SR-A Mice Research Articles

Hypertension in mice with transgenic activation of the brain renin-angiotensin system is vasopressin dependent

An indispensable role for the brain renin-angiotensin system (RAS) has been documented in most experimental animal models of hypertension. To identify the specific efferent pathway activated by the brain RAS that mediates hypertension, we examined the hypothesis that elevated arginine vasopressin (AVP) release is necessary for hypertension in a double-transgenic model of brain-specific RAS hyperactivity (the "sRA" mouse model). sRA mice experience elevated brain RAS activity due to human angiotensinogen expression plus neuron-specific human renin expression. Total daily loss of the 4-kDa AVP prosegment (copeptin) into urine was grossly elevated (≥8-fold). Immunohistochemical staining for AVP was increased in the supraoptic nucleus of sRA mice (~2-fold), but no quantitative difference in the paraventricular nucleus was observed. Chronic subcutaneous infusion of a nonselective AVP receptor antagonist conivaptan (YM-087, Vaprisol, 22 ng/h) or the V(2)-selective antagonist tolvaptan (OPC-41061, 22 ng/h) resulted in normalization of the baseline (~15 mmHg) hypertension in sRA mice. Abdominal aortas and second-order mesenteric arteries displayed AVP-specific desensitization, with minor or no changes in responses to phenylephrine and endothelin-1. Mesenteric arteries exhibited substantial reductions in V(1A) receptor mRNA, but no significant changes in V(2) receptor expression in kidney were observed. Chronic tolvaptan infusion also normalized the (5 mmol/l) hyponatremia of sRA mice. Together, these data support a major role for vasopressin in the hypertension of mice with brain-specific hyperactivity of the RAS and suggest a primary role of V(2) receptors.

Abstract 126: Transgenic Hyperactivity of the Brain Renin-Angiotensin System Causes Vasopressin-Dependent Hypertension

Hypertension in many animal models is sensitive to inhibition of the brain renin-angiotensin system (RAS). We examined mice with transgenic hyperactivity of the brain RAS (sRA mice) to determine whether this manipulation is sufficient to cause hypertension, and to identify the causative mechanism. sRA mice exhibit brain-specific increases in angiotensin (ANG) peptide production through neuron-specific expression of human renin (synapsin promoter) and expression of human angiotensinogen through its own promoter. We determined both through tail-cuff and radiotelemetric methods that sRA mice are hypertensive (SBP; control 112±2 vs sRA 127±5 mmHg, P=0.02). Despite normal plasma osmolality and moderate hyponatremia, sRA mice exhibit double the number of vasopressin-expressing neurons in the supraoptic nucleus (P=0.002), as detected by immunohistochemistry. Plasma levels of the vasopressin pro-segment, copeptin, were reduced in sRA mice (140±19 vs 68±21 pg/mL, P=0.02), which correlated with severe polyuria (1.8±0.3 vs 12.3±1.6 mL/day, P<0.001). Indeed, total daily copeptin loss in the urine was significantly increased almost twenty-fold in sRA mice (7.9±4.3 vs 154.4±62.4 pg/day, P=0.03), highlighting an increase in vasopressin secretion per unit time. The baseline hypertension of sRA mice was completely reversed by chronic infusion of the dual V 1A / V 2 receptor antagonist, conivaptan (22 ng/hr, 10 days, s.c.; 113±5 mmHg, P=0.02). Preliminary experiments demonstrate that infusion of the selective V 2 receptor antagonist, tolvaptan (22 ng/hr, 10 days, s.c.), has similar effects. Further, while abdominal aorta and mesenteric arteries demonstrate selective desensitization to vasopressin and down-regulation of the V 1A receptor (38% of control, P<0.05), renal V 2 receptor expression remained normal in sRA mice. Together, these data demonstrate major roles for vasopressin and its V 2 receptor in the hypertension caused by elevated brain RAS activity.

Abstract 20: Angiotensin Suppresses Thermogenic Capacity Through Adipose AT2 Receptors

Selective induction of the brain renin-angiotensin system (RAS) results in hypertension, polydipsia, and elevated resting metabolic rate. Transgenic sRA mice exhibit brain-specific RAS hyperactivity through expression of human renin via the synapsin promoter, and human angiotensinogen via its own promoter. The circulating RAS in these animals is suppressed due to chronic hypertension, and the elevated resting metabolic rate of sRA mice is sensitive to peripheral propranolol or angiotensin replacement. Cultured 3T3L1 adipocytes exhibited a dose-dependent induction of uncoupling protein 1 (UCP1) by the AT 2 receptor (AT 2 R) antagonist, PD-123,319, and suppression by the AT 2 R agonist, CGP-42112a (CGP). UCP1 mRNA was specifically induced within inguinal adipose of sRA mice (25-fold, P=0.02). Treatment of sRA mice with CGP (50 ng/kg/min, 8 weeks, s.c.) normalized metabolic rate (control 2.8±0.2, sRA 3.6±0.2, sRA+CGP 3.1±0.2 mL O 2 /100g/min) and inguinal UCP1 mRNA (sRA 24.6 vs sRA+CGP 4.6-fold of control, P=0.02), blood pH (control 7.26±0.02, sRA 7.37±0.03, sRA+CGP 7.28±0.06), bicarbonate (control 22±1, sRA 31±1, sRA+CGP 21±6 mM) and perigenital adipose mass (control 317±100, sRA 127±16, sRA+CGP 590±127 mg). Fluid and sodium intake, urine volume and electrolytes, and heart and kidney masses were different in sRA mice but unchanged by CGP treatment, and food intake was unchanged in all groups, highlighting the specificity of effects elicited by CGP. We conclude that AT 2 R activation blunts the thermogenic response to increased brain RAS activity. Interestingly, sRA gain weight at the same rate as control mice when fed a high fat diet, prompting the hypothesis that an elevated circulating and/or adipose RAS may result in attenuated thermogenic responses during obesity. This may suggest a positive-feedback loop between the circulating RAS and adiposity, and identify adipose AT 2 R as a novel therapeutic target for obesity.

Abstract 84: Angiotensin II Type 1a Receptor in the Subfornical Organ is Essential for Blood Pressure Regulation and Hydromineral Effects in Mouse Models of Elevated Brain Renin-Angiotensin System Activity.

Elevated brain renin-angiotensin system (RAS) activity is necessary to increase blood pressure in many animal models of hypertension. We tested the hypothesis that AT1A receptors (AT1AR) within the subfornical organ (SFO) are required for the phenotypes that result from an increased brain RAS. We examined the effect of SFO-targeted Cre-recombinase mediated ablation of AT1A in mice treated with DOCA-salt (deoxycorticosterone acetate, 50 mg s.c. + ad lib 0.15 M NaCl). Mice homozygous for a conditional allele of the endogenous AT1A gene (AT1ARflox) were administered an adenovirus encoding Cre-recombinase and eGFP (AdCre), or eGFP alone (AdGFP) into the lateral cerebral ventricle, then treated for 3 weeks with DOCA-salt. AdCre reduced DOCA-salt hypertension (AdGFP baseline: 108±3 mmHg; AdGFP pre-DOCA: 104±3; AdGFP post-DOCA: 136±6 vs AdCre baseline: 116±2; Adcre pre-DOCA: 109±3, Adre post-DOCA: 118±5; P≤0.01), polydipsia (AdGFP+DOCA: 20.6±2.1 mL/day; AdCre+DOCA: 11.6±1.1, P<0.05), and sodium intake (AdGFP+DOCA: 2.6±0.3 mEq/day; AdCre+DOCA: 1.8±0.2, P<0.05). AdCre reduced AT1AR mRNA in the SFO (0.4±0.3 fold of AdGFP), without significant effect in the paraventricular or arcuate nuclei, or cortex; this was paralleled by SFO-specific AT1AR genomic DNA recombination. AdCre also caused SFO-specific recombination in ROSA-TdTomato reporter mice. Complementing the DOCA-salt model, we also examined the effect of AT1AR ablation in the SFO of double-transgenic sRA mice. sRA mice exhibit life-long brain-specific angiotensin overproduction via expression of human angiotensinogen via its own promoter and neuron-specific expression of human renin via the synapsin promoter. In sRA mice bred onto the AT1A conditional genetic background, AdCre significantly attenuated the polydipsia (AdGFP: -0.2±2; AdCre: -9.7±2.6 mL/day) and sodium intake (AdGFP: +0.2±0.7; AdCre: -1.3±0.4 mEq/day). Blood pressure measures are in progress. Together, these data highlight the involvement of SFO AT1A receptors in blood pressure in DOCA-salt model and additionally in hydromineral balance in two different models of increased brain RAS activity.

Abstract 409: Endoplasmic Reticulum Stress Mediates Fluid Balance and Metabolic Effects of the Brain Renin-Angiotensin System

Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response (UPR) have been identified as important contributors to various neural diseases. We examined the role of ER stress and the UPR in the metabolic and fluid balance effects of brain angiotensin in two mouse models. The ER stress-reducing chemical chaperone tauroursodeoxycholic acid (TUDCA, 5.28 ug/day) or aCSF vehicle was infused into the lateral cerebral ventricle (ICV) of either C57Bl/6J mice treated for three weeks with high dietary sodium and chronic subcutaneous deoxycorticosterone acetate (the “DOCA-salt” model), or transgenic “sRA” mice that express human renin in neurons via the synapsin promoter and human angiotensinogen via its own promoter. Both the DOCA-salt and sRA models exhibit hyperactivity of the brain RAS, suppression of circulating RAS, hypertension, polydipsia, and an elevated resting metabolic rate. Forebrain, midbrain, and hindbrain regions of sRA mice exhibited a significant elevation of CHOP mRNA, a marker of long-term UPR. There were no changes in CHOP expression in the cortex and cerebellum. ICV TUDCA significantly attenuated the polydipsia (DOCA-aCSF 20.7±0.9 vs DOCA-TUDCA 10.8±1.0 mL/day, P<0.05) and metabolic rate (DOCA-aCSF, 3.38±0.07 vs DOCA-TUDCA 3.16±0.06 mL O 2 /100g/min, P<0.05) in the DOCA-salt model. ICV TUDCA had similar effects on the polydipsia in the sRA model (51±10% of aCSF control, P<0.05). Metabolic studies in the sRA model, and blood pressure in the sRA and DOCA models are ongoing. Together these data mechanistically implicate ER stress in the fluid and metabolic responses to increased brain RAS activity.

Deletion of scavenger receptor A gene in mice resulted in protection from septic shock and modulation of TLR4 signaling in isolated peritoneal macrophages

Scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a surface glycoprotein preferentially present in macrophages that plays a primary role in innate immunity. Previous studies have shown that Sra is a modifier gene for the response to bacterial LPS in mice at the level of IL-10 production, in particular. In the present study, we found that Sra(-/-) mice are more resistant to septic shock induced by cecal ligation and puncture than wild-type C57BL/6 J (B6) mice. In addition, Sra(-/-) mice displayed initial elevated high density lipoprotein (HDL) circulating levels. Naïve peritoneal macrophages (PMs) were isolated from Sra(-/-) mice to understand the possible protective mechanism. Incubation of these cells with LPS was found to modulate TLR4 signaling, leading to a reduction in IL-10 and IL-6 mRNA levels, but not TNF-α expression, at low concentrations of LPS in comparison with PMs isolated from B6 mice. No differences were found in LPS binding between PMs derived from Sra(-/-) or B6 mice. The lack of Sra binding to LPS was confirmed after transfection of Chinese hamster ovary (CHO) cells with the Sra gene. The contribution of Sra to the outcome of sepsis may be a combination of changes in TLR4 signaling pathway and elevated levels of HDL in circulation, but also LPS toxicity.

Angiotensinergic signaling in the brain controls whole‐body metabolism: Role of adipose AT2 receptors

Transgenic “sRA” mice exhibit elevated brain renin‐angiotensin system (RAS) activity through expression of a human angiotensinogen transgene and a neuron‐specific human renin transgene. sRA mice exhibit elevated metabolic rate, partially mediated through a suppression of the circulating RAS. Uncoupling protein‐1 (UCP1; 24.6‐fold, n=5, P<0.01), and PPARγ‐coactivator‐1α (PGC‐1α; 2.8‐fold, n=5, P=0.01) mRNA were increased selectively in subcutaneous adipose (scWAT) of sRA mice, suggesting an increased brown adipocyte content. Treating differentiated 3T3‐L1 preadipocytes with the AT2 antagonist PD‐123,319 induced PGC1α (2.4‐fold) and the PGC1α target, catalase (3.4‐fold), while the AT1 antagonist Losartan had no effect. Treating sRA mice with the AT2 agonist CGP‐42112a (90 ng/hr, s.c., 8 weeks) normalized scWAT UCP1 mRNA (vehicle 24.6 vs CGP 2.1‐fold of control, n=5,7, P<0.05), suppressed resting metabolic rate (3.59±0.22 vs 3.12±0.17 mL O2/100g/min, P<0.01), and increased body weight (17.8±0.5 vs 23.1±2.1 grams, P=0.01), but had no effect on food intake (3.1±0.2 vs 3.6±0.3 g/day, P=0.72). These data support a model where AT2 receptor‐mediated suppression of adipose transdifferentiation mediates increased thermogenic capacity in animals with elevated brain RAS activity. Selective antagonism of AT2 receptors in adipose tissue may therefore represent a novel therapeutic strategy for obesity.

MTOR/S6K Signaling: A Novel Effector of Neuronal Action of Angiotensin II

The renin‐angiotensin system (RAS) is critically involved in arterial pressure regulation. Angiotensin II (Ang II), the main effector of the RAS, acts in several brain nuclei including the subfornical organ (SFO) to increase arterial pressure by stimulating Ang II type 1 receptors (AT1R). However, the signaling mechanisms of neuronal AT1R remain poorly defined. Neuronal mammalian target of rapamycin (mTOR) and its downstream effector, p70S6 kinase (S6K), have emerged as important regulators of arterial pressure. Thus, we hypothesized that mTOR/S6K signaling mediates brain Ang II actions. In hypothalamic neuronal cell line (GT1‐7), transfected with AT1aR, Ang II (100 nM) caused a time‐dependent increase in S6K activity with a maximum effect at 15 min (3.4‐fold; P<0.05 vs. vehicle). Pre‐treatment with AT1R blocker (1 μM losartan) completely abolished Ang II‐induced activation of S6K. We also examined, by immunohistochemistry, the level of phospho‐S6 (a readout of S6K activity) in the brain of transgenic sRA mice which have overactive brain RAS. The SFO of female sRA mice exhibited significantly (P=0.02) elevated phospho‐S6 positive cells (64±6) relative to littermate controls (36±8). This increase was specific to the SFO as there was no difference in phospho‐S6 levels in other forebrain nuclei in sRA vs. control mice. This study has identified mTOR/S6K as a novel mechanism of AT1R signaling in neurons.

Abstract 17594: Brain Microglial Cytokines are Critical for Renin-Angiotensin-Aldoterone-System(RAAS)-Induced Neurogenic Hypertension

Purpose: Our previous studies have established that activation of microglial cells and production of proinflammatory cytokines (PIC) within the PVN are two critical events in the establishment of chronic angiotensin (Ang) II-induced neurogenic hypertension. These have led us to hypothesize that brain microglial cells are a primary target of Ang II, initiating a sequence of cellular and physiological events resulting in sympathetic nervous system activation and hypertension. Methods: Microglial cells from the hypothalamus of Sprague-Dawley rats were established in primary culture. Their phenotype was confirmed by the presence of IBa1 immunoreactivity and high levels of CD11b mRNA, markers for microglia. They were used to determine the effects of, Ang II, prorenin and aldosterone (ALDO) on cytokine production. Neurogenic hypertensive mice were generated using decoxycorticosterone acetate (DOCA)-salt model (DOCA 50 mg s.c., 0.15 M NaCl in drinking water for 3 weeks). The PVN was dissected to determine PIC expression at the end of the protocol. Results: Treatment of cultures with Ang II (100 nM) or ALDO (1 nM) for 24 hr increased the expression of PIC mRNAs, e.g. for tumor necrosis factor (TNF)α. Ang II and ALDO elicited respective 2- and 3-fold increases (P<0.05). More strikingly, we found that prorenin (20 nM, 24 hr) elicited highly significant 300- and 400-fold increases in the expression of TNFα and interleukin (IL)-1β mRNAs, respectively (P<0.05). These effects of prorenin were associated with respective 16- and 14-fold increases in NFκb1 and NFκbia mRNA levels, which are two subunits of the transcription factor NFκb complex. We previously demonstrated that DOCA mice exhibit a similar hypertensive phenotype as double transgenic hypertensive sRA mice, which display increased levels of PIC and activation of microglia in the PVN. Consistently, DOCA-salt mice exhibit respective 1.5, 1.08 and 1.09 fold increases in the expression of mRNAs for IL-1β, TNFα and IL-6 in the PVN compared to control mice (n=3-4). Conclusion: These data indicate that brain microglial cells are the primary target of RAAS components in promoting PIC production, which in turn contribute to hypertension development.

Class A scavenger receptor attenuates myocardial infarction-induced cardiomyocyte necrosis through suppressing M1 macrophage subset polarization

Classically (M1) and alternatively (M2) activated macrophage subsets play differential roles in left ventricular remodeling after myocardial infarction (MI). The precise mechanism underlying the regulation of M1/M2 polarization during MI is unknown. We hypothesized that class A scavenger receptor (SR-A), a key modulator of inflammation, may steer macrophage polarization, which in turn influences cardiomyocytes necrosis after MI. MI was induced in wild type (WT) and SR-A deficient (SR-A(-/-)) mice by left anterior descending coronary artery ligation. Cardiac function deterioration, ventricular dilatation and fibrosis were all exacerbated in SR-A(-/-) mice following MI compared to WT littermates. Meanwhile, enhanced M1 macrophage polarization was observed in SR-A(-/-) mice, along with increased production of M1 signature cytokines including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) as demonstrated by immunohistochemistry, flow cytometry, quantitative real-time PCR, and ELISA assays. Moreover, activation of the activated apoptosis signal regulating kinase 1 (ASK1)/p38 mitogen-activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) signaling pathway was markedly elevated in SR-A(-/-) animals post-MI. Most importantly, transplantation using bone marrow from SR-A(+/+) mice partially restored M1 macrophages and significantly augmented left ventricular fractional shortening in SR-A(-/-) mice. SR-A attenuated MI-induced cardiac remodeling by suppressing macrophage polarization toward a skewed M1 phenotype, reducing secretion of IL-1β, IL-6, and TNF-α, and dampening the ASK1/p38/NF-κB signaling pathway. Therefore, SR-A may exert a protective effect against MI, which may represent a new interventional target for treatment of post-infarct remodeling and subsequent heart failure.

MICROGLIAL ACTIVATION BY THE BRAIN RENIN‐ANGIOTENSIN SYSTEM

Recently we demonstrated the necessity of microglial activation, and subsequent secretion of proinflammatory cytokines (PIC) within the brain, in peripheral-angiotensin-infusion hypertension. Here, we examine the involvement of microglial activation in a model of low-renin hypertension. Mice expressing human renin under a neuron-specific synapsin promoter were crossed with mice expressing human angiotensinogen under its own promoter. Resulting double-transgenic (“sRA”) mice exhibit moderate hypertension (30 mmHg), extreme polydipsia and polyuria (8x controls), elevated basal metabolic rate (1.25x controls), and increased sodium intake due to elevated brain renin-angiotensin system (RAS) activity (2x controls) and suppressed circulating RAS activity (0.3x controls). sRA mice exhibited enhanced microglial activation and significant increases in angiotensin type 1 receptor (1.6-fold, P<0.05) and interleukin-1β (1.5-fold, P<0.05) mRNA levels in the hypothalamic paraventricular nucleus when compared to littermate control mice. Together, our data suggest that increased brain-RAS signaling is associated with mciroglial activation and PIC, independent of the status of the circulating RAS. Ongoing studies are examining the necessity of mciroglial activation and PIC in the blood pressure, hydromineral, and metabolic phenotypes of the sRA model. AHA 09POST2230164 to PS; HL098276 To JLG; HL076803 to CS.

Acute Acalculous Cholecystitis-Like Phenotype in Scavenger Receptor A Knock-Out Mice

Sepsis is a major health problem in the United States that affects more than three-quarters of a million people every year. Previous studies have shown that scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a modifier of interleukin 10 (IL-10) expression after injection of bacterial lipopolysaccharide (LPS). Therefore, we investigated the response to sepsis in Sra knock out mice. C57BL/6J (B6) (n = 88) and Sra (-/-) mice (n = 88) were subjected to cecal ligation and puncture (CLP) using 18G or 16G needles, sham operation, or non-operated controls. At the end, mice were autopsied for the determination of abnormalities after the procedure. Cytokine gene expression was examined in lung and liver samples by quantitative RT-PCR (qRT-PCR), and circulating cholesterol levels were also measured. Sra (-/-) mice displayed an enlargement of the gallbladder after CLP that was not detected in sham or non-operated mice or in B6 mice (wild-type) after CLP. The enlarged gallbladder resembles a condition of acute acalculous cholecystitis observed in humans. Sra (-/-) mice presented high cholesterol levels in circulation as opposed to wild type B6 mice. Moreover, Sra (-/-) mice exhibited a reduction in IL-10 mRNA levels in lungs compared to wild-type B6 mice after CLP. The development of acute acalculous cholecystitis may be the combination of pre-existing conditions, such as hypercholesterolemia associated with a defect in Sra (Msr1) and a robust inflammation induced by sepsis.

The Brain Renin-Angiotensin System Controls Divergent Efferent Mechanisms to Regulate Fluid and Energy Balance

The renin-angiotensin system (RAS), in addition to its endocrine functions, plays a role within individual tissues such as the brain. The brain RAS is thought to control blood pressure through effects on fluid intake, vasopressin release, and sympathetic nerve activity (SNA), and may regulate metabolism through mechanisms which remain undefined. We used a double-transgenic mouse model that exhibits brain-specific RAS activity to examine mechanisms contributing to fluid and energy homeostasis. The mice exhibit high fluid turnover through increased adrenal steroids, which is corrected by adrenalectomy and attenuated by mineralocorticoid receptor blockade. They are also hyperphagic but lean because of a marked increase in body temperature and metabolic rate, mediated by increased SNA and suppression of the circulating RAS. β-adrenergic blockade or restoration of circulating angiotensin-II, but not adrenalectomy, normalized metabolic rate. Our data point to contrasting mechanisms by which the brain RAS regulates fluid intake and energy expenditure.

Delayed growth of EL4 lymphoma in SR‐A‐deficient mice is due to upregulation of nitric oxide and interferon‐γ production by tumor‐associated macrophages

Class A scavenger receptors (SR-A, CD204) are highly expressed in tumor-associated macrophages (TAM). To investigate the function of SR-A in TAM, wild-type and SR-A-deficient (SR-A(-/-)) mice were injected with EL4 cells. Although these groups of mice did not differ in the numbers of infiltrating macrophages and lymphocytes and in neovascularization, SR-A(-/-) mice had delayed growth of EL4 tumors. Expression of inducible nitric oxide (NO) synthase and interferon (IFN)-gamma mRNA increased significantly in tumor tissues from SR-A(-/-) mice. Engulfment of necrotic EL4 cells induced upregulation of NO and IFN-gamma production by cultured macrophages, and production of NO and IFN-gamma increased in SR-A(-/-) macrophages in vitro. IFN-beta production by cultured macrophages was also elevated in SR-A(-/-) macrophages in vitro. These results suggested that the antitumor activity of macrophages increased in SR-A(-/-) mice because of upregulation of NO and IFN-gamma production. These data indicate an important role of SR-A in regulating TAM function by inhibiting toll-like receptor (TLR)4-IFN-beta signaling.

Adrenal hormones are necessary for the hydromineral phenotypes of transgenic mice with central renin‐angiotensin system overactivity

To examine the roles of the central renin‐angiotensin system (RAS) in blood pressure and fluid volume homeostasis, our lab previously developed the sRA mouse model, in which human renin is expressed via the neuron‐specific synapsin promoter, and human angiotensinogen via its endogenous promoter. Due to species‐specificity of this reaction, RAS overactivity is restricted to the brain. sRA mice exhibit polydipsia (control, 0.04±0.01 vs sRA, 0.31±0.12 mL/g/d), polyuria (0.08±0.02 vs 0.92±0.04 g/g/d), and a robust sodium appetite (0.94±0.18 vs 3.93±0.07 mEq/d). Here, we examined whether elevated HPA‐axis activity is necessary for these phenotypes. Four weeks after total adrenalectomy, the sodium appetite of sRA mice was completely normalized (0.72±0.21 mEq/d), and the polydipsia (0.11±0.01 mL/g/d) and polyuria (0.15±0.01 g/g/d) were significantly attenuated. In a separate cohort, acute spironolactone treatment partially blocked the sodium intake of sRA mice (3.03±0.31 down to 2.33±0.44 mEq/d), suggesting the involvement of multiple adrenal hormones in this behavior. From these data, we conclude that multiple adrenal hormones are necessary for the dipsogenic and sodium appetite behaviors of mice with chronic overactivity of the brain RAS. Ongoing work is dissecting the contribution of glucocorticoids in hydromineral regulation in sRA mice. (JLG supported by the APS; CLG, AKJ, CDS supported by NIH)

The brain renin‐angiotensin system promotes a negative energy balance in mice

The role of the renin‐angiotensin system (RAS) in the control of energy homeostasis has long been debated, and evidence suggests opposing actions of the central versus the circulating and adipose RAS. We previously developed double‐transgenic (sRA) mice consisting of the human renin gene under the transcriptional control of the neuron‐specific synapsin promoter, and human angiotensinogen under the control of its own endogenous promoter, which results in a CNS‐specific over‐activation of the RAS. Here, we investigated the hypothesis that a centrally overactive RAS would increase energy utilization, and possibly decrease food intake. sRA mice (n=8) exhibited a sustained significant (15%, P&lt;0.001) decrease in body mass compared to littermate controls, which was accompanied by a significantly increased metabolic rate during both sleep (20%, P&lt;0.001) and stationary wakefulness (18%, P=0.002) when assessed indirectly through oxygen consumption (ages 10–12 weeks). sRA mice paradoxically exhibited increased food consumption (20%, P=0.005). Together, these results indicate a negative energy balance in sRA mice, which is predominantly mediated through increased energy expenditure. These findings prompt further studies into the CNS‐RAS in metabolic disorders such as diabetes and the metabolic syndrome. This work was supported by grants (CDS) and postdoctoral fellowships (JLG, CLG) from the NIH and the APS.

Local production of angiotensin II in the subfornical organ causes elevated drinking

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 (hAGT(flox)), so that deletions could be induced with Cre recombinase. We targeted SFO-specific ablation of hAGT(flox) by microinjection of an adenovirus encoding Cre recombinase (AdCre). SRA(flox) 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.

Class A scavenger receptor (CD204) attenuates hyperoxia‐induced lung injury by reducing oxidative stress

To clarify the role of macrophage class A scavenger receptors (SR-A, CD204) in oxidative lung injury, we examined lung tissue of SR-A deficient (SR-A(-/-)) and wild-type (SR-A(+/+)) mice in response to hyperoxic treatment. Protein levels of bronchoalveolar lavage fluid (BALF) and pulmonary oedema (wet : dry weight ratios) were higher in SR-A(-/-) mice than those in SR-A(+/+) mice. Cumulative survival was significantly decreased in SR-A(-/-) mice. However, there were no differences in BALF macrophage and neutrophil count between the two groups. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that messenger RNA (mRNA) levels of the inducible nitric oxide synthase (iNOS) were increased during hyperoxic injury, and this increase was more prominent in SR-A(-/-) mice. Expression levels of iNOS in alveolar macrophages after hyperoxia in vivo and in vitro were higher in SR-A(-/-) macrophages compared with SR-A(+/+) macrophages. Immunohistochemistry using anti-nitrotyrosine antibodies revealed distinctive oxidative stress in the injured lung in both groups, but it was more remarkable in the SR-A(-/-) mice. After hyperoxic treatment, pulmonary mRNA levels of tumour necrosis factor-alpha(TNF-alpha) were elevated more rapidly in SR-A(-/-) mice than in SR-A(+/+) mice. Together these results suggest that SR-A expression attenuates hyperoxia-induced lung injury by reducing macrophage activation.

A CD36-dependent signaling cascade is necessary for macrophage foam cell formation

Accumulation of macrophage foam cells in atherosclerotic blood vessel intima is a critical component of atherogenesis mediated by scavenger receptor-dependent internalization of oxidized LDL. We demonstrated by coimmunoprecipitation and pull-down assays that the macrophage scavenger receptor CD36 associates with a signaling complex containing Lyn and MEKK2. The MAP kinases JNK1 and JNK2 were specifically phosphorylated in macrophages exposed to oxLDL. Using cells isolated from SRA, TLR2, or CD36 null mice, and phospholipid ligands specific for either SRA or CD36, we showed that JNK activation was mediated by CD36. Both foam cell formation and activation of JNK2 in hyperlipidemic mice were diminished in the absence of CD36. Furthermore, inhibition of Src or JNK blocked oxLDL uptake and inhibited foam cell formation in vitro and in vivo. These findings show that a specific CD36-dependent signaling pathway initiated by oxLDL is necessary for foam cell formation and identify potential targets for antiatherosclerosis therapy.