Toll-like receptor 4 inhibition within the paraventricular nucleus attenuates blood pressure and inflammatory response in a genetic model of hypertension

© 2015 Dange et al. Background: Despite the availability of several antihypertensive medications, the morbidity and mortality caused by hypertension is on the rise, suggesting the need for investigation of novel signaling pathways involved in its pathogenesis. Recent evidence suggests the role of toll-like receptor (TLR) 4 in various inflammatory diseases, including hypertension. The role of the brain in the initiation and progression of all forms of hypertension is well established, but the role of brain TLR4 in progression of hypertension has never been explored. Therefore, we investigated the role of TLR4 within the paraventricular nucleus (PVN; an important cardioregulatory center in the brain) in an animal model of human essential hypertension. We hypothesized that a TLR4 blockade within the PVN causes a reduction in mean arterial blood pressure (MAP), inflammatory cytokines and sympathetic drive in hypertensive animals. Methods: Spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats were administered either a specific TLR4 blocker, viral inhibitory peptide (VIPER), or control peptide in their PVN for 14 days. MAP was recorded continuously by radiotelemetry. PVN and blood were collected for the measurement of pro-inflammatory cytokines (Tumor Necrosis Factor (TNF)-α, interleukin (IL)-1β), anti-inflammatory cytokine IL-10, inducible nitric oxide synthase (iNOS), TLR4, nuclear factor (NF) ΚB activity and plasma norepinephrine (NE) and high mobility group box (HMGB)1 expression, respectively. Results: Hypertensive rats exhibited significantly higher levels of TLR4 in the PVN. TLR4 inhibition within the PVN attenuated MAP, improved cardiac hypertrophy, reduced TNF-α, IL-1β, iNOS levels, and NFΚB activity in SHR but not in WKY rats. These results were associated with a reduction in plasma NE and HMGB1 levels and an increase in IL-10 levels in SHR. Conclusions: This study demonstrates that TLR4 upregulation in PVN plays an important role in hypertensive response. Our results provide mechanistic evidence that hypertensive response in SHR are mediated, at least in part, by TLR4 in the PVN and that inhibition of TLR4 within the PVN attenuates blood pressure and improves inflammation, possibly via reduction in sympathetic activity

Angiotensin II causes imbalance between pro- and anti-inflammatory cytokines by modulating GSK-3β in neuronal culture

BACKGROUND AND PURPOSE: Emerging evidence indicates that the balance between pro-inflammatory cytokines (PICs) and anti-inflammatory cytokines (AICs) within the brain is an important determinant in the outcome of hypertension. However, the mechanism by which this dysregulation occurs is not known. We aimed to investigate whether AngII induces imbalance between PIC and AIC by modulating downstream transcription factors, NFκB and cyclic AMP response element-binding protein (CREB), and whether AngII-induced effects are mediated by glycogen synthase kinase-3β (GSK-3β). EXPERIMENTAL APPROACH: CATH.a neurons were exposed to AngII (10 nM-1 μM) over a preset time course. In another set of experiments, GSK-3β was knock down by using lentivirus containing short hairpin RNA targeting GSK-3β (L-sh-GSK3β) before AngII exposure. Cell extracts were subjected to RT-PCR, immunoblot and immunoprecipitation. KEY RESULTS: AngII caused time-dependent increase in PICs (TNF-α and IL-1β) and reduction in AIC (IL-10). AngII exposure caused reduced phosphorylated CREB(Ser-133) and increased p-NFκB(Ser-276) levels, leading to reduced CREB-CBP and increased NFκB-CBP binding. These results were accompanied by increased activation of GSK-3β, as indicated by increased p-GSK3(Tyr-216) to p-GSK3(Ser-9) ratio. In a subsequent study, pretreatment with L-sh-GSK3β attenuated AngII-induced alterations in PICs and IL-10 by augmenting CREB-CBP and attenuating NFκB-CBP binding. CONCLUSIONS AND IMPLICATIONS: Collectively, these findings are the first to provide direct evidence that AngII-induced dysregulation in cytokines is mediated by GSK-3β-mediated alterations in downstream transcription factors in neuronal cells. Our data also reveal that AngII-induced effects could be alleviated by GSK-3β inhibition, suggesting GSK-3β as an important therapeutic target for hypertension that is characterized by increased PICs and NFκB activation

Detraining differentially preserved beneficial effects of exercise on hypertension: effects on blood pressure, cardiac function, brain inflammatory cytokines and oxidative stress.

AIMS: This study sought to investigate the effects of physical detraining on blood pressure (BP) and cardiac morphology and function in hypertension, and on pro- and anti-inflammatory cytokines (PICs and AIC) and oxidative stress within the brain of hypertensive rats. METHODS AND RESULTS: Hypertension was induced in male Sprague-Dawley rats by delivering AngiotensinII for 42 days using implanted osmotic minipumps. Rats were randomized into sedentary, trained, and detrained groups. Trained rats underwent moderate-intensity exercise (ExT) for 42 days, whereas, detrained groups underwent 28 days of exercise followed by 14 days of detraining. BP and cardiac function were evaluated by radio-telemetry and echocardiography, respectively. At the end, the paraventricular nucleus (PVN) was analyzed by Real-time RT-PCR and Western blot. ExT in AngII-infused rats caused delayed progression of hypertension, reduced cardiac hypertrophy, and improved diastolic function. These results were associated with significantly reduced PICs, increased AIC (interleukin (IL)-10), and attenuated oxidative stress in the PVN. Detraining did not abolish the exercise-induced attenuation in MAP in hypertensive rats; however, detraining failed to completely preserve exercise-mediated improvement in cardiac hypertrophy and function. Additionally, detraining did not reverse exercise-induced improvement in PICs in the PVN of hypertensive rats; however, the improvements in IL-10 were abolished. CONCLUSION: These results indicate that although 2 weeks of detraining is not long enough to completely abolish the beneficial effects of regular exercise, continuing cessation of exercise may lead to detrimental effects

Central blockade of TLR4 improves cardiac function and attenuates myocardial inflammation in angiotensin II-induced hypertension

AIMS: Understanding the novel signalling pathways involved in the pathogenesis of hypertension is vital for the development of effective therapeutic strategies. Recent evidence suggests a role for Toll-like receptor (TLR) 4 in the development of cardiovascular diseases. Although brain has been implicated in the pathogenesis of hypertension, the role of brain TLR4 in hypertension is largely unexplored. Therefore, we investigated the role of brain TLR4 in angiotensin (Ang) II-induced hypertension and whether central TLR4 blockade has cardioprotective effects in hypertension. METHODS AND RESULTS: Hypertension was induced in male Sprague-Dawley rats by delivering AngII for 14 days. The rats were administered either specific TLR4 blocker, viral inhibitory peptide (VIPER), or control peptide, intracerebroventricularly. Blood pressure, and cardiac hypertrophy and function, was evaluated by radiotelemetry and echocardiography, respectively. Blood and paraventricular nucleus were collected for measurement of plasma norepinephrine (NE), tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and TLR4 expression, respectively. Heart was analysed for TNF-α, IL-1β, inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NFκB), and renin-angiotensin system (RAS) components. Hypertensive rats had dramatically increased TLR4 expression compared with normotensive rats. Central blockade of TLR4 delayed progression of hypertension and improved cardiac hypertrophy and function in hypertensive rats. TLR4 blockade significantly reduced myocardial TNF-α, IL-1β, iNOS levels, NFκB activity, and altered RAS components in hypertensive rats. These results were associated with reduced circulating NE levels in VIPER-treated hypertensive rats. CONCLUSION: These results provide mechanistic evidence that AngII-induced hypertensive effects are mediated, at least in part, by brain TLR4, and that brain TLR4 blockade attenuates AngII-induced hypertensive response, possibly via down-regulation of myocardial inflammatory molecules and sympathetic activity

Effects of exercise on Cu/ZnSOD in the PVN of normotensive and hypertensive rats.

<p>Densitometric analysis of protein expression (upper panel) and a representative Western blot (lower panel) showed that detraining completely abolished exercise-mediated increase in Cu/ZnSOD levels.Values are mean±SE. n = 6 per group. *p<i><</i>0.05 Sal+Sed versus AngII+Sed; ^#p<0.05 AngII+Sed versus AngII+Ex and AngII+Sed versus AngII+Det; ^@p<0.05 AngII+Ex versus AngII+Det; ^$AngII+Sed versus AngII+Det.</p

Time course of mean arterial pressure (MAP, in millimeters of mercury) in normotensive and hypertensive rats.

<p>MAP was significantly increased in AngII+Sed compared with Sal+Sed rats from day 8 of AngII infusion (empty arrow). MAP was significantly reduced in AngII+Ex compared with AngII+Sed rats from day 16 of exercise (filled arrow). 2 weeks of detraining did not abolish the exercise-induced reduction in MAP in AngII-infused rats. Values are mean±SE; n = 6 per group. *p<i><</i>0.05 Sal+Sed versus AngII+Sed; ^#p<0.05 AngII+Sed versus AngII+Ex; ^$p<0.05 AngII+Sed versus AngII+Det.</p

Baseline characteristic of studied rats: BW, MAP, and Echocardiographic Analysis of Cardiac Hypertrophy and Function.

<p>Values are mean ±SE. Sal+Sed, saline+sedentary; Sal+Ex, saline+exercise; Sal+Det, saline+detraining; AngII+Sed, angiotensionII+sedentary; AngII+Ex, angiotensinII+exercise; AngII+Det, angiotensinII+detraining. BW(g), body weight (grams); MAP, mean arterial pressure (mmHg). LVIDd and LVIDs indicate left ventricular internal diameter at diastole and systole, respectively; IVSTd and IVSTs, interventricular septal thickness at diastole and systole, respectively; LVPWTd and LVPWTd, left ventricle posterior wall thickness at diastole and systole, respectively; FS, fractional shortening (%); EF (%), ejection fraction; HR, heart rate; bpm, beats per minute.</p

Rat primers used for real-time RT-PCR.

<p>IL, Interleukin; TNF-α, Tumor necrosis factor-alpha; gp91^phox, NADPH oxidase subunit; iNOS, Inducible nitric oxide synthase; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.</p

Effect of Exercise and Detraining on Weights, MAP, and HR of rats.

<p>Values are mean ±SE. Sal+Sed, saline+sedentary; Sal+Ex, saline+exercise; Sal+Det, saline+detraining; AngII+Sed, angiotensinII+sedentary; AngII+Ex, angiotensinII+exercise; AngII+Det, angiotensinII+detraining. BW(g), body weight (grams); HM (g), heart mass (grams); HM/BW (mg/g), heart mass to body weight ratio; MAP, mean arterial pressure (mmHg); HR, heart rate; bpm, beats per minute.</p>*<p>p<0.05 Sal+Sed vs AngII+Sed;</p>#<p>p<0.05 AngII+Sed vs AngII+Ex.</p