AT1 Receptor Research Articles

Cross-Talk between the (Endo)Cannabinoid and Renin-Angiotensin Systems: Basic Evidence and Potential Therapeutic Significance.

This review is dedicated to the cross-talk between the (endo)cannabinoid and renin angiotensin systems (RAS). Activation of AT1 receptors (AT1Rs) by angiotensin II (Ang II) can release endocannabinoids that, by acting at cannabinoid CB1 receptors (CB1Rs), modify the response to AT1R stimulation. CB1R blockade may enhance AT1R-mediated responses (mainly vasoconstrictor effects) or reduce them (mainly central nervous system-mediated effects). The final effects depend on whether stimulation of CB1Rs and AT1Rs induces opposite or the same effects. Second, CB1R blockade may diminish AT1R levels. Third, phytocannabinoids modulate angiotensin-converting enzyme-2. Additional studies are required to clarify (1) the existence of a cross-talk between the protective axis of the RAS (Ang II—AT2 receptor system or angiotensin 1-7—Mas receptor system) with components of the endocannabinoid system, (2) the influence of Ang II on constituents of the endocannabinoid system and (3) the (patho)physiological significance of AT1R-CB1R heteromerization. As a therapeutic consequence, CB1R antagonists may influence effects elicited by the activation or blockade of the RAS; phytocannabinoids may be useful as adjuvant therapy against COVID-19; single drugs acting on the (endo)cannabinoid system (cannabidiol) and the RAS (telmisartan) may show pharmacokinetic interactions since they are substrates of the same metabolizing enzyme of the transport mechanism.

Клинико-фармакологические особенности и место ирбесартана в лечении сердечно-сосудистых заболеваний

Irbesartan belongs to a group of angiotensin II receptor blockers that are used worldwide for the treatment of patients with hypertension, type 2 diabetes, and impaired renal function. Irbesartan can be administered alone or in combination with other antihypertensive drugs (for example, hydrochlorothiazide), if blood pressure control cannot be achieved with monotherapy. The mechanism of action of Irbesartan is mainly due to the selective blockade of AT1 receptors and, as a consequence, a decrease in the vasopressor effect of angiotensin II. The review article summarizes pharmacokinetic and pharmacodynamic properties and clinical data on the tolerability and safety of Irbesartan.

PERMISSIVE ROLE OF ANGIOTENSIN II IN MANTENANCE OF ENDOTHELIUM-DEPENDENT VASODILATION IN CEREBRAL MICROCIRCULATION

Objective: A number of data suggest, rather controversial, permissive role of angiotensin II (ANGII) in maintaining vasodilator responses in microcirculation, via its interaction with AT1 receptors (AT1R). This study aimed to determine the role of ANGII and AT1R in the mechanisms of flow-induced dilation (FID) in middle cerebral arteries (MCA) and incidence of oxidative stress at the functional, cellular and molecular level in the cerebral vasculature and in the serum of Sprague-Dawley (SD) rats fed high salt diet (HS). Design and method: Eleven-week old, male SD rats were randomly assigned to control group (CTRL, 0.4% NaCl in rat chow); Losartan group (rats on a standard diet given the AT1R blocker losartan (1 mg/mL) in drinking water); HS group (7 days 4% NaCl in rat chow) or HS+ANGII group (7 days HS with 3 days ANGII administration via osmotic minipumps (100 ng/kg/min on days 4–7)). FID of MCA was determined in absence/presence of the NOS inhibitor L-NAME, the non-selective cyclooxygenases inhibitor indomethacin, and the superoxide dismutase mimetic TEMPOL. Gene and protein expression of antioxidative enzymes and enzymes involved in FID mechanisms were determined by RT-qPCR and western blot. Vascular NO and superoxide/ROS levels were assessed by direct fluorescence. Serum systemic oxidative stress parameters was measured by spectrophotometry. All experimental procedures conformed to the EU Directive 2010/63/EU. Results: HS or AT1R blockade impaired FID and increased oxidative stress. Supplementation of ANGII restored the mechanisms of FID in MCA of rats fed HS diet to the ones present in CTRL rats without affecting arterial blood pressure by decreasing systemic oxidative stress and decreasing superoxide/ROS levels and increased NO bioavailability in the vascular wall of HS treated rats. The AT1R blockade resulted in significantly attenuated endothelium-dependent dilation, changed mechanisms of FID, increased vascular and systemic oxidative stress and decreased expression/ activity of antioxidative enzymes. Conclusions: ANGII is crucial in the preservation of vascular oxidative stress-antioxidant system balance and via AT1R, in maintenance of physiological vasodilation mechanisms of MCA. Suppression of ANGII is a key link between HS intake and development of endothelial dysfunction, and impaired regulation of cerebral blood flow.

COMPOUND 21 STIMULATES THE ACTIVATION OF AKT AND ERK1/2 IN MOUSE ADIPOSE TISSUE IN VIVO

Objective: To analyze signaling events triggered by in vivo stimulation of the AT2 receptor. Design and method: Male B57Bl/6 mice were used as model at the age of 3 month. Mice were subjected to a bolus in vivo intravenous injection (cava vein) of saline buffer containing either the AT2 agonist compound 21 (0.25 mg/Kg), a mixture of C21 (0.25 mg/Kg) and the AT2 antagonist PD123319 (2.5 mg/Kg) or saline for baseline assesment under anesthesia. A group of animals that received insulin (0.25 mg/Kg) were used as positive control. The heart and the inguinal adipose tissue were removed after 1 and 3 min respectively, flash-frozen and kept at -70 C until analysis. Proteins from heart and adipose tissue were solubilized in a buffer containing 1% Triton together withprotease and phosphatase inhibitors. Protein content was measured by the BCA method and homogenates were subjected to Western Blotting using specific anti-phospho Akt or anti-phospho ERK1/2 antibodies. The content of Akt and ERK content was detected through incubation with either anti-Akt or anti-ERK1/2 antibodies. Specific bands were detected by chemiluminescence. Protein loading control was checked by coomassie blue staining of every PVDF membrane employed. Results: Injection of C21 resulted in the activation of both Akt and ERK1/2 in mouse adipose tissue. The extent of activation of Akt and ERK1/2 attained was equivalent to approximately 30–40% of the levels of activation of these enzymes obtained after an injection of a dose of insulin known to generate maximum activation of these signaling components. Co-infusion of C21 and PD123319 abolished the in vivo activation of Akt and ERK1/2, indicating that these events were mediated by the AT2 receptor. Conclusions: In vivo administration of the AT2 receptor agonist C21 resulted in the stimulation of the phosphorylation of both Akt and ERK1/2 at stimulatory sites indicative of activation of these enzymes. Co-infusion of C21 with the specific AT2 receptor antangonist blunted this effect. It is concluded that the AT2 receptor is capable of recruiting both Akt as ERK1/2 as signaling molecules in mouse adipose tissue in vivo.

Type 1 Angiotensin Receptors on CD11c-Expressing Cells Protect Against Hypertension by Regulating Dendritic Cell-Mediated T Cell Activation.

Type 1 angiotensin (AT1) receptors are expressed on immune cells, and we previously found that bone marrow-derived AT1 receptors protect against Ang (angiotensin) II-induced hypertension. CD11c is expressed on myeloid cells derived from the bone marrow, including dendritic cells (DCs) that activate T lymphocytes. Here, we examined the role of AT1 receptors on CD11c+ cells in hypertension pathogenesis. Mice lacking the dominant murine AT1 receptor isoform, AT1a, on CD11c+ cells (dendritic cell [DC] AT1aR knockout [KO]) and wild-type (WT) littermates were subjected to Ang II-induced hypertension. Blood pressures were measured by radiotelemetry. DC AT1aR KO mice had exaggerated hypertensive responses to chronic Ang II infusion with enhanced renal accumulation of effector memory T cells and CD40+ DCs. CCL5 (C-C motif chemokine ligand 5) recruits T cells into injured tissues, and CCR7 (C-C motif chemokine receptor 7) facilitates DC and T cell interactions in the kidney lymph node to allow T cell activation. DCs from the hypertensive DC AT1aR KO kidneys expressed higher levels of CCL5 and CCR7. mRNA expressions for CCR7 and tumor necrosis factor-α were increased in CD4+ T cells from the renal lymph nodes of DC AT1aR KO mice. During the second week of Ang II infusion when blood pressures between groups diverged, DC AT1aR KO mice excreted less sodium than WTs. Expressions for epithelial sodium channel subunits were increased in DC AT1aR KO kidneys. Following activation of the renin angiotensin system, AT1aR stimulation on DCs suppresses renal DC maturation and T cell activation with consequent protection from sodium retention and blood pressure elevation.

CLOFIBRATE TREATMENT IMPROVED THE STRUCTURAL DAMAGE ELICITED BY MYOCARDIAL ISCHEMIA THROUGH MODULATION OF RENIN-ANGIOTENSIN SYSTEM

Objective: Myocardial ischemia is an acute condition that, if not treated in a timely manner, produces the loss of heart tissue which can lead to heart failure or death of the patients. Our group has studied fibrates, exogenous ligands of PPAR, as an alternative to reduce processes associated with inflammation and we interested to know the exogenous ligand effect over damage in the cardiac tissue by RAS system. To study the effect of PPAR alpha ligand; clofibrate, on renin angiotensin system and on the structural morphology of the heart in myocardial infarcted subjects. Design and method: Male Wistar rats were separated in two groups: 1.- Sham, 2.- Myocardial infarct by coronary artery ligation. Seven days after, the rats were subdivided in vehicle (Vh)- or clofibrate treatment (Clof 100 mg/kg/day) administered for seven more days. At the end of the treatment (14 days in total), serum and left ventricle were analyzed. Results: Angiotensin converting enzyme (ACE), AT1 receptor and Ang II decreased in MI-Clof vs. MI-Vh, whereas non-classic RAS system components: ACE2, AT2 receptor, bradykinin, and Ang-(1–7) augmented in MI-Clof with respect to MI-Vh. VEGF also raised in clofibrate-treated animals. Clofibrate treatment preserved the structure of the sarcomere (fiber). Conclusions: Clofibrate treatment to MI rats exerts an antifibrotic effect and augmented the expression of non-classical axis of RAS system.

Prolonged NHE Activation may be both Cause and Outcome of Cytokine Release Syndrome in COVID-19.

The release of cytokines and chemokines such as IL-1β, IL-2, IL-6, IL-7, IL-10, TNF-α, IFN-γ, CCL2, CCL3, and CXCL10 is increased in critically ill patients with COVID-19. Excessive cytokine release during COVID-19 is related to increased morbidity and mortality. Several mechanisms are put forward for cytokine release syndrome during COVID-19. Here we have mentioned novel pathways. SARS-CoV-2 increases angiotensin II levels by rendering ACE2 nonfunctional. Angiotensin II causes cytokine release via AT1 and AT2 receptors. Moreover, angiotensin II potently stimulates the Na+/H+ exchanger (NHE). It is a pump found in the membranes of many cells that pumps Na+ inward and H+ outward. NHE has nine isoforms. NHE1 is the most common isoform found in endothelial cells and many cells. NHE is involved in keeping the intracellular pH within physiological limits. When the intracellular pH is acidic, NHE is activated, bringing the intracellular pH to physiological levels, ending its activity. Sustained NHE activity is highly pathological and causes many problems. Prolonged NHE activation in COVID-19 may cause a decrease in intracellular pH through H+ ion accumulation in the extracellular area and subsequent redox reactions. The activation reduces the intracellular K+ concentration and leads to Na+ and Ca2+ overload. Increased ROS can cause intense cytokine release by stimulating NF-κB and NLRP3 inflammasomes. Cytokines also cause overstimulation of NHE. As the intracellular pH decreases, SARS-CoV-2 rapidly infects new cells, increasing the viral load. This vicious circle increases morbidity and mortality in patients with COVID-19. On the other hand, SARS-CoV-2 interaction with NHE3 in intestinal tissue is different from other tissues. SARS-CoV-2 can trigger CRS via NHE3 inhibition by disrupting the intestinal microbiota. This review aimed to help develop new treatment models against SARS-CoV-2- induced CRS by revealing the possible effects of SARS-CoV-2 on the NHE.

THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM IN THE PREGNANT RAT

Objective: Preeclampsia is the leading cause of maternal and fetal morbidity and mortality in the United States, affecting ~2–5% of all pregnancies. Women with a history of preeclampsia have a 4-fold increased risk for developing hypertension, 2-fold increased risk for developing stroke, and 2-fold increased risk for developing ischemic heart disease later in life. Although it is known that intact renin-angiotensin-aldosterone system (RAAS) signaling is critical to a healthy pregnancy and that the RAAS is altered in preeclampsia the pathogenic mechanisms are largely unknown and a comprehensive examination of RAAS metabolism and receptor expression is lacking in the literature. The purpose of this study is to understand how the vasculature becomes refractory to the reported high circulating angiotensin II (ANGII) during pregnancy. This work provides basic information needed to understand blood pressure regulation and volume expansion in normal pregnancy, and why it fails to occur in women with preeclampsia, resulting in hypertension and fetal growth restriction. Design and method: We performed AT1 receptor autoradiography in kidneys of virgin (V) and late pregnant (LP) rats. We also performed equilibrium RAAS Fingerprinting in the plasma and kidneys of LP rats and V rats using a new LC/MS technique. Results: Using this new method, ANGII levels were decreased in the plasma and unchanged in the kidney of LP animals. This was surprising as it is inconsistent with historical data, however, other methods of measuring ANGII are problematic and often unreliable. There was a very high Aldosterone/ANGII-ratio (AA2) in both plasma and kidney of LP animals. The AA2-ratio provides information about the adrenal aldosterone-releasing response to ANGII. We also found that AT1R expression is decreased in the kidneys of LP compared to V rats. Conclusions: These results suggest a systemic down regulation of renal ANGII signaling with increased adrenal signaling. Continuing studies are assessing the RAAS signaling cascade to further validate these data, which may revolutionize the way we think about the RAAS in pregnancy, leading to improved treatment of women with preeclampsia.

A NOVEL VASOACTIVE PENTAPEPTIDE “PG1’’ FROM BUFFALO ICE-CREAM PROTECTS FROM ANGIOTENSIN-EVOKED HIGH BLOOD PRESSURE

Objective: Arterial hypertension is the most important risk factor for cardiovascular diseases, myocardial infarction, heart failure, renal failure and peripheral vascular disease. In the last decade, milk-derived bioactive peptides have attracted attention for their beneficial cardiovascular properties. Identify new vasoactive natural peptides able to fight arterial hypertension that can be used as a preventive strategy. Design and method: Here we combined in-vitro chemical assay such as LC-MS / MS analysis of buffalo ice cream, ex-vivo vascular studies evaluating endothelial and smooth muscle responses using pressure myograph, and translational assay testing in-vivo the vascular actions of PG1 administration in murine models. Results: We demonstrate that a novel buffalo ice-cream-derived pentapeptide “QKEPM’’, namely PG1, is a stable peptide that can be obtained at higher concentration after gastro-intestinal digestions (GID) of buffalo ice-cream (BIC). It owns potent vascular effect in counteract the effects of angiotensin II-evoked vasoconstriction and high blood pressure levels. Its effects are mediated by the inhibitory effect on AT1 receptor leading to a downregulation of p-ERK½ / Rac1-GTP and consequent reduction of oxidative stress. Conclusions: These results strongly candidate PG1, as a novel bioactive peptide for the prevention and management of hypertension, thus expanding the armamentarium of preventive strategies aimed at reducing the incidence and progression of hypertension and its related cardiovascular complicications.

INHIBITION OF RAS COMPONENTS ATTENUATES PROGRESSION OF MYOCARDIAL REMODELING IN BOTH NORMOTENSIVE AND HYPERTENSIVE RATS IN EXPERIMENTAL HEART FAILURE

Objective: New findings in the pathogenesis of heart failure (HF) with a focus on extracellular matrix (ECM) and communication in the heart are still relevant for more effective treatment. We focused on signaling pathways involved in cardiac structural remodeling in HF due to pressure and volume overload, as well as on investigating the efficacy of substances with possible antifibrotic potential. Design and method: We used as experimental model 28-week-old hypertensive (TGR) and normotensive (HSD) rats with created aortocaval fistula (ACF), supplemented with ACE inhibitor (ACEi) (Trandolapril, 15 weeks, 6 mg/L) and AT1 receptor blocker (ARB) (Losartan, 15 weeks, 200 mg/L). Results: Echocardiography confirmed an increase in cardiac output and a decrease in ejection fraction in both strains of rats. BW was higher in TGR vs HSD and it was not affected by VO. ACF-induced cardiac volume overload, elevated markers of heart failure and oxidative damage, ANP, BNP-45, and TBARS, were elevated in the blood serum of both strains. ACF induced profibrotic signaling by increasing TGF-, and mRNA collagen I, and collagen I protein expression in the left and right ventricles of HSD rats. In TGR rats ACF decreased TGF- and SMAD2 protein expression in the right ventricle similar to HSD rats, in which ACF also suppressed MMP-2 protein expression. Heart failure affected protein kinase signaling by reducing the protein expression of PKC, PKG, and in part, PKC in both chambers of the heart of both strains. ACF decreased the protein expression of Cx43 and its phosphorylated form, which is significantly proarrhythmic. Hypertension in TGR rats increased mRNA TGF- expression and collagen I, but decreased PKC and PKC protein expression and increased PKC. Conclusions: A notable finding was that the signaling pathways involved in cardiac remodeling were more normalized by the ARB, while proteins involved in intercellular communication, ACEi. The results of analyzes of both experiments indicate attenuated responses of the signaling pathways monitored by us in the heart of hypertensive strains reflecting adaptation to primarily induced pathophysiology. Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA 2/0158/19, 2/0002/20

Enalapril Diminishes the Diabetes-Induced Changes in Intestinal Morphology, Intestinal RAS and Blood SCFA Concentration in Rats.

Evidence suggests that microbiota-derived metabolites, including short-chain fatty acids (SCFAs) and trimethylamine-oxide (TMAO), affect the course of diabetic multiorgan pathology. We hypothesized that diabetes activates the intestinal renin–angiotensin system (RAS), contributing to gut pathology. Twelve-week-old male rats were divided into three groups: controls, diabetic (streptozotocin-induced) and diabetic treated with enalapril. Histological examination and RT-qPCR were performed to evaluate morphology and RAS expression in the jejunum and the colon. SCFA and TMAO concentrations in stools, portal and systemic blood were evaluated. In comparison to the controls, the diabetic rats showed hyperplastic changes in jejunal and colonic mucosa, increased plasma SCFA, and slightly increased plasma TMAO. The size of the changes was smaller in enalapril-treated rats. Diabetic rats had a lower expression of Mas receptor (MasR) and angiotensinogen in the jejunum whereas, in the colon, the expression of MasR and renin was greater in diabetic rats. Enalapril-treated rats had a lower expression of MasR in the colon. The expression of AT1a, AT1b, and AT2 receptors was similar between groups. In conclusion, diabetes produces morphological changes in the intestines, increases plasma SCFA, and alters the expression of renin and MasR. These alterations were reduced in enalapril-treated rats. Future studies need to evaluate the clinical significance of intestinal pathology in diabetes.

Angiotensin Receptors - Affinity and Beyond

This commentary on the article "Relative affinity of angiotensin peptides and novel ligands at AT1 and AT2 receptors" by Sanja Bosnyak et al. (Clini. Sci. (Lond.) (2011) 121(7): 297-303. https://doi.org/10.1042/CS20110036) summarises the main findings of the study, followed by a discussion of the findings and their relevance for various aspects of the biology of receptors of the renin-angiotensin system in the context of the current state of knowledge.

Vasoconstrictor and Pressor Effects of Des-Aspartate-Angiotensin I in Rat.

This study investigated the vasoactive effects of des-aspartate-angiotensin-I (DAA-I) in male Wistar rats on whole body vascular bed, isolated perfused kidneys, and aortic rings. Dose–response curves to DAA-I were compared with those to angiotensin II (Ang II). The Ang II-type-1 (AT1) receptor blocker, losartan, was used to evaluate the role of AT1 receptors in the responses to DAA-I. Studies were also conducted of the responsiveness in aortic rings after endothelium removal, nitric oxide synthase inhibition, or AT2 receptor blockade. DAA-I induced a dose-related systemic pressor response that was shifted to the right compared with Ang II. Losartan markedly attenuated the responsiveness to DAA-I. DAA-I showed a similar pattern in renal vasculature and aortic rings. In aortic rings, removal of endothelium and nitric oxide inhibition increased the sensitivity and maximal response to DAA-I and Ang II. AT2 receptor blockade did not significantly affect the responsiveness to DAA-I. According to these findings, DAA-I increases the systemic blood pressure and vascular tone in conductance and resistance vessels via AT1 receptor activation. This vasoconstrictor effect of DAA-I participates in the homeostatic control of arterial pressure, which can also contribute to the pathogenesis of hypertension. DAA-I may therefore be a potential therapeutic target in cardiovascular disease.

2-Alkyl substituted benzimidazoles as a new class of selective AT2 receptor ligands

Ligands comprising a benzimidazole rather than the imidazole ring that is common in AT2R ligands e.g. in the AT2R agonist C21, can provide both high affinity and receptor selectivity. In particular, compounds encompassing benzimidazoles, substituted in the 2-position with small bulky groups such as an isopropyl (Ki = 4.0 nM) or a tert-butyl (Ki = 5.3 nM) or alternatively a thiazole heterocycle (Ki = 5.1 nM) demonstrate high affinity and AT2R selectivity. An n-butyl chain, as found in the AT1R selective sartans, makes the ligand less receptor selective. The isobutyl group on the biaryl scaffold present in most AT2R selective ligands reported so far was originally derived from the nonselective potent AT1R/AT2R ligand L-162,313. Notably, in all ligands discussed herein, the isobutyl group was substituted by an n-propyl group and ligands with high affinity to AT2R were provided and in addition the majority of them demonstrate a favorable AT2R/AT1R selectivity. The introduction of fluoro atoms in various positions had no pronounced effect on the affinity data. Ligands with a thiazole or a tert-butyl group attached to the 2-position and with a terminal trifluoromethyl butoxycarbonyl sidechain exhibited a similar stability as C21 in human liver microsomes, while other ligands examined were less stable in the microsome assay.

Cross-Talk between CB1, AT1, AT2 and Mas Receptors Responsible for Blood Pressure Control in the Paraventricular Nucleus of Hypothalamus in Conscious Spontaneously Hypertensive Rats and Their Normotensive Controls.

We have previously shown that in urethane-anaesthetized rats, intravenous injection of the angiotensin II (Ang II) AT1 receptor antagonist losartan reversed the pressor effect of the cannabinoid CB1 receptor agonist CP55940 given in the paraventricular nucleus of hypothalamus (PVN). The aim of our study was to determine the potential interactions in the PVN between CB1 receptors and AT1 and AT2 receptors for Ang II and Mas receptors for Ang 1–7 in blood pressure regulation in conscious spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. The pressor effects of Ang II, Ang 1–7 and CP55940 microinjected into the PVN were stronger in SHRs than in WKYs. Increases in blood pressure in response to Ang II were strongly inhibited by antagonists of AT1 (losartan), AT2 (PD123319) and CB1 (AM251) receptors, to Ang 1–7 by a Mas antagonist (A-779) and AM251 and to CP55940 by losartan, PD123319 and A-779. Higher (AT1 and CB1) and lower (AT2 and Mas) receptor expression in the PVN of SHR compared to WKY may partially explain the above differences. In conclusion, blood pressure control in the PVN depends on the mutual interaction of CB1, AT1, AT2 and Mas receptors in conscious spontaneously hypertensive rats and their normotensive controls.

Angiotensin II AT2 receptor ligands with phenylthiazole scaffolds

The syntheses and the AT1R and AT2R binding data of a series of new small molecule ligands are reported. These ligands comprise a phenylthiazole scaffold rather than the biphenyl or phenylthiophene scaffolds found in essentially all of the previously described ligands originating from the nonselective AT1R/AT2R ligand L-162,313 and the AT2R selective agonist C21, the latter now in Phase II/III clinical trials. A phenylthiazole rather than the phenylthiophene scaffold that is present in the AT2R selective agonist C21 and in the AT2R selective antagonist C38 had a deleterious effect on the affinity to AT2R. Nevertheless, a significant improvement could be accomplished by introduction of a small bulky alkyl group in the 2-position of the imidazole ring attached through a methylene group bridge to the phenylthiazole scaffold. Hence, a combination of a 2-tert-butyl or a 2-isopropyl group and a butoxycarbonyl furnished potent AT2R selective ligands. Furthermore, a high affinity ligand derived from L-162,313 and exhibiting a > 35 fold selectivity for AT1R was identified (10). The ligand 21 with the 2-tert-butyl group and ∼ 35 fold selectivity for AT2R, demonstrated high stability in human, rat and mouse liver microsomes and a very attractive profile with regard to the inhibition of common drug-metabolizing CYP enzymes. Thus, very low levels of inhibition of CYP 3A (5%), 2D6 (12%), 2C8 (26%), 2C9 (23%) and 2B6 (24%) were observed with the 2-tert-butyl derivative comprising the methoxycarbonyl sulfonamide function, levels that are significantly lower than those obtained with C21 under the same experimental conditions.

Genetic Evidence for A Critical Role of Intratubular Angiotensin II AT1aReceptors in The Proximal Tubules of The Kidney in Two‐Kidney, One‐Clip Goldblatt Hypertension in PT‐Agtr1a‐/‐Mice

It was previously reported the activation of the renin‐angiotensin system (RAS) in the clipped kidney plays a critical role in the development of two‐kidney, one‐clip Goldblatt hypertension (2K1C). However, most of previous studies used the global angiotensin II (Ang II) type 1a (AT1a) receptor (Agtr1a‐/‐) knockout model or used systemic AT1receptor blockers (ARB) to determine renal mechanisms of 2K1C hypertension. The roles of intratubular AT1 (AT1a) receptors or its downstream target Na+/H+ exchanger 3 (NHE3) in the proximal tubules in the development of 2K1C hypertension has not been determined previously. In the present study, we tested the hypothesis that genetic deletion of AT1 (AT1a) receptors selectively in the proximal tubules attenuates the development of 2K1C hypertension via AT1 (AT1a) receptor‐mediated, Na+/H+ exchanger 3 (NHE3)‐dependent mechanisms. To test the hypothesis, 2K1C Goldblatt hypertension was induced by placing a silver clip, 0.12 mm internal diameter, on the left renal artery for 4 weeks in adult male wild‐type (WT), global Agtr1a‐/‐, proximal tubule (PT)‐specific PT‐Agtr1a‐/‐, or PT‐Nhe3‐/‐ (Na+/H+ exchanger 3) mice, respectively. As expected, systolic blood pressure increased in a time‐dependent manner in wild‐type mice, reaching a maximal response by Week 3 (Basal: 112 ± 2 vs. 2K1C: 149 ± 4 mmHg, n=12, P<0.01). 2K1C hypertension in WT mice was associated with increases in renin mRNA expression in the clipped kidney (Control: 2066 ± 255 vs. Clipped: 3144 ± 569 copies/ng RNA, P<0.01), and decreases in renin mRNA expression in the nonclipped right kidney (1738 ± 341 copies/ng RNA, P<0.05). Plasma Ang II levels were significantly increased in WT mice with 2K1C hypertension (Control: 50.2 ± 7.2 vs. 2K1C: 109.7 ± 17.2 pg/ml, P<0.05). Glomerular and tubulointerstitial fibrotic responses were also significantly increased in the clipped kidney (P<0.01). By comparisons, the development of 2K1C hypertension was entirely prevented in Agtr1a‐/‐ (Basal: 88 ± 4 vs. 2K1C: 92 ± 2 mmHg, n=9, n.s.), or significantly attenuated in PT‐Agtr1a‐/‐ mice (Basal: 101 ± 2 vs. 2K1C: 104 ± 4 mmHg, n=12, n.s.) or in PT‐Nhe3‐/‐ mice (Basal: 103 ± 3 vs. 109 ± 5 mmHg, n.s.). Renin mRNA expression was not different in clipped and nonclipped kidney of Agtr1a‐/‐ mice, but it was decreased in the nonclipped kidney of PT‐Agtr1a‐/‐ mice (P<0.05). Furthermore, glomerular and tubulointerstitial fibrotic responses were also markedly attenuated in the clipped kidney in PT‐Agtr1a‐/‐ mice (P<0.05). We conclude that the results of the present study provide strong evidence for a critical role of AT1a receptors and NHE3 in the proximal tubules of the kidney in the development of 2K1C Goldblatt hypertension in mice.

AT1 Receptor as a Potential Target in Amphetamine-induced Neuroinflammation.

AT1 Receptor as a Potential Target in Amphetamine-induced Neuroinflammation.

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 endogenous sm22α promoter (Kock‐In, KIsm22α‐Cre). Male AT1a flox/flox KIsm22α‐Cre+/‐ (SMKO) and control (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 control mice after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKO mice. Control mice (n=5) showed vascular medial hypertrophy and perivascular fibrosis in coronary arteries, whereas these phenotypic changes were attenuated in SMKO mice (96.8 µm vs. 56.4 µm, respectively, p<0.0001). In conclusion, AT1a receptors expressed in 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. Our data also contrast with the past manuscript showing a protective effect in AT1a flox/flox S100A4‐Cre+/‐ (fibroblast silencing) but not in AT1a flox/flox sm22α‐Cre+/‐ mice with Ang II infusion.

Abstract 543: Inhibition Of Renin-angiotensin System Failed To Suppress Beta-aminopropionitrile-induced Thoracic Aortopathies In Mice

Objective: Cross-linking of elastin and collagen fibers is a vital process in aortic development. Pharmacological inhibition of aortic cross-linking by β-aminopropionitrile (BAPN) leads to thoracic aortopathies, such as thoracic aortic aneurysm and dissection, in mice. Although the renin angiotensin system (RAS) contributes to several types of thoracic aortopathies, it remains unclear whether inhibition of the RAS protects against aortopathy formation caused by the impairment of elastin and collagen cross-linking in mice. Approach and Results: Either vehicle or losartan (15 mg/kg/d, AT1 receptor blocker) were administered through osmotic pumps for 4 weeks in male C57BL/6J mice (3-4-weeks-old, n=30/group). BAPN (0.5% wt/vol) was given through drinking water to induce aortopathies. Losartan increased plasma renin concentrations significantly compared to vehicle-infused mice, indicating effective blockade of at1 receptors. However, losartan did not suppress BAPN-induced aortic rupture and dilatation. Since losartan is a surmountable inhibitor, we also tested the effects of irbesartan, an insurmountable inhibitor of AT1 receptor blocker, on BAPN-induced aortopathy formation (50 mg/kg/day, diet, n=30/group). Despite the significant increase of plasma renin concentrations, irbesartan did not ameliorate aortic rupture and dilatation in BAPN-administered mice. Thus, BAPN-induced thoracic aortopathies were refractory to angiotensin receptor blockade. We next inhibited angiotensinogen, the unique substrate for angiotensin II, using an antisense oligonucleotide targeting angiotensinogen (AGT-ASO). AGT-ASO decreased plasma angiotensinogen concentrations, while aortic death and dilatations were not attenuated. Since hepatocytes are the major source of angiotensinogen, BAPN-induced thoracic aortopathy formation was also examined in mice with hepatocyte-specific angiotensinogen deletion. Although plasma angiotensinogen concentrations displayed 90% reduction compared to wild type littermates, aortic rupture and aneurysm formation were not suppressed. Conclusion: Inhibition of the RAS does not attenuate BAPN-induced thoracic aortopathy formation in mice.