Takuya Kishi

Increased Reactive Oxygen Species in Rostral Ventrolateral Medulla Contribute to Neural Mechanisms of Hypertension in Stroke-Prone Spontaneously Hypertensive Rats

Background—Oxidative stress increases in hypertension. The aim of this study was to determine whether reactive oxygen species (ROS) are increased in the rostral ventrolateral medulla (RVLM) in the brainstem, where the vasomotor center is located, in stroke-prone spontaneously hypertensive rats (SHRSP), and, if so, to determine whether the increased ROS contribute to neural mechanisms of hypertension in SHRSP. Methods and Results—We measured ROS levels in the RVLM of SHRSP and compared them with those in Wistar-Kyoto rats (WKY). Thiobarbituric acid–reactive substances were increased in SHRSP compared with WKY. ROS were measured by electron spin resonance (ESR) spectroscopy. The ESR signal decay rate in the RVLM of SHRSP was significantly increased compared with that in WKY, and this increase was abolished by dimethylthiourea (a hydroxyl radical scavenger). The increased ESR signal decay rate was reduced to the same extent in the presence of desferrioxamine, catalase, and Tiron, indicating that hydroxyl radicals are derived from superoxide anions and hydrogen peroxide. In addition, total superoxide dismutase (SOD) activity in the RVLM was decreased in SHRSP compared with WKY. Furthermore, bilateral microinjection of tempol into the RVLM decreased blood pressure in SHRSP but not in WKY, and MnSOD overexpression in the RVLM of SHRSP decreased blood pressure and inhibited sympathetic nerve activity. Conclusions—These results suggest that superoxide anions in the RVLM, which generate hydroxyl radicals, are increased in SHRSP and contribute to the neural mechanisms of hypertension in SHRSP.

Extracorporeal cardiac shock wave therapy ameliorates myocardial ischemia in patients with severe coronary artery disease

ObjectivePrognosis of severe coronary artery disease with no indication of percutaneous coronary intervention or coronary artery bypass grafting remains poor. We have recently demonstrated that shock wave therapy effectively induces neovascularization and improves myocardial ischemia in a porcine model in vivo. MethodsWith permission from the Ethical Committee of our Institute, we treated nine patients with end-stage coronary artery disease with no indication of percutaneous coronary intervention or coronary artery bypass grafting (55–82 years old, five men and four women) with our cardiac shock wave therapy (200 shots/spot at 0.09 mJ/mm2 for 20–40 spots, 3 times a week/series). We followed-up the patients at 1, 3, 6, and 12 months after the therapy to examine the amelioration of myocardial ischemia. When needed, shock wave therapy was performed up to three series at 0, and 1, 3 or 6 months. ResultsThe cardiac shock wave therapy improved symptoms (Canadian Cardiovascular Society functional class score, from 2.7±0.2 to 1.8±0.2, P<0.01) and reduced nitroglycerin use (from 5.4±2.5 to 0.3±0.3/week, P<0.05). The treatment also improved myocardial perfusion as assessed by dipyridamole stress thallium scintigraphy (severity score, 25.2±7.2% improvement, P<0.05; extent score, 23.3±9.0% improvement, P=0.10; washout rate, 20±3 to 34±3, P<0.05). Myocardial perfusion was improved only in the ischemic area treated with the therapy. These beneficial effects persisted for 12 months. No procedural complications or adverse effects were noted. ConclusionThese results indicate that our extracorporeal cardiac shock wave therapy is an effective and non-invasive treatment for end-stage coronary artery disease, although further careful evaluation is needed.

Rho-Kinase Inhibitor Improves Increased Vascular Resistance and Impaired Vasodilation of the Forearm in Patients With Heart Failure

Background—Rho-kinase is suggested to have an important role in enhanced vasoconstriction in animal models of heart failure (HF). Patients with HF are characterized by increased vasoconstriction and reduced vasodilator responses to reactive hyperemia and exercise. The aim of the present study was to examine whether Rho-kinase is involved in the peripheral circulation abnormalities of HF in humans with the Rho-kinase inhibitor fasudil. Methods and Results—Studies were performed in patients with HF (HF group, n=26) and an age-matched control group (n=26). Forearm blood flow was measured with a strain-gauge plethysmograph during intra-arterial infusion of graded doses of fasudil or sodium nitroprusside. Resting forearm vascular resistance (FVR) was significantly higher in the HF group than in the control group. The increase in forearm blood flow evoked by fasudil was significantly greater in the HF group than in the control group. The increased FVR was decreased by fasudil in the HF group toward the level of the control group. By contrast, FVR evoked by sodium nitroprusside was comparable between the 2 groups. Fasudil significantly augmented the impaired ischemic vasodilation during reactive hyperemia after arterial occlusion of the forearm in the HF group but not in the control group. Fasudil did not augment the increased FVR evoked by phenylephrine in the control group significantly. Conclusions—These results indicate that Rho-kinase is involved in increased FVR and impaired vasodilation of the forearm in patients with HF.

Imbalance of central nitric oxide and reactive oxygen species in the regulation of sympathetic activity and neural mechanisms of hypertension

Nitric oxide (NO) and reactive oxygen species (ROS) play important roles in blood pressure regulation via the modulation of the autonomic nervous system, particularly in the central nervous system (CNS). In general, accumulating evidence suggests that NO inhibits, but ROS activates, the sympathetic nervous system. NO and ROS, however, interact with each other. Our consecutive studies and those of others strongly indicate that an imbalance between NO bioavailability and ROS generation in the CNS, including the brain stem, activates the sympathetic nervous system, and this mechanism is involved in the pathogenesis of neurogenic aspects of hypertension. In this review, we focus on the role of NO and ROS in the regulation of the sympathetic nervous system within the brain stem and subsequent cardiovascular control. Multiple mechanisms are proposed, including modulation of neurotransmitter release, inhibition of receptors, and alterations of intracellular signaling pathways. Together, the evidence indicates that an imbalance of NO and ROS in the CNS plays a pivotal role in the pathogenesis of hypertension.

Overexpression of Inducible Nitric Oxide Synthase in Rostral Ventrolateral Medulla Causes Hypertension and Sympathoexcitation via an Increase in Oxidative Stress

The present study examined the role of inducible nitric oxide synthase (iNOS) in the rostral ventrolateral medulla (RVLM) of the brain stem, where the vasomotor center is located, in the control of blood pressure and sympathetic nerve activity. Adenovirus vectors encoding iNOS (AdiNOS) or &bgr;-galactosidase (Ad&bgr;gal) were transfected into the RVLM in Wistar-Kyoto (WKY) rats. Blood pressure and heart rate were monitored using a radiotelemetry system. iNOS expression in the RVLM was confirmed by immunohistochemical staining or Western blot analysis. Mean arterial pressure significantly increased from day 6 to day 11 after AdiNOS transfection, but did not change after Ad&bgr;gal transfection. Urinary norepinephrine excretion was significantly higher in AdiNOS-transfected rats than in Ad&bgr;gal-transfected rats. Microinjection of aminoguanidine or S-methylisothiourea, iNOS inhibitors, or tempol, an antioxidant, significantly attenuated the pressor response evoked by iNOS gene transfer. The levels of thiobarbituric acid-reactive substances, a marker of oxidative stress, were significantly greater in AdiNOS-transfected rats than in Ad&bgr;gal-transfected rats. Dihydroethidium fluorescence in the RVLM was increased in AdiNOS-transfected rats. In addition, nitrotyrosine-positive cells were observed in the RVLM only in AdiNOS-transfected rats. Intracisternal infusion of tempol significantly attenuated the pressor response and the increase in the levels of thiobarbituric acid–reactive substances induced by AdiNOS transfection. These results suggest that overexpression of iNOS in the RVLM increases blood pressure via activation of the sympathetic nervous system, which is mediated by an increase in oxidative stress.

Heart failure as an autonomic nervous system dysfunction

In heart failure, it has been recognized that the sympathetic nervous system (SNS) is activated and the imbalance of the activity of the SNS and vagal activity interaction occurs. The abnormal activation of the SNS leads to further worsening of heart failure. Many previous clinical and basic studies have demonstrated that the abnormal activation of the SNS is caused by the enhancement of excitatory inputs including changes in: (1) peripheral baroreceptor and chemoreceptor reflexes; (2) chemical mediators that control sympathetic outflow; and (3) central sites that integrate sympathetic outflow. In particular, the abnormalities in central SNS regulation due to the renin angiotensin system-oxidative stress axis have recently been in focus. In the treatment of heart failure, the inhibition of the activated SNS, such as with beta-blockers and/or exercise training, is important. Furthermore, many experimental studies have demonstrated that vagal stimulation has beneficial effects on heart failure, and recently several clinical studies have also demonstrated that vagal stimulation is a possible novel therapy for heart failure. In conclusion, we must recognize that heart failure is a complex syndrome with an autonomic nervous system dysfunction, and that the autonomic imbalance with the activation of the SNS and the reduction of vagal activity should be treated.

Rho/Rho-Kinase Pathway in Brain Stem Contributes to Blood Pressure Regulation via Sympathetic Nervous System: Possible Involvement in Neural Mechanisms of Hypertension

Abstract— Recent studies have demonstrated that the Rho/Rho-kinase pathway plays an important role in various cellular functions, including actin cytoskeleton organization and vascular smooth muscle contraction. This pathway is also present in the central nervous system and is involved in the maintenance of dendritic spines and axon outgrowth and in the regulation of neurotransmitter release. However, its role in central blood pressure regulation is unknown. In the present study, blockade of the Rho/Rho-kinase pathway in the nucleus tractus solitarii (NTS) of the brain stem by microinjection of a specific Rho-kinase inhibitor decreased blood pressure, heart rate, and renal sympathetic nerve activity in both Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). However, the magnitude of decreases in these variables was greater in SHR than in WKY rats. In addition, an adenovirus vector encoding dominant-negative Rho-kinase decreased blood pressure, heart rate, and urinary norepinephrine excretion in both WKY rats and SHR in an awake and free-moving state. The magnitude of decreases in these variables was also greater in SHR than in WKY rats. Furthermore, membrane RhoA expression and Rho-kinase activity in the NTS were enhanced in SHR compared with WKY rats. These observations indicate that the Rho/Rho-kinase pathway in the NTS contributes to blood pressure regulation via the sympathetic nervous system in vivo and suggest that activation of this pathway is involved in the central mechanisms of hypertension.

Atorvastatin causes depressor and sympatho-inhibitory effects with upregulation of nitric oxide synthases in stroke-prone spontaneously hypertensive rats

Objective Recent studies have suggested that statins decrease blood pressure in hypertensive animals and upregulate endothelial nitric oxide synthase (eNOS) expression. However, the effects of statins on the expression of nitric oxide synthase (NOS) in the brain and the sympathetic nervous system remain to be elucidated. The aim of this study was thus to examine the effects of atorvastatin on blood pressure, sympathetic nerve activity, and the expression of NOS in stroke-prone spontaneously hypertensive rats (SHRSP) as well as in Wistar–Kyoto (WKY) rats. Methods The animals received atorvastatin (50 mg/kg per day) for 30 days. Systolic blood pressure and heart rate were evaluated using the tail-cuff method. Urinary norepinephrine excretion was measured for 24 h. The expression of eNOS, neuronal NOS (nNOS), and inducible NOS (iNOS) in the brain (cortex, cerebellum, hypothalamus and brainstem), aorta and heart were determined by Western blot analysis. Results Systolic blood pressure and 24-h urinary norepinephrine excretion were significantly decreased in SHRSP, but not in WKY, after the treatment with atorvastatin. The eNOS and iNOS expression in the brain and aorta was significantly increased in atorvastatin-treated SHRSP and WKY. However, the nNOS expression in the brain was not altered in the atorvastatin-treated group. Conclusions These results suggest that atorvastatin decreases blood pressure, at least in part via the reduction of sympathetic nervous system activity in SHRSP. They also suggest that this sympatho-inhibitory effect may be mediated by an increase in NO production, with the upregulation of eNOS expression in the brain.

Mitochondria-derived reactive oxygen species mediate sympathoexcitation induced by angiotensin II in the rostral ventrolateral medulla.

Objectives Reactive oxygen species (ROS) in the central nervous system are thought to contribute to sympathoexcitation in cardiovascular diseases such as hypertension and heart failure. Nicotinamide adenine dinucleotide phosphate oxidase is a major source of ROS in the central nervous system, which acts as a key mediator (mediators) of angiotensin II (AngII). It is not clear, however, whether mitochondria-derived ROS in the central nervous system also participate in sympathoexcitation. Methods In an in-vivo study, we investigated whether the AngII-elicited pressor response in the rostral ventrolateral medulla, which controls sympathetic nerve activity, is attenuated by adenovirus-mediated gene transfer of a mitochondria-derived antioxidant (Mn-SOD). In an in-vitro study, using differentiated PC-12 cells with characteristics similar to those of sympathetic neurons, we examined whether AngII increases mitochondrial ROS production. Results Overexpression of Mn-SOD attenuated the AngII-induced pressor response and also suppressed AngII-induced ROS production, as evaluated by microdialysis in the rostral ventrolateral medulla. Using reduced MitoTracker red, we showed that AngII increased mitochondrial ROS production in differentiated PC-12 cells in vitro. Overexpression of Mn-SOD and rotenone, a mitochondrial respiratory complex I inhibitor, suppressed AngII-induced ROS production. Depletion of extracellular Ca2+ with ethylene glycol bis-N,N,N′,N′-tetraacetate (EGTA) and administration of p-trifluoromethoxycarbonylcyanide phenylhydrazone, which prevents further Ca2+ uptake into the mitochondria, blocked AngII-elicited mitochondrial ROS production. Conclusion These results indicate that AngII increases the intracellular Ca2+ concentration and that the increase in mitochondrial Ca2+ uptake leads to mitochondrial ROS production.

Inhibition of Rac1-Derived Reactive Oxygen Species in Nucleus Tractus Solitarius Decreases Blood Pressure and Heart Rate in Stroke-Prone Spontaneously Hypertensive Rats

Reactive oxygen species (ROS) in the brain are thought to contribute to the neuropathogenesis of hypertension by enhancing sympathetic nervous system activity. The nucleus tractus solitarius (NTS), which receives afferent input from baroreceptors, has an important role in cardiovascular regulation. reduced nicotinamide-adenine dinucleotide phosphate oxidase is thought to be a major source of ROS in the NTS. Rac1 is a small G protein and a key component of reduced nicotinamide-adenine dinucleotide phosphate oxidase. The role of Rac1-derived ROS in the NTS in cardiovascular regulation of hypertension is unknown. Therefore, we examined whether inhibition of Rac1 in the NTS decreases ROS generation, thereby reducing blood pressure in stroke-prone spontaneously hypertensive rats (SHRSPs). The basal Rac1 activity level in the NTS was greater in SHRSPs than in Wistar-Kyoto rats. Inhibition of Rac1, induced by transfecting adenovirus vectors encoding dominant-negative Rac1 into the NTS, decreased blood pressure, heart rate, and urinary norepinephrine excretion in SHRSPs but not in Wistar-Kyoto rats. Inhibition of Rac1 also reduced nicotinamide-adenine dinucleotide phosphate oxidase activity and ROS generation. In addition, Cu/Zn-superoxide dismutase activity in the NTS of SHRSPs was decreased compared with that of Wistar-Kyoto rats, despite the increased ROS generation. Overexpression of Cu/Zn-superoxide dismutase in the NTS decreased blood pressure and heart rate in SHRSPs. These results indicate that the activation of Rac1 in the NTS generates ROS via reduced nicotinamide-adenine dinucleotide phosphate oxidase in SHRSPs, and this mechanism might be important for the neuropathogenesis of hypertension in SHRSPs.