Jeffrey R. Peterson

Genetic Silencing of Nox2 and Nox4 Reveals Differential Roles of These NADPH Oxidase Homologues in the Vasopressor and Dipsogenic Effects of Brain Angiotensin II

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.

Central Cardiovascular Circuits Contribute to the Neurovascular Dysfunction in Angiotensin II Hypertension

Hypertension, a powerful risk factor for stroke and dementia, has damaging effects on the brain and its vessels. In particular, hypertension alters vital cerebrovascular control mechanisms linking neural activity to cerebral perfusion. In experimental models of slow-developing hypertension, free radical signaling in the subfornical organ (SFO), one of the forebrain circumventricular organs, is critical for the hormonal release and sympathetic activation driving the elevation in arterial pressure. However, the contribution of this central mechanism to the cerebrovascular alterations induced by hypertension remains uncertain. We tested the hypothesis that free radical production in the SFO is involved in the alterations in cerebrovascular regulation produced by hypertension. In a mouse model of gradual hypertension induced by chronic administration of subpressor doses of angiotensin II (AngII), suppression of free radicals in the SFO by overexpression of CuZn-superoxide dismutase (CuZnSOD) prevented the alteration in neurovascular coupling and endothelium-dependent responses in somatosensory cortex induced by hypertension. The SFO mediates the dysfunction via two signaling pathways. One involves SFO-dependent activation of the paraventricular hypothalamic nucleus, elevations in plasma vasopressin, upregulation of endothelin-1 in cerebral resistance arterioles and activation of endothelin type A receptors. The other pathway depends on activation of cerebrovascular AngII type 1 (AT1) receptors by AngII. Both pathways mediate vasomotor dysfunction by inducing vascular oxidative stress. The findings implicate for the first time the SFO and its efferent hypothalamic pathways in the cerebrovascular alterations induced by AngII, and identify vasopressin and endothelin-1 as potential therapeutic targets to counteract the devastating effects of hypertension on the brain.

Angiotensin II-dependent hypertension requires cyclooxygenase 1-derived prostaglandin E2 and EP1 receptor signaling in the subfornical organ of the brain.

Cyclooxygenase (COX)-derived prostanoids have long been implicated in blood pressure (BP) regulation. Recently prostaglandin E2 (PGE2) and its receptor EP1 (EP1R) have emerged as key players in angiotensin II (Ang II)–dependent hypertension (HTN) and related end-organ damage. However, the enzymatic source of PGE2, that is, COX-1 or COX-2, and its site(s) of action are not known. The subfornical organ (SFO) is a key forebrain region that mediates systemic Ang II–dependent HTN via reactive oxygen species (ROS). We tested the hypothesis that cross-talk between PGE2/EP1R and ROS signaling in the SFO is required for Ang II HTN. Radiotelemetric assessment of blood pressure revealed that HTN induced by infusion of systemic “slow-pressor” doses of Ang II was abolished in mice with null mutations in EP1R or COX-1 but not COX-2. Slow-pressor Ang II–evoked HTN and ROS formation in the SFO were prevented when the EP1R antagonist SC-51089 was infused directly into brains of wild-type mice, and Ang-II-induced ROS production was blunted in cells dissociated from SFO of EP1R−/− and COX-1−/− but not COX-2−/− mice. In addition, slow-pressor Ang II infusion caused a ≈3-fold increase in PGE2 levels in the SFO but not in other brain regions. Finally, genetic reconstitution of EP1R selectively in the SFO of EP1R-null mice was sufficient to rescue slow-pressor Ang II–elicited HTN and ROS formation in the SFO of this model. Thus, COX 1–derived PGE2 signaling through EP1R in the SFO is required for the ROS-mediated HTN induced by systemic infusion of Ang II and suggests that EP1R in the SFO may provide a novel target for antihypertensive therapy.

COX-1-derived PGE2 and PGE2 type 1 receptors are vital for angiotensin II-induced formation of reactive oxygen species and Ca2+ influx in the subfornical organ

Regulation of blood pressure by angiotensin II (ANG II) is a process that involves the reactive oxygen species (ROS) and calcium. We have shown that ANG-II type 1 receptor (AT1R) and prostaglandin E2 (PGE2) type 1 receptors (EP1R) are required in the subfornical organ (SFO) for ROS-mediated hypertension induced by slow-pressor ANG-II infusion. However, the signaling pathway associated with this process remains unclear. We sought to determine mechanisms underlying the ANG II-induced ROS and calcium influx in mouse SFO cells. Ultrastructural studies showed that cyclooxygenase 1 (COX-1) codistributes with AT1R in the SFO, indicating spatial proximity. Functional studies using SFO cells revealed that ANG II potentiated PGE2 release, an effect dependent on AT1R, phospholipase A2 (PLA2) and COX-1. Furthermore, both ANG II and PGE2 increased ROS formation. While the increase in ROS initiated by ANG II, but not PGE2, required the activation of the AT1R/PLA2/COX-1 pathway, both ANG II and PGE2 were dependent on EP1R and Nox2 as downstream effectors. Finally, ANG II potentiated voltage-gated L-type Ca(2+) currents in SFO neurons via the same signaling pathway required for PGE2 production. Blockade of EP1R and Nox2-derived ROS inhibited ANG II and PGE2-mediated Ca(2+) currents. We propose a mechanism whereby ANG II increases COX-1-derived PGE2 through the AT1R/PLA2 pathway, which promotes ROS production by EP1R/Nox2 signaling in the SFO. ANG II-induced ROS are coupled with Ca(2+) influx in SFO neurons, which may influence SFO-mediated sympathoexcitation. Our findings provide the first evidence of a spatial and functional framework that underlies ANG-II signaling in the SFO and reveal novel targets for antihypertensive therapies.

Longitudinal noninvasive monitoring of transcription factor activation in cardiovascular regulatory nuclei using bioluminescence imaging

The ability to monitor transcription factor (TF) activation in the central nervous system (CNS) has the potential to provide novel information regarding the molecular mechanisms underlying a wide range of neurobiological processes. However, traditional biochemical assays limit the mapping of TF activity to select time points. In vivo bioluminescence imaging (BLI) has emerged as an attractive technology for visualizing internal molecular events in the same animal over time. Here, we evaluated the utility of BLI, in combination with virally mediated delivery of reporter constructs to cardiovascular nuclei, for monitoring of TF activity in these discrete brain regions. Following viral gene transfer of NF-kappaB-driven luciferase reporter to the subfornical organ (SFO), BLI enabled daily measurements of baseline TF activity in the same animal for 1 mo. Importantly, systemic endotoxin, a stimulator of NF-kappaB activity, induced dramatic and dose-dependent increases in NF-kappaB-dependent bioluminescence in the SFO up to 30 days after gene transfer. Cotreatment with a dominant-negative IkappaBalpha mutant significantly prevented endotoxin-dependent NF-kappaB activation, confirming the specificity of the bioluminescence signal. NF-kappaB-dependent luminescence signals were also stable and inducible 1 mo following delivery of luciferase reporter construct to the paraventricular nucleus or rostral ventrolateral medulla. Lastly, using targeted adenoviral delivery of an AP-1 responsive luciferase reporter, we showed similar baseline and endotoxin-induced AP-1 activity in these same brain regions as with NF-kappaB reporters. These results demonstrate that BLI, in combination with virally mediated gene transfer, is a powerful method for longitudinal monitoring and quantification of TF activity in targeted CNS nuclei in vivo.