Masanobu Yamazato

Overexpression of Angiotensin-Converting Enzyme 2 in the Rostral Ventrolateral Medulla Causes Long-Term Decrease in Blood Pressure in the Spontaneously Hypertensive Rats

The rostral ventrolateral medulla (RVLM) is a relay point that provides supraspinal excitatory input to sympathetic preganglionic neurons in the regulation of blood pressure. The importance of the RVLM is further highlighted by observations that an increase of RVLM sensitivity to angiotensin II and enhanced sympathetic activity are associated with hypertension. Angiotensin-converting enzyme 2 (ACE2) has been shown to be central in maintaining the balance between vasoconstrictor activity of angiotensin II with vasoprotective action of angiotensin-(1-7) in the peripheral system. However, its role in central control of blood pressure in the RVLM is yet to be investigated. Thus, our objective in this study was to compare ACE2 expression in the RVLM of Wistar–Kyoto rats and spontaneously hypertensive rats and to determine whether RVLM ACE2 is involved in blood pressure control. ACE2 immunoreactivity was diffusely distributed in many cardiovascular regulatory neurons, including the RVLM. Western blot analysis revealed a 40% decrease in ACE2 in the RVLM of spontaneously hypertensive rat compared with Wistar–Kyoto rats. Lentiviral-mediated overexpression of ACE2 (lenti-ACE2) was used to determine whether a decrease in ACE2 in the RVLM is associated with hypertensive state. Bilateral injection of lenti-ACE2 resulted in a long-term expression of transgenic ACE2. This was associated with a decrease in mean arterial pressure exclusively in the spontaneously hypertensive rat (141±4 mm Hg in lenti-GFP versus 124±5 mm Hg in lenti-ACE2) and heart rate (304±7 bpm in lenti-GFP versus 285±5 bpm in lenti-ACE2). These observations demonstrate that overexpression of ACE2 overcomes its intrinsic decrease in the RVLM and decreases high blood pressure in the spontaneously hypertensive rat.

Prevention of Pulmonary Hypertension by Angiotensin-Converting Enzyme 2 Gene Transfer

In spite of recent advancements in the treatment of pulmonary hypertension, successful control has yet to be accomplished. The abundant presence of angiotensin-converting enzyme 2 (ACE2) in the lungs and its impressive effect in the prevention of acute lung injury led us to test the hypothesis that pulmonary overexpression of this enzyme could produce beneficial outcomes against pulmonary hypertension. Monocrotaline (MCT) treatment of mice for 8 weeks resulted in significant increases in right ventricular systolic pressure, right ventricle:left ventricle plus septal weight ratio, and muscularization of pulmonary vessels. Administration of a lentiviral vector containing ACE2, 7 days before MCT treatment prevented the increases in right ventricular systolic pressure (control: 25±1 mm Hg; MCT: 44±5 mm Hg; MCT+ACE2: 26±1 mm Hg; n=6; P<0.05) and right ventricle:left ventricle plus septal weight ratio (control: 0.25±0.01; MCT: 0.31±0.01; MCT+ACE2: 0.26±0.01; n=8; P<0.05). A significant attenuation in muscularization of pulmonary vessels induced by MCT was also observed in animals overexpressing ACE2. These beneficial effects were associated with an increase in the angiotensin II type 2 receptor:angiotensin II type 1 receptor mRNA ratio. Also, pulmonary hypertension–induced increases in proinflammatory cytokines were significantly attenuated by lentiviral vector–containing ACE2 treatment. Furthermore, ACE2 gene transfer in mice after 6 weeks of MCT treatment resulted in a significant reversal of right ventricular systolic pressure. These observations demonstrate that ACE2 overexpression prevents and reverses right ventricular systolic pressure and associated pathophysiology in MCT-induced pulmonary hypertension by a mechanism involving a shift from the vasoconstrictive, proliferative, and fibrotic axes to the vasoprotective axis of the renin-angiotensin system and inhibition of proinflammatory cytokines.

Increased PI3-kinase in presympathetic brain areas of the spontaneously hypertensive rat.

Existing evidence led us to hypothesize that increases in p85α, a regulatory subunit of PI3-kinase, in presympathetic brain areas contribute to hypertension. PI3-kinase p85α, p110α, and p110δ mRNA was 1.5- to 2-fold higher in the paraventricular nucleus (PVN) of spontaneously hypertensive rats (SHR) compared with their controls, Wistar Kyoto rats (WKY). The increase in p85α/p110δ was attenuated in SHR treated with captopril, an angiotensin (Ang)-converting enzyme inhibitor, from in utero to 6 months of age. In the rostral ventrolateral medulla (RVLM), p110δ mRNA was ≈2-fold higher in SHR than in WKY. Moreover, the increases in mRNA were associated with higher PI3-kinase activity in both nuclei. The functional relevance was studied in neuronal cultures because SHR neurons reflect the augmented p85α mRNA and PI3-kinase activity. Expression of a p85 dominant-negative mutant decreased norepinephrine (NE) transporter mRNA and [3H]NE uptake by ≈60% selectively in SHR neurons. In summary, increased p85α/p110δ expression in the PVN and RVLM is associated with increased PI3-kinase activity in the SHR. Furthermore, normalized PI3-kinase p85α/p110δ expression within the PVN might contribute to the overall effect of captopril, perhaps attributable to a consequent decrease in NE availability.

Pioglitazone, a Thiazolidinedione Derivative, Attenuates Left Ventricular Hypertrophy and Fibrosis in Salt-Sensitive Hypertension

Thiazolidinediones, which stimulate peroxisome proliferator–activated receptor γ, have been shown to prevent cardiovascular injury. However, little is known about their effects on salt-sensitive hypertension. We thus investigated whether or not pioglitazone affects left ventricular (LV) hypertrophy in Dahl salt-sensitive rats, then compared its effects to those of an angiotensin II receptor blocker, candesartan. Rats were used at 16 weeks of age after they had been fed either a low-salt (0.3%; DSL) or high-salt (8%; DSH) diet for 10 weeks; some of the DSH rats were treated with pioglitazone (10 mg/kg/day) or candesartan (4 mg/kg/day). Both drugs decreased the elevated blood pressure in DSH rats, although it was still higher than in DSL rats. Both drugs decreased plasma insulin levels, but neither affected plasma glucose levels. The thiobarbituric acid reactive substance level in the LV was decreased by both drugs. LV hypertrophy evaluated by echocardiography in DSH rats was nearly normalized by both drugs, whereas only candesartan decreased LV diameter. In histological analysis, both drugs ameliorated LV fibrosis and myocardial cell hypertrophy. Both drugs decreased elevated gene expression levels of transforming growth factor-β1 and collagen type I, although the pioglitazone action was slightly modest. The metalloproteinase activity was increased in DSH rats, but both drugs decreased this level. Taken together, these findings indicate that pioglitazone reduced LV hypertrophy and fibrosis in salt-sensitive hypertension. Improvement in blood pressure, insulin level, and oxidative stress may be associated with this beneficial action of pioglitazone.

Gene transfer of angiotensin-converting enzyme 2 in the nucleus tractus solitarius improves baroreceptor heart rate reflex in spontaneously hypertensive rats

The renin–angiotensin system (RAS) in the nucleus tractus solitarius (NTS) is an important modulator of the baroreceptor heart rate reflex. This study tested the hypothesis that angiotensin-converting enzyme 2 (ACE2) expression is decreased in the NTS of spontaneously hypertensive rats (SHRs) and that its gene transfer in this nucleus would lead to beneficial effects on baroreflex function since this enzyme is key in the regulation of the vasoprotective axis of the RAS. ACE2 protein levels and its activity were significantly decreased in the NTS of SHRs compared to normotensive Wistar-Kyoto (WKY) control rats. Rats instrumented with radio-telemetry transducers received NTS microinjection of either Lenti-ACE2 (Lentiviral vector-mediated gene transfer of ACE2) or lenti-GFP (green fluorescent protein). The ACE2 gene transfer into the NTS resulted in long-term overexpression of ACE2. This was associated with a 60% increase in heart rate baroreflex sensitivity in the lenti-ACE2 injected SHRs compared with the lenti-GFP injected control SHRs (0.27 ± 0.02 ms/mmHg in lenti-GFP rats vs. 0.44 ± 0.07 ms/mmHg in lenti-ACE2 rats). These observations demonstrate that ACE2 gene transfer overcomes its intrinsic decrease in the NTS of SHRs and improves baroreceptor heart rate reflex.

Prolonged NOS inhibition in the brain elevates blood pressure in normotensive rats

Systemic inhibition of nitric oxide synthase (NOS) evokes hypertension, which is enhanced by salt loading, partly via augmented sympathetic activity. We investigated whether inhibition of brain NOS elevates blood pressure (BP) in normotensive rats and, if so, whether the BP elevation is enhanced by salt loading. After a 2-wk low-salt (0.3%) diet, male Sprague-Dawley (SD) rats were divided into four groups. Groups 1 and 2 received a chronic intracerebroventricular infusion of 0.5 mg . kg-1 . day-1 of NG-monomethyl-L-arginine (L-NMMA), and groups 3 and 4 were given artificial cerebrospinal fluid (aCSF). Groups 1 and 3 were placed on a high-salt (8%) diet, whereas groups 2 and 4 were on a low-salt diet. On day 9 or 10, group 1 showed significantly higher mean arterial pressure (MAP) in a conscious unrestrained state (129 +/- 3 mmHg vs. 114 +/- 3, 113 +/- 1, and 108 +/- 3 mmHg in groups 2, 3, and 4, respectively, P < 0.05). On a high-salt diet, response of renal sympathetic nerve activity but not of BP to air-jet stress was significantly larger in rats given L-NMMA than in rats given aCSF (29 +/- 4% vs. 19 +/- 3%, P < 0.05). When the intracerebroventricular infusions were continued for 3 wk, MAP was significantly higher in rats given L-NMMA than in rats given aCSF irrespective of salt intake, although the difference was approximately 7 mmHg. Thus chronic inhibition of NOS in the brain only slightly elevates BP in SD rats. Salt loading causes a more rapid rise in BP. The mechanisms of the BP elevation and its acceleration by salt loading remain to be elucidated.Systemic inhibition of nitric oxide synthase (NOS) evokes hypertension, which is enhanced by salt loading, partly via augmented sympathetic activity. We investigated whether inhibition of brain NOS elevates blood pressure (BP) in normotensive rats and, if so, whether the BP elevation is enhanced by salt loading. After a 2-wk low-salt (0.3%) diet, male Sprague-Dawley (SD) rats were divided into four groups. Groups 1 and 2 received a chronic intracerebroventricular infusion of 0.5 mg ⋅ kg-1 ⋅ day-1of N G-monomethyl-l-arginine (l-NMMA), and groups 3 and 4 were given artificial cerebrospinal fluid (aCSF). Groups 1 and 3 were placed on a high-salt (8%) diet, whereas groups 2 and 4 were on a low-salt diet. On day 9or 10, group 1 showed significantly higher mean arterial pressure (MAP) in a conscious unrestrained state (129 ± 3 mmHg vs. 114 ± 3, 113 ± 1, and 108 ± 3 mmHg in groups 2, 3, and 4, respectively, P < 0.05). On a high-salt diet, response of renal sympathetic nerve activity but not of BP to air-jet stress was significantly larger in rats givenl-NMMA than in rats given aCSF (29 ± 4% vs. 19 ± 3%, P < 0.05). When the intracerebroventricular infusions were continued for 3 wk, MAP was significantly higher in rats givenl-NMMA than in rats given aCSF irrespective of salt intake, although the difference was ∼7 mmHg. Thus chronic inhibition of NOS in the brain only slightly elevates BP in SD rats. Salt loading causes a more rapid rise in BP. The mechanisms of the BP elevation and its acceleration by salt loading remain to be elucidated.

Effects of xanthine oxidase inhibitors on renal function and blood pressure in hypertensive patients with hyperuricemia

Hyperuricemia may promote the progression of hypertension and renal dysfunction. However, the effects of hyperuricemia treatment on blood pressure and renal function in adult hypertensive patients with hyperuricemia remain unclear. A total of 137 hypertensive patients with hyperuricemia (96 men and 41 women; mean age of 67 years) who recently started taking xanthine oxidase inhibitors (allopurinol or febuxostat) as outpatients were recruited. Serum uric acid level, estimated glomerular filtration rate (eGFR, ml min−1 per 1.73 m2) and blood pressure (mm Hg) were retrospectively compared immediately before and shortly after starting treatment with xanthine oxidase inhibitors. The mean blood pressure and the eGFR immediately before starting treatment were 128/71 mm Hg and 44.6 ml min−1 per 1.73 m2, respectively. Although the eGFR decreased from 46.6 to 44.6 ml min−1 per 1.73 m2 before starting treatment with xanthine oxidase inhibitors, it increased to 46.2 ml min−1 per 1.73 m2 (P=0.001, compared with immediately before treatment) without any significant changes in blood pressure after the administration of xanthine oxidase inhibitors. Multiple regression analysis revealed that the increase in eGFR after starting xanthine oxidase inhibitor treatment positively correlated with the changes in systolic blood pressure and negatively correlated with the changes in uric acid levels and the use of renin–angiotensin system inhibitors. These results suggest that xanthine oxidase inhibitors may delay the progression of renal dysfunction in adult hypertensive patients with hyperuricemia.

Azelnidipine Attenuates Cardiovascular and Sympathetic Responses to Air-Jet Stress in Genetically Hypertensive Rats

Azelnidipine is a new dihydropyridine calcium channel blocker that causes minimal stimulation of the sympathetic nervous system despite its significant depressor effect. In the present study, we examined the effects of oral or intravenous administration of azelnidipine on cardiovascular and renal sympathetic nerve activity (RSNA) responses to air-jet stress in conscious, unrestrained stroke-prone spontaneously hypertensive rats. Oral administration of high-dose azelnidipine (10 mg/kg per day) or nicardipine (150 mg/kg per day) for 10 days caused a significant and comparable decrease in blood pressure, but low-dose azelnidipine (3 mg/kg per day) did not. Air-jet stress increased mean arterial pressure (MAP), heart rate (HR) and RSNA. High-dose azelnidipine significantly attenuated the increases in MAP, HR and RSNA in response to air-jet stress while nicardipine did not. Low-dose azelnidipine significantly attenuated the pressor response with a trend of decrease in RSNA. Intravenous injection of azelnidipine induced a slowly developing depressor effect. To obtain a similar time course of decrease in MAP by azelnidipine, nicardipine was continuously infused at adjusted doses. Both drugs increased HR and RSNA significantly, while the change in RSNA was smaller in the azelnidipine group. In addition, intravenous administration of azelnidipine attenuated the responses of MAP, HR, and RSNA to air-jet stress; by comparison, the inhibitory actions of nicardipine were weak. In conclusion, oral or intravenous administration of azelnidipine inhibited cardiovascular and sympathetic responses to air-jet stress. This action of azelnidipine may be mediated at least in part by the inhibition of the sympathetic nervous system.

Contribution of α2-adrenoceptors in caudal ventrolateral medulla to cardiovascular regulation in rat

The inhibitory action of α2-agonists on the cardiovascular neurons has been elucidated in the rostral ventrolateral medulla (RVLM) but not in the caudal ventrolateral medulla (CVLM). Our study aimed to clarify whether microinjection of clonidine into the CVLM elicits any cardiovascular effect and whether endogenous α2-adrenoceptor-mediated mechanisms contribute to the tonic activity of the CVLM neurons. In male Sprague-Dawley rats (7-9 wk old, 270-320 g) anesthetized with urethan, unilateral microinjection of 8 nmol of clonidine into the CVLM ( n = 10) increased mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) by 12.1 ± 1.8 mmHg (mean ± SE, P < 0.01) and 25.8 ± 4.8% ( P < 0.01), while heart rate (HR) remained unaltered. Unilateral microinjection of 2 nmol of SKF-86466, a selective blocker of the α2-adrenoceptors, into the CVLM ( n = 10) decreased MAP, HR, and RSNA (-11.6 ± 2.6 mmHg, -26 ± 7 beats/min, and -15.3 ± 1.7%, respectively, P < 0.01 for each). Artificial cerebrospinal fluid caused neither a cardiovascular effect nor a sympathetic response. Prior injection of SKF-86466 into the ipsilateral CVLM attenuated the effects of clonidine. Bilateral microinjection of muscimol into the RVLM abolished the effects of both clonidine and SKF-86466 injected into the CVLM. The pressor and sympathoexcitatory effects of clonidine injected into the CVLM suggest a neuroinhibitory action of the drug on the CVLM neurons. In addition,the depressor and sympathoinhibitory effects of SKF-86466 injected into the CVLM indicated that activation of α2-adrenoceptors by endogenous ligand inhibits CVLM neurons. The effects of clonidine and the α2-adrenoceptor antagonist in the CVLM require the integrity of the RVLM.The inhibitory action of alpha 2-agonists on the cardiovascular neurons has been elucidated in the rostral ventrolateral medulla (RVLM) but not in the caudal ventrolateral medulla (CVLM). Our study aimed to clarify whether microinjection of clonidine into the CVLM elicits any cardiovascular effect and whether endogenous alpha 2-adrenoceptor-mediated mechanisms contribute to the tonic activity of the CVLM neurons. In male Sprague-Dawley rats (7-9 wk old, 270-320 g) anesthetized with urethan, unilateral microinjection of 8 nmol of clonidine into the CVLM (n = 10) increased mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) by 12.1 +/- 1.8 mmHg (mean +/- SE, P < 0.01) and 25.8 +/- 4.8% (P < 0.01), while heart rate (HR) remained unaltered. Unilateral microinjection of 2 nmol of SKF-86466, a selective blocker of the alpha 2-adrenoceptors, into the CVLM (n = 10) decreased MAP, HR, and RSNA (-11.6 +/- 2.6 mmHg, -26 +/- 7 beats/min, and -15.3 +/- 1.7%, respectively, P < 0.01 for each). Artificial cerebrospinal fluid caused neither a cardiovascular effect nor a sympathetic response. Prior injection of SKF-86466 into the ipsilateral CVLM attenuated the effects of clonidine. Bilateral microinjection of muscimol into the RVLM abolished the effects of both clonidine and SKF-86466 injected into the CVLM. The pressor and sympathoexcitatory effects of clonidine injected into the CVLM suggest a neuroinhibitory action of the drug on the CVLM neurons. In addition, the depressor and sympathoinhibitory effects of SKF-86466 injected into the CVLM indicated that activation of alpha 2-adrenoceptors by endogenous ligand inhibits CVLM neurons. The effects of clonidine and the alpha 2-adrenoceptor antagonist in the CVLM require the integrity of the RVLM.

Beneficial Effect of Switching from a Combination of Angiotensin II Receptor Blockers other than Losartan and Thiazides to a Fixed Dose of Losartan/Hydrochlorothiazide on Uric Acid Metabolism in Hypertensive Patients

Among the angiotensin II receptor blockers (ARBs), losartan (LOS) has uricosuric action. The clinical benefits of LOS compared with those of other ARBs may be apparent when it is combined with diuretics, which have an unfavorable influence on serum uric acid (SUA). The effects of switching from combinations of ARBs other than LOS and thiazides to a fixed-dose combination comprising 50 mg LOS and 12.5 mg hydrochlorothiazide on blood pressure (BP), SUA, percent fractional excretion of UA (FEUA), and urine pH were assessed in 57 hypertensive outpatients. A significant reduction in BP was observed after 6 months (P < .01). The switching therapy significantly decreased SUA level (6.0 ± 1.3 vs. 5.7 ± 1.3 mg/dL, P < .01), which was accompanied by increases in FEUA (P < .01) and urine pH (P < .01). The change in SUA was negatively correlated with the changes in FEUA (P < .004) and estimated glomerular filtration rate (P < .05). The change in FEUA was positively correlated with the changes in urine pH (P < .05) but not with BP or estimated glomerular filtration rate. In a separate group of patients treated with ARBs other than LOS (n = 82), a significant BP reduction was observed, but no change in SUA or FEUA was observed. In conclusion, switching therapy decreased SUA level, which was accompanied by an increase in FEUA. This result may depend on the balance between LOS-induced inhibitory action of urate transporter 1 and hydrochlorothiazide-induced plasma volume reduction. The increase in urine pH plays a role in UA urinary excretion.