SIRT1 exerts anti-hypertensive effect via FOXO1 activation in the rostral ventrolateral medulla.

The rostral ventrolateral medulla (RVLM) is a pivotal region in the central regulation of blood pressure (BP). It has been documented that silent information regulator 2 homolog 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD+)-dependent multifunctional transcription regulatory factor, has many cardiovascular protective effects. However, the role and significance of SIRT1 in the central regulation of cardiovascular activity, especially in RVLM, remains unknown. Therefore, the aim of this study was to explore the role and underlying mechanism of SIRT1 in the central regulation of cardiovascular activity in hypertension. Spontaneously hypertensive rats (SHRs) were given resveratrol (RSV) via intracerebroventricular (ICV) infusion or injected with SIRT1-overexpressing lentiviral vectors into the RVLM. In vitro experiments, angiotensin II (Ang II)-induced rat pheochromocytoma cell line (PC12 cells) were transfected with forkhead box protein O1 (FOXO1) small interfering RNA (siRNA) before treatment with RSV. Our results showed that SIRT1 activation with RSV or overexpression in the RVLM significantly decreased BP and sympathetic outflow of SHRs. Furthermore, SIRT1 overexpression in the RVLM significantly decreased reactive oxygen species (ROS) production and facilitated the forkhead box protein O1 (FOXO1) activation, accompanied by upregulation of the ROS-detoxifying enzyme superoxide dismutases 1 (SOD1) in the RVLM of SHRs. In PC12 cells, it was found that Ang II could induce oxidative stress and downregulate the SIRT1-FOXO1-SOD1 signaling pathway, which indicated that the suppressed expression of SIRT1 in the RVLM of SHRs might relate to the elevated central Ang II level. Furthermore, the enhanced oxidative stress and decreased SIRT1-FOXO1-SOD1 axis induced by Ang II were restored by treatment with RSV. However, these favorable effects mediated by SIRT1 activation were blocked by FOXO1 knockdown. Based on these findings, we concluded that SIRT1 activation or overexpression in the RVLM exerts anti-hypertensive effect through reducing oxidative stress via SIRT1-FOXO1-SOD1 signaling pathway, which providing a new target for the prevention and intervention of hypertension.

Constant Light Exerted Detrimental Cardiovascular Effects Through Sympathetic Hyperactivity in Normal and Heart Failure Rats.

It has been documented that constant light exposure exerts complicated cardiovascular effects. However, a mounting collection of conflicting results did not make it any easier for researchers and physicians to consider the role of light on cardiovascular function. This study was designed to investigate how constant light exposure (24 h light/day) influences the cardiac function in normal and heart-failure (HF) rats. In normal rats, two groups of SD rats were accustomed in 12 h light/12 h dark (LD) or 24 h light (constant light, CL) for 4 weeks. In HF rats which was induced by myocardial infarction (MI) was let recover in LD for 4 weeks. Interestingly, compared with rats in LD environment (ejection fraction, EF%: 93.64 ± 2.02 in LD, 14.62 ± 1.53 in HF-LD), constant light (2 weeks) weakened the cardiac function in normal and HF rats (EF%: 79.42 ± 2.91 in CL, 11.50 ± 1.08 in HF-CL). The levels of renal sympathetic nerve activity and c-fos expression in the rostral ventrolateral medulla (RVLM), a key region controlling sympathetic outflow, were significantly increased in normal and HF rats after constant light (RSNA, Max%: 8.64 ± 0.48 in LD, 20.02 ± 1.24 in CL, 20.10 ± 1.16 in HF-LD, 26.82 ± 1.69 in HF-CL). In conclusion, it is suggested that constant light exposure exerts detrimental cardiovascular effects, which may be associated with the RVLM-related sympathetic hyperactivity.

The asymmetric dimethylarginine-mediated inhibition of nitric oxide in the rostral ventrolateral medulla contributes to regulation of blood pressure in hypertensive rats.

Nitric oxide (NO) contributes to the central control of cardiovascular activity. The rostral ventrolateral medulla (RVLM) has been recognized as a pivotal region for maintaining basal blood pressure (BP) and sympathetic tone. It is reported that asymmetric dimethylarginine (ADMA), characterized as a cardiovascular risk marker, is an endogenous inhibitor of nitric oxide synthesis. The present was designed to determine the role of ADMA in the RVLM in the central control of BP in hypertensive rats. In Sprague Dawley (SD) rats, microinjection of ADMA into the RVLM dose-dependently increased BP, heart rate (HR), and renal sympathetic never activity (RSNA), but also reduced total NO production in the RVLM. In central angiotensin II (Ang II)-induced hypertensive rats and spontaneously hypertensive rat (SHR), the level of ADMA in the RVLM was increased and total NO production was decreased significantly, compared with SD rats treated vehicle infusion and WKY rats, respectively. These hypertensive rats also showed an increased protein level of protein arginine methyltransferases1 (PRMT1, which generates ADMA) and a decreased expression level of dimethylarginine dimethylaminohydrolases 1 (DDAH1, which degrades ADMA) in the RVLM. Furthermore, increased AMDA content and PRMT1 expression, and decreased levels of total NO production and DDAH1 expression in the RVLM in SHR were blunted by intracisternal infusion of the angiotensin II type 1 receptor (AT1R) blocker losartan. The current data indicate that the ADMA-mediated NO inhibition in the RVLM plays a critical role in involving in the central regulation of BP in hypertension, which may be associated with increased Ang II.

The phosphoinositide-3 kinase signaling is involved in neuroinflammation in hypertensive rats.

AIMS: It has been demonstrated that neuroinflammation is associated with cardiovascular dysfunction. The phosphoinositide-3 kinase (PI3K) signaling in the rostral ventrolateral medulla (RVLM), a key region for sympathetic outflow, is upregulated and contributes to increased blood pressure (BP) and sympathetic outflow in hypertension. This study was designed to determine the role of the PI3K signaling in neuroinflammation in the RVLM of hypertension. METHODS: The normotensive WKY rats were performed by intracisternal infusion of lipopolysaccharide (LPS) or angiotensin II (Ang II) for inducing neuroinflammation. Elisa was used to determine the level of proinflammatory cytokines. Western blot was employed to detect the protein expression of PI3K signaling pathway. Gene silencing of PI3K p110δ subunit and overexpression of angiotensin-converting enzyme 2 (ACE2) were realized by injecting related lentivirus into the RVLM. RESULTS: In the spontaneously hypertensive rats (SHR), the PI3K signaling in the RVLM was upregulated compared with WKY, gene silencing of PI3K in the RVLM significantly reduced BP and renal sympathetic nerve activity (RSNA), but also decreased the levels of proinflammatory cytokines. In the WKY rats, central infusion of LPS and Ang II significantly elevated BP and RSNA, but also increased the levels of proinflammatory cytokines and PI3K signaling activation in the RVLM. These changes in the Ang II-induced hypertension were effectively prevented by gene silencing of PI3K in the RVLM. Furthermore, overexpression of ACE2 in the RVLM significantly attenuated high BP and neuroinflammation, as well as decreased the activation of PI3K signaling in hypertensive rats. CONCLUSION: This study suggests that the PI3K signaling in the RVLM is involved in neuroinflammation in hypertension and plays an important role in the renin-angiotensin system-mediated changes in neuroinflammation in the RVLM.

Diurnal Variation of the Peripheral Cholinergic Antiinflammatory Function in Mice.

AIMS: Cholinergic antiinflammatory (CAI) pathway functions importantly in inflammation via α7 nicotinic acetylcholine receptors (α7nAChR). The present work tested circadian rhythm in peripheral CAI activity and validities of CAI activity and glucocorticoids in chronotherapy for lipopolysaccharide (LPS)-induced shock. METHODS: Vesicular acetylcholine transporter (VAChT) expressed in liver and kidney was examined every 3 h in C57BL/6 mice. Proinflammatory cytokines in serum and survival time in shock were monitored after LPS injection every 3 h. Mifepristone, antagonist of glucocorticoid receptors, and methyllycaconitine (MLA), antagonist of α7nAChR, were administrated before LPS to block antiinflammatory function of endogenous glucocorticoids and acetylcholine. RESULTS: Both levels of tumor necrosis factor α, interleukin 1ß, and interleukin 6 and mortality exhibited diurnal variations with prominent peaks when LPS was given at 15:00, and the minimum mortality occurred at 00:00. Expression of VAChT increased during resting period. MLA increased serum proinflammatory cytokines slightly, but not affected survival rate. Both differences in cytokines and in survival times between LPS injection at 15:00 and 00:00 were eliminated by mifepristone, but not by MLA. CONCLUSION: Peripheral CAI pathway exerts more powerful antiinflammatory effect during resting period. Glucocorticoids appear to be efficient in chronotherapy for septic shock.

Centrally acting drug moxonidine decreases reactive oxygen species via inactivation of the phosphoinositide-3 kinase signaling in the rostral ventrolateral medulla in hypertensive rats.

OBJECTIVE: Centrally acting antihypertensive action of moxonidine is a result of activation of Imidazoline-1 receptor (I1R) in the rostral ventrolateral medulla (RVLM). Hypertension shows an increase in reactive oxygen species (ROS) in the RVLM. The present objective was to determine the phosphoinositide-3 kinase (PI3K) signaling pathway involved in the effect of moxonidine on ROS generation in the RVLM of spontaneously hypertensive rat (SHR). METHODS: Wistar-Kyoto rats and SHR received intracisternal infusion (2 weeks) of tested agents which were subjected to subsequent experiments. In-situ ROS in the RVLM was evaluated by the oxidative fluorescence dye. Western blot and PCR analysis were performed to detect the expression levels of PI3K signaling pathway. Lentivirus was injected bilaterally into the RVLM for silencing PI3K signaling. RESULTS: ROS production in the RVLM was dose-dependently reduced in SHRs treated with infusion of moxonidine (20 nmol/day), which was prevented by the I1R antagonist efaroxan but not by the α2-adrenoceptor antagonist yohimbine. Moxonidine pretreatment significantly blunted cardiovascular sensitivity to injection of tempol (5 nmol) or angiotensin II (10 pmol) into the RVLM in SHR. Expression levels of PI3K/Akt, nuclear factor kappa-B (NFκB), NADPHase (NOX4), and angiotensin type I receptor (AT1R) in the RVLM were markedly decreased in SHR treated with moxonidine. Infection of lentivirus containing PI3K shRNA in the RVLM effectively prevented effects of moxonidine on cardiovascular activity and expression levels of Akt, NFκB, NOX4, and AT1R. CONCLUSION: The centrally antihypertensive drug moxonidine decreases ROS production in the RVLM through inactivation of the PI3K/Akt signaling pathway in hypertension.

Exercise Training Improves the Altered Renin-Angiotensin System in the Rostral Ventrolateral Medulla of Hypertensive Rats.

The imbalance between angiotensin II (Ang II) and angiotensin 1-7 (Ang 1-7) in the brain has been reported to contribute to cardiovascular dysfunction in hypertension. Exercise training (ExT) is beneficial to hypertension and the mechanism is unclear. This study was aimed to determine if ExT improves hypertension via adjusting renin angiotensin system in cardiovascular centers including the rostral ventrolateral medulla (RVLM). Spontaneously hypertensive rats (SHR, 8 weeks old) were subjected to low-intensity ExT or kept sedentary (Sed) for 12 weeks. Blood pressure elevation coupled with increase in age was significantly decreased in SHR received ExT compared with Sed. The results in vivo showed that ExT significantly reduced or increased the cardiovascular responses to central application of sarthran (antagonist of Ang II) or A779 (antagonist of Ang 1-7), respectively. The protein expression of the Ang II acting receptor AT1R and the Ang 1-7 acting receptor Mas in the RVLM was significantly reduced and elevated in SHR following ExT, respectively. Moreover, production of reactive oxygen species in the RVLM was significantly decreased in SHR following ExT. The current data suggest that ExT improves hypertension via improving the balance of Ang II and Ang 1-7 and antioxidative stress at the level of RVLM.

The PI3K signaling-mediated nitric oxide contributes to cardiovascular effects of angiotensin-(1-7) in the nucleus tractus solitarii of rats.

Angiotensin-1-7 [Ang-(1-7)], acting via the Mas receptor in the central nervous system, is involved in the regulation of cardiovascular activity. Nitric oxide (NO) is implicated as an important modulator in the nucleus tractus solitarii (NTS), a key region involved in control of cardiovascular activity. The aim of the present study was to determine the role of phosphatidylinositol 3-kinase (PI3K) signaling in mediating the effect of Ang-(1-7) on NO generation in the NTS. In Sprague-Dawley rats, acute injection of Ang-(1-7) into the NTS significantly increased NO generation and neuronal/endothelial NO synthase (n/eNOS) activity, which were abolished by the selective Mas receptor antagonist d-Alanine-[Ang-(1-7)] (A-779), the PI3K inhibitor LY294002, or the Akt inhibitor triciribine (TCN). Western blotting analysis further demonstrated that Ang-(1-7) significantly increased levels of Akt/NOS phosphorylation in the NTS, and Ang-(1-7)-induced e/nNOS phosphorylation was antagonized by LY294002 or TCN. Furthermore, gene knockdown of PI3K by lentivirus containing small hairpin RNA in the NTS prevented the Ang-(1-7)-induced increases in NOS/Akt phosphorylation and NO production. The physiological (in vivo) experiments showed that pretreatment with the NOS inhibitor l-NAME, LY294002, or TCN abolished the decreases in blood pressure, heart rate, and renal sympathetic nerve activity induced by Ang-(1-7) injected into the NTS. Our findings suggest that nitric oxide release meditated by the Mas-PI3K-NOS signaling pathway is involved in the cardiovascular effects of Ang-(1-7) in the NTS.

Estrogen Replacement Reduces Oxidative Stress in the Rostral Ventrolateral Medulla of Ovariectomized Rats.

Cardiovascular disease prevalence rises rapidly after menopause, which is believed to be derived from the loss of estrogen. It is reported that sympathetic tone is increased in postmenopause. The high level of oxidative stress in the rostral ventrolateral medulla (RVLM) contributes to increased sympathetic outflow. The focus of this study was to determine if estrogen replacement reduces oxidative stress in the RVLM and sympathetic outflow in the ovariectomized (OVX) rats. The data of this study showed that OVX rat increased oxidative stress in the RVLM and sympathetic tone; estrogen replacement improved cardiovascular functions but also reduced the level of oxidative stress in the RVLM. These findings suggest that estrogen replacement decreases blood pressure and sympathoexcitation in the OVX rats, which may be associated with suppression in oxidative stress in the RVLM through downregulation of protein expression of NADPHase (NOX4) and upregulation of protein expression of SOD1. The data from this study is beneficial for our understanding of the mechanism of estrogen exerting cardiovascular protective effects on postmenopause.

Overexpression of angiotensin-converting enzyme 2 attenuates tonically active glutamatergic input to the rostral ventrolateral medulla in hypertensive rats.

The rostral ventrolateral medulla (RVLM) plays a key role in cardiovascular regulation. It has been reported that tonically active glutamatergic input to the RVLM is increased in hypertensive rats, whereas angiotensin-converting enzyme 2 (ACE2) in the brain has been suggested to be beneficial to hypertension. This study was designed to determine the effect of ACE2 gene transfer into the RVLM on tonically active glutamatergic input in spontaneously hypertensive rats (SHRs). Lentiviral particles containing enhanced green fluorescent protein (lenti-GFP) or ACE2 (lenti-ACE2) were injected bilaterally into the RVLM. Both protein expression and activity of ACE2 in the RVLM were increased in SHRs after overexpression of ACE2. A significant reduction in blood pressure and heart rate in SHRs was observed 6 wk after lenti-ACE2 injected into the RVLM. The concentration of glutamate in microdialysis fluid from the RVLM was significantly reduced by an average of 61% in SHRs with lenti-ACE2 compared with lenti-GFP. ACE2 overexpression significantly attenuated the decrease in blood pressure and renal sympathetic nerve activity evoked by bilateral injection of the glutamate receptor antagonist kynurenic acid (2.7 nmol in 100 nl) into the RVLM in SHRs. Therefore, we suggest that ACE2 overexpression in the RVLM attenuates the enhanced tonically active glutamatergic input in SHRs, which may be an important mechanism underlying the beneficial effect of central ACE2 to hypertension.

Synergic effects of levamlodipine and bisoprolol on blood pressure reduction and organ protection in spontaneously hypertensive rats.

AIMS: Stroke is a major cause of disability and death worldwide. Hypertension is one of the most important risk factors for stroke. The objective of this work was to study the synergic effects of levamlodipine and bisoprolol on blood pressure reduction and organ protection in spontaneously hypertensive rats (SHR). METHODS: Blood pressure was continuously monitored in conscious SHR. For acute study, a single dose of drugs was administrated via an intragastric catheter. For chronic study (4 months), drugs were delivered via rat chow. RESULTS: A single dose of levamlodipine (from 1 mg/kg), bisoprolol (from 0.125 mg/kg), and their combinations significantly decreased blood pressure. The levamlodipine-induced tachycardia and the bisoprolol-induced bradycardia were temporized by the combination of these two drugs. Upon chronic treatment, this combination also decreased blood pressure variability and reduced organ damage. CONCLUSION: Levamlodipine and bisoprolol produce synergic effects on blood pressure reduction and organ protection in SHR.

GABAergic mechanism in the rostral ventrolateral medulla contributes to the hypotension of moxonidine.

AIMS: The depressor action of the centrally antihypertensive drug moxonidine has been attributed to activation of I(1)-imidazoline receptor in the rostral ventrolateral medulla (RVLM). The objective of this study was to determine the role of the γ-aminobutyric acid (GABA) mechanisms in the RVLM in mediating the effect of moxonidine in anaesthetized normotensive rats. METHODS AND RESULTS: The relationship between the effects of microinjection or picoinjection of moxonidine and the functional state of GABA receptors at the level of the RVLM or pre-sympathetic neuron was determined. Microdialysis was performed to detect the effect of moxonidine on the release of GABA in the RVLM. Western blot analysis was carried out to test the effect of chronic intracerebroventricular injection of moxonidine on the protein expression of GABA receptors in the RVLM. Pre-treatment with the GABA(A) or GABA(B) receptor antagonist bicuculline (5 pmol) or CGP35348 (200 pmol), respectively, microinjected into the RVLM significantly attenuated the decrease in blood pressure and renal sympathetic nerve activity induced by moxonidine. In 22 moxonidine-sensitive pre-sympathetic neurons in the RVLM, picoinjection of bicuculline (100 fmol/5 nL) significantly attenuated the neuronal inhibition evoked by moxonidine (100 pmol/5 nL). The release of GABA in the RVLM was increased after intravenous moxonidine (50 µg/kg). Central infusion of moxonidine upregulated the protein expression of both GABA(A) and GABA(B) receptors in the RVLM. CONCLUSION: The current data demonstrate that GABAergic mechanisms in the RVLM are responsible for the hypotension and sympathoinhibition of moxonidine.

Low dose of moxonidine within the rostral ventrolateral medulla improves the baroreflex sensitivity control of sympathetic activity in hypertensive rat.

AIM: To determine the effects of the centrally antihypertensive drug moxonidine injected into the rostral ventrolateral medulla (RVLM) on baroreflex function in spontaneously hypertensive rats (SHR). METHODS: Baroreflex sensitivity control of renal sympathetic nerve activity (RSNA) and barosensitivity of the RVLM presympathetic neurons were determined following application of different doses of moxonidine within the RVLM. RESULTS: Three doses (0.05, 0.5, and 5 nmol in 50 nL) of moxonidine injected bilaterally into the RVLM dose-dependently reduced the baseline blood pressure (BP) and RSNA in SHR. At the highest dose (5 nmol) of moxonidine injection, the maximum gain (1.24%+/-0.04%/mmHg) of baroreflex control of RSNA was significantly decreased. However, the lower doses (0.05 and 0.5 nmol) of moxonidine injection into the RVLM significantly enhanced the baroreflex gain (2.34%+/-0.08% and 2.01%+/-0.07%/mmHg). The moxonidine-induced enhancement in baroreflex function was completely prevented by the imidazoline receptor antagonist efaroxan but not by the alpha(2)-adrenoceptor antagonist yohimbine. A total of 48 presympathetic neurons were recorded extracellularly in the RVLM of SHR. Iontophoresis of applied moxonidine (30-60 nA) dose-dependently decreased the discharge of RVLM presympathetic neurons but also significantly increased the barosensitivity of RVLM presympathetic neurons. CONCLUSION: These data demonstrate that a low dose of moxonidine within the RVLM has a beneficial effect on improving the baroreflex function in SHR via an imidazoline receptor-dependent mechanism.