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RGS (regulator of G protein signaling) family members catalyze the termination of G protein signaling cascades. Single nucleotide polymorphisms in theTo study cell-specific functions of RGS2, a novel gene-targeted mouse harboring a conditional allele for theWhereasThese results demonstrate the development of a novel mouse with conditional expression of

Low-renin hypertension is common among the elderly, subjects of black African ancestry, those with heart or renal failure, and women with preeclampsia. Increased understanding of the causes and consequences of low-renin hypertension is necessary for the development of new efficacious and personalized therapeutics. One commonly employed preclinical model is “DOCA-salt” (DOCA), which causes robust hypertension, proteinuria, and organ damage. The arcuate nucleus of the hypothalamus (ARC) has been implicated in control of metabolic rate and blood pressure responses to DOCA. To characterize the DOCA related changes within the ARC, we used single nucleus RNA sequencing (snRNA-Seq) to individually interrogate the transcriptomes of numerous mouse ARC cell types. Male C57BL/6J mice 7 weeks-of-age underwent sham surgery or subcutaneous implantation of a 50mg DOCA pellet. All mice had water; DOCA mice were also provided 0.15 M NaCl drink ad libitum. Body composition was assessed by nuclear magnetic resonance. After 3 weeks, tissues were weighed and blood chemistries assessed. Features of DOCA treatment were consistent with previous studies, such as suppressed growth trajectory (sham n=7, DOCA n=8; -0.8±0.4g/3wk; p

Activation of central AT 1 Rs (angiotensin type 1 receptors) is required for the increased blood pressure, polydipsia, and salt intake in deoxycorticosterone acetate (DOCA)–salt hypertension. TRV120027 (TRV027) is an AT 1 R-biased agonist that selectively acts through β-arrestin. We hypothesized that intracerebroventricular administration of TRV027 would ameliorate the effects of DOCA-salt. In a neuronal cell line, TRV027 induced AT 1a R internalization through dynamin and clathrin-mediated endocytosis. We next evaluated the effect of chronic intracerebroventricular infusion of TRV027 on fluid intake. We measured the relative intake of water versus various saline solutions using a 2-bottle choice paradigm in mice subjected to DOCA with a concomitant intracerebroventricular infusion of either vehicle, TRV027, or losartan. Sham mice received intracerebroventricular vehicle without DOCA. TRV027 potentiated DOCA-induced water intake in the presence or absence of saline. TRV027 and losartan both increased the aversion for saline—an effect particularly pronounced for highly aversive saline solutions. Intracerebroventricular Ang (angiotensin) II, but not TRV027, increased water and saline intake in the absence of DOCA. In a separate cohort, blood pressure responses to acute intracerebroventricular injection of vehicle, TRV, or losartan were measured by radiotelemetry in mice with established DOCA-salt hypertension. Central administration of intracerebroventricular TRV027 or losartan each caused a significant and similar reduction of blood pressure and heart rate. We conclude that administration of TRV027 a selective β-arrestin biased agonist directly into the brain increases aversion to saline and lowers blood pressure in a model of salt-sensitive hypertension. These data suggest that selective activation of AT 1 R β-arrestin pathways may be exploitable therapeutically.

Estrogen receptor alpha (ERα)-mediated estrogen signaling play a pivotal role in both reproductive and non-reproductive functions. Transcriptional regulation of ERα gene is highly complex, with multiple transcript variants being differentially produced across the tissues. However, tissue-specific variation and physiological specificity of the ERα variants are not yet fully understood. In an attempt to generate a Cre-dependently restorable ERα-null mice for functional investigation of the genetic sufficiency, we unexpectedly produced ERα hypomorphic mice with biased downregulation of a previously unappreciated long ERα isoform that is enriched in the female reproductive organs (uterus and ovaries) and the pituitary but minimally expressed in the brain. Female homozygous mutant mice were capable of pregnancy but displayed irregular estrus cycle and rarely maintained alive newborns without significant morphological and pathological changes in reproductive system and the disruption of body weight homeostasis, indicating the vital role of this long isoform in female reproductive function. Collectively, our results define a tissue-specifically enriched long ERα isoform and its preferential role in female reproductive function over body weight homeostasis.

Prolonged obesity is associated with blunted feeding and thermogenic autonomic responses to leptin, but cardiovascular responses to leptin are maintained. This state of selective leptin resistance is, therefore, proposed to contribute to the pathogenesis and maintenance of obesity-associated hypertension. Cells of the arcuate nucleus of the hypothalamus detect leptin, and although the cellular and molecular mechanisms remain unclear, altered arcuate nucleus biology is hypothesized to contribute to selective leptin resistance. Male C57BL/6J mice were fed a high-fat diet (HFD) or chow from 8 to 18 weeks of age, as this paradigm models selective leptin resistance. Nuclei were then isolated from arcuate nucleus for single-nucleus RNA sequencing. HFD caused expected gains in adiposity and circulating leptin. Twenty-three unique cell-type clusters were identified, and Ingenuity Pathway Analysis was used to explore changes in gene expression patterns due to chronic HFD within each cluster. Notably, gene expression signatures related to leptin signaling exhibited suppression predominantly in neurons identified as the Agouti-related peptide ( Agrp ) subtype. Ingenuity Pathway Analysis results were also consistent with alterations in CREB (cAMP response element-binding protein) signaling in Agrp neurons after HFD, and reduced phosphorylated CREB was confirmed in arcuate nucleus after prolonged HFD by capillary electrophoresis-based Western blotting. These findings support the concept that prolonged HFD-induced obesity is associated with selective changes in Agrp neuron biology, possibly secondary to altered CREB signaling.

Angiotensin II (ANG II) Agtr1a receptor (AT1A) is expressed in cells of the arcuate nucleus of the hypothalamus that express the leptin receptor ( Lepr) and agouti-related peptide ( Agrp). Agtr1a expression in these cells is required to stimulate resting energy expenditure in response to leptin and high-fat diets (HFDs), but the mechanism activating AT1Asignaling by leptin remains unclear. To probe the role of local paracrine/autocrine ANG II generation and signaling in this mechanism, we bred mice harboring a conditional allele for angiotensinogen ( Agt, encoding AGT) with mice expressing Cre-recombinase via the Lepr or Agrp promoters to cause cell-specific deletions of Agt ( AgtLepr-KOand AgtAgrp-KOmice, respectively). AgtLepr-KOmice were phenotypically normal, arguing against a paracrine/autocrine AGT signaling mechanism for metabolic control. In contrast, AgtAgrp-KOmice exhibited reduced preweaning survival, and surviving adults exhibited altered renal structure and steroid flux, paralleling previous reports of animals with whole body Agt deficiency or Agt disruption in albumin ( Alb)-expressing cells (thought to cause liver-specific disruption). Surprisingly, adult AgtAgrp-KOmice exhibited normal circulating AGT protein and hepatic Agt mRNA expression but reduced Agt mRNA expression in adrenal glands. Reanalysis of RNA-sequencing data sets describing transcriptomes of normal adrenal glands suggests that Agrp and Alb are both expressed in this tissue, and fluorescent reporter gene expression confirms Cre activity in adrenal gland of both Agrp-Cre and Alb-Cre mice. These findings lead to the iconoclastic conclusion that extrahepatic (i.e., adrenal) expression of Agt is critically required for normal renal development and survival.

Objective The paraventricular nucleus of hypothalamus (PVN), an integrative center in the brain, orchestrates a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ connectivity and its role in other physiological regulations are incompletely understood. Here we aimed to better characterize the input–output organization of PVNMC4R+ neurons and test their physiological functions beyond feeding. Methods Using a combination of viral tools, we mapped PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermoregulation, cardiovascular control, and other behavioral responses beyond feeding. Results We found that PVNMC4R+ neurons innervate many different brain regions that are known to be important not only for feeding but also for neuroendocrine and autonomic control of thermoregulation and cardiovascular function, including but not limited to the preoptic area, median eminence, parabrachial nucleus, pre-locus coeruleus, nucleus of solitary tract, ventrolateral medulla, and thoracic spinal cord. Contrary to these broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs mainly from other hypothalamic nuclei (preoptic area, arcuate and dorsomedial hypothalamic nuclei, supraoptic nucleus, and premammillary nucleus), the circumventricular organs (subfornical organ and vascular organ of lamina terminalis), the bed nucleus of stria terminalis, and the parabrachial nucleus. Consistent with their broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure, and brown adipose tissue temperature. These physiological changes accompanied acute transient hyperactivity followed by hypoactivity and resting-like behavior. Conclusions Our results elucidate the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermoregulation, cardiovascular function, and biphasic behavioral activation., Highlights • PVNMC4R+ neurons innervate brain regions involved in feeding and autonomic regulation. • Activation of PVNMC4R+ neurons increases BAT thermogenesis. • Activation of PVNMC4R+ neurons increases blood pressure and heart rate. • Activation of PVNMC4R+ neurons lead to transient hyperactivity followed by hypoactivity and resting-like behavior.

ObjectiveThe paraventricular nucleus of hypothalamus (PVN) is an integrative center in the brain orchestrating a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is undoubtedly involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ pathway and its role in diverse physiological and behavioral regulations have not been fully understood. Here we aimed to better characterize the input-output organization of PVNMC4R+ neurons and further test their potential functional roles beyond feeding.MethodsUsing a combination of viral tools, we performed a comprehensive mapping of PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermogenesis, cardiovascular control and other behavioral regulations beyond feeding.ResultsWe found that PVNMC4R+ neurons broadly innervate many different brain regions known to be important not only for feeding but also for neuroendocrine and autonomic control of thermogenesis and cardiovascular function, including but not limited to preoptic area, median eminence, parabrachial nucleus, locus coeruleus, nucleus of solitary tract, ventrolateral medulla and thoracic spinal cord. Contrary to broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs from limited brain regions, including medial preoptic nucleus, arcuate and dorsomedial hypothalamic nuclei, and supraoptic nucleus. Consistent with broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure and brown adipose tissue thermogenesis. Strikingly, these physiological changes accompanied an unexpected repetitive bedding-removing behavior followed by hypoactivity and resting-like behavior.ConclusionsOur results clarify the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermogenesis, cardiovascular function and other behavioral regulations.

Activation of central AT

Cascade-specific termination of G protein signaling is catalyzed by the RGS (regulator of G protein signaling) family members, including RGS2 . Angiotensin, vasopressin, and endothelin are implicated in preeclampsia, and RGS2 is known to inhibit G protein cascades activated by these hormones. Mutations in RGS2 are associated with human hypertension and increased risk of developing preeclampsia and its sequelae. RGS family members are known to influence maternal vascular function, but the role of RGS2 within the placenta has not been explored. Here, we hypothesized that reduced expression of RGS2 within the placenta represents a risk factor for the development of preeclampsia. Although cAMP/CREB signaling was enriched in placentas from human pregnancies affected by preeclampsia compared with clinically matched controls and RGS2 is known to be a CREB-responsive gene, RGS2 mRNA was reduced in placentas from pregnancies affected by preeclampsia. Experimentally reducing Rgs2 expression within the feto-placental unit was sufficient to induce preeclampsia-like phenotypes in pregnant wild-type C57BL/6J mice. Stimulation of RGS2 transcription within immortalized human HTR8/SVneo trophoblasts by cAMP/CREB signaling was discovered to be dependent on the activity of histone deacetylase activity, and more specifically, HDAC9 (histone deacetylase-9), and HDAC9 expression was reduced in placentas from human pregnancies affected by preeclampsia. We conclude that reduced expression of RGS2 within the placenta may mechanistically contribute to preeclampsia. More generally, this work identifies RGS2 as an HDAC9 -dependent CREB-responsive gene, which may contribute to reduced RGS2 expression in placenta during preeclampsia.

Selective leptin resistance (SLR) is a pathological state caused by prolonged high fat diet (HFD) feeding in which cardiovascular responses to leptin are preserved, but metabolic effects are attenuated. Thus, SLR is hypothesized to contribute to obesity-associated hypertension, but the mechanisms causing SLR are unclear. Desensitization of leptin signaling in cells of the arcuate nucleus (ARC) of the hypothalamus has been proposed as one of potential mechanisms contributing to SLR. Tanycytes, located along the walls of the third ventricle, play critical roles in the regulation of energy balance through the transport of blood-borne metabolic hormones such as leptin and ghrelin into the cerebrospinal fluid. Previous studies have reported that 10 weeks of 45% HFD (D12451) feeding is sufficient to cause SLR in wildtype C57BL/6J mice. To examine the transcriptomic changes induced by prolonged HFD exposure in tanycytes, we performed single-nucleus RNA-sequencing on nuclei isolated from ARC of ad libitum fed male C57BL/6J mice fed chow diet (7013; CD, n=7) or HFD (n=8) from 8 to 18 weeks of age. At 18 weeks, fat mass (+10.1±1.4 vs +3.2±0.4g, pAgrp ) neurons (z-score -1.6, p=0.009) and unchanged in neurons expressing proopiomelanocortin ( Pomc ) (p=0.2). Collectively, these results lead us to hypothesize that SLR is associated with normal transport of circulating leptin into the arcuate nucleus, while the primary dysfunction that causes SLR instead occurs within selected neuronal populations such as Agrp neurons.

Purpose of review Obesity represents the primary challenge to improving cardiovascular health, and suppression of resting metabolic rate (RMR) is implicated in the maintenance of obesity. Increasing evidence supports a major role for the renin-angiotensin system (RAS) within the brain in the control of RMR. Recent findings The angiotensin II (ANG) Agtr1a receptor colocalizes with the leptin receptor (Lepr) primarily within cells of the arcuate nucleus (ARC) of the hypothalamus that also express Agouti-related peptide (Agrp). This sub-population of Agtr1a receptors is required for stimulation of thermogenic sympathetic nervous activity and RMR, but not the suppression of food intake or increasing blood pressure, in response to various stimuli including high-fat diet, deoxycorticosterone acetate and salt, and leptin. Agtr1a is localized to a specific subset (SST3) of Agrp neurons within the ARC. Summary The RAS within the ARC is implicated specifically in RMR control, primarily through Agtr1a localized to the SST3 subset of Agrp neurons. Ongoing research is focused on understanding the unique anatomical projections, neurotransmitter utilization, and signal transduction pathways of Agtr1a within this subset of neurons. Understanding these projections and molecular mechanisms may identify therapeutic targets for RMR and thus obesity, independent of blood pressure and appetite.

Copeptin, a marker of arginine vasopressin (AVP) secretion, is elevated throughout human pregnancies complicated by preeclampsia (PE), and AVP infusion throughout gestation is sufficient to induce the major phenotypes of PE in mice. Thus, we hypothesized a role for AVP in the pathogenesis of PE. AVP infusion into pregnant C57BL/6J mice resulted in hypertension, renal glomerular endotheliosis, intrauterine growth restriction, decreased placental growth factor (PGF), altered placental morphology, placental oxidative stress, and placental gene expression consistent with human PE. Interestingly, these changes occurred despite a lack of placental hypoxia or elevations in placental fms-like tyrosine kinase-1 (FLT1). Coinfusion of AVP receptor antagonists and time-restricted infusion of AVP uncovered a mid-gestational role for the AVPR1A receptor in the observed renal pathologies, versus mid- and late-gestational roles for the AVPR2 receptor in the blood pressure and fetal phenotypes. These findings demonstrate that AVP is sufficient to initiate phenotypes of PE in the absence of placental hypoxia, and indicate that AVP may mechanistically (independently, and possibly synergistically with hypoxia) contribute to the development of clinical signs of PE in specific subtypes of human PE. Additionally, they identify divergent and gestational time-specific signaling mechanisms that mediate the development of PE phenotypes in response to AVP.

TRV120027 (TRV) is biased agonist at the angiotensin AT1 receptor that activates the β-arrestin pathway, causing receptor internalization. AT1 signaling within the hypothalamus is required for the blood pressure, polydipsia, and sodium appetite phenotypes in the deoxycorticosterone acetate (DOCA)-salt model of salt-sensitive hypertension (HTN). We hypothesized that TRV action within the hypothalamus would antagonize the effects of DOCA-salt. Using a novel luciferase-based HiBit tagging assay, we confirmed that TRV decreased AT1 surface density in immortalized mouse hypothalamic N43/5 cells by 35±4% compared to vehicle (n=4, pad libitum . DOCA increased fluid intake at baseline (CTL 4±0 vs 15±1 mL/day DOCA+aCSF, p0.05), 0.15 mol/L NaCl (27±4 vs 20±2 mL/day, p>0.05), 0.3 mol/L (25±4 vs 26±2 mL/day, p>0.05), or 0.45 mol/L (25±3 vs 28±3 mL/day, p>0.05). However, TRV prevented DOCA-induced increase of NaCl preference at NaCl 0.15 mol/L (DOCA+aCSF 29±1 vs 14±2% DOCA+TRV, p

Genetic disruption of angiotensin (ANG) type 1A receptors (AT1A) in cells which express Agouti-related peptide (AgRP) abolishes thermogenic sympathetic nerve activity (SNA) and resting metabolic rate (RMR) responses to leptin and high fat diet (HFD). Although it is established that glia secrete angiotensinogen (AGT), in silico reanalysis of published single-cell RNAseq datasets describing transcriptomes of cells from mouse hypothalamus (GSE74672) and RNAscope fluorescent in situ hybridization independently confirm that AgRP neurons of the hypothalamic arcuate nucleus (ARC) in adult wildtype mice express AGT mRNA. We therefore sought to explore the functional significance of this expression pattern. Why, if AgRP neurons are bathed in AGT in the interstitial space, would they synthesize AGT? Mice were generated which lack AGT specifically in AgRP cells (AGT AgRP-KO mice) using the Cre-Lox approach (sire: AgRP-Cre + , AGT F/wt x dam: AgRP-Cre — , AGT F/F ). Of the first 116 offspring, this resulted in expected overall distributions across sex (53% female), Cre (47% Cre + ) and AGT (46% AGT F/F ) genotypes, but an underrepresentation of targeted AGT AgRP-KO mice at weaning (n=5 females + 1 male; χ 2 =52.0, df=3, pin utero lethality of the genotype. Before and at 8 weeks of age, female AGT AgRP-KO mice exhibited normal body mass (n=5, 17.3±0.4 vs n=12 littermates 17.3±0.3 g); 5 weeks of 45% HFD caused significant weight gain in all mice (pAgRP-KO mice gained less (body mass: 18.3±0.4 vs 19.7±0.3 g, p

Obesity is a major risk factor for development of hypertension, and evidence supports a role for the suppression of resting metabolic rate (RMR) in the development and maintenance of obesity. Angiotensin II (ANG) AT1A receptors, localized to cells expressing the leptin receptor and Agouti-related peptide (AgRP) within the arcuate nucleus of the hypothalamus (ARC), are involved in transcriptional control of AgRP and γ-aminobutyric acid (GABA) synthetic genes ( Gad65 , Gad67 , Slc32a1 ) within the ARC, as well as the integrative control of thermogenic sympathetic nerve activity (SNA) and RMR responses in vivo to various stimuli including leptin and high fat diet. These data led us to hypothesize that AT1A functions to suppress GABA signaling in AgRP neurons, ultimately to disinhibit SNA and RMR. Treatment of cultured mouse hypothalamic N43/5 cells (which express endogenous Agtr1a , AgRP and Gad67 ) with 100 nM ANG for 1 hour (n=6) resulted in reduced AgRP (0.57-fold of control, p=0.03) and Gad67 (0.72-fold of control, p=0.09) mRNA. Mice with targeted disruption of Slc32a1 (vesicular GABA transporter; VGAT) in AgRP neurons (VGAT AgRP-KO mice) were generated on a mixed background. Compared to littermates (male n=9-17, female n=7-8), VGAT AgRP-KO mice (male n=8-13, female n=2-3) at 8 weeks of age exhibited normal body mass (genotype p=ns, sex p

We recently demonstrated that leptin stimulates resting metabolic rate (RMR) through a mechanism that requires angiotensin (ANG) type 1A receptors (AT 1A ) localized to neurons of the arcuate nucleus (ARC) which express Agouti-related peptide (AgRP). Genetic disruption of AT 1A in AgRP neurons results in the loss of RMR responses to leptin, high fat diet, and various other stimuli which correlates with the disinhibition of AgRP, neuropeptide Y (NPY), and production enzymes and transporters (GAD1, GAD2, VGAT) for γ-aminobutyric acid (GABA) within the ARC. We hypothesize that AT 1A activation in AgRP neurons causes disinhibition of AgRP, NPY, GAD1, GAD2 and VGAT expression and thus increased inhibitory neurotransmission to pre-autonomic nuclei, and we seek to understand the second-messenger network activated by AT 1A which mediates transcriptional control of these genes. Intracellular calcium ([Ca 2+ ] i , determined by FURA-2 fluorescence), cyclic AMP (cAMP, by LANCE assay), receptor surface localization (by HiBiT-tagging), and gene expression responses (by qPCR) to ANG were examined in immortalized mouse hypothalamic cells (N43/5 and N39 cells) that express typical markers of AgRP neurons. ANG (0.1 and 1 μM, n = 3) had no effect to modulate [Ca 2+ ] i , but caused a dose-dependent reduction in forskolin-stimulated cAMP (EC 50 = 43 nM) which could be blocked by losartan (1 μM). ANG increased GTP-bound Gαi (PBS vehicle = 0.57±0.08, ANG 1 μM = 0.94±0.07 ratio vs IgG, p < 0.05, n = 3), reduced cell-surface localization of HiBiT-tagged AT 1A (PBS vehicle = 0.22±0.02, ANG 0.1 μM = 0.15±0.01, p < 0.05, n = 4), and significantly (p < 0.05, n = 6) reduced AgRP and GAD1 expression by 43% and 27%, respectively. Lastly, preliminary studies suggest that pretreatment with pertussis toxin (PTX, 100 ng/mL), an inhibitor of Gαi, abrogated AgRP and GAD1 suppression by ANG (0.01 μM). Collectively these findings support the novel concept that within immortalized cells that express markers of AgRP neurons, AT 1A couples to Gαi to reduce cAMP, which suppresses AgRP, NPY and GABA. This should disinhibit pre-motor, pre-autonomic circuits within the hypothalamus, which results in increased thermogenic sympathetic nerve activity, ultimately increasing RMR and energy expenditure.

To date, the early-gestational mechanisms driving the pathogenesis of preeclampsia (PreE) remain largely unclear. However, altered G protein signaling has been implicated in PreE, including alterations in GPCR agonists such as vasopressin, endothelin, and angiotensin. Regulator of G protein Signaling 2 (RGS2) is an endogenous terminator of Gαq signaling, and mutations in the Rgs2 gene are linked to hypertension and increased risk of developing PreE. Therefore, we hypothesized reduced placental RGS2 may increase the risk of developing PreE by disinhibiting Gαq signaling. In silico reanalysis of a publicly-available microarray dataset (GSE75010) revealed a significant reduction in Rgs2 mRNA in placentas from PreE pregnancies compared to controls (Con n=35, PreE n=49, pRgs2 mRNA by qPCR using human placental tissue samples (PreE 19% of Con, n=11 vs 9, pRgs2 -deficient ( Rgs2 -KO) sires or wildtype littermate sires. Dams mated with Rgs2 -KO sires developed diastolic hypertension (Con 92 ± 2 vs Rgs2 -KO 98.2 ± 2 [24 hr avg mmHg]; pRgs2 -KO) and increased proteinuria compared to dams mated with littermate sires (18.2 ± 2.2, n=7 vs 28.4 ± 2.8, n=10 mg/day, pRgs2 -KO sire suggest decreased spiral artery number and diameter (SA Number: Con 7.7 ± 0.2 vs Rgs2 -KO 5.6 ± 0.4, SA Diameter: Con 128 ± 3 vs Rgs2 -KO 86 ± 12). Previous studies have outlined a CRE element in the Rgs2 promoter that is critical for transcriptional regulation of Rgs2 . Thus, we hypothesized loss of cAMP/CREB regulation may lead to reduced RGS2 in human PreE. Indeed, reduced phosphorylated CREB (p-CREB) binding was observed in PreE placentas (Con 0.146 ± 0.024 vs PreE 0.070 ± 0.021 p

Preeclampsia (PreE), a cardiovascular disorder of pregnancy, remains a major cause of maternal and fetal mortality worldwide. The early etiology of the disorder is unclear, though increased G protein-coupled receptor signaling has been implicated. Regulator of G protein Signaling-2 (RGS2) is a negative regulator of G protein signaling, and mutations of RGS2 have been associated with increased risk for PreE in humans. Our ongoing work has demonstrated that breeding wildtype C57BL/6J dams with Rgs2 -deficient ( Rgs2 -KO; B6.129P2- Rgs2 tm1Dgen /Mmnc) sires is sufficient to cause ~50% reduced placental expression of Rgs2 mRNA, and to induce hypertension, renal and placental morphological phenotypes typical of PreE in the wildtype dams. To test the hypothesis that reduced RGS2 (via paternal contribution of an Rgs2 -null allele) is sufficient to cause molecular changes within the placenta that are typical of PreE, we performed RNA sequencing on placentas collected on gestational day 12.5 from C57BL/6J dams mated with either Rgs2 -KO sires or wildtype littermates of these sires. RNA was isolated and sequenced using an Illumina HiSeq 4000. Transcript counts were quantified and aligned to the mouse genome (UCSC genome browser) using Kallisto, followed by differential gene expression analysis using DEseq2 (FDR = 0.1). 726 genes were differentially expressed (479 up, 247 down), including some ( Hsd11b2 (up), Adm (down)), that are similarly changed in human PreE placenta (PMID: 28618048 & 26268791). Ingenuity Pathway Analysis indicated that reduced placental Rgs2 expression was associated with mitochondrial dysfunction, unfolded protein response, and oxidative stress (all p < 1e-3), which also parallels findings in human PreE placenta. Collectively these data support the conclusion that reduced Rgs2 expression in the feto-placental unit is sufficient to induce transcriptomic changes within the placenta that are typical of PreE.

G protein-coupled receptor signaling mechanisms are implicated in many aspects of cardiovascular control, and dysfunction of such signaling mechanisms is commonly associated with disease states. Investigators have identified a large number of regulator of G protein signaling (RGS) proteins that variously contribute to the modulation of intracellular second-messenger signaling kinetics. These many RGS proteins each interact with a specific set of second-messenger cascades and receptor types and exhibit tissue-specific expression patterns. Increasing evidence supports the contribution of RGS proteins, or their loss, in the pathogenesis of cardiovascular dysfunctions. This review summarizes the current understanding of the functional contributions of RGS proteins, particularly within the B/R4 family, in cardiovascular disorders of pregnancy including gestational hypertension, uterine artery dysfunction, and preeclampsia.

The renin-angiotensin system (RAS), originally described as a circulating hormone system, is an enzymatic cascade in which the final vasoactive peptide angiotensin II (ANG) regulates cardiovascular, hydromineral, and metabolic functions. The RAS is also synthesized locally in a number of tissues including the brain, where it can act in a paracrine fashion to regulate blood pressure, thirst, fluid balance, and resting energy expenditure/resting metabolic rate (RMR). Recent studies demonstrate that ANG AT1Areceptors ( Agtr1a) specifically in agouti-related peptide (AgRP) neurons of the arcuate nucleus (ARC) coordinate autonomic and energy expenditure responses to various stimuli including deoxycorticosterone acetate (DOCA)-salt, high-fat feeding, and leptin. It remains unclear, however, how these disparate stimuli converge upon and activate this specific population of AT1Areceptors in AgRP neurons. We hypothesize that these stimuli may act to stimulate local expression of the angiotensinogen (AGT) precursor for ANG, or the expression of AT1Areceptors, and thereby local activity of the RAS within the (ARC). Here we review mechanisms that may control AGT and AT1Aexpression within the central nervous system, with a particular focus on mechanisms activated by steroids, dietary fat, and leptin.

Human preeclampsia (PE) is associated with elevated secretion of arginine vasopressin (AVP), and chronic infusion of AVP into pregnant mice is sufficient to model PE by causing hypertension, renal glomerular endotheliosis, proteinuria, fetal placental hypoxia, and growth restriction. Early stages of PE are associated with defective trophoblast invasion of maternal spiral arteries, leading to decreased artery diameter and placental oxygenation. AVP infusion (24 ng/hr, sc) into pregnant mice caused placental hypoxia (chromatin-bound HIF1α on gestational day (GD)17.5; saline n=5, 0.31±0.01; AVP n=5, 0.34±0.01 AU, p

Recent studies from our laboratory have demonstrated that angiotensin II (ANG) type 1A receptors (AT1A) within the arcuate nucleus, expressed in the subset of neurons which express both the leptin receptor (LepR) and agouti-related peptide (AgRP), are critically involved in the control of thermogenic adipose sympathetic nerve activity (SNA) and resting metabolic rate (RMR) by leptin. This mechanism appears to involve AT1A-mediated suppression of gamma-aminobutyric acid (GABA) synthesis and packaging in AgRP neurons of the arcuate nucleus (ARC). It remains unclear, however, how leptin/LepR signaling results in AT1A activation within AgRP neurons. We hypothesize that LepR signaling results in increased transcription and local release of angiotensinogen (AGT) within the ARC, and consequently increased autocrine or paracrine ANG signaling within the ARC. Fluorescent in situ hybridization (RNAscope) uncovered expression of AGT mRNA in various cells of the ARC of C57BL/6J mice, including cells expressing mRNA for LepR, AgRP, proopiomelanocortin (POMC), insulin II, or glial fibrillary acidic protein, supporting local generation of AGT within the ARC. In silico re-analysis of a publically-available gene expression dataset interrogating individual cell types of the mouse hypothalamus (GSE74672) similarly uncovered expression of AGT in astrocytes, microglia, and many neuronal cell types of the ARC, including those expressing AgRP and POMC. Stimulation of immortalized mouse hypothalamic AgRP-like cell cultures (N47) with leptin may increase AGT mRNA (100 nM, 4 hrs; n=4 passages, 1.0 (0.6-1.8) vs 2.0 (1.2-3.6) fold, p=0.19). Further, using Cre-lox technology, mice lacking AGT in LepR- or AgRP-expressing cells (AGT LepR-KO or AGT AgRP-KO mice) were generated. In females at 8 weeks of age fed a chow diet (Teklad 7013), trends toward increased fat mass were noted (control n=28, 0.7±0.1; AGT LepR-KO n=2, 1.0±0.5; AGT AgRP-KO n=3, 1.0±0.2 g), despite normal food intake (13.5±0.6, 16.3±0.0, 12.4±0.8 kcal/d) and digestive efficiency (79±1, 83±0, 80±1 %). Collectively, these findings support local production of ANG peptides within the ARC, and may indicate transcriptional regulation of AGT by leptin and a role for ARC AGT in the control of energy expenditure.

Preeclampsia (PE) is a cardiovascular disorder of late pregnancy characterized by pregnancy-specific onset of hypertension and proteinuria. Although initiating events leading to PE development remain unclear, emerging studies suggest arginine vasopressin (AVP) signaling may contribute to PE pathogenesis, as elevated circulating copeptin (a stable biomarker of AVP secretion) levels precede the onset of human PE, and chronic infusion of AVP into wildtype mice phenocopies PE. Here, we tested the hypothesis that AVP signaling is dysregulated in PE placenta by performing in-silico reanalysis of a publically available gene expression dataset derived from placentas of human PE and normal pregnancies (GSE75010). Significant increases in expression [log 2 values] of AVP (Con: 6.96 ± 0.02, PE: 7.03 ± 0.17, p=0.005) and its receptors AVPR1a (Con: 5.73 ± 0.02, PE: 5.80 ± 0.02, p=0.03) and OXTR (Con: 6.58 ± 0.02, PE: 6.65 ± 0.02, p=0.003) were observed in PE placentas (n=80) compared to normal placentas (n=77). Preeclamptic placentas also displayed an enrichment in a hypoxic gene signature, as shown by gene set enrichment analyses (NES: 1.75, FDR qAVPR1a expression as well as OXTR is positively and significantly correlated with the expression of two hypoxia-inducible genes HK2 and DDIT4 ( AVPR1a v HK2 : r 2 =0.04, p=0.02, AVPR1a v DDIT4 : r 2 =0.06, p=0.003, OXTR v HK2 : r 2 =0.05, p=0.007, OXTR v DDIT4 : r 2 =0.04, p=0.008). To further elucidate the role hypoxia may play in promoting AVP signaling, HTR8/SVNeo cells (immortalized human first trimester trophoblasts) were treated with the hypoxia mimetic dimethyloxallyl glycine (DMOG). DMOG incubation did not significantly alter the expression of AVP and AVPR1a . Unexpectedly, a marked reduction in OXTR expression was observed upon DMOG treatment. In summary, these data suggest that 1) components of the AVP signaling pathway are aberrantly expressed in human PE placentas, 2) PE placentas exhibit a gene expression signature consistent with increased canonical hypoxia signaling, and 3) hypoxia may not be the cause of elevated placental AVP , AVPR1a , and OXTR expression in human PE.

The early mechanisms and genetic risk factors driving the pathogenesis of preeclampsia (PE), a cardiovascular disorder of pregnancy, remain largely unclear. Various hormone activators of Gα q second-messenger signaling pathways have been implicated in PE. Regulator of G-protein Signaling 2 (RGS2) acts as an endogenous terminator of Gα q signaling and previous studies identified a SNP (rs4606), which results in reduced RGS2 mRNA, as a risk factor for development of PE and its sequelae. We hypothesized reduced placental expression of RGS2 may precipitate the development of PE due to disinhibited Gα q signaling. In silico reanalysis of publically available dataset GSE75010 revealed RGS2 mRNA is reduced in placentas from pre-term PE pregnancies compared to normal pre-term pregnancies (con: 8.73 ± 0.07 n=35, PE: 8.37 ± 0.055 n=49, p

The development of peptides as therapeutic agents has progressed such that these small molecules of less than fifty amino acids are currently in use for the treatment of a variety of pathologies. This review focuses on the pre-clinical studies and clinical trials assessing the anti-cancer properties of angiotensin-(1-7) [Ang-(1-7)], an endogenous heptapeptide hormone of the renin-angiotensin system. Ang-(1-7) mediates biological responses by activating mas, a unique G protein- coupled receptor, thereby providing specific targeted actions when used as a therapeutic agent. Studies in in vitro as well as in vivo mouse models demonstrated that the heptapeptide hormone reduced proliferation of human cancer cells and xenograft tumors. This attenuation was concomitant with decreased angiogenesis, cancer associated fibrosis, osteoclastogenesis, tumor-induced inflammation and metastasis as well as altered regulation of growth promoting cellular signaling pathways. In three clinical trials, Ang-(1-7) was well tolerated with limited toxic or quality-of-life side effects and showed clinical benefit in cancer patients with solid tumors. Taken together, these studies suggest that Ang-(1-7) may serve as a first-in-class peptide chemotherapeutic agent, reducing cancer growth and metastases by pleiotrophic mechanisms as well as targeting the tumor microenvironment.

Patients with triple negative breast cancer often develop metastatic brain disease and have limited treatment options due to the lack of targeted therapeutics that efficiently cross the blood brain barrier. In the current study, we investigated the effect of angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone with anti-proliferative and anti-metastatic activities, on the growth of murine brain tumors using brain trophic BR.5-4T1 cells in a model of metastatic triple negative breast tumor growth. BR.5-4T1 cells (1.25 x 104) were injected into the internal carotid artery of female BALB/c mice; after two days, mice were implanted with osmotic mini-pumps to deliver 24 µg/kg/h Ang-(1-7) and all mice were sacrificed after 20 days of treatment. Ang-(1-7) treatment significantly reduced metastatic tumor number in the brain; an average of 20 metastatic lesions per mouse was observed in the brains of an untreated control cohort while an average of 3 metastatic tumors per mouse was observed in the Ang-(1-7)-treated cohort. Tumor size was also reduced 17-fold by treatment with Ang-(1-7). Ki67, a marker of proliferation, was decreased from 29.1±6.7% to 6.4±2.2% in tumors from mice treated with Ang-(1-7), indicating that the heptapeptide hormone decreased metastatic tumor burden in the brain. Aberrant activation of the c-Met receptor tyrosine kinase signaling pathway facilitates tumor cell invasion and metastasis. Treatment with Ang-(1-7) significantly reduced the percentage of metastatic tumor cells exhibiting phospho-c-Met immunoreactivity, from 20.8±3.4% in tumors from untreated mice to 6.7±2.6% in mice treated with Ang-(1-7). Protein phosphatase 1b (PTB1b), which dephosphorylates and negatively regulates phospho-c-Met, was increased in the tumor cells of mice treated with Ang-(1-7) compared to tumor cells from untreated animals, from 4.1±1.4% in sections of tumors from control mice compared to 8.8±0.9% in sections of tumors from mice treated with Ang-(1-7). These results suggest that Ang-(1-7) increases PTP1b to antagonize c-Met signaling and decrease triple negative breast cancer brain metastasis. In support of a role for c-Met signaling in the regulation of metastatic brain disease in breast cancer patients, analysis of publicly available breast cancer cohort databases demonstrated a significant correlation between low c-Met/high PTP1b expression and improved survival outcome, including an increase in brain metastasis-free survival and distant-metastasis free survival. This is the first report to demonstrate that Ang-(1-7) reduces metastatic triple negative breast cancer localized to the brain in association with increased protein phosphatase PTP1b and decreased phospho-c-Met, suggesting that the heptapeptide hormone may serve as a novel targeted therapy to reduce metastatic brain tumor burden in triple negative breast cancer patients. Citation Format: E. Ann Tallant, Guorui Deng, Wenhong Chen, Linda J. Metheny-Barlow, Patricia E. Gallagher. Angiotensin-(1-7) reduces triple negative breast cancer brain metastatic growth in association with decreased c-Met signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1227. doi:10.1158/1538-7445.AM2017-1227