Distal-less 3 (Dlx3)-/- mice die at E9.5 presumably due to an abnormal placental phenotype including reduced placental vasculature and secretion of placental growth factor. To examine the role of Dlx3 specifically within the epiblast, Dlx3 conditional knockout mice were generated using an epiblast-specific Meox2CreSor allele. Dlx3-/fl, Meox2CreSor animals were born at expected frequencies and survived to weaning providing indirect evidence that loss of Dlx3 within the trophoectoderm plays a critical role in fetal survival in the Dlx3-/- mouse. We next examined the hypothesis that loss of a single Dlx3 allele would have a negative impact on placental and fetal fitness. Dlx3+/- mice displayed reduced fetal growth beginning at E12.5 compared with Dlx3+/+ controls. Altered fetal growth trajectory occurred coincident with elevated oxidative stress and apoptosis within Dlx3+/- placentas. Oral supplementation with the superoxide dismutase mimetic, Tempol, rescued the fetal growth and placental cell death phenotypes in Dlx3+/- mice. To determine the potential mechanisms associated with elevated oxidative stress on the Dlx3+/- placentas, we next examined vascular characteristics within the feto-placental unit. Studies revealed reduced maternal spiral artery luminal area in the Dlx3+/- mice receiving water; Dlx3+/- mice receiving Tempol displayed maternal spiral artery luminal area similar to control Dlx3+/+ mice. We conclude that reduced Dlx3 gene dose results in diminished fetal fitness associated with elevated placental cell oxidative stress and apoptosis coincident with altered vascular remodeling. Administration of antioxidant therapy ameliorated this feto-placental phenotype, suggesting that Dlx3 may be required for adaptation to oxidative stresses within the intrauterine environment.
Placenta; fetal growth; oxidative stress; labyrinth; vascular remodeling; mouse
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.
two-kidney; one-clip (2K1C); Goldblatt; CuZnSOD; superoxide; adenovirus-mediated gene transfer
Current strategies to help tobacco smokers quit have limited success as a result of the addictive properties of the nicotine in cigarette smoke. We hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector expressing high levels of an anti-nicotine antibody would persistently prevent nicotine from reaching its receptors in the brain. To test this hypothesis, we constructed an AAV.rh10 vector that expressed a full length, high affinity, anti-nicotine antibody derived from the Fab fragment of the anti-nicotine monoclonal antibody NIC9D9 (AAVantiNic). In mice treated with this vector, blood concentrations of the anti-nicotine antibody were dose-dependent, and the antibody showed high specificity and affinity for nicotine. The antibody shielded the brain from systemically administered nicotine, reducing brain nicotine concentrations to 15% of those in naive mice. The amount of nicotine sequestered in the serum of vector-treated mice was over 7 times greater than that in non-treated mice, with 83% of serum nicotine bound to IgG. Treatment with the AAVantiNic vector blocked nicotine-mediated alterations in arterial blood pressure, heart rate and locomotor activity. In summary, a single administration of a gene transfer vector expressing a high affinity anti-nicotine monoclonal antibody elicited persistent (18 weeks), high titers of an anti-nicotine antibody that obviated the physiologic effects of nicotine. If this degree of efficacy translates to humans, AAVantiNic could be an effective preventative therapy for nicotine addiction.
Cyclooxygenase (COX)-derived prostanoids have long been implicated in blood pressure (BP) regulation. Recently prostaglandin E2 (PGE2) and its receptor 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, ie 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 BP 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 AngII-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.
Prostanoids; PGE2; COX; reactive oxygen species; blood pressure; central nervous system
Obstructive sleep apnea, a condition resulting in chronic intermittent hypoxia (CIH), is an independent risk factor for stroke and dementia, but the mechanisms of the effect are unknown. We tested the hypothesis that CIH increases cerebrovascular risk by altering critical mechanisms regulating cerebral blood flow thereby lowering cerebrovascular reserves. Male C57Bl6/J mice were subjected to CIH (10% O2 for 90 seconds/room air for 90 seconds; during sleep hours) or sham treatment for 35 days. Somatosensory cortex blood flow was assessed by laser Doppler flowmetry in anesthetized mice equipped with a cranial window. CIH increased mean arterial pressure (from 74±2 to 83±3 mm Hg; P<0.05) and attenuated the blood flow increase produced by neural activity (whisker stimulation; −39±2%; P<0.05) or neocortical application of endothelium-dependent vasodilators (acetylcholine response: −41±3%; P<0.05). The cerebrovascular dysfunction was associated with oxidative stress in cerebral resistance arterioles and was abrogated by free radical scavenging or NADPH oxidase inhibition. Furthermore, cerebrovascular dysfunction and free radical increase were not observed in mice lacking the NOX2 subunit of NADPH oxidase. CIH markedly increased endothelin 1 in cerebral blood vessels, whereas cerebrovascular dysfunction and oxidative stress were abrogated by neocortical application of the endothelin type A receptor antagonist BQ123. These data demonstrate for the first time that CIH alters key regulatory mechanisms of the cerebral circulation through endothelin 1 and NADPH oxidase– derived radicals. The ensuing cerebrovascular dysfunction may increase stroke risk in patients with sleep apnea by reducing cerebrovascular reserves and increasing the brain’s susceptibility to cerebral ischemia.
obstructive sleep apnea; reactive oxygen species; neurovascular coupling; endothelium-dependent vasodilation
Although endoplasmic reticulum (ER) stress is a pathologic mechanism in a variety of chronic diseases, it is unclear what role it plays in chronic hypertension (HTN). Dysregulation of brain mechanisms controlling arterial pressure is strongly implicated in HTN, particularly in models involving angiotensin II (Ang II). We tested the hypothesis that ER stress in the brain is causally linked to Ang II–dependent HTN. Chronic systemic infusion of low-dose Ang II in C57BL/6 mice induced slowly developing HTN, which was abolished by co-infusion of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) into the lateral cerebroventricle. Investigations of the brain regions involved revealed robust increases in ER stress biomarkers and profound ER morphological abnormalities in the circumventricular subfornical organ (SFO), a region outside the blood-brain barrier and replete with Ang II receptors. Ang II–induced HTN could be prevented in this model by selective genetic supplementation of the ER chaperone 78-kDa glucose-regulated protein (GRP78) in the SFO. These data demonstrate that Ang II–dependent HTN is mediated by ER stress in the brain, particularly the SFO. To our knowledge, this is the first report that ER stress, notably brain ER stress, plays a key role in chronic HTN. Taken together, these findings may have broad implications for the pathophysiology of this disease.
The renin-angiotensin system (RAS) plays a critical role in cardiovascular and fluid homeostasis. The major biologically active peptide of the RAS is angiotensin II, which acts through G protein–coupled receptors of two pharmacological classes, AT1 and AT2. AT1 receptors, expressed in brain and peripheral tissues, mediate most classically recognized actions of the RAS, including blood pressure homeostasis and regulation of drinking and water balance. In rodents, two highly homologous AT1 receptor isoforms, termed AT1A and AT1B receptors, are expressed at different levels in major forebrain cardiovascular and fluid regulatory centers, with AT1A expression generally exceeding AT1B expression, but the relative contributions of these receptor subtypes to central angiotensin II responses are not known. We used gene targeting in combination with a unique system for maintaining catheters in the cerebral ventricles of conscious mice to test whether there are differential roles for AT1A and AT1B receptors in responses elicited by angiotensin II in the brain. Here we show that the blood pressure increase elicited by centrally administered angiotensin II can be selectively ascribed to the AT1A receptor. However, the drinking response requires the presence of AT1B receptors. To our knowledge, this is the first demonstration of a primary and nonredundant physiological function for AT1B receptors.
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 sub-pressor doses of angiotensin II (AngII), suppression of free radicals in the SFO by overexpression of 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 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.
Obesity and hypertension are strongly associated and neural dysfunction has been implicated in both. The hypothalamus integrates signals regulating blood pressure and energy homeostasis. A recent paper in Nature Medicine (Purkayastha et al, 2011) suggests that obesity and hypertension are caused by inflammation in distinct hypothalamic neuronal populations.
The renin-angiotensin system (RAS) and its active peptide angiotensin II (AngII) have major involvements not only in hypertension but also in mood and anxiety disorders. Substantial evidence supports the notion that AngII acts as a neuromodulator in the brain. In this review, we provide an overview of the link between the RAS and anxiety or mood disorders, and focus on recent advances in the understanding of AngII-linked, NADPH oxidase-derived oxidative stress in the central nervous system, which may underlie pathogenesis of mood and anxiety disorders.
Renin-angiotensin system; angiotensin II; anxiety disorder; bipolar disorder; major depressive disorder; reactive oxygen species; NADPH oxidase
An imbalance in circulating pro-angiogenic and anti-angiogenic factors is postulated to play a causal role in pre-eclampsia (PE). We have described an inbred mouse strain, BPH/5, which spontaneously develops a PE-like syndrome including late-gestational hypertension, proteinuria, and poor feto-placental outcomes. Here we tested the hypothesis that an angiogenic imbalance during pregnancy in BPH/5 mice leads to the development of PE-like phenotypes in this model. Similar to clinical findings, plasma from pregnant BPH/5 showed reduced levels of free vascular endothelial growth factor (VEGF) and placental growth factor (PGF) compared to C57BL/6 controls. This was paralleled by a marked decrease in VEGF protein and Pgf mRNA in BPH/5 placentae. Surprisingly, antagonism by the soluble form of the FLT1 receptor (sFLT1) did not appear to be the cause of this reduction, as sFLT1 levels were unchanged or even reduced in BPH/5 compared to controls. Adenoviral-mediated delivery of VEGF121 (Ad-VEGF) via tail vein at e7.5 normalized both the plasma free VEGF levels in BPH/5 and restored the in vitro angiogenic capacity of serum from these mice. Ad-VEGF also reduced the incidence of fetal resorptions and prevented the late-gestational spike in blood pressure and proteinuria observed in BPH/5. These data underscore the importance of dysregulation of angiogenic factors in the pathogenesis of PE, and suggest the potential utility of early pro-angiogenic therapies in treating this disease.
hypertension; proteinuria; PGF; sFLT1; angiogenesis; placenta
Myocardial infarction (MI)-induced heart failure (HF) is characterized by central nervous system (CNS)-driven sympathoexcitation and deteriorating cardiac function. The paraventricular nucleus (PVN) of the hypothalamus is a key regulator of sympathetic nerve activity and is implicated in HF. Redox signaling in the PVN and other CNS sites is a primary mechanism of neuro-cardiovascular regulation, and excessive oxidant production by activation of NADPH oxidases (Nox) is implicated in some neuro-cardiovascular diseases.
We tested the hypothesis that Nox-mediated redox signaling in the PVN contributes to MI-induced sympathoexcitation and cardiac dysfunction in mice.
Methods and Results:
Real-time PCR revealed that Nox4 was the most abundantly expressed Nox in PVN under basal conditions. Coronary arterial ligation (MI) caused a selective upregulation of this homologue compared to Nox1 and Nox2. Adenoviral gene transfer of Nox4 siRNA (AdsiNox4) to PVN (bilateral) attenuated MI-induced superoxide formation in this brain region (day 14) to the same level as that produced by PVN-targeted gene transfer of cytoplasmic superoxide dismutase (AdCu/ZnSOD). MI mice treated with AdsiNox4 or AdCu/ZnSOD in the PVN showed marked improvement in cardiac function as assessed by echocardiography and left ventricular hemodynamic analysis. This was accompanied by significantly diminished sympathetic outflow and apoptosis in the peri-infarct region of the heart.
These results suggest that MI causes dysregulation of Nox4-mediated redox signaling in the PVN, which leads to sympathetic overactivation and a decline in cardiac function. Targeted inhibition of oxidant signaling in the PVN could provide a novel treatment for MI-induced HF.
heart; reactive oxygen species; NADPH oxidase; brain; sympathetic nerves
Internalization of activated receptors to a compartment enriched with NAPDH oxidase and associated signaling molecules is expected to facilitate regulation of redox-mediated signal transduction. The aim of this study was to test the hypothesis that endocytosis is necessary for generation of reactive oxygen species (ROS) by Nox1 and for redox-dependent signaling in smooth muscle cells (SMCs). Within minutes of treatment with tumor necrosis factor (TNF)-α or thrombin, SMCs increased cellular levels of ROS that was inhibited by shRNA to Nox1. Treatment of SMC with TNF-α induced a dynamin-dependent endosomal generation of ROS, whereas thrombin-mediated ROS production did not occur within endosomes and was not prevented by dominant-negative dynamin (dn-dynamin), but instead required transactivation of the epidermal growth factor receptor (EGFR). Activation of the phosphatidylinositol 3-kinase (PI3K)-Akt-activating transcription factor-1 (ATF-1) pathway by TNF-α and thrombin were both Nox1- and dynamin-dependent. In conclusion, we show that formation of specific ligand–receptor complexes results in spatially distinct mechanisms of Nox1 activation and generation of ROS. These findings provide novel insights into the role of compartmentalization for integrating redox-dependent cell signaling. Antioxid. Redox Signal. 12, 583–593.
The renin angiotensin system (RAS) exerts a tremendous influence over fluid balance and arterial pressure. Angiotensin II (Ang-II), the effector peptide of the RAS, acts in the CNS to regulate neurohumoral outflow and thirst. Dysregulation of Ang-II signaling in the CNS 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 (SFO) is critical in the physiologic 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 siRNA to selectively silence Nox2 or Nox4 expression in the SFO. 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 CNS, and are the first to reveal differential involvement of these Nox enzymes in the various physiologic effects of central Ang-II.
hypertension; blood pressure; water intake; subfornical organ; adenovirus; siRNA
Peripheral chemoreflex sensitivity is potentiated in both clinical and experimental chronic heart failure (CHF). NADPH oxidase-derived superoxide mediates angiotensin II (Ang II)-enhanced carotid body (CB) chemoreceptor sensitivity in CHF rabbits, and tempol, the superoxide dismutase (SOD) mimetic, inhibits this Ang II- and CHF-enhanced superoxide anion effect. Here we investigated the role of cytoplasmic SOD [CuZn superoxide dismutase (CuZnSOD)] in the CB on chemoreceptor activity and function in CHF rabbits.
Methods and results
CuZnSOD protein expression was decreased in CBs from CHF rabbits vs. sham (P < 0.05). Adenoviral CuZnSOD (Ad CuZnSOD) gene transfer to the CBs increased CuZnSOD protein expression and significantly reduced the baseline renal sympathetic nerve activity (RSNA) and the response of RSNA to hypoxia in the CHF rabbits (P < 0.05). Single-fibre discharge from CB chemoafferents during normoxia (baseline, at ∼100 mmHg PO2) and in response to hypoxia were enhanced in CHF vs. sham rabbits (P < 0.05). Ad CuZnSOD decreased the baseline discharge (7.6 ± 1.3 vs. 12.6 ± 1.7 imp/s at ∼100 mmHg PO2) and the response to hypoxia (22.4 ± 1.6 vs. 32.3 ± 1.2 imp/s at ∼40 mmHg PO2, P < 0.05) in CHF rabbits. Ad CuZnSOD also normalized the blunted outward K+ current (IK) in CB glomus cells from CHF rabbits (369 ± 14 vs. 565 ± 31 pA/pF at +70 mV, P < 0.05). In addition, Ad CuZnSOD reduced the elevation of superoxide level in CBs from CHF rabbits.
Downregulation of CuZnSOD in the CB contributes to the enhanced activity of CB chemoreceptors and chemoreflex function in CHF rabbits.
Superoxide dismutase; Adenoviral vector; Carotid body; Sympathetic nerve activity; Chemoreceptor; Glomus cell; Chronic heart failure
Essential hypertension has devastating effects on the brain, being the major cause of stroke and a leading cause of dementia. Hypertension alters the structure of cerebral blood vessels and disrupts intricate vasoregulatory mechanisms that assure an adequate blood supply to the brain. These alterations threaten the cerebral blood supply and increase the susceptibility of the brain to ischemic injury as well as Alzheimer’s disease. This review focuses on the mechanisms by which hypertension disrupts cerebral blood vessels, highlighting recent advances and outstanding issues.
More than 90% of Duchenne muscular dystrophy (DMD) patients develop cardiomyopathy, and many die of cardiac failure. Despite tremendous progress in skeletal muscle gene therapy, few attempts have been made to treat cardiomyopathy. Microdystrophin genes are shown to correct skeletal muscle pathological lesions in the mdx mouse model for DMD. Here, we tested the therapeutic potential of adeno-associated virus (AAV)–mediated microdystrophin gene therapy in the mdx mouse heart.
Methods and Results
AAV was delivered to the newborn mdx mouse cardiac cavity. The procedure was rapid and well tolerated. Efficient expression was achieved in the inner and the outer layers of the myocardium. The ubiquitous cytomegalovirus promoter resulted in substantially higher expression than the muscle-specific CK6 promoter. The therapeutic effects of microdystrophin were evaluated at 10 months after infection. Immunostaining demonstrated extensive microdystrophin expression and successful restoration of the dystrophin-glycoprotein complex. Importantly, AAV-mediated microdystrophin expression improved the sarcolemma integrity in the mdx heart.
We established a simple gene transfer method for efficient and persistent transduction of the mdx mouse heart. AAV-mediated microdystrophin expression restored the critical dystrophin-glycoprotein complex and improved sarcolemma integrity of the mdx heart. Our results revealed the promise of AAV-microdystrophin gene therapy for cardiomyopathy in DMD.
muscular dystrophy; genes; viruses; gene therapy; microdystrophin
ACE2 is a newly discovered carboxy-peptidase responsible for the formation of vasodilatory peptides such as angiotensin-(1-7). We hypothesized that ACE2 is part of the brain renin-angiotensin system (RAS) and its expression regulated by the other elements of this system. ACE2 immuno-staining was performed in transgenic mouse brain sections from NSE-AT1A (overexpressing AT 1A receptors), R+A+ (overexpressing angiotensinogen, and renin) and control (non transgenic littermates) mice. Results show that ACE2 staining is widely distributed throughout the brain. Using cell type-specific antibodies, we observed that ACE2 staining is present in the cytoplasm of neuronal cell bodies but not in glial cells. In the subfornical organ, an area lacking the blood brain barrier and sensitive to blood borne angiotensin-II, ACE2 was significantly increased in transgenic mice. Interestingly, ACE2 mRNA and protein expression were inversely correlated in the nucleus of tractus solitarius/dorsal motor nucleus of the vagus and the ventrolateral medulla, when comparing transgenic to non-transgenic mice. These results suggest that ACE2 is localized to the cytoplasm of neuronal cells in the brain, and that ACE2 levels appear highly regulated by other components of the RAS, confirming its involvement in this system. Moreover, ACE2 expression in brain structures involved in the control of cardiovascular function suggests that the carboxypeptidase may have a role in the central regulation of blood pressure and diseases involving the autonomic nervous system such as hypertension.
central nervous system; circumventricular organs; volume homeostasis; blood pressure; carboxypeptidase
The mechanism controlling cell-specific Ang II production in the brain remains unclear despite evidence supporting neuron-specific renin and glial- and neuronal-specific angiotensinogen (AGT) expression. We generated double-transgenic mice expressing human renin (hREN) from a neuron-specific promoter and human AGT (hAGT) from its own promoter (SRA mice) to emulate this expression. SRA mice exhibited an increase in water and salt intake and urinary volume, which were significantly reduced after chronic intracerebroventricular delivery of losartan. Ang II–like immunoreactivity was markedly increased in the subfornical organ (SFO). To further evaluate the physiological importance of de novo Ang II production specifically in the SFO, we utilized a transgenic mouse model expressing a floxed version of hAGT (hAGTflox), so that deletions could be induced with Cre recombinase. We targeted SFO-specific ablation of hAGTflox by microinjection of an adenovirus encoding Cre recombinase (AdCre). SRAflox mice exhibited a marked increase in drinking at baseline and a significant decrease in water intake after administration of AdCre/adenovirus encoding enhanced GFP (AdCre/AdEGFP), but not after administration of AdEGFP alone. This decrease only occurred when Cre recombinase correctly targeted the SFO and correlated with a loss of hAGT and angiotensin peptide immunostaining in the SFO. These data provide strong genetic evidence implicating de novo synthesis of Ang II in the SFO as an integral player in fluid homeostasis.