Abstract P252: Smooth Muscle At1a Receptor Mediates Perivascular Fibrosis In Angiotensin Ii-infused Mice

Angiotensin II (Ang II) signaling via AT1 receptor has been shown to play a critical role in the pathogenesis of hypertension, cardiovascular hypertrophy and fibrosis. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates EGF receptor activation and promotes cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. In a recent study, deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affects hypertensive fibrosis in vasculature. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis during Ang II stimulation using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the sm22α promoter (KIsm22α-Cre). Male AT1a flox/flox KIsm22α-Cre+/- (SMKO) and Controls (AT1a flox/flox KIsm22α-Cre-/-) mice were infused with Ang II (1 μg/kg/min) for 2 weeks via osmotic mini-pump. In Control mice, Ang II infusion for 2 weeks induced cardiac hypertrophy indicated by heart-to-body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart-to-body weight ratios were significantly increased in Control mice compared with AT1a SMKO mice (6.04 versus 4.89, respectively, p=0.032). Cardiac wall hypertrophy was seen in Controls after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKOs. Control mice (n=5) showed vascular medial hypertrophy and perivascular fibrosis, whereas these phenotypic changes were attenuated in SMKO mice (n=4). In conclusion, AT1a receptors from VSMC could mediate Ang II-induced cardiovascular hypertrophy and perivascular fibrosis. Whether the data can be fully explained by the prevention of hypertension remains to be determined, the data contrast to the past manuscript showing a protective effect in AT1a flox/flox S100A4-Cre+/- mice (fibroblast silencing) with Ang II infusion.

Abstract P249: Angiotensin At1a Receptor Signaling In Agrp Neurons In Metabolism Control

The adverse cardiovascular responses to leptin are preserved, but beneficial metabolic effects, such asincreases in resting metabolic rate (RMR) are lost in obesity induced by high fat diet (HFD) feeding. Wepreviously reported angiotensin II (ANG) type 1a ( Agtr1a ; AT1A) receptors in a subset of AgRP neurons arecritically required for the integrative control of RMR. Understanding the mechanisms in this unique subset ofRMR controlling- AgRP neurons is the goal of this study and is critical to decipher the pathogenesis of obesity-associated hypertension. Male C57BL/6J mice were maintained on chow or HFD from 8-18 weeks of age. Cell-attached voltage clamp recording of AgRP neurons in ARC sections of Ai9(tdTomato) AgRP mice showed that ANGapplication results in three distinct cellular responses (n=65 neurons; 8 mice). In chow fed mice, ANG suppressedelectrical activity in 1/3 of AgRP neurons (ie. “Type 1” AgRP neurons) via a losartan-sensitive mechanism,indicating involvement of AT1 receptors (Firing rate; aCSF:1.23 ± 0.10 vs ANG:0.60 ± 0.08 vs ANG+LOS:1.37 ±0.11 Hz, p<0.05). ANG caused excitation in 1/3 of AgRP neurons (“Type 2” AgRP cells) which was mediatedthrough a PD-123,319-sensitive mechanism, implicating AT2 receptors (ANG:1.69 ± 0.12 vs ANG+PD:0.86 ±0.06 Hz, p<0.05). Finally, 1/3 of AgRP neurons did not respond to ANG (“Type 0” cells). Complementary to thesefindings, mice with AT1A deleted from AgRP cells (ie, Agrp -Cre x Agtr1a Flox x Ai9 mice) exhibited a completeloss of Type 1 responses but maintenance of Types 0 and 2 (n=23 cells; 4 mice). Further, pharmacologicaldissection of signaling cascades implicated a pertussis toxin-sensitive mechanism (Gαi cascade; ANG+PTX:1.00± 0.04 Hz, p<0.05) and multiple potassium + chloride channels in the ANG-mediated inhibition of Type 1 cells.Most intriguingly, the relative proportion of AgRP neurons exhibiting Type 1 (ANG -inhibited) vs Type 0 or 2responses was decreased with HFD (Type 1 cells proportion- chow: 35%,23 out of 65; HFD: 18%, 10 out of 56).These results establish a specific ANG-inhibited subtype of AgRP neuron and implicate the AT1A/Gαi signalingcascade in the inhibitory effect of ANG. Switch of Type 1 AgRP cells to Type 0 or 2 in response to HFD suggestsHFD-induced neural plasticity/adaptation.

AT1a dependent GABAA inhibition in the MnPO following chronic intermittent hypoxia

Chronic intermittent hypoxia (CIH) is associated with diurnal hypertension, increased sympathetic nerve activity (SNA), and increases in circulating angiotensin II (ANG II). In rats, CIH increases angiotensin type 1 (AT1a) receptor expression in the median preoptic nucleus (MnPO), and pharmacological blockade or viral knockdown of this receptor prevents CIH dependent increases in diurnal blood pressure. The current study investigates the role of AT1a receptor in modulating the activity of MnPO neurons following 7 days of CIH. Male Sprague-Dawley rats received MnPO injections of an adeno-associated virus with a shRNA against the AT1a receptor or a scrambled control. Rats were then exposed to CIH 8 h a day for 7 days. In vitro loose patch recordings of spontaneous action potential activity were made from labeled MnPO neurons in response to brief focal application of ANG II or the GABAA receptor agonist muscimol. Additionally, MnPO KCC2 protein expression was assessed using Western blot. CIH impaired the duration but not the magnitude of ANG II mediated excitation in the MnPO. Both CIH and AT1a knockdown also impaired GABAA mediated inhibition and CIH with AT1a knockdown produced GABAA mediated excitation. Recordings using the ratiometric Cl- indicator ClopHensorN showed CIH was associated with Cl- efflux in MnPO neurons that was associated with decreased KCC2 phosphorylation. The combination of CIH and AT1a knockdown attenuated reduced KCC2 phosphorylation seen with CIH alone. The current study shows that CIH, through the activity of AT1a receptors, can impair GABAA mediated inhibition in the MnPO contributing sustained hypertension.

Not so dry after all – DRY mutants of the AT1A receptor and H1 receptor can induce G protein-dependent signaling

ABSTRACTGPCRs are seven transmembrane spanning receptors that regulate a wide array of intracellular signaling cascades in response to various stimuli. To do so, they couple to different heterotrimeric G proteins and adaptor proteins, including arrestins. Importantly, arrestins were shown to regulate GPCR signaling through G proteins, as well as promote G protein-independent signaling events. Several research groups have reported successful isolation of exclusively G protein-dependent and arrestin-dependent signaling downstream of GPCR activation using biased agonists or receptor mutants incapable of coupling to either arrestins or G proteins. In the latter category, the DRY mutant of the angiotensin II type 1 receptor was extensively used to characterize functional selectivity downstream of AT1AR. In an attempt to understand histamine 1 receptor signaling, we characterized the signaling capacity of the H1R DRY mutant in a panel of dynamic, live cell biosensor assays, including arrestin recruitment, heterotrimeric G-protein activation, Ca2+ signaling, protein kinase C activity, GTP binding of RhoA, and activation of ERK1/2. Here we show that both H1R DRY mutant and the AT1AR DRY mutant (used as a reference) are capable of efficient activation of G protein-mediated signaling. Therefore, contrary to common belief, they do not constitute suitable tools for dissection of arrestin-mediated, G protein-independent signaling downstream of these receptors.

Angiotensin type 1a receptors in the median preoptic nucleus support intermittent hypoxia-induced hypertension

Chronic intermittent hypoxia (CIH) is a model of the hypoxemia from sleep apnea that causes a sustained increase in blood pressure. Inhibition of the central renin-angiotensin system or FosB in the median preoptic nucleus (MnPO) prevents the sustained hypertensive response to CIH. We tested the hypothesis that angiotensin type 1a (AT1a) receptors in the MnPO, which are upregulated by CIH, contribute to this hypertension. In preliminary experiments, retrograde tract tracing studies showed AT1a receptor expression in MnPO neurons projecting to the paraventricular nucleus. Adult male rats were exposed to 7 days of intermittent hypoxia (cycling between 21% and 10% O2 every 6 min, 8 h/day during light phase). Seven days of CIH was associated with a FosB-dependent increase in AT1a receptor mRNA without changes in the permeability of the blood-brain barrier in the MnPO. Separate groups of rats were injected in the MnPO with an adeno-associated virus containing short hairpin (sh)RNA against AT1a receptors to test their role in intermittent hypoxia hypertension. Injections of shRNA against AT1a in MnPO blocked the increase in mRNA associated with CIH, prevented the sustained component of the hypertension during normoxia, and reduced circulating advanced oxidation protein products, an indicator of oxidative stress. Rats injected with shRNA against AT1a and exposed to CIH had less FosB staining in MnPO and the rostral ventrolateral medulla after intermittent hypoxia than rats injected with the control vector that were exposed to CIH. Our results indicate AT1a receptors in the MnPO contribute to the sustained blood pressure increase to intermittent hypoxia.