Genetic silencing of Nox2 and Nox4 reveals differential roles of these NADPH oxidase homologues in the vasopressor and dipsogenic effects of brain angiotensin II

Abstract

The renin-angiotensin system exerts a tremendous influence over fluid balance and arterial pressure. Angiotensin II (Ang-II), the effector peptide of the renin-angiotensin system, acts in the central nervous system to regulate neurohumoral outflow and thirst. Dysregulation of Ang-II signaling in the central nervous system is implicated in cardiovascular diseases; however, the mechanisms remain poorly understood. Recently we established that NADPH oxidase (Nox)-derived superoxide acting in the forebrain subfornical organ is critical in the physiological responses to central Ang-II. In addition, we have found that Nox2 and Nox4 are the most abundantly expressed Nox homologues within Ang-II-sensitive sites in the forebrain. To dissect out the functional importance and unique roles of these Nox enzymes in the pressor and dipsogenic effects of central Ang-II, we developed adenoviral vectors expressing small interfering RNA to selectively silence Nox2 or Nox4 expression in the subfornical organ. Our results demonstrate that both Nox2 and Nox4 are required for the full vasopressor effects of brain Ang-II but that only Nox2 is coupled to the Ang-II-induced water intake response. These studies establish the importance of both Nox2- and Nox4-containing NADPH oxidases in the actions of Ang-II in the central nervous system and are the first to reveal differential involvement of these Nox enzymes in the various physiological effects of central Ang-II.

Figure 1. Ad-mediated delivery of RNAi effectively silences Nox homologue expression in the SFO

A) Real-time RT-PCR data showing the effects of AdsiGFP, AdsiNox2, or AdsiNox4 treatment on endogenous Nox1, Nox2, and Nox4 transcript levels in primary neonatal cardiomyocytes. Levels of Nox1, Nox2, and Nox4 mRNA were first normalized to β-actin, and expressed relative to levels in vehicle-treated cells. Experiments were performed in triplicate. B) ICV viral delivery results in robust and widespread transgene expression in the SFO. One week after adenoviral delivery, coronal brain sections through the SFO were visualized for reporter gene expression. The SFO is depicted in the upper left panel. Representative light and fluorescence microscopy images of sections from AdsiGFP(LacZ) (upper right panel), AdsiNox2(GFP) (lower left panel), or AdsiNox4(GFP) (lower right panel) treated mice demonstrate robust LacZ or GFP expression in the SFO. C) SFO-targeted delivery of AdsiNox2 or AdsiNox4 significantly silences Nox2 and Nox4 expression, respectively. Representative western blot and summary data of Nox2 (left) and Nox4 (right) expression in the lamina terminalis of mice microinjected with saline, AdsiGFP, or AdsiRNA targeted against Nox2 or Nox4 one week earlier. Brain tissue was pooled from 3 animals per group. Experiment was performed in triplicate. Data are expressed as mean ±SEM. *P<0.05 vs. saline and AdsiGFP. †P<0.05 vs. AdsiNox4. ‡P<0.05 vs. AdsiNox2.

Figure 2. ICV delivery of virus results in robust and localized transgene expression in the SFO

Serial coronal sections (30 µm) throughout the CNS from 4 mice injected ICV with AdsiNox2 + AdsiNox4 7 days earlier were processed for GFP expression using confocal microscopy. Both AdsiNox2 and AdsiNox4 harbor GFP as a reporter gene. Robust GFP expression was seen in the SFO (top left) and along the lining of the lateral ventricle (left middle). While sparse expression was found in the OVLT of one animal (left bottom), expression could not be detected in the median preoptic nucleus (MnPO), paraventricular nucleus (PVN), or area postrema (AP) (right). LV, lateral ventricle; 3V, 3^rd ventricle.

Figure 3. Silencing Nox2 or Nox4 prevents Ang-II-induced ROS production in neurons cultured from the lamina terminalis

A) Representative confocal images of DHE (5µM, 30 min)–loaded cells cultured from the lamina terminalis showing the effects of Ang-II (1µM, 30 min) on production of reactive oxygen species in noninfected cells and cells treated with AdsiGFP, AdsiNox2 alone, AdsiNox4 alone, or AdsiNox2 and AdsiNox4 together. Effective transduction of AdsiNox2 and/or AdsiNox4 was verified by visualization of expression of the reporter gene GFP. B) Summary of relative DHE fluorescence in individual cells before Ang-II stimulation (0 min) and after 30 minutes of Ang-II stimulation. Cells were stimulated with Ang-II 24 hours after treatment with AdsiGFP, AdsiNox2, AdsiNox4, or AdsiNox2 + AdsiNox4 together. Vehicle-treated cells served as a control. Data are expressed as mean±SEM (n=10–14 cells/group over 3 separate experiments) and expressed relative to DHE fluorescence prior to Ang-II treatment (0 min). *P<0.05 vs. 0 minutes. †P<0.05 vs. 30 min saline & 30 min & AdsiGFP.

Figure 4. Inhibiting both Nox2 and Nox4 expression in the SFO prevents the pressor and bradycardic responses to central Ang-II

A) Representative recordings of the effects of ICV Ang-II (200ng, 200nl) on MAP and HR in mice that underwent SFO-targeted delivery of saline, AdsiGFP, AdsiNox2, AdsiNox4, or AdsiNox2 and AdsiNox4 concomitantly 7 days earlier. Arrows indicate Ang-II injection. B) Summary data of the peak changes in MAP and HR in response to ICV Ang-II in mice that received brain microinjections of saline (n=7), AdsiGFP (n=11), AdsiNox2 (n=12), AdsiNox4 (n=6), or AdsiNox2 and AdsiNox4 concomitantly (AdsiNox2 + AdsiNox4, n=6) 7 days earlier. Data are expressed as mean±SEM. *P<0.05 vs. saline and AdsiGFP; †P<0.05 vs. saline, AdsiGFP, AdsiNox2 and AdsiNox4.

Figure 5. Inhibiting Nox2 expression in the SFO prevents the dipsogenic response to central Ang-II

Summary of the drinking response to ICV AngII (200ng, 200nl) in mice administered saline (n=11), AdsiGFP (n=17), AdsiNox2 (n=16), AdsiNox4 (n=13), or AdsiNox2 and AdsiNox4 concomitantly (AdsiNox2 + AdsiNox4, n=12) in the brain 7 days earlier. Data are expressed as the total time spent licking the water bottle (seconds) for 15 minutes after ICV injection of Ang-II. *P<0.05 vs. saline, AdsiGFP, and AdsiNox4.