Toll-like receptor 4 inhibition within the paraventricular nucleus attenuates blood pressure and inflammatory response in a genetic model of hypertension - PubMed (original) (raw)
Toll-like receptor 4 inhibition within the paraventricular nucleus attenuates blood pressure and inflammatory response in a genetic model of hypertension
Rahul B Dange et al. J Neuroinflammation. 2015.
Abstract
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.
Figures
Figure 1
An immunofluorescence double labeling image (x 20) showing the effects of intra-PVN infusion of VIPER on protein expression of TLR4 and NeuN in the PVN of WKY and SHR rats. n = 5/group. SHR + CP rats showed higher levels of immunofluorescence for TLR4 within the neurons of PVN, whereas, VIPER infusion in these rats caused significant reduction in TLR4 expression. Arrow indicates double- labeled cells.VIPER infusion in saline-infused rats did not have any effects. Scale bar 20 μm: CP, control peptide; NeuN, neuronal nuclei; PVN, paraventricular nucleus; SHR, spontaneously hypertensive rat; TLR4, Toll-like receptor 4; VIPER, viral inhibitory peptide of TLR4; WKY, wistar-Kyoto.
Figure 2
An immunofluorescence double labeling image (x 40) showing the effects of intra-PVN infusion of VIPER on protein expression of TLR4 and CD11B in the PVN of WKY and SHR rats. SHR + CP rats showed modest expression of TLR4 within the microglia of PVN, whereas, VIPER infusion in these rats caused significant reduction in TLR4 expression. Arrow indicates double-labeled cells.VIPER infusion in saline-infused rats did not have any effects. n = 5/group. Scale bar 20 μm : CD11B, cluster of differentiation molecule 11B; CP, control peptide; PVN, paraventricular nucleus; SHR, spontaneously hypertensive rat; TLR4, Toll-like receptor 4; VIPER, viral inhibitory peptide of TLR4; WKY, wistar-Kyoto.
Figure 3
An immunofluorescence double labeling image (x 20) showing the effects of intra-PVN infusion of VIPER on protein expression of TLR4 and GFAP in the PVN of WKY and SHR rats. n = 5/group. Scale bar 20 μm : GFAP, glial fibrillary acidic protein; PVN, paraventricular nucleus; SHR, spontaneously hypertensive rat; TLR4, Toll-like receptor 4; VIPER, viral inhibitory peptide of TLR4; WKY, wistar-Kyoto.
Figure 4
Effects of PVN infusion of VIPER on gene and protein expression of TLR4 and HMGB1 in WKY and SHR rats. (A) mRNA expression of TLR4; (B) protein expression of TLR4; (C) A representative immunoblot; (D) mRNA expression of HMGB1; (E) protein expression of HMGB1; and (F) plasma levels of HMGB1. Values are means ± SE. n = 8 in each group for mRNA analysis, n = 5 in each group for western blot, and n = 8 for plasma HMGB1. ***P <0.001 : HMGB1, high mobility group box 1; PVN, paraventricular nucleus; SHR, spontaneously hypertensive rat; TLR4, Toll-like receptor 4; VIPER, viral inhibitory peptide of TLR4; WKY, wistar-Kyoto.
Figure 5
Effect of bilateral intra-PVN infusion of VIPER on mean arterial blood pressure (MAP, in millimeters of mercury) and cardiac hypertrophy in WKY and SHR rats. (A) SHR + CP group had significantly increased MAP when compared to WKY + CP rats. Interestingly, infusion of VIPER in PVN of SHR rats for 14 days resulted in significant decrease in MAP, starting from day 4 of VIPER infusion; (B) heart weight to body weight ratio; and (C) mRNA expression of atrial natriuretic peptide (ANP) in tissue obtained from left ventricle. Values are mean ± SE; n = 8/group for mRNA analysis and n = 14 for MAP. *P <0.05 SHR + CP versus WKY + CP; #P < 0.05 SHR + VIPER versus SHR + CP; ***P <0.001 : ANP, Atrial Natriuretic Peptide; CP, control peptide; MAP, mean arterial pressure; PVN, paraventricular nucleus; SHR, spontaneously hypertensive rat; TLR4, Toll-like receptor 4; VIPER, viral inhibitory peptide of TLR4; WKY, wistar-Kyoto.
Figure 6
Effects of bilateral intra-PVN infusion of VIPER on gene and protein expression of pro- and anti-inflammatory cytokines and iNOS in WKY and SHR rats. (A) mRNA expression of TNF-α; (B) mRNA expression of IL1-β; (C) A representative immunoblot; (D) densitometric analysis of protein expression of TNF-α, IL1-β, IL10 and iNOS; (E) mRNA expression of IL10; and (F) mRNA expression of iNOS. Values are means ± SE. n = 8 in each group for mRNA analysis and n = 5 in each group for western blot. ***P <0.001; *P <0.05 SHR + CP versus WKY + CP; #P <0.05 SHR + VIPER versus SHR + CP: CP, control peptide; IL-1β, interleukin-1 beta; IL10, interleukin10; iNOS, inducible nitric oxide; PVN, paraventricular nucleus; SHR, spontaneously hypertensive rat; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor-alpha; VIPER, viral inhibitory peptide of TLR4; WKY, wistar-Kyoto. Furthermore, IL-10 gene and protein expression in the PVN tissue was measured to assess the effect of TLR4 blockade on anti-inflammatory axis. A significant reduction in IL-10 gene and protein expression in the SHR + CP compared with the WKY + CP rats was evident (Figure 6 C and E). Interestingly, the SHR + VIPER group had significantly higher levels of IL-10 in comparison with the SHR + CP rats, whereas there was no difference between the WKY + CP and WKY + VIPER groups.
Figure 7
Effects of bilateral intra-PVN infusion of VIPER on (A) NFκB activity of PVN tissue and (B) plasma levels of norepinephrine (NE) in WKY and SHR rats. NFκB activity assay showing increased activity in PVN of SHR + CP rats when compared to WKY + CP rats, whereas, VIPER infusion in SHR rats resulted in significant reduction in the NFκB activity. Similar trends were observed with regard to plasma norepinephrine levels as well. Values are means ± SE. n = 6/group for NFκB activity; n = 8 for plasma NE analysis. ***P <0.001. CP, control peptide; NE, norepinephrine; NFĸB, nuclear factor-kappa B; PVN, paraventricular nucleus; SHR, spontaneously hypertensive rat; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor-alpha; VIPER, viral inhibitory peptide of TLR4; WKY, wistar-Kyoto.
Figure 8
Schematic illustrating proposed mechanism of TLR4 activation and downstream signaling in the PVN hypertension. One of the mechanisms of sustained elevation of blood pressure in essential hypertension could be due to increased activation of TLR4 in the PVN and subsequent activation of NFκB pathway to produce inflammatory alterations, sympathoexcitation, and cardiac hypertrophy. Blockade of TLR4 in the PVN attenuates hypertensive response and prevents detrimental inflammatory changes associated with hypertension. Besides reduction in MAP in VIPER treated SHR rats, we also observed reduction in plasma NE levels. Therefore these beneficial effects might be also due to reduction in plasma NE and HMGB1 levels as observed in this study: HMGB1, high mobility group box 1; MAP, mean arterial pressure; NE, norepinephrine; NFĸB, nuclear factor-kappa B; PVN, paraventricular nucleus; SHR, spontaneously hypertensive rat; TLR4, Toll-like receptor 4; VIPER, viral inhibitory peptide of TLR4.
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