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. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: Hypertension. 2011 Nov 21;59(1):113–121. doi: 10.1161/HYPERTENSIONAHA.111.182154

Angiotensin II-Induced Hypertension is Modulated by Nuclear Factor-kappaB in the Paraventricular Nucleus

Jeffrey P Cardinale 1, Srinivas Sriramula 1, Nithya Mariappan 1, Deepmala Agarwal 1, Joseph Francis 1
PMCID: PMC3268075  NIHMSID: NIHMS341982  PMID: 22106405

Abstract

Hypertension is considered a low-grade inflammatory condition and understanding the role of transcription factors in guiding this response is pertinent. A prominent transcription factor that governs inflammatory responses and has become a focal point in hypertensive research is Nuclear Factor-kappaB (NFκB). Within the hypothalamic paraventricular nucleus (PVN), a known brain cardio-regulatory center, NFκB becomes potentially even more important in ultimately coordinating the systemic hypertensive response. To definitively demonstrate the role of NFκB in the neurogenic hypertensive response, we hypothesized that PVN NFκB blockade would attenuate angiotensin II (AngII)-induced hypertension. Twelve-week old Sprague-Dawley rats were implanted with radio-telemetry probes for blood pressure measurement and allowed a 7-day recovery. Following baseline blood pressure recordings, rats were administered either continuous NFκB decoy oligodeoxynucleotide infusion or microinjection of a serine mutated Adenoviral Inhibitory-kappaB (AdIκB) vector, or their respective controls, bilaterally into the PVN to inhibit NFκB at two levels of its activation pathway. Simultaneously, rats were implanted subcutaneously with an AngII or saline-filled 14-day osmotic minipump. Following the 2-week treatments, rats were sacrificed and brain tissues collected for PVN analysis. Bilaterally inhibited NFκB rats had a decreased blood pressure, NFκB p65 subunit activity, proinflammatory cytokines and reactive oxygen species, including the AngII type-1 receptor, ACE, TNF and superoxide, in AngII-treated rats. Moreover, following NFκB blockade, key protective anti-hypertensive renin-angiotensin system components were up-regulated. This demonstrates the important role that transcription factor NFκB plays within the PVN in modulating and perpetuating the hypertensive response via renin-angiotensin system modulation.

Keywords: Angiotensin II, hypertension, cytokines, transcription factors, superoxide

Introduction

Hypertension is a condition closely associated with the renin-angiotensin system (RAS) and increased expression of proinflammatory cytokines (PICs) and reactive oxygen species (ROS), in both systemic and local hypertensive responses.1–6 Studies from our laboratory and others have shown that in hypertension, angiotensin II (AngII), PICs and ROS can increase the activity of the transcription factor Nuclear Factor-kappaB (NFκB), which in turn, can further increase PIC and ROS expression in a positive feed-forward manner.5, 7–10

Within the brain, multiple cardio-regulatory regions exhibit a local RAS, including the hypothalamic paraventricular nucleus (PVN), which can synthesize and release both pro- and anti-hypertensive RAS component peptides.2, 11, 12 The PVN is widely recognized as a central integration site for the coordination of autonomic and neuroendocrine responses that regulates thirst, salt appetite and sympathetic outflow.13–15 AngII is a large peptide that cannot cross the blood-brain barrier (BBB). Therefore, it exerts its roles by acting on the circumventricular organs (CVOs), where the BBB is either weak or absent.16, 17 Signals from these CVOs subsequently activate neurons within the various cardio-regulatory centers of the hypothalamus and brainstem, including the PVN17, which can respond by locally producing components of the RAS and via sympathetic signals to the periphery.5

Findings from our laboratory and others have shown that, in hypertension, PICs are increased within discrete brain sites such as the PVN, and that signals from both the systemic and local RASs increase PICs and oxidative stress.5, 18 Within the PVN, RAS components, PICs and ROS have been linked to increased sympathoexcitation and perpetuation of the hypertensive state.19, 20 Based upon the preceding evidence, we hypothesized that bilateral PVN blockade of NFκB would attenuate these observed regional changes which propagate the AngII-induced hyppertensive response, including increases in PICs and ROS. To test this hypothesis, we blocked NFκB within the PVN using two approaches: bilateral PVN NFκB decoy oligodeoxynucleotide infusion, or bilateral PVN microinjection of an Adenoviral vector containing a serine mutated Inhibitory-kappaB (IκB) (AdIκB) insert. These techniques block separate locations in the NFκB transcription activation pathway. Our results demonstrate that blocking NFκB attenuates hypertension through a reduction of PIC and ROS actions within the PVN. NFκB also appears to mediate the balance between the pro-hypertensive and the anti-hypertensive arms of the RAS. This data indicates that PVN specific NFκB plays a role in controlling hypertension through increased PICs and ROS via RAS modulation.

Materials and Methods

Animals

Male Sprague-Dawley rats (12 weeks old, 250–350gms) were used in this study. Animals were housed in a temperature- (25±1°C) and light-controlled (12:12 hour light:dark cycle) room with free access to water and normal rat chow (0.4% salt content). All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Louisiana State University in accordance with NIH guidelines.

Experimental protocol

All experimental rats were anaesthetized and implanted with radio-telemetry transmitters. Following a 7-day recovery, bilateral cannulae were placed into the PVN. Fourteen-day osmotic minipumps (Alzet, model 2002; infusion rate of 0.5 μl/h) were filled with AngII (Bachem, 200ng/kg/min) dissolved in 0.9% saline, or saline alone, and implanted subcutaneously in the retroscapular area. Simultaneously, osmotic minipumps (Alzet, model 1004; infusion rate of 0.11 μl/h) were filled with NFκB decoy or control scrambled decoy oligodeoxynucleotide (2ng/kg/min; Sigma), dissolved in aCSF, implanted subcutaneously in the retroscapular area and connected to the cannula. The NFκB decoy concentration was determined from a previous pilot study in rats using three different doses, 200pg/kg/min, 2ng/kg/min and 200ng/kg/min. The 2ng/kg/min dose was found to be optimal, while the highest dose caused increased mortality and the lowest dose did not produce complete NFκB inhibition as measured using an NFκB (p65) activity assay. Rats were divided into 4 groups: 1) No treatment (Controls; n=11); 2) Saline minipump + bilateral PVN NFkB decoy (Saline+NFκB decoy; n=18); 3) AngII minipump + bilateral PVN scrambled decoy (AngII+Scramble decoy; n=18); and 4) AngII minipump + bilateral PVN NFκB decoy (AngII+NFκB decoy; n=20). To determine the potential effect of NFκB decoy leakage into the brain’s ventricular system, rats (n=7) were administered intracerebroventricularly (ICV) the same dose (2ng/kg/min) at the same flow rate.

Another group of rats were injected (2×1010 pfu/ml, 100nL) bilaterally intra-PVN with an Adenoviral vector (Ad) containing IκB serine mutated at the S23A/S36A positions (AdIκB), or a control Ad with an empty cassette region (AdEmpty; both adenoviruses obtained from Gene Transfer Vector Core, University of Iowa, Carver College of Medicine) using a 1 μl Hamilton syringe, as previously described.12, 21 Fourteen-day osmotic minipumps (Alzet, model 2002; 0.5 μl/h) were filled with AngII dissolved in 0.9% saline or saline alone and simultaneously implanted subcutaneously into the retroscapular area. These rats were divided into 4 groups: 1) No treatment (Controls; n=10); 2) Saline minipump + bilateral PVN AdIκB (Saline+AdIκB; n=19); 3) AngII minipump + bilateral PVN AdEmpty (AngII+AdEmpty; n=18); and 4) AngII minipump + bilateral PVN AdIκB (AngII+AdIκB; n=21). A final group of rats were treated with AngII alone (n=15) and used for western blot, immunohistochemical and electron paramagnetic resonance (EPR) analysis. After 14 days of blood pressure recordings, rats were euthanized using a high ketamine+xylazine dose and brain tissue was collected for mRNA and protein analysis. Blood plasma was used to determine circulating norepinephrine (NE) via HPLC. Rats that received treatment unilaterally into the PVN or had malfunctioning pumps (i.e. tube detachment from pump or cannula, based upon post-mortem analysis) were excluded from the final analysis (success rate: bilateral cannulation ~78%; bilateral microinjection ~65%). Chow salt content did not appear to have any effect on pressure response. A p<0.05 was considered statistically significant. An expanded Methods section can be found in the online data supplement, available at http://hyper.ahajournals.org.

Results

NFκB blockade in the PVN reduces p65 subunit binding activity

The PVN was bilaterally infused with NFκB decoy oligodeoxynucleotide via a fixed cannula (Figure 1A), or bilaterally microinjected with an Adenovirus encoding serine mutated IκB to overexpress IκB and inhibit NFκB within the PVN. Localization of injection sites of all rats are schematically represented in Figure S1 (please see http://hyper.ahajournals.org), with those receiving unilateral or no treatment excluded from the subsequent final analyses. The localization of IκB gene overexpression specifically within the PVN following bilateral PVN AdIκB microinjection was indicated by enhanced IκB fluorescence (Figure 1B; S2A, please see http://hyper.ahajournals.org). To determine the efficacy of the two methods of inhibiting NFκB activity, an NFκB p65 subunit activity assay was conducted following the PVN 14-day treatments. The p65 subunit activity was dramatically increased in the PVN in the two AngII-treated groups versus their respective controls (Figure 1C). This increase in activity was attenuated in both AngII+NFκB decoy and Ang II+AdIκB-treated rats. However, p65 subunit activity was unaltered in the lateral hypothalamus of any group (Figure S2B, please see http://hyper.ahajournals.org), indicating that the effect of NFκB and its blockade was localized to the PVN and not the surrounding regions. This data shows that NFκB is increased within the PVN during AngII-induced hypertension and that the use of NFκB decoy or AdIκB can potently, and site specifically, inhibit NFκB activity.

Figure 1.

Figure 1

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PVN specific inhibition of NFκB. (A) Schematic showing cannulae placement/microinjection stereotaxic location coordinates. (B) Representative immunofluorescence image for IκB is localized in the PVN following AdIκB bilateral microinjection into control rat. (C) NFκB p65 activity assay showing increased activity in the PVN of the AngII-treated groups when compared to control groups. p65 activity is decreased in AngII-treated rats following bilateral PVN NFκB decoy infusion or AdIκB microinjection. n=5–6/group, *p<0.05 vs respective AngII-treated rats, #p<0.05 vs respective control-treated rats.

NFκB blockade in the PVN attenuates the AngII-induced blood pressure response

Chronic 14-day AngII infusion significantly increased the mean arterial pressure (MAP) in rats that received scramble decoy or AdEmpty treatment versus their respective saline-infused controls (Saline+NFκB decoy and Saline+AdIκB) (Figure 2). Conversely, the MAP of AngII+NFκB decoy and AngII+AdIκB rats had a significantly reduced MAP from their AngII-treated counterparts, though the MAP was not normalized. Rats receiving either treatment unilaterally, though showing a reduced MAP versus the AngII control groups, was not reduced as effectively as bilateral treatments (Figure S3, please see http://hyper.ahajournals.org), indicating compensation from the untreated side of the PVN. Furthermore, AngII+ICV NFκB decoy-treated rats had a slightly, though not significantly, reduced MAP versus the AngII+Scramble decoy group (Figure S3A, please see http://hyper.ahajournals.org), demonstrating that the potential effect from ventricle decoy leakage on additional central cardio-regulatory sites is minimal. This data indicates that PVN NFκB plays an important role in regulating blood pressure response in AngII-induced hypertension.

Figure 2.

Figure 2

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Effects of NFκB inhibition on AngII-induced increases in MAP. AngII increased MAP in centrally treated control rats. Bilateral NFκB decoy infusion or AdIκB microinjection into the PVN decreased this AngII-induced increase in MAP. n=7–8/group, *p<0.05 vs respective AngII-treated rats, #p<0.05 vs respective control-treated rats.

NFκB blockade decreases PIC expression in the PVN

AngII infusion significantly increased the mRNA expression of TNF-α, IL-1β, IL-6, and the chemokine MCP-1 in the PVN versus control rats. However, bilateral PVN NFκB decoy infusion or bilateral microinjection of AdIκB into the PVN attenuated these changes in PIC gene expression (Figure 3A). Furthermore, immunohistochemistry against TNF showed an increased staining in AngII-treated rats (AngII alone) versus Controls (no treatment) (Figure 3B; S4, please see http://hyper.ahajournals.org). This protein expression was reduced via bilateral PVN NFκB decoy infusion or bilateral AdIκB microinjection into the PVN, demonstrating that through bilateral PVN NFκB inhibition, PIC levels are reduced within this hypothalamic region.

Figure 3.

Figure 3

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Effects of NFκB inhibition on AngII-induced increases in PIC expression. (A) mRNA expression of the PICs TNF, IL-1β, IL-6 and MCP-1 in the PVN of AngII+Scramble decoy and AngII+AdEmpty-infused rats are increased versus control groups. Expression is decreased following bilateral PVN NFκB decoy infusion or AdIκB microinjection (n=6–8/group). (B) Immunohistochemical staining for TNF is increased in the PVN in AngII-infused rats (AngII alone) versus Controls (no treatment). Staining was decreased following NFκB decoy infusion or AdIκB microinjection in rats treated bilaterally intra-PVN. Representative images from preparations of 5–6 rats. *p<0.05 vs respective AngII-treated rats, #p<0.05 vs respective control-treated rats.

NFκB blockade effects RAS component expression in the PVN

AngII infusion significantly increased the mRNA expression of the pro-hypertensive AT1R and ACE in the PVN when compared with control rat groups, and decreased the anti-hypertensive ACE2 and MasR expression versus their respective control groups (Figure 4A). Bilateral NFκB decoy infusion or microinjection of AdIκB into the PVN reversed these gene expression changes. AngII also decreased the Mas/AT1R ratio within the PVN, which was reversed in AngII+NFκB decoy and AngII+AdIκB groups (Figure S5A, please see http://hyper.ahajournals.org). These results were further confirmed for AT1R and MasR protein levels in the PVN by western blot (Figure 4B) and densitometric (Figure S5B and C, please see http://hyper.ahajournals.org) analysis. These results show that NFκB plays a modulatory role in differential RAS component expression in the PVN during AngII-induced hypertension.

Figure 4.

Figure 4

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Effects of NFκB inhibition on AngII-induced changes in RAS component expression. (A) mRNA expression of AT1R and ACE was increased, while ACE2 and the MasR were decreased in the AngII-treated rat groups versus controls. Bilateral PVN NFκB decoy infusion or AdIκB microinjection reversed these changes. (B) Western blots show increased AT1R and decreased MasR in AngII-treated rats (AngII alone) versus Controls (no treatment). Bilateral PVN NFκB decoy infusion or AdIκB microinjection reversed these changes. n=6–8/group, *p<0.05 vs respective AngII-treated rats, #p<0.05 vs respective control-treated rats.

NFκB blockade reduces ROS production in the PVN

Total ROS, superoxide (O2•−) and peroxynitrite (OONO•) levels were significantly increased in the PVN of AngII-treated rats (AngII alone) versus Controls (no treatment) (Figure 5). These ROS levels were decreased to normal following chronic bilateral NFκB decoy infusion or AdIκB microinjection into the PVN, indicating the role that NFκB plays in increasing ROS during the hypertensive response.

Figure 5.

Figure 5

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Effects of NFκB inhibition on AngII-induced changes in ROS levels. AngII-infused rats (AngII alone) had an increase in total ROS, superoxide and peroxynitrite within the PVN versus Control rats (no treatment). Bilateral PVN NFκB decoy infusion or AdIκB microinjection decreased these ROS changes within the PVN. EPR spectra (right) for total ROS and superoxide. Graphical data when compared to total protein/sample. n=5–6/group, *p<0.05 vs AngII-treated rats, #p<0.05 vs Controls.

NFκB blockade increases nNOS expression in the PVN and decreased circulating plasma norepinephrine (NE)

Neuronal NOS (nNOS) expression is an indirect indicator of neuronal activity. In AngII-treated rat groups, nNOS mRNA was significantly decreased when compared to the control groups (Figure 6A). Bilateral NFκB decoy infusion or AdIκB microinjection into the PVN reversed and elevated these AngII-induced changes above that of the normotesive controls. These results were further confirmed with immunohistochemistry against nNOS (Figure 6B; S6A, please see http://hyper.ahajournals.org), where AngII-infused rats (AngII alone) had decreased nNOS presence versus Controls (no treatment), which was reversed by NFκB blockade. Furthermore, plasma NE was increased in AngII-treated rats when compared to controls, but normalized following bilateral NFκB decoy infusion or AdIκB microinjection into the PVN (Figure S6B, please see http://hyper.ahajournals.org). These results indicate that NFκB within the PVN potentially augments neuronal activity and sympathoexcitation, and that NFκB inhibition within this region can attenuate these AngII-induced hypertensive changes.

Figure 6.

Figure 6

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Effects of NFκB inhibition on AngII-induced increases in nNOS expression. (A) mRNA expression of nNOS in the PVN of AngII-infused rat groups is decreased versus control groups, but increased following bilateral PVN NFκB decoy infusion or AdIκB microinjection (n=6–8/group). (B) Immunohistochemical staining for nNOS is increased in the PVN in AngII-infused rats (AngII alone) versus Controls (no treatment). Staining was increased in rats treated bilaterally intra-PVN via NFκB decoy infusion or AdIκB microinjection. Representative images from preparations of 5–6 rats. *p<0.05 vs respective AngII-treated rats, #p<0.05 vs respective control-treated rats.

Discussion

In the present study, we investigated the effects of bilateral hypothalamic PVN specific blockade of NFκB on the AngII-induced hypertensive response. The salient findings of this study are as follows: 1) peripheral AngII infusion increases MAP, which is attenuated by bilateral PVN NFκB blockade; 2) peripheral AngII infusion increases PICs, ROS and pro-hypertensive (ACE and AT1R) RAS components, and decreases the anti-hypertensive (ACE2 and Mas) RAS components within the PVN; 3) bilateral PVN specific NFκB inhibition of AngII-infused rats not only decreases PVN PICs, but also modulates RAS component expression, such as decreasing ACE and AT1R expression and increasing ACE2 and MasR expression; 4) PVN blockade of NFκB leads to an increase in nNOS expression and a decrease in superoxide, peroxynitrite and circulating NE levels. These results indicate that bilateral PVN NFκB blockade decreases the deleterious pro-hypertensive RAS arm and increases the protective anti-hypertensive RAS arm, possibly through a ROS-mediated mechanism, thereby attenuating the AngII-induced hypertensive response.

Upon stimulation, NFκB is released following IκB phosphorylation, ubiquination and degradation, freeing NFκB and allowing its nuclear translocation to act on κB binding sites and commence transcription.22 In this study, two approaches were utilized to block NFκB within the PVN. AdIκB binds to NFκB similar to endogenous IκB, but serine mutations at the S32A/S36A position prevent IκB phosphorylation and the ensuing NFκB release, thereby inhibiting its capability to translocate into the nucleus and transcribe target genes.22 Decoy oligodeoxynucleotides act by targeting and adhering to the cis-element binding sites of free NFκB, preventing its attachment to κB binding sites and blocking subsequent gene transcription.23, 24 By blocking NFκB at two separate activation pathway locations, it effectively demonstrates the role of PVN NFκB in AngII-induced hypertension and potentially signifies that there are no secondary pathways activated between NFκB/IκB release and its nuclear translocation/binding. Previous findings from our lab investigating the PVN’s role in AngII-induced hypertension demonstrated that NFκB within the PVN was differentially regulated along with PIC and oxidative stress genes and proteins.5 This work presented the involvement of brain NFκB in regulating the hypertensive response, a previously novel proposal. However, ICV infusion of PDTC, a known antioxidant25, was used to study NFκB in the PVN, signifying that NFκB and its subsequent actions could have been reduced through an antioxidant-driven mechanism rather than through direct NFκB intervention. Also, due to ICV administration, we could not rule out the possible involvement of other affected cardio-regulatory regions for the observed reduction in the hypertensive response. Therefore, the exact role and proposed involvement/mechanism of NFκB within the PVN remains uncertain. The current study looks at NFκB blockade specifically within the PVN in the AngII-induced hypertensive response, delineates the involvement of PVN NFκB in regulating ROS and PIC expression, and perhaps more importantly, shows that NFκB serves as a potential tipping point between the injurious pro-hypertensive and protective anti-hypertensive RAS axes within the PVN.

Extensive evidence implicates the RAS within the brain in hypertension.2, 12, 26, 27 In the PVN, AngII is increased in multiple hypertensive models, and PVN blockade of the AT1R partially inhibits the effects of AngII-induced hypertension.5, 14, 26–28 In the current study, NFκB blockade within the PVN decreases ACE and the AT1R, thereby limiting the effect that AngII could have on perpetuating AngII-induced hypertension. Moreover, NFκB inhibition also increased ACE2 and MasR expression, as well as improved the MasR/AT1R ratio, indicating enhancement of the protective anti-hypertensive RAS axis. ACE converts AngI to AngII. ACE2, however, converts AngI and AngII to Ang(1–7),29 which acts on the MasR to elicit actions opposing those of ACE/AngII, including vasorelaxation and decreased sympathetic activity.30–32 Our results agree with other findings that detail the anti-hypertensive actions of ACE2 and the MasR,30, 32 including their decreased activity following, and interplay with, AT1R activation within the brain.12, 21, 31, 33 Combined, these results suggest NFκB as an important balance point between the protective (ACE2/Ang(1–7)/Mas) and non-protective (ACE/AngII/AT1R) arms of the RAS, and that NFκB blockade promotes the more beneficial actions of ACE2 and the MasR.

The association between the RAS and elevated PICs in hypertension has often been explored.1, 15, 19, 20 NFκB is a key regulator of the PIC expression and inflammatory response observed in hypertension.5, 8, 34 Here, we show that bilateral NFκB inhibition in the PVN reduces PIC expression, establishing not only the definitive involvement of NFκB within the PVN in regulating the AngII-induced hypertensive pressure response, but also that PICs may play a role in RAS modulation. Recently, ROS, especially O2•−, have been shown as important signaling factors within the brain for enhancement of the neurogenic hypertensive response through both AngII and PIC mechanisms.9, 10, 35 Here, bilateral NFκB blockade reduced the ROS response, including O2•−, thus potentially inhibiting one of the mechanistic pathways by which the hypertensive response and sympathoexcitation (as indicated by decreased plasma NE), is modulated. These results are further confirmed by previous experiments in our lab showing that ICV administration of the ROS scavenger, Tempol, reduced renal sympathetic nerve activity in AngII-treated rats.5 This shows that PVN specific NFκB blockade reduces the PIC and ROS reactions typically associated with AngII-induced hypertension and highlights the central position that PVN NFκB plays in regulating the neurogenic component of hypertension.

Symapthoexcitation is a component of hypertension, and increased levels of AngII in the central nervous system (CNS) and PVN can enhance sympathetic outflow through increased ROS, AT1R and NE activation and a subsequent down-regulation of nNOS.4, 36–39 The expression and presence of nNOS, an indirect indicator of neuronal activity and downstream sympathetic activity, is inversely proportional to the level of sympathetic outflow,40–42 and NO, a well known sympatho-inhibitory neurotransmitter, when blocked, results in elevated MAP and sympathoexcitation.37, 43 Furthermore, increased superoxide can interact with the decreasing nNOS-produced NO, forming OONO•, further reducing NO bioavailability and resulting in enhanced sympathoexcitation.44 Recently, Ang(1–7) and MasR activation have been shown to increase nNOS activity and NO release.45 The current study shows that following AngII treatment, nNOS is decreased within the PVN, potentially indicating increased sympathetic outflow. Also, bilateral NFκB inhibition decreased elevated AngII-induced OONO•. This OONO• decrease paralleled that of O2•− and was concurrent with the nNOS increase, indicating that NFκB potentially plays a deciding role in regulating NO availability and sympathoexcitation. These results were reinforced by circulating NE, which was normalized in AngII-infused rats following bilateral NFκB blockade. Therefore, bilateral NFκB inhibition increased nNOS and reduced O2•−, OONO• and plasma NE, possibly through balancing RAS components, and thus potentially reducing sympathoexcitation.

In conclusion, this study shows that following the AngII-activation of the pro-hypertensive RAS arm, PICs are increased, which separately and together, can act to increase the activity of NFκB and lead to the transcription of additional pro-hypertensive modulators in a positive feed-forward manner. NFκB acts to increase, along with AngII, the presence of ROS, such as O2•−, which subsequently affects the present NO levels, thereby effecting neuronal activity/function and NE release. Blockade of NFκB at two separate locations in its activation pathway prevents these changes, restores the RAS balance and promotes the anti-hypertensive RAS arm, including ACE2 and the MasR. It also reduces PIC and ROS expression and elevates nNOS, all of which contributes to a reduction in MAP and an improvement in the AngII-induced hypertensive state. However, this signaling mechanism must be further studied to delineate the manner by which NFκB and O2•− interact within the PVN in AngII-induced hypertension. We propose that AngII activation of NFκB increases PICs and O2•−, tipping the balance of the RAS in favor of the pro-hypertensive arm and decreasing the anti-hypertensive arm, resulting in a further increase in PIC and ROS expression, in a vicious positive feed-forward mechanism (Figure S7, please see http://hyper.ahajournals.org). Limitations for this study include the use of the AngII hypertensive model, since this does not represent all modes of hypertension. Also, we only explored the PVN region, though there are multiple cardio-relevant sites in the brain that can play a role in modulating the hypertensive response; however, we feel that the PVN is of importance due to its recognized integrative functions. Moreover, as the literature suggests, Adenoviuses are well known for their lack of cell specificity and can be expressed by neurons and glia as well as participate in retrograde transport.46 For this reason, we employed NFκB decoy oligodeoxynucleotides to further verify our results. Thus, in the current clinical environment where novel hypertensive therapeutic measures are being sought, this study provides a conceptual basis for including NFκB inhibitors that can specifically act within the brain as a possible future pharmacological approach for the treatment of hypertension.

Perspectives

Increasing evidence indicates that CNS mechanisms play an important role in the pathogenesis of cardiovascular disease. In this study, we demonstrate that inflammatory molecules, specifically transcription factor NFκB, within the PVN, can modulate the hypertensive response. Additionally, we demonstrate that inflammation is a double-edged weapon that not only up-regulates the deleteriou pro-hypertensive RAS axis, but also down-regulates the protective anti-hypertensive RAS axis. Thus, inflammatory-mediated modulation of the brain RAS might be an important critical contributor to neurogenic hypertension. Since inflammation and the RAS are potent inducers of oxidative stress, and NFκB has been shown to respond to and induce oxidative stress, it may be advantageous to target NFκB to better treat hypertension. While the current methods used within this study are impractical for current clinical administration, one can explore the use of NFκB small molecule inhibitors that cross the BBB, thereby targeting the brain’s source of inflammation and oxidative stress, for controlling and treating this debilitating condition.

Supplementary Material

1

Acknowledgments

The authors thank Dr. Romain Pariaut for his extensive criticisms and suggestions regarding this manuscript and Dr. Philip J. Ebenezer for his assistance with the HPLC.

Sources of Funding

These studies were supported by National Heart, Lung and Blood Institute Grant HL-80544 (to J. Francis).

Footnotes

Disclosures

None

References

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