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. Author manuscript; available in PMC: 2012 Feb 1.
Published in final edited form as: Hypertension. 2010 Dec 20;57(2):289–297. doi: 10.1161/HYPERTENSIONAHA.110.160564

In Vivo Bioluminescence Imaging Reveals Redox-regulated AP-1 Activation in Paraventricular Nucleus of Mice with Renovascular Hypertension

Melissa A Burmeister 1, Colin N Young 1, Valdir A Braga 1, Scott D Butler 1, Ram V Sharma 1,2, Robin L Davisson 1,2
PMCID: PMC3026319  NIHMSID: NIHMS259741  PMID: 21173341

Abstract

Renovascular hypertension (RVH) in mice is characterized by an elevation in hypothalamic angiotensin-II (Ang-II) levels. The paraventricular nucleus (PVN) is a major cardioregulatory site implicated in the neurogenic component of RVH. Increased superoxide (O2−·) production in the PVN is involved in Ang-II-dependent neuro-cardiovascular diseases such as hypertension and heart failure. Here we tested the hypothesis that excessive O2−· production and activation of the redox-regulated transcription factor activator protein-1 (AP-1) in PVN contributes to the development and maintenance of RVH. Male C57Bl/6 mice underwent implantation of radiotelemeters, bilateral PVN injections of an adenovirus (Ad) encoding superoxide dismutase (AdCuZnSOD) or a control gene (LacZ), and unilateral renal artery clipping (2K1C) or sham surgery. AP-1 activity was longitudinally monitored in vivo by bioluminescence imaging in 2K1C or sham mice that had undergone PVN-targeted microinjections of an Ad encoding the firefly luciferase (Luc) gene downstream of AP-1 response elements (AdAP-1Luc). 2K1C evoked chronic hypertension and an increase in O2−· production in the PVN. Viral delivery of CuZnSOD to the PVN not only prevented the elevation in O2−·, but also abolished RVH. 2K1C also caused a surge in AP-1 activity in the PVN, which paralleled the rise in O2−· production in this brain region, and this was prevented by treatment with AdCuZnSOD. Finally, Ad-mediated expression of a dominant-negative inhibitor of AP-1 activity in the PVN prevented 2K1C-evoked hypertension. These results implicate oxidant signaling and AP-1 transcriptional activity in the PVN as key mediators in the pathogenesis of RVH.

Keywords: two-kidney, one-clip (2K1C), Goldblatt, CuZnSOD, superoxide, adenovirus-mediated gene transfer

INTRODUCTION

The central nervous system (CNS) is strongly implicated in the pathogenesis of renovascular hypertension (RVH), a disease that is characterized by renal artery stenosis most commonly caused by atherosclerosis1. The stenotic kidney responds to reduction in perfusion pressure by secreting renin from juxtaglomerular cells, which leads to an initial increase in circulating levels of angiotensin II (Ang-II) and an elevation in blood pressure2. However, as demonstrated in experimental animal models of RVH such as two-kidney, one-clip (2K1C) Goldblatt hypertension, initial elevations in circulating Ang-II levels subside and renal homeostasis returns to normal in later phases; nevertheless, pathologic hypertension is maintained3, 4. While a number of potential mechanisms may contribute (e.g., altered renal reflexes), several laboratories, including ours, have identified the brain renin-angiotensin system (RAS), i.e., Ang-II that is produced and acts specifically in the brain, as a critical mediator of chronic hypertension in this model3–5.

Abundant evidence now suggests that a key mechanism in the neurogenic control of blood pressure is the production of NADPH-oxidase (Nox)-derived reactive oxygen species (ROS) such as superoxide (O2−·) in the CNS6–8. Work from our laboratory and others has shown that Ang-II, administered either directly into the CNS or systemically in subpressor doses over weeks (i.e. “slow-pressor”) causes hypertension along with Ang-II receptor 1 (AT1R)- and Nox-mediated ROS production in central cardioregulatory nuclei9–11. Both the ROS production and hypertension can be prevented by genetic or chemical ROS scavengers or Nox inhibitors administered in the brain 9, 12, 13, suggesting a causative role of brain ROS signaling in Ang-II-dependent hypertension.

The paraventricular nucleus (PVN) of the hypothalamus is a key site of autonomic and neurohumoral regulation, and both Ang-II and ROS are known to be potent mediators of these processes. Either local production of Ang-II in the PVN or activation of angiotensinergic signaling in the PVN through blood-borne Ang-II binding AT1R in upstream circumventricular organs can cause alterations in sympathetic reflexes/activity, baroreflex sensitivity and/or secretion of arginine vasopressin (AVP)14. Recently, oxidant signaling has been shown to be important in at least some of these Ang-II-mediated effects in the PVN. For example, the cardiac sympathetic afferent reflex involves Ang-II-mediated ROS formation in the PVN15. In addition, both acute central16 and chronic systemic pressor effects of Ang-II17 are linked to Nox-dependent ROS formation in the PVN. Recent work from our laboratory shows that Nox4-mediated O2−· production in the PVN mediates sympathoexcitation and cardiac dysfunction in a mouse model of heart failure18. Blood-borne Ang-II signaling through the circumventricular subfornical organ (SFO) is a likely upstream mediator of this response19, 20. In 2K1C RVH, aberrant Ang-II signaling in the PVN has been implicated in the hypertension, increased sympathetic discharge and attenuation of baroreflex function observed in this model4, 21. However, the underlying signaling mechanisms are largely unknown.

The molecular pathways by which ROS mediate long-term changes in central neural circuits involved in diseases such as hypertension and heart failure are yet to be elucidated. Emerging evidence suggests that redox-regulated transcription factor (TF) activation and ensuing alterations in gene profiles in these circuits may be involved17, 22. The redox-regulated activator protein 1 (AP-1) transcription complex may have particular relevance in this regard, as two of its members, c-Fos and c-Jun, have been used extensively as marker proteins for activated CNS neurons in models of chronic cardiovascular disease19, 23. Furthermore, abundant evidence shows that Ang-II increases AP-1 family member levels and activity in the PVN and other central cardiovascular regions in vivo17, 24–26.

Considering the importance of ROS signaling in the PVN in a number of neuro-cardiovascular diseases, we hypothesized that oxidant signaling in the PVN plays an important role in the hypertension evoked by renal artery stenosis. Furthermore, given recent evidence that redox-regulated TFs mediate Ang-II induced changes in CNS circuits involved in chronic diseases such as hypertension, we hypothesized that AP-1 activation in the PVN would be associated with redox signaling and elevations in blood pressure induced by renal artery clipping. We employed the 2K1C model of RVH in mice, and modulated O2−· levels selectively in the PVN by targeted adenoviral (Ad) delivery of cytoplasmic superoxide dismutase (AdCuZnSOD) to this brain region. In addition, we utilized an Ad encoding a luciferase reporter downstream of AP-1 response elements (AdAP-1Luc) in conjunction with bioluminescence imaging (BLI)22 to longitudinally track AP-1 activity in the PVN in vivo during the development and progression of RVH. Our data show that hypertension in this model is associated with increased O2−· production and AP-1 activation in the PVN. PVN-targeted overexpression of CuZnSOD abolished these increases in O2−· and AP-1 activation. Furthermore, expression of either CuZnSOD or a dominant-negative inhibitor of AP-1 activity in the PVN protected against RVH in this model.

METHODS

An expanded Methods section is available in the online supplement at http://hypertension.ahajournals.org.

Animals

Adult C57Bl/6 mice (8–12 wks) were used. All procedures were approved by the Institutional Animal Care and Use Committee at Cornell University.

Adenoviral vectors

Ad vectors encoding for human cytoplasmic superoxide dismutase (AdCuZnSOD) and bacterial β-galactosidase (AdLacZ) were obtained from the University of Iowa Gene Transfer Vector Core. Ad vectors encoding an AP-1-responsive luciferase reporter construct (AdAP-1Luc) and a dominant –negative c-Jun NH2-terminal kinase 1 JNK1 (Ad-dnJNK1) were kindly provided by Dr. John F. Engelhardt (The University of Iowa).

Radiotelemeter implantation and gene transfer to the PVN

Mice were instrumented with radiotelemeters as described4, 9, followed by stereotaxic bilateral PVN microinjections of AdLacZ, AdCuZnSOD or Ad-dnJNK1. For in vivo BLI studies, non-telemetered mice underwent bilateral PVN microinjection of a 1:1 mixture of AdAP-1Luc/AdLacZ or AdAP-1Luc/AdCuZnSOD as described18.

2K1C RVH model

One week following telemeter implantation and PVN microinjection, mice were anesthetized with isofluorane. The right renal artery was exposed through a midline abdominal incision, and a silver clip was placed over the vessel as described4. A sham procedure served as control.

In vivo BLI

To longitudinally track AP-1 activation in the PVN in vivo, AdAP-1Luc was utilized in conjunction with in vivo BLI as described22. Animals underwent sham or 2K1C surgery 2 wks following AdAP-1Luc gene transfer, after which daily bioluminescence images were acquired with the IVIS®200 daily until 28 days post-surgery. At the end of the study, systemic endotoxin-induced activation of AP-1 was assessed by injection of lipopolysaccharide (LPS)22.

ROS measurements in brain sections

Dihydroethidium (DHE) staining was performed as described9, 18. On days 5 and 14 post-surgery, brains were removed, cryosectioned and treated with DHE. DHE fluorescence was visualized by confocal microscopy and quantified using ImageJ as described9.

CuZnSOD and luciferase immunohistochemistry

Mice were perfused, and brains were removed and cryosectioned. Free-floating sections were processed for immunofluorescence with antibodies to luciferase, CuZnSOD, neuronal nuclei (Neu-N) or glial fibrillary acidic protein (GFAP) as described9, 18, 19. Sections were analyzed by confocal microscopy.

Body and kidney weights

Mice were weighed and euthanized at the end of the telemetry experiments (day 28), and kidneys were removed and weighed to confirm atrophy of the clipped kidney and hypertrophy of the contralateral unclipped kidney.

Statistical analyses

Results are expressed as mean±SEM. All data were analyzed by one-way analysis of variance (ANOVA) and Newman-Keuls post-tests, except changes in MAP and HR from baseline were assessed by repeated measures ANOVA with Tukey’s multiple comparison post-test. Significance was defined as p<0.05.

RESULTS

PVN-targeted scavenging of ctyoplasmic O2−· protects against 2K1C hypertension

Changes in mean arterial pressure (MAP) and heart rate (HR) induced by unilateral renal artery clipping (or sham surgery) are presented in Figure 1. In mice treated with the control AdLacZ vector in the PVN, renal artery occlusion resulted in a gradual rise in MAP that became significantly elevated above baseline and sham controls by day 5 post-clip, and reached maximum by 1 week. Blood pressure in this group remained at this level throughout the rest of the experimental period (3 weeks). In AdCuZnSOD-treated mice that underwent renal artery clipping, MAP was elevated above sham controls for 1 day post-clip (day 2). However, on all days thereafter, MAP in this group returned to sham levels and was significantly attenuated compared to control virus-treated 2K1C mice (days 6–28; 2K1C+AdLacZ vs. 2K1C+AdCuZnSOD, p<0.05). Blood pressure was not significantly altered in sham mice treated with AdLacZ at any time during the study, nor was HR different between the three groups throughout the experiment. Baseline pre-clip MAP (Sham+AdlacZ=101±5, n=6; 2K1C+AdlacZ=109±1, n=4; 2K1C+AdCuZnSOD =105±6, n=6; p>0.05) and HR (Sham+AdlacZ=563±24, n=6; 2K1C+AdlacZ=582±28, n=4; 2K1C+AdCuZnSOD =554±17, n=6; p>0.05) were also not different between the treatment groups, suggesting that O2−· scavenging in the PVN does not alter basal cardiovascular function. Taken together, these results suggest that O2−· signaling in the PVN is critical in mediating 2K1C hypertension, both during the early development and chronic phases.

Figure 1. Scavenging of cytoplasmic O2−· selectively in the PVN ameliorates 2K1C-induced hypertension.

Figure 1

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Summary of MAP and HR recorded by radiotelemetry before (3 days) and after (28 days) unilateral renal artery clipping and treatment with AdLacZ (n=4) or AdCuZnSOD (n=6) in the PVN 10 days earlier. Sham-operated mice received PVN-targeted control vector AdLacZ (n=6). *p<0.05 2K1C+AdLacZ vs. Sham+AdLacZ and baseline; †p<0.05 2K1C+AdCuZnSOD vs. 2K1C + AdLacZ; ‡ p<0.05 2K1C+AdCuZnSOD vs. Sham+AdLacZ.

Renal artery clipping induces CuZnSOD-sensitive AP-1 transcriptional activity in the PVN

Given that AP-1 activation is redox-sensitive27 and may be important in mediating long-term changes in CNS cardiovascular networks17, 24–26, we hypothesized that RVH involves ROS-dependent AP-1 activation in the PVN. To test this, the AP-1 reporter virus Ad-AP-1Luc was microinjected selectively into PVN. This was then coupled with in vivo BLI for non-invasive real-time monitoring of AP-1 activation following renal artery occlusion. Representative BLI images (panel A) and a summary of AP-1-dependent photon flux over time (grouped in 2-day bins, panel B) are shown in Figure 2. Baseline photon flux, averaged over 4 consecutive days prior to 2K1C or sham surgery, was at low levels and not different between the groups. This level of background flux is consistent with what we have shown previously using this technology22. Starting 3–4 days after clipping, the AP-1-dependent luminescent signal began to rise significantly compared to sham animals (Fig. 2A and B, 2K1C+AdLacZ vs. Sham+AdLacZ, p<0.05). By 5–6 days post-surgery, AP-1 activation in the PVN of the 2K1C group surged to ~5-fold greater levels compared to sham controls (Fig. 2A and B). AP-1-dependent photon flux in clipped mice remained significantly elevated compared to sham animals until 13–14 days post-clip (2K1C+LacZ vs. Sham+AdLacZ, p<0.05), albeit at lower levels compared to the surge at 5–6 days (Fig. 2B). To examine the role of redox signaling in this 2K1C-induced AP-1 activation, photon flux was investigated in mice that had undergone PVN-selective injections of Ad-AP-1 + AdCuZnSOD. In these mice, the 2K1C-induced increases in AP-1 activity observed in AdLacZ-treated mice on days 3 to14 were significantly attenuated (Fig. 2B, 2K1C+AdCuZnSOD vs. 2K1C+AdLacZ, p<0.05). After the 2 week time-point and for the duration of the study, AP-1 activity was not significantly different between the groups. In addition, photon flux was not altered at any time-point in sham animals compared to baseline. On the final day of the study (day 28; ~6 weeks after initial gene transfer), we verified that AP-1Luc had retained the functional capacity for activation in vivo using systemic challenge with LPS (4 μg/g, i.p.). AP-1 is known to be activated in the PVN upon systemic LPS administration22. LPS induced profound increases in AP-1-dependent luminescence in both sham and 2K1C groups, confirming functional expression of AP-1Luc until the end of the study (Fig. 2B, far right panel).

Figure 2. Renovascular hypertension induces CuZnSOD-sensitive AP-1 transcriptional activity in the PVN.

Figure 2

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(A) Representative BLI images at key time-points showing AP-1-dependent photon emission in the PVN of control (Sham+AdLacZ) and 2K1C mice (2K1C+LacZ). The pseudocolor scale bar represents high (red) vs. low photon emission (blue). (B) Summary of temporal changes in AP-1 activity (grouped into 2-day bins) quantified as the total flux (photons/sec) of the bioluminescent signal in Sham+Ad-LacZ (n=21), 2K1C+Ad-LacZ (n=20) and 2K1C+AdCuZnSOD (n=12) mice. Changes in AP-1 activity in response to systemic LPS at the end of the studies is shown on the right; note the different scale. *p<0.05 vs. Sham+AdLacZ and baseline; †p<0.05 vs. 2K1C+AdLacZ. (C) Representative photomicrograph showing luciferase immunoreactivity, confirming robust bilateral expression of AP-1Luc in the PVN. 3V, third ventricle.

It should be noted that in addition to temporal resolution, spatial localization of AP-1 activation in PVN is verified by virtue of the fact that the Ad-AP-1 reporter was injected site-selectively22. This is an important point since the scattering of visible light through tissue prevents the surface-weighted signal itself from strictly informing of the three-dimensional position of the luminescence source22. However, since the luciferase reporter was delivered selectively to PVN, we have independent confirmation of spatial localization of AP-1 activation. To provide further evidence of this, immunohistochemical staining of luciferase was performed at day 28. As seen in Fig. 2C, luciferase was expressed at high levels bilaterally in the PVN. Together, these results suggest that renal artery clipping induces O2−·-dependent AP-1 transcriptional activity in the PVN during the initiation and early phase of hypertension in this model.

AdCuZnSOD prevents 2K1C-induced ROS formation in the PVN

Data in Figure 1 suggest that 2K1C-induced increases in O2−· levels in the PVN are associated with increased MAP in this model. BLI results in Figure 2 show that AP-1 activation in the PVN in response to renal artery clipping is CuZnSOD-sensitive, and with a time-course that parallels the rise in MAP. These results implicate 2K1C-induced increases in O2−· in the PVN, and here we sought to confirm this directly by measuring ROS levels in this brain region at 5 and 14 days post-clip using DHE confocal microscopy. As shown in representative images of DHE fluorescence (day 14, Fig. 3A) and summary data at both time-points (Fig. 3B), renal artery clipping increased ROS levels in the PVN of mice treated with the control virus. This occurred during the early phase post-clip, coinciding with the start of the rise in MAP and the peak AP-1 activation response (day 5). Elevated ROS levels in the PVN were maintained at day 14, a time when MAP was maximum in the AdLacZ group but back to baseline in the AdCuZnSOD-treated animals (Fig. 1). 2K1C-induced increases in PVN ROS levels were significantly blunted at both time-points by AdCuZnSOD (Fig. 3A and B), implicating O2−· as the radical species involved in both the initiation and maintenance of hypertension in this model.

Figure 3. Unilateral renal artery clipping induces robust increases in ROS production in the PVN, which are abolished by AdCuZnSOD.

Figure 3

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(A) Representative confocal images showing DHE fluorescence in the PVN at day 14 following renal artery clipping (or sham surgery) in mice that had previously received PVN injections of either AdLacZ or AdCuZnSOD. (B) Summary of DHE fluorescence intensity in the PVN at 5 and 14 days following sham surgery (n=5) or renal artery clipping in AdLacZ-treated (n=6) and AdCuZnSOD-treated (n=6) mice. Data are expressed relative to Sham controls. *p<0.05 vs. Sham+AdLacZ; †p<0.05 vs. 2K1C+AdLacZ. 3V, third ventricle.

Localization of CuZnSOD in PVN

We have shown previously that AdCuZnSOD induces robust, localized and stable CuZnSOD expression and SOD activity in cardiovascular nuclei of the CNS9, 18, 19. As shown in Figure 4A, we have confirmed this here, and demonstrate that the stereotaxic coordinates used resulted in viral injections in the parvocellular region of PVN. In addition, to determine the subcellular targeting of CuZnSOD in the PVN of mice injected with AdCuZnSOD, double immunohistochemistry was performed for CuZnSOD with either the neuronal cell marker Neu-N or the glial cell marker GFAP. As shown in Figure 4B, double immuno-labeling revealed CuZnSOD expression in both neurons and glia in this region. This confirms what we and others have shown previously – that Ad vectors transduce both cell types in the hypothalamus and other central cardioregulatory nuclei28, 29.

Figure 4. Regional and cellular localization of CuZnSOD in the PVN.

Figure 4

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(A, left) Map of the location of microinjection sites for AdLacZ (left, triangles) and AdCuZnSOD (left, circles) in the PVN relative to the third ventricle by −4.5 mm (ventral), −0.7 mm (posterior), and −0.3 mm (lateral). Subpopulations of PVN nuclei are outlined: mm, medial magnocellular; mpd, medial parvocellular; pml, posterior magnocellular; pv, parvocellular; 3V, third ventricle (adapted from the Allen Mouse Brain Atlas, Allen Institute for Brain Science). Representative photomicrographs of coronal brain sections showing CuZnSOD immunoreactivity in the PVN of AdCuZnSOD- or control-treated mice are shown on the right. (B) Representative confocal images of coronal brain sections showing CuZnSOD immunoreactivity (left), GFAP or NeuN (center), and merged images (right). Arrows indicate neurons or glia that are co-localized with CuZnSOD.

PVN-targeted expression of dnJNK1 ameliorates 2K1C hypertension

Data presented in Figures 1 and 2 demonstrate that 2K1C-induced hypertension is associated with AP-1 activation in the PVN. To determine whether there is a causal link between these two responses, we injected an adenovirus encoding a dominant-negative inhibitor of AP-1 transcriptional activation (Ad-dnJNK1) into the PVN prior to renal artery clipping. Summary data in Figure 5 demonstrate that PVN-targeted dnJNK1 expression prevented the 2K1C-induced rise in MAP in both the early and chronic phases. Similar to Figure 1, blood pressure was not significantly altered in sham mice treated with AdLacZ, nor was HR different between the three groups at any time-point. Baseline pre-clip MAP (Sham+AdlacZ=102±4, n=3; 2K1C+AdlacZ=98±2, n=4; 2K1C+Ad-dnJNK1 =98±2, n=5; p>0.05) and HR (Sham+AdlacZ=485±17, n=4; 2K1C+AdlacZ=487±21, n=4; 2K1C+Ad-dnJNK1 =488±5, n=5; p>0.05) were also not different between the treatment groups.

Figure 5. Inhibition of AP-1 transcriptional activity in the PVN amelioriates 2K1C-induced hypertension.

Figure 5

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Summary of MAP and HR recorded by radiotelemetry before (2 days) and after (28 days) unilateral renal artery clipping and treatment with AdLacZ (n=4) or Ad-dnJNK1 (n=5) in the PVN 10 days earlier. Sham-operated mice received PVN-targeted control vector AdLacZ (n=3). *p<0.05 2K1C+AdLacZ vs. Sham+AdLacZ and baseline; †p<0.05 2K1C + AdLacZ vs. 2K1C + Ad-dnJNK1.

Renal atrophy induced by renal artery clipping

Kidneys were weighed at the end of the 4 week period in a subset of sham, 2K1C+AdLacZ, 2K1C+AdCuZnSOD and 2K1C+Ad-dnJNK1 animals to confirm atrophy of the clipped kidney and hypertrophy of the contralateral unclipped kidney30. As shown in Table 1, there were significant decreases and increases in renal mass of right (clipped) and left (non-clipped) kidneys, respectively, compared to shams. This was unaffected by PVN-targeted AdLacZ, AdCuZnSOD or Ad-dnJNK1 treatment. Finally, body weights were not different between the groups.

Table 1.

Body and kidney weights 4 weeks following surgery.

Parameters Sham+AdLacZ (n=9) 2K1C+Ad LacZ (n=8) 2K1C+AdCuZn SOD (n=6) 2K1C+Ad-dnJNK1 (n=5)
Body weight, g 23.3 ± 0.9 25.4 ± 0.7 23.5 ± 0.8 25.3 ± 1.0
Left kidney weight, mg 135.0 ± 5.3 211.2 ± 7.9* 202.5 ± 11.5* 202.1 ± 12.6*
Right kidney weight, mg 154.3 ± 5.8 64.0 ± 5.8* 66.5 ± 6.9* 74.0 ± 7.5*
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*

p<0.05 vs. Sham + AdLacZ

DISCUSSION

Using a combination of brain site-directed viral gene transfer, longitudinal monitoring of TF activation in vivo by BLI, and radiotelemetry in the 2K1C model of hypertension in mice, this study reveals that redox-regulated AP-1 activation in the PVN of the hypothalamus is causally linked to RVH. The time-course and magnitude of hypertension observed in this study are consistent with what we and others have reported for this model in a variety of species4, 30, 31; that is, blood pressure rose gradually over the first week, ie early phase, and was sustained at this level over the following weeks after clipping, ie chronic phase. Also consistent with what others have reported, HR was not significantly altered at any time post-clip, likely due to impaired baroreflex sensitivity32, 33. The initial rise in MAP coincided with increases in ROS production and a dramatic surge in AP-1 transactivation in the PVN. Each of these PVN responses was sustained through 2 weeks post-clip, coinciding with the chronic phase of hypertension. AdCuZnSOD targeted to the PVN abolished both the ROS and AP-1 responses. This was associated with an attenuation of both the early and chronic phases of hypertension. Similarly, dominant-negative inhibition of AP-1 transcription in PVN with Ad-dnJNK1 prevented both phases of 2K1C-evoked hypertension. These results implicate O2−· signaling and AP-1 transcriptional activity in the PVN as key mediators of RVH.

The upstream signaling events that lead to increased O2−· production in the PVN following renal artery stenosis remain unclear, although locally generated Ang-II is a leading candidate given its well established role in a number of neuro-cardiovascular diseases including RVH34, as well as previous data showing that Ang-II is elevated in the hypothalamus during both the early and chronic phases of 2K1C hypertension4, with AT1R mRNA levels also being upregulated in the PVN21. Given that a major mechanism through which Ang-II exerts its powerful effects on central control of blood pressure is redox signaling6, we first tested the hypothesis that 2K1C hypertension involves oxidant stress in the PVN. Indeed, renal artery clipping induced CuZnSOD-sensitive ROS increases in the PVN during the initial rise in MAP (day 5), suggesting that O2−· signaling in this brain region may be involved in the development of RVH. To test this directly, PVN-targeted AdCuZnSOD treatment was employed prior to placement of the clip. This led to attenuation of 2K1C-induced hypertension during the early phase, suggesting that the development of hypertension in this model does involve cytoplasmic O2−· signaling in the PVN. Since AdCuZnSOD targeted to this brain region also ameliorated hypertension during the chronic phase, in addition to normalizing ROS levels in the PVN at 2 weeks post-clip, these data suggest that O2−· signaling in the PVN is also critical in the maintenance phase of RVH. Importantly, this occurred despite sustained 2K1C-induced renal atrophy. These results are consistent with those of Oliveira-Sales et al. whose findings support a role for ROS in the PVN in RVH, however their studies used acute microinjection of Tempol unilaterally into PVN of anesthetized rats and showed that established hypertension (after 6 weeks) could be transiently reversed by this treatment21. Our experimental design involving long-term gene transfer of CuZnSOD to PVN prior to renal artery clipping leads us to conclude that cytoplasmic O2−· signaling in the PVN plays a causal role in RVH. Studies utilizing PVN-specific ablation of AT1R through Cre-loxP technology35 will be important to verify whether Ang-II signaling in the PVN is indeed functionally linked to 2K1C-induced oxidant stress and RVH. In addition, since Ad-mediated CuZnSOD transduction occurred in both neurons and glia of the PVN, further studies will be required to determine the relative role of O2−· signaling in these two cell types.

Additional future studies should aim to determine the enzymatic source of 2K1C-induced ROS formation in the PVN. It is certainly well established that NADPH oxidase is a primary source of ROS mediating many cardiovascular responses including central neural control of blood pressure and related parameters6, 9–12, 36. Recently, Nox-derived ROS have been implicated in RVH. A number of studies have shown that systemic treatment with the antioxidants Tempol or apocynin relieve a number of peripheral pathologies associated with RVH in rats including endothelial dysfunction, impairment in renal hemodynamics and increased renal sympathetic nerve activity31, 37, 38. In addition, Wang et al. demonstrated that Ang-II stimulated NADPH oxidase contributes to the development of 2K1C hypertension and cardiac remodeling in rats39. Interestingly, NADPH oxidase has also recently been implicated in the CNS component of RVH. Oliveria-Sales et al. showed that both Nox2 and p47phox were markedly upregulated in PVN of rats with 2K1C hypertension21. Using viruses encoding siRNAs targeted against Nox1, Nox2 or Nox412, 18, 40, it should be possible to dissect the relative functional role of each of these homologues in ROS formation in the PVN and 2K1C RVH.

The downstream molecular mechanisms by which increased O2−· in the PVN leads to chronic hypertension in this model was the next focus of this study. Effectors of O2−· in CNS cardioregulatory nuclei have been the subject of a number of recent studies, with evidence that kinase cascades, calcium transients and delayed rectifier potassium channels may be involved6, 11, 41–43. However, many of these studies focused on the effects of relatively short-term stimulation of ROS production. Since O2−· signaling in the context of our study was involved in chronic regulation of blood pressure, we focused on ROS-sensitive TFs because of their well known role in long-term modulation of a wide variety of CNS parameters44, and an emerging role in CNS cardiovascular circuits. For example, Liu et al. have shown that Ang-II induces AT1 receptor upregulation in the rostral ventrolateral medulla (RVLM) by an increase in ROS-dependent activation of AP-1 in brains of chronic heart failure rabbits26. In addition, a recent report by Kang et al. shows that chronic systemic infusion of Ang-II in rats causes ROS-dependent activation of NFκB in the PVN17. Both of these studies utilized gel mobility shift assays to examine TF activation in brain nuclei at a few select time-points during these chronic conditions. However, since we were interested in the full time-course of TF activation in the PVN during the development and progression of RVH, we turned to in vivo BLI technology for longitudinal real-time monitoring of AP-1 activation. Our results demonstrate that renal artery clipping caused a small but significant increase in AP-1 transcriptional activation in the PVN as early as 3–4 days post-clip. This transitioned to a marked surge in activity over the following 2 days, followed by sustained elevations compared to shams for another week, albeit at lower levels compared to the early surge. AP-1 activity in the PVN then returned to basal levels for the remaining 2 weeks of the experiment. The sensitivity of these increases in AP-1-dependent photon flux to AdCuZnSOD suggest that activity of this TF in PVN is O2−·-regulated.

It is interesting to speculate about the time-course of AP-1 activation in the context of the different phases of 2K1C-induced RVH in this study. Genes encoding Fos and Jun family proteins – which form the AP-1 complex that binds AP-1 sites in gene promoters – are termed immediate early genes because of their classic quick and transient induction, ie hours, in response to a variety of stimuli45. However, more recently some family members such as isoforms of FosB, which are important in CNS adaptation, were shown to be induced only by more chronic stimuli and persist for longer periods of time, ie days to weeks to months46. Although our strategy for measuring AP-1 activation in the current study does not allow for resolution of the various family members involved, we speculate that the early robust increases in AP-1 transcriptional activity leads to changes in PVN neural networks that play a key role in initiating the long-term changes in blood pressure in this model. This was supported by the finding that AdCuZnSOD injection into PVN blocked both the AP-1 transactivation and chronic hypertension in this model. Furthermore, since dominant-negative inhibition of AP-1 in the PVN prevented 2K1C-evoked rises in MAP, this suggests that its transcriptional activity is functionally linked to hypertension in this model.

The physiological effectors in the PVN that lead to 2K1C hypertension have been the subject of many investigations. On the one hand, increased AVP release has been implicated in this model over the years47, 48, and there are strong links between Ang-II, ROS and AP-1 with increased activation of vasopressinergic neurons in the PVN49–51. However, there are other reports that AVP in not involved in 2K1C hypertension52. More recently, it is thought that PVN involvement in RVH is through activation of RVLM-projecting parvocellular neurons in this region, leading to increased sympathoexcitation21. Interestingly, ROS are now also implicated in this pathway21. Further studies will be required to determine the physiological effectors that are downstream of ROS and AP-1 activation in PVN during 2K1C hypertension.

Perspectives

Our data suggest that 2K1C-induced RVH in mice is mediated by oxidative stress-induced AP-1 activation in the PVN. This is supported by the following observations: 1) the development of 2K1C hypertension is associated with a marked increase in O2−· production in the PVN; 2) PVN-targeted overexpression of the CuZnSOD prevents both the increase in PVN O2−· levels and hypertension in this model; 3) AP-1 is robustly activated in the PVN with a time-course that is consistent with its involvement in the development of hypertension of this model, and its inhibition by O2−· scavenging parallels the inhibition of hypertension; 4) dominant-negative inhibition of AP-1 transcriptional activity in the PVN prevents RVH. The present findings have the potential to fundamentally advance our understanding of the molecular mechanisms underlying RVH, and could have important implications in the design of novel and innovative therapeutic approaches targeting the neurogenic component of this disease.

Supplementary Material

Supp1

Acknowledgments

We thank Dr. Carlos Alberto Aguiar da Silva (University of São Paulo Ribeirão Preto) for providing the renal artery clips.

SOURCES OF FUNDING

These studies were supported by grants from NIH to RLD (HL063887, HL084624) and an American Heart Association Established Investigator Award to RLD (0540114N).

Footnotes

DISCLOSURES

None.

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