreover, olmesartan significantly decreased the number of vascular SA b-gal positive cells in aged SHR. These results suggest that ARBs may ameliorate the progression of vascular senescence caused by aging. Furthermore, taken together with the findings that oxidative stress plays a central role in the pathogenesis of vascular senescence [18,36,37], the amelioration of vascular senescence by ARBs appears to be mediated by the reduction of oxidative stress. In the present study, olmesartan ameliorated aging-related impairment of autonomic function, enhancement of sympathetic activity, BP variability, vascular endothelial dysfunction, vascular senescence, and oxidative stress to a greater extent than valsartan. The present study did not allow us to elucidate the reason for the
differences between olmesartan and valsartan regarding the effect on aging-related disorders. Continuous monitoring of BP in aged SHR with telemetry indicated the BP lowering effect caused by olmesartan was significantly longer than the effect caused by valsartan. Thus, the extended duration of BP lowering effects was associated with a greater reduction of 24-h BP by olmesartan. These findings demonstrate inhibition of the AT1 receptor in vivo in aged SHR by olmesartan lasted longer than valsartan. Furthermore, angiotensin II activates the AT1 receptor and is responsible for the impairment of autonomic function, vascular senescence and injury. Collectively, the amelioration of aging-associated autonomic dysfunction, BP variability, vascular senescence and injury by olmesartan was better than valsartan because of the longer lasting AT1 receptor suppression by olmesartan. 4.1. Study limitation There are several study limitations in this study. First, we did not examine the effect of angiotensin-converting enzyme (ACE) inhibitor in this study, since the main purpose of our work was to examine the effect of specific angiotensin blockade in aged SHR. Accumulating evidence establishes that cardiovascular protective effects and blood pressure lowering effects of ACE inhibitor is significantly mediated by the increase in bradykinin, independently of angiotensin II blockade [38]. Therefore, in the present study, we did not use ACE inhibitor. However, the investigation on the effect of ACE inhibitor in aged SHR is an important future issue since ACE inhibitor is one of the major cardiovascular protective drugs. Second, we did not measure blood concentrations of olmesartan and valsartan in this study. Olmesartan and valsartan have different pharmacokinetics [29]. Therefore, it cannot be excluded that longer-lasting AT1 receptor blockade by olmesartan than valsartan in this study might be attributed to the potential difference in blood concentrations between these drugs as well as the differential AT1 receptor affinity between the drugs. Third, our present work did not allow us to provide the detailed mechanisms underlying more
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attenuation of vascular oxidative stress and urinary norepinephrine excretion by olmesartan than valsartan, since our work was in vivo experiments on aged SHR. Further study including in vitro experiment is warranted to elucidate the detailed mechanisms responsible for the effects of ARBs on these parameters. In conclusion, aging and hypertension additively cause the enhancement of sympathetic activity, the impairment of autonomic function, BP variability, and vascular senescence and dysfunction. BP lowering by ARBs ameliorates these aging-related disorders. The magnitude of amelioration of these aging-related disorders may depend on the duration of AT1 receptor inhibition. However, a clinical study is warranted to investigate this hypothesis. Disclosures H.O. received lecture fees and research grant from Astellas, AstraZeneca, Bayer, Boehringer Ingelheim, Chugai, Daiichi Sankyo, Dainippon Sumitomo Pharma, Eisai, Kowa, Kyowa Hakko Kirin, Mitsubishi Tanabe, MSD, Novartis, Otsuka, Pfizer, Sanofi, Sionogi, Takeda.S. S. K-M received lecture fees and research grant from Astellas, AstraZeneca, Boehringer Ingelheim, Daiichi Sankyo, Novartis, Sionogi, Takeda, Kyowa Hakko Kirin. Conflict of interest The authors report no relationships that could be construed as a conflict of interest. Acknowledgments This work was in part supported by Daiichi Sankyo (Tokyo, Japan). We would like to thank Erina Haraguchi, Yusei Kawahara, Koji Ichiki, Miho Kataoka, Michie Uchikawa, Keiko Morozumi, Yuriko Shimamura, Shuhei Hasebe, Noriko Yoshimura, Kazuko Noda and Tomoko Moriyama for their kind support during the study. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2014.06.016. References [1] Drayer JI, Weber MA, DeYoung JL, Wyle FA. Circadian blood pressure patterns in ambulatory hypertensive patients: effects of age. Am J Med 1982;73(4): 493e9. [2] Gribbin B, Pickering TG, Sleight P, Peto R. Effect of age and high blood pressure on baroreflex sensitivity in man. Circ Res 1971;29(4):424e31. [3] O'Rourke MF, Hashimoto J. Mechanical factors in arterial aging: a clinical perspective. J Am Coll Cardiol 2007;50(1):1e13. [4] Shimada K, Kitazumi T, Sadakane N, Ogura H, Ozawa T. Age-related changes of baroreflex function, plasma norepinephrine, and blood pressure. Hypertension 1985;7(1):113e7. [5] Verdecchia P, Angeli F, Mazzotta G, Garofoli M, Ramundo E, Gentile G, et al. Day-night dip and early-morning surge in blood pressure in hypertension: prognostic implications. Hypertension 2012;60(1):34e42. [6] Verdecchia P, Porcellati C, Schillaci G, Borgioni C, Ciucci A, Battistelli M, et al. Ambulatory blood pressure. An independent predictor of prognosis in essential hypertension. Hypertension 1994;24(6):793e801. [7] Hastie CE, Jeemon P, Coleman H, McCallum L, Patel R, Dawson J, et al. Long-term and ultra long-term blood pressure variability during follow-up and mortality in 14,522 patients with hypertension. Hypertension 2013;62(4):698e705. [8] Hata J, Arima H, Rothwell PM, Woodward M, Zoungas S, Anderson C, et al. Effects of visit-to-visit variability in systolic blood pressure on macrovascular and microvascular complications in patients with type 2 diabetes mellitus: the ADVANCE trial. Circulation 2013;128(12):1325e34. [9] Rothwell PM. Limitations of the usual blood-pressure hypothesis and importance of variability, instability, and episodic hypertension. Lancet 2010;375(9718):938e48.
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Blood pressure variability, impaired autonomic function and vascular senescence in aged spontaneously hypertensive rats are ameliorated by angiotensin blockade Daisuke Sueta a, Nobutaka Koibuchi a, Yu Hasegawa a, Kensuke Toyama a, Ken Uekawa a, Tetsuji Katayama a, MingJie Ma a, Takashi Nakagawa a, Hidefumi Waki b, Masanobu Maeda b, Hisao Ogawa c, Shokei Kim-Mitsuyama a, * a
Department of Pharmacology and Molecular Therapeutics, Kumamoto University Graduate School of Medical Sciences, 1-1-1 Honjo, Kumamoto 860-8556, Japan b Department of Physiology, Wakayama Medical University School of Medicine, Wakayama, Japan c Department of Cardiovascular Medicine, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 January 2014 Received in revised form 29 May 2014 Accepted 17 June 2014 Available online 26 June 2014
Objective: Elderly hypertensive patients are characterized by blood pressure (BP) variability, impaired autonomic function, and vascular endothelial dysfunction and stiffness. However, the mechanisms causing these conditions are unclear. The present study examined the effect of angiotensin receptor blockers (ARBs) on aged spontaneously hypertensive rats (SHR). Methods: We surgically implanted telemetry devices in SHR and WKY at the age of 15 weeks (Young) and 80 weeks (Aged). Aged SHR were orally administered either olmesartan or valsartan once daily at 19:00 h (at the beginning of the dark period (active phase)) for 4 weeks to examine the effects on BP variability, impaired autonomic function, and vascular senescence. Results: Aging and hypertension in SHR additively caused the following: increased low frequency (LF) power of systolic BP, a decreased spontaneous baroreceptor reflex gain (sBRG), increased BP variability, increased urinary norepinephrine excretion, increased vascular senescence-related beta-galactosidase positive cells and oxidative stress. Treatment with olmesartan or valsartan significantly ameliorated these changes in aged SHR. However, olmesartan ameliorated these changes in aged SHR better than valsartan. The reductions in BP caused by olmesartan in aged SHR were sustained longer than reductions by valsartan. This result indicates longer-lasting inhibition of the AT1 receptor by olmesartan than by valsartan. Conclusion: ARBs ameliorated autonomic dysfunction, BP variability, and vascular senescence in aged SHR. Olmesartan ameliorated the aging-related disorders better than valsartan and was associated with longer-lasting AT1 receptor inhibition by olmesartan. Thus, the magnitude of improvement of these aging-related abnormalities differs for ARBs. © 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Circadian blood pressure Blood pressure variability Vascular senescence Angiotensin Oxidative stress
1. Introduction Most elderly hypertensive patients display blood pressure (BP) circadian rhythm abnormality, BP variability, impaired autonomic function, and vascular senescence such as vascular endothelial dysfunction, vascular remodeling, vascular stiffness, or enhanced oxidative stress [1e4]. Importantly, abnormal BP circadian rhythm
* Corresponding author. Tel./fax: þ81 96 373 5082. E-mail address: [email protected] (S. Kim-Mitsuyama). http://dx.doi.org/10.1016/j.atherosclerosis.2014.06.016 0021-9150/© 2014 Elsevier Ireland Ltd. All rights reserved.
[5,6], BP variability [7e12], impaired autonomic function [13e16], and vascular senescence-related abnormality [17e19] are associated with increased risk of cerebrovascular or cardiovascular events. However, the mechanism underlying these abnormalities in elderly patients has not been elucidated. Several clinical findings [9,20e22] demonstrated that the effects on these abnormalities in elderly hypertensive patients differ for different classes of antihypertensive drugs. There is substantial evidence [18,23e27] that angiotensin II is involved in not only the elevation of BP but also the activation of sympathetic nerve activity, vascular senescence, and vascular
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endothelial dysfunction. We previously examined the effects of an angiotensin receptor blocker (ARB) on impaired autonomic function in a rat model of metabolic syndrome. We found that ARBs significantly reduce sympathetic activity and ameliorate the impairment of baroreceptor reflex function [27]. Furthermore, angiotensin II is involved in cardiac remodeling aggravated by combination of BP variability with hypertension [28]. However, there is little information regarding the effects of ARBs on aged hypertensive rats with respect to autonomic function, BP variability, and vascular senescence. Eight ARBs are clinically available and ARBs differ in chemical structure, physicochemical, pharmacological and pharmacokinetic properties [29,30]. ARBs vary in their binding affinity to AT1 receptor [29,30]. Olmesartan is shown to have higher affinity to AT1 receptor than valsartan [29,30]. However, it remains to be determined whether different affinity to AT1 receptor can affect the effects of ARB on aged hypertensive rats. To address this issue, we examined the comparative effects of olmesartan and valsartan on aged spontaneously hypertensive rats (SHR). We analyzed their effects on autonomic function, BP variability, and vascular senescence caused by aging. Our data indicate that
these ARBs improve aging-related abnormalities in SHR and suggest that the magnitude of these beneficial effects differs for each ARB. 2. Materials and methods Supplemental Materials Supplemental appendixes.
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3. Results 3.1. Effects of aging on circadian rhythms of BP, HR, locomotor activity, autonomic function, and vascular function in WKY As shown in Supplemental Fig. 2(A), there was a significant difference in 24-h-averaged systolic BP between 85-week-old and 15-week-old WKY (126 ± 1 vs 118 ± 1 mmHg). The HR in 85-weekold WKY was significantly lower during both dark and light periods than in 15-week-old WKY (Supplemental Fig. 2(B)). The locomotor activity during the dark period in 85-week-old WKY was less than that observed in 15-week-old WKY (Supplemental Fig. 2(C)).
Fig. 1. Circadian rhythms of systolic BP (A), HR (B), and locomotor activity (C) in SHR revealed by 24-h recording with telemetry. In (A), the left panel shows hourly recordings of systolic BP over 24 h, and the right panel indicates 24-h-averaged systolic BP. In (B), the left panel shows hourly recordings of HR over 24 h, and the right panel indicates 24-haveraged HR. In (C), the left panel shows hourly recordings of locomotor activity over 24 h, and the right panel indicates 24-h-averaged locomotor activity. Values are expressed as the means ± SEM (n ¼ 5 in each group). Statistical analysis was performed by 1-factor analysis of variance (ANOVA) followed by post hoc Fisher's protected least significant difference test. Abbreviations used: Young, 15-week-old SHR; aged, 85-week-old SHR; Veh vehicle-treated 85-week-old SHR; Ol, olmesartan-treated 85-week-old SHR; Va, valsartan-treated 85-week-old SHR.
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As shown in Supplemental Fig. 3(A), LF of systolic BP in 85week-old WKY was significantly greater during both dark and light periods than in 15-week-old WKY. sBRG in 85-week-old WKY was significantly smaller during both dark and light periods than in 15-week-old WKY (Supplemental Fig. 3(B)). Supplemental Fig. 3(C) shows the CV of systolic BP in 85-weekold WKY were greater than in 15-week-old WKY. The 24-h urinary norepinephrine excretions were greater in 85week-old WKY than in 15-week-old WKY (Supplemental Fig. 3(D)). As shown in Supplemental Table 1, left ventricular weight corrected for tibia length in 85-week-old WKY was greater than 15-week-old WKY (P < 0.01). As shown in Supplemental Fig. 4(A), vascular endotheliumdependent relaxation by acetylcholine was significantly impaired in 85-week-old WKY compared with 15-week-old WKY (P < 0.01). Supplemental Fig. 4(B) demonstrates that pretreatment with LNAME almost completely abolished acetylcholine-induced vascular relaxation in both groups of WKY. There was no significant difference in vascular endothelium-independent relaxation with sodium nitroprusside between groups (Supplemental Fig. 4(C)). The 85-week old WKY displayed greater aortic SA b-galactosidase positive cell numbers (P < 0.01), greater vascular superoxide levels (P < 0.01) and greater vascular p22phox levels (P < 0.01) than 15-week-old WKY (Supplemental Fig. 4(D)e(F), respectively).
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3.2. Effect of olmesartan and valsartan on BP, autonomic function, and BP variability, in aged SHR As shown in Fig. 1(A), systolic BP over 24 h was higher in 85-week-old SHR than in 15-week-old SHR (172 ± 1 vs 154 ± 1 mmHg; P < 0.01). The once-daily oral dosing of olmesartan and valsartan significantly decreased BP in 85-week-old SHR. There was no significant difference in systolic BP between olmesartan and valsartan groups until 5 h after oral administration of each drug (during 5-h dark period). However, systolic BP after the 5-h dark period was lower in the olmesartan group than in the valsartan group (P < 0.01). As shown in Fig. 1(B), the HR in 85-week-old SHR was higher than the HR in 15-week-old SHR over 24 h (P < 0.01). The HR in the olmesartan group was significantly lower than in the valsartan group over 24 h (P < 0.01). Fig. 1(C) indicates there was no significant difference in locomotor activity over 24 h between the olmesartan and valsartan groups. Furthermore, no significant difference was noted in food intake, water intake, or urine volume between animals treated with the two drugs (data not shown). As shown in Fig. 2(A), LF of systolic BP over 24 h in 85-week-old SHR was higher than in 15-week-old SHR (P < 0.01). Once-daily oral dosing of olmesartan and valsartan significantly decreased LF of systolic BP
Fig. 2. Circadian rhythms of low-frequency (LF) power of systolic BP (A) and spontaneous baroreceptor reflex gain (sBRG) (B), systolic BP variability (C), and urinary norepinephrine excretion (D) in 15-week-old and 85-week-old SHR. In (A), the left panel indicates hourly recordings of LF of systolic BP over 24 h, and the right panel indicates 24-h-averaged LF of systolic BP. In (B), the left panel indicates hourly recordings of sBRG over 24 h, and the right panel indicates 24-h-averaged sBRG. In (C), left and right panels indicate CV-systolic BP at 0:00 h and 12:00 h, respectively. (D) Indicates 24-h-averaged urinary norepinephrine excretions. Values are the means ± SEM (n ¼ 5 in each group). Abbreviations used are the same as in Fig. 1. Statistical analysis was performed by one-factor analysis of variance (ANOVA) followed by post hoc Fisher's protected least significant difference test ((A)e(D)).
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in 85-week-old SHR. There was no significant difference in LF of systolic BP over 24 h between the olmesartan and valsartan groups. As shown in Fig. 2(B), sBRG over 24 h was lower in the 85-weekold SHR than in 15-week-old SHR (P < 0.01). The treatment with olmesartan and valsartan significantly increased sBRG in 85-weekold SHR. The sBRG over 24 h was higher for the olmesartan group than the valsartan group (P < 0.01). 3.3. Effect of olmesartan and valsartan on systolic BP variability in aging SHR As shown in Fig. 2(C), CV of systolic BP measured at 0:00 h or 12:00 h were greater in 85-week-old SHR than those in 15-weekold SHR. Olmesartan decreased CV of systolic BP in aged SHR more than valsartan. 3.4. Effect of olmesartan and valsartan on urinary norepinephrine in aged SHR Eighty five-week-old SHR exhibited greater 24-h-urinary norepinephrine excretion than 15-week-old SHR (Fig. 2(D)). Both olmesartan and valsartan significantly reduced 24-h-urinary norepinephrine excretions in 85-week-old SHR. However, olmesartan treatment led to a greater reduction of norepinephrine excretions than valsartan. 3.5. Effect of olmesartan and valsartan on vascular endothelial function, cardiac weight, and aortic thickness in aged SHR As shown in Fig. 3(A), vascular endothelium-dependent relaxation by acetylcholine was significantly impaired in aged SHR compared to
young SHR (P < 0.01). Olmesartan ameliorated the impairment of acetylcholine-induced vascular relaxation in 85-week-old SHR more than valsartan. As shown in Fig. 3(B), pretreatment with L-NAME almost completely abolished acetylcholine-induced vascular relaxation in all groups of rats. The data in Fig. 3(C) show there was no significant difference in sodium nitroprusside-induced endotheliumindependent vascular relaxation. As shown in Supplemental Table 2, the left ventricular weight of 85-week-old SHR was significantly greater than that of 15-weekold SHR (P < 0.01). The LV weight in olmesartan-treated SHR was significantly lower than in valsartan-treated SHR (P < 0.05). Olmesartan tended to reduce aortic thickness compared to vehicle or valsartan, although the difference did not reach statistical significance. 3.6. Effects of olmesartan and valsartan on vascular senescence, superoxide, and AT1 receptor in aged SHR As shown in Fig. 4, aortic SA b-galactosidase positive cell numbers in 85-week-old SHR were greater than those in 15-weekold SHR. Although once-daily oral dosing of olmesartan and valsartan significantly decreased aortic SA b-galactosidase positive cell numbers in 85-week-old SHR, aortic SA b-galactosidase positive cell numbers in the olmesartan group were significantly less than in the valsartan group (P < 0.05). As shown in Fig. 5, 85-week-old SHR showed greater aortic superoxide levels (P < 0.01) and greater NADPH oxidase subunit p22phox levels (P < 0.01) than 15-week-old SHR. Olmesartan and valsartan significantly attenuated aortic superoxide (P < 0.01) and p22phox (P < 0.01) levels of aged SHR. However, olmesartan caused a more significant reduction of aortic superoxide levels and p22phox
Fig. 3. Vascular endothelium-dependent relaxation with acetylcholine without L-NAME pretreatment (A) and with L-NAME pretreatment (B), and endothelium-independent relaxation with sodium nitroprusside (C) in SHR. Acetylcholine-induced vascular relaxation was examined without pretreatment with L-NAME (A), and with pretreatment with L-NAME (100 mmol/l) (B). Abbreviations used are the same as in Fig. 1. Values are the means ± SEM (n ¼ 7). Statistical analysis was performed by one-factor analysis of variance (ANOVA) with repeated measurements followed by post hoc Fisher's protected least significant difference test (A). Acetylcholine-induced vascular relaxation without pretreatment with L-NAME was significantly influenced by acetylcholine (P < 0.01) and age (P < 0.01). Acetylcholine-induced vascular relaxation with pretreatment with L-NAME was significantly influenced by acetylcholine (P < 0.01) but not influenced by age (P ¼ 0.0756). Sodium nitroprusside-induced vascular relaxation was significantly influenced by acetylcholine (P < 0.01) but not influenced by age (P ¼ 0.2146).
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Fig. 4. Effect of olmesartan and valsartan on senescence-associated beta-galactosidase (SA b-gal) positive cell numbers in the aortas in aging SHR. Upper panels indicate representative photomicrographs of aortic sections stained with senescence-associated b-galactosidase (SA b-gal). Values are the means ± SEM (n ¼ 7). Statistical analysis was performed by 1-factor analysis of variance (ANOVA) followed by post hoc Fisher's protected least significant difference test. Abbreviations used: Young, 15-week-old SHR; Aged, 85-week-old SHR; Veh, vehicle-treated 85-week-old SHR; Ol, olmesartan-treated 85-week-old SHR; Va, valsartan-treated 85-week-old SHR.
levels in 85-week-old SHR than valsartan. Aortic extracellular-SOD, copperezinc-SOD, and manganese-SOD levels in 85-week-old SHR were not significantly changed by olmesartan or valsartan (data not shown). Vascular AT1 receptor was significantly greater in 85-week-old SHR than 15-week-old SHR (P < 0.01) (Fig. 5(D)). 4. Discussion The major findings of this work are the following: (1) aging led to increased BP variability, enhancement of sympathetic activity, impairment of autonomic function, and the increase in vascular senescence and oxidative stress; (2) hypertension exacerbated these changes caused by aging; (3) ARBs ameliorated these changes in aged SHR; (4) the magnitude of improvements of these agingrelated changes may differ across ARBs. It is well recognized that elderly hypertensive patients are generally characterized by the abnormality of BP circadian rhythm, BP variability, the impairment of autonomic function, and vascular endothelial dysfunction and stiffness. However, it is unclear whether aging or high BP contributes more to such changes in elderly hypertensive patients. To address this issue, we compared BP circadian rhythm, BP variability, autonomic function, and vascular function in young and old WKY and SHR. The comparison between young and old normotensive WKY showed that the impairment of
autonomic function, BP variability, vascular endothelial dysfunction, vascular senescence and oxidative stress were significantly enhanced in old WKY compared with young WKY. These results demonstrate that aging itself causes the impairment of autonomic function, BP variability, vascular endothelial dysfunction, and vascular senescence. The comparison between young and old SHR indicated that the abnormalities caused by aging were more pronounced in SHR than in WKY. Therefore, aging and hypertension seem to additively enhance the impairment of autonomic function, BP variability, and vascular senescence. Aged SHR are regarded as a useful animal model of elderly hypertensive patients. There is no convincing evidence supporting an optimal hypertensive therapy for elderly patients. Thus, it is unclear which antihypertensive drug is optimal for the treatment of elderly patients. Accumulating experimental evidence [18,24,25,27] indicates that angiotensin II is involved in the activation of the sympathetic nervous system [31] and the pathogenesis of vascular senescence. Therefore, we investigated the effects of two ARBs, olmesartan and valsartan, on aged SHR to determine the potential benefit of angiotensin receptor inhibition in improvement of aging-associated disorder. We found that treatment with olmesartan or valsartan significantly reduced HR, and ameliorated sympathetic activity as shown by the significant reduction of LF of systolic BP and urinary norepinephrine excretion, the most popular marker of sympathetic activity. Additionally, the treatments also improved baroreceptor
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Fig. 5. Effects of olmesartan and valsartan on vascular superoxide levels (A), NADPH oxidase subunit p22phox (C) and AT1 receptor (D) of aged SHR. Upper panels in (A) indicate representative photomicrographs of aortic sections stained with dihydroethidium. (B) indicate representative photographs of western blot. Statistical analysis was performed by 1factor analysis of variance (ANOVA) followed by post hoc Fisher's protected least significant difference test. Abbreviations used are the same as in Fig. 4. DHE, dihydroethidium. Values are the means ± SEM (n ¼ 7).
reflex function, as shown by the significant increase in sBRG. These findings suggest that ARB may improve aging-related autonomic dysfunction. Furthermore, both ARBs ameliorated BP variability in aged SHR, as shown by the reduction of CV of systolic BP. Taken together with the findings that the enhancement of sympathetic activity contributes to the pathogenesis of BP variability, our results suggest that the amelioration of BP variability [32] by ARBs may be attributable to the suppression of sympathetic activity. Vascular senescence plays a causative role in vascular diseases such as vascular endothelial dysfunction, vascular remodeling, and atherosclerosis [18,19]. Of note, vascular AT1 receptor levels in aged SHR was significantly higher than in young SHR, supporting the notion that vascular renin-angiotensin system may be enhanced in aged SHR. Previous in vitro and in vivo studies indicate that angiotensin II, through activation of AT1 receptor, increases vascular oxidative stress by increasing p22phox, a key subunit of NADPH oxidase [33e35]. However, there is no report comparing the effects of different ARBs on vascular oxidative stress in aged SHR. In the present study, both olmesartan and valsartan significantly ameliorated the impairment of vascular endothelial function in aged SHR and reduced vascular superoxide levels and NADPH oxidase subunit p22phox in aged SHR. Thus, the amelioration of vascular oxidative stress by these ARBs appears to be at least in part mediated by the reduction of p22phox. Moreover, olmesartan significantly decreased the number of vascular SA b-gal positive cells in aged SHR. These results suggest that ARBs may ameliorate the progression of vascular senescence caused by aging. Furthermore, taken together with the findings that oxidative stress plays a central role in the pathogenesis of vascular senescence [18,36,37], the amelioration of vascular senescence by ARBs appears to be mediated by the reduction of oxidative stress. In the present study, olmesartan ameliorated aging-related impairment of autonomic function, enhancement of sympathetic activity, BP variability, vascular endothelial dysfunction, vascular senescence, and oxidative stress to a greater extent than valsartan. The present study did not allow us to elucidate the reason for the
differences between olmesartan and valsartan regarding the effect on aging-related disorders. Continuous monitoring of BP in aged SHR with telemetry indicated the BP lowering effect caused by olmesartan was significantly longer than the effect caused by valsartan. Thus, the extended duration of BP lowering effects was associated with a greater reduction of 24-h BP by olmesartan. These findings demonstrate inhibition of the AT1 receptor in vivo in aged SHR by olmesartan lasted longer than valsartan. Furthermore, angiotensin II activates the AT1 receptor and is responsible for the impairment of autonomic function, vascular senescence and injury. Collectively, the amelioration of aging-associated autonomic dysfunction, BP variability, vascular senescence and injury by olmesartan was better than valsartan because of the longer lasting AT1 receptor suppression by olmesartan. 4.1. Study limitation There are several study limitations in this study. First, we did not examine the effect of angiotensin-converting enzyme (ACE) inhibitor in this study, since the main purpose of our work was to examine the effect of specific angiotensin blockade in aged SHR. Accumulating evidence establishes that cardiovascular protective effects and blood pressure lowering effects of ACE inhibitor is significantly mediated by the increase in bradykinin, independently of angiotensin II blockade [38]. Therefore, in the present study, we did not use ACE inhibitor. However, the investigation on the effect of ACE inhibitor in aged SHR is an important future issue since ACE inhibitor is one of the major cardiovascular protective drugs. Second, we did not measure blood concentrations of olmesartan and valsartan in this study. Olmesartan and valsartan have different pharmacokinetics [29]. Therefore, it cannot be excluded that longer-lasting AT1 receptor blockade by olmesartan than valsartan in this study might be attributed to the potential difference in blood concentrations between these drugs as well as the differential AT1 receptor affinity between the drugs. Third, our present work did not allow us to provide the detailed mechanisms underlying more
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attenuation of vascular oxidative stress and urinary norepinephrine excretion by olmesartan than valsartan, since our work was in vivo experiments on aged SHR. Further study including in vitro experiment is warranted to elucidate the detailed mechanisms responsible for the effects of ARBs on these parameters. In conclusion, aging and hypertension additively cause the enhancement of sympathetic activity, the impairment of autonomic function, BP variability, and vascular senescence and dysfunction. BP lowering by ARBs ameliorates these aging-related disorders. The magnitude of amelioration of these aging-related disorders may depend on the duration of AT1 receptor inhibition. However, a clinical study is warranted to investigate this hypothesis. Disclosures H.O. received lecture fees and research grant from Astellas, AstraZeneca, Bayer, Boehringer Ingelheim, Chugai, Daiichi Sankyo, Dainippon Sumitomo Pharma, Eisai, Kowa, Kyowa Hakko Kirin, Mitsubishi Tanabe, MSD, Novartis, Otsuka, Pfizer, Sanofi, Sionogi, Takeda.S. S. K-M received lecture fees and research grant from Astellas, AstraZeneca, Boehringer Ingelheim, Daiichi Sankyo, Novartis, Sionogi, Takeda, Kyowa Hakko Kirin. Conflict of interest The authors report no relationships that could be construed as a conflict of interest. Acknowledgments This work was in part supported by Daiichi Sankyo (Tokyo, Japan). We would like to thank Erina Haraguchi, Yusei Kawahara, Koji Ichiki, Miho Kataoka, Michie Uchikawa, Keiko Morozumi, Yuriko Shimamura, Shuhei Hasebe, Noriko Yoshimura, Kazuko Noda and Tomoko Moriyama for their kind support during the study. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2014.06.016. References [1] Drayer JI, Weber MA, DeYoung JL, Wyle FA. Circadian blood pressure patterns in ambulatory hypertensive patients: effects of age. Am J Med 1982;73(4): 493e9. [2] Gribbin B, Pickering TG, Sleight P, Peto R. Effect of age and high blood pressure on baroreflex sensitivity in man. Circ Res 1971;29(4):424e31. [3] O'Rourke MF, Hashimoto J. Mechanical factors in arterial aging: a clinical perspective. J Am Coll Cardiol 2007;50(1):1e13. [4] Shimada K, Kitazumi T, Sadakane N, Ogura H, Ozawa T. Age-related changes of baroreflex function, plasma norepinephrine, and blood pressure. Hypertension 1985;7(1):113e7. [5] Verdecchia P, Angeli F, Mazzotta G, Garofoli M, Ramundo E, Gentile G, et al. Day-night dip and early-morning surge in blood pressure in hypertension: prognostic implications. Hypertension 2012;60(1):34e42. [6] Verdecchia P, Porcellati C, Schillaci G, Borgioni C, Ciucci A, Battistelli M, et al. Ambulatory blood pressure. An independent predictor of prognosis in essential hypertension. Hypertension 1994;24(6):793e801. [7] Hastie CE, Jeemon P, Coleman H, McCallum L, Patel R, Dawson J, et al. Long-term and ultra long-term blood pressure variability during follow-up and mortality in 14,522 patients with hypertension. Hypertension 2013;62(4):698e705. [8] Hata J, Arima H, Rothwell PM, Woodward M, Zoungas S, Anderson C, et al. Effects of visit-to-visit variability in systolic blood pressure on macrovascular and microvascular complications in patients with type 2 diabetes mellitus: the ADVANCE trial. Circulation 2013;128(12):1325e34. [9] Rothwell PM. Limitations of the usual blood-pressure hypothesis and importance of variability, instability, and episodic hypertension. Lancet 2010;375(9718):938e48.
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