Angiotensin-(1-9) [Ang-(1-9)], generated from Ang I by Ang II converting enzyme 2, has been reported to have protective effects on cardiac and vascular remodeling. However, there is no report about the effect of Ang-(1-9) on pulmonary hypertension. The aim of the present study is to investigate whether Ang-(1-9) improves pulmonary vascular remodeling in monocrotaline (MCT)-induced pulmonary hypertensive rats. Sprague-Dawley rats received Ang-(1-9) (576 µg/kg/day) or saline via osmotic mini-pumps for 3 weeks. Three days after implantation of osmotic mini-pumps, 50 mg/kg MCT or vehicle were subcutaneously injected. MCT caused increases in right ventricular weight and systolic pressure, which were reduced by co-administration of Ang-(1-9). Ang-(1-9) also attenuated endothelial damage and medial hypertrophy of pulmonary arterioles as well as pulmonary fibrosis induced by MCT. The protective effects of Ang-(1-9) against pulmonary hypertension were inhibited by Ang type 2 receptor (AT2R) blocker, but not by Mas receptor blocker. Additionally, the levels of LDH and inflammatory cytokines, such as TNF-α, MCP-1, IL-1β, and IL-6, in plasma were lower in Ang-(1-9) co-treated MCT group than in vehicle-treated MCT group. Changes in expressions of apoptosis-related proteins such as Bax, Bcl-2, Caspase-3 and -9 in the lung tissue of MCT rats were attenuated by the treatment with Ang-(1-9). These results indicate that Ang-(1-9) improves MCT-induced pulmonary hypertension by decreasing apoptosis and inflammatory reaction via AT2R.

Angiotensin-(1-9) [Ang-(1-9)] and Ang-(1-7) are cleaved by Ang converting enzyme 2 forming Ang I and Ang II, respectively, and the truncated Angs play a role in regulating atrial natriuretic peptide (ANP) secretion. Previously, we found that Ang-(1-7) stimulates ANP secretion via the Mas receptor. However, the effect of Ang-(1-9) on ANP secretion is still unknown. The aim of the present study is to determine whether Ang-(1-9) stimulates ANP secretion and to characterize the signaling pathway involved in stimulating secretion.

Angiotensin II (Ang II) is a major component of the renin-angiotensin or renin-angiotensin-aldosterone system, which is the main element found to be involved in cardiopathology. Recently, long-term metabolomics studies have linked high levels of angiotensin plasma to inflammatory conditions such as coronary heart disease, obesity, and type 2 diabetes. Monocyte/macrophage cellular function and phenotype orchestrate the inflammatory response in various pathological conditions, most notably cardiometabolic disease. An activation of the Ang II system is usually associated with inflammation and cardiovascular disease; however, the direct effect on monocyte/macrophages has still not been well elucidated. Herein, we have evaluated the cellular effects of Ang II on THP-1-derived macrophages. Ang II stimulated the expression of markers involved in monocyte/macrophage cell differentiation (e.g., CD116), as well as adhesion, cell-cell interaction, chemotaxis, and phagocytosis (CD15, CD44, CD33, and CD49F). Yet, Ang II increased the expression of proinflammatory markers (HLA-DR, TNF-α, CD64, CD11c, and CD38) and decreased CD206 (mannose receptor), an M2 marker. Moreover, Ang II induced cytosolic calcium overload, increased reactive oxygen species, and arrested cells in the G1 phase. Most of these effects were induced via the angiotensin II type 1 receptor (AT1R). Collectively, our results provide new evidence in support of the effect of Ang II in inflammation associated with cardiometabolic diseases.

Angiotensin II type 1 receptor antibody (AT1R-Ab), is a non-human leukocyte antigen (HLA) antibody implicated in poor renal allograft outcomes, although its actions may be mediated through a different pathway than HLA donor-specific antibodies (DSAs). Our aim was to examine serum cytokine profiles associated with AT1R-Ab and distinguish them from those associated with HLA DSA in serially collected blood samples from a cohort of pediatric renal transplant recipients.

The counter-regulatory axis of the renin angiotensin system peptide angiotensin-(1-7) [Ang-(1-7)] has been identified as a potential therapeutic target in cardiac remodelling, acting via the mas receptor. Furthermore, we recently reported that an alternative peptide, Ang-(1-9) also counteracts cardiac remodelling via the angiotensin type 2 receptor (AT(2)R). Here, we have engineered adenoviral vectors expressing fusion proteins which release Ang-(1-7) [RAdAng-(1-7)] or Ang-(1-9) [RAdAng-(1-9)] and compared their effects on cardiomyocyte hypertrophy in rat H9c2 cardiomyocytes or primary adult rabbit cardiomyocytes, stimulated with angiotensin II, isoproterenol or arg-vasopressin. RAdAng-(1-7) and RAdAng-(1-9) efficiently transduced cardiomyocytes, expressed fusion proteins and secreted peptides, as demonstrated by western immunoblotting and conditioned media assays. Furthermore, secreted Ang-(1-7) and Ang-(1-9) inhibited cardiomyocyte hypertrophy (Control = 168.7±8.4 µm; AngII = 232.1±10.7 µm; AngII+RAdAng-(1-7) = 186±9.1 µm, RAdAng-(1-9) = 180.5±9 µm; P<0.05) and these effects were selectively reversed by inhibitors of their cognate receptors, the mas antagonist A779 for RAdAng-(1-7) and the AT(2)R antagonist PD123,319 for RAdAng-(1-9). Thus gene transfer of Ang-(1-7) and Ang-(1-9) produces receptor-specific effects equivalent to those observed with addition of exogenous peptides. These data highlight that Ang-(1-7) and Ang-(1-9) can be expressed via gene transfer and inhibit cardiomyocyte hypertrophy via their respective receptors. This supports applications for this approach for sustained peptide delivery to study molecular effects and potential gene therapeutic actions.

Renin-angiotensin system (RAS) components exert diverse physiological functions and have been sub-grouped into deleterious angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/angiotensin type 1 receptor (AT1R) and protective ACE2/angiotensin (1-7) (Ang-(1-7))/Mas receptor (MasR) axes. We have reported that chronic activation of angiotensin type 2 receptor (AT2R) alters RAS components and provides protection against obesity-related kidney injury.

We previously showed that Angiotensin (Ang) II stimulated pro-inflammatory and mitogenic actions in astrocytes suggesting that astrocytes are emerging as key players in neuroinflammation. Evidence suggests that neuroinflammation may contribute to central sympathetic overactivity and elevated blood pressure. Further, cyclooxygenase (Cox)-derived prostanoids were implicated in Ang II-dependent hypertension. Cox2 is one of two Cox isoenzymes that is responsible for the formation of prostanoids from arachidonic acid. Constitutively expressed Cox2 has a protective and homeostatic role in the cardiovascular and renal systems. Inducible Cox2 has been associated with pathogenic stimuli resulting in inflammatory conditions and cancers. In this study, we investigated the effect of Ang II on Cox2 protein and mRNA expression in brainstem and cerebellum astrocytes, and determined whether any differences in Cox2 expression exist in spontaneously hypertensive rat (SHR) astrocytes compared to their normotensive control Wistar rats. We demonstrated that Ang II increased Cox2 protein and mRNA levels relative to untreated controls in a time-dependent manner, in Wistar and SHR brainstem and cerebellum astrocytes. Increases in Cox2 protein expression were evident within 4 h, with subsequent sustained elevation for several hours followed by a decline at 48 h. Ang II-induced Cox2 protein levels were higher in Wistar compared to SHRs in both brainstem and cerebellum astrocytes for the majority of time points examined. The Ang II-induced Cox2 mRNA levels increased within 8 h followed by a rapid decline to almost basal levels at later time points. At the earlier time points, Cox2 mRNA elevation were higher in SHR compared to Wistar rat astrocytes. These Ang II actions were mediated by the Ang type I receptor. Our results corroborate previous reports of Ang II's ability to stimulate neuroinflammatory mediators in astrocytes. Cox2-derived prostaglandins might play a role in brain-renin angiotensin system associated hypertension, and astrocytes could be significant players.

The kidney is easily affected by aging-associated changes, including glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Particularly, renal tubulointerstitial fibrosis is a final common pathway in most forms of progressive renal disease. Angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP), which was originally identified as a molecule that binds to AT1R, is highly expressed in the kidney. Previously, we have shown that ATRAP suppresses hyperactivation of AT1R signaling, but does not affect physiological AT1R signaling.

Browning of white adipose tissue (WAT) has been highlighted as a new possible therapeutic target for obesity, diabetes and lipid metabolic disorders, because WAT browning could increase energy expenditure and reduce adiposity. The new clusters of adipocytes that emerge with WAT browning have been named 'beige' or 'brite' adipocytes. Recent reports have indicated that the renin-angiotensin system (RAS) plays a role in various aspects of adipose tissue physiology and dysfunction. The biological effects of angiotensin II, a major component of RAS, are mediated by two receptor subtypes, angiotensin II type 1 receptor (AT1R) and type 2 receptor (AT2R). However, the functional roles of angiotensin II receptor subtypes in WAT browning have not been defined. Therefore, we examined whether deletion of angiotensin II receptor subtypes (AT1aR and AT2R) may affect white-to-beige fat conversion in vivo. AT1a receptor knockout (AT1aKO) mice exhibited increased appearance of multilocular lipid droplets and upregulation of thermogenic gene expression in inguinal white adipose tissue (iWAT) compared to wild-type (WT) mice. AT2 receptor-deleted mice did not show miniaturization of lipid droplets or alteration of thermogenic gene expression levels in iWAT. An in vitro experiment using adipose tissue-derived stem cells showed that deletion of the AT1a receptor resulted in suppression of adipocyte differentiation, with reduction in expression of thermogenic genes. These results indicate that deletion of the AT1a receptor might have some effects on the process of browning of WAT and that blockade of the AT1 receptor could be a therapeutic target for the treatment of metabolic disorders.

Kidney fibrosis is a common pathway that leads to chronic kidney disease. Angiotensin II type-1 receptor (AT1R)-associated protein (ATRAP) was originally identified as an AT1R-binding protein. Previously, we reported that systemic knockout of ATRAP exacerbates kidney fibrosis in aged mice. Although these effects of ATRAP appeared to be AT1R-independent actions, the molecular mechanism remains poorly understood. To elucidate the molecular mechanism of ATRAP independent of AT1R, we explored novel ATRAP-interacting proteins. Mass spectrometric analysis of the immunoprecipitants of a Flag-tagged ATRAP complex revealed 376 candidate proteins that potentially interact with ATRAP. Gene ontology analysis revealed that proteins related to vesicle trafficking, membrane transport, and many membrane proteins, including transferrin receptor 1 (TfR1), were enriched. Because TfR1 promotes cellular iron uptake and iron is a key factor involved in kidney fibrosis, we focused on TfR1 and confirmed that it interacts with ATRAP. In addition, our findings revealed that enhanced ATRAP expression decreased cell-surface TfR1 expression without altering the overall cellular TfR1 expression levels. Furthermore, enhanced ATRAP expression attenuated cellular iron levels. Together, our results highlight the role of ATRAP as a suppressor of TfR1 that functions by facilitating TfR1 internalization, which affects iron metabolism and oxidative stress signaling.

Recently, our group has developed a therapeutic hypertensive vaccine against angiotensin (Ang) II type 1 receptor (AT1R) named ATRQβ-001. To explore its potential effectiveness on streptozotocin-induced diabetic nephropathy, male Sprague Dawley rats were randomly divided into two groups: a control and a diabetic model. After 1 week, the diabetic rats were divided into four subgroups (each with 15 rats) for 14-week treatments with saline, olmesartan, ATRQβ-001, and Qβ virus-like particle (VLP), respectively. In addition to lower blood pressure, ATRQβ-001 vaccination ameliorated biochemical parameter changes of renal dysfunction, mesangial expansion, and fibrosis through inhibiting oxidative stress, macrophage infiltration, and proinflammatory factor expression. Furthermore, ATRQβ-001 vaccination suppressed renal Ang II-AT1R activation and abrogated the downregulation of angiotensin-converting enzyme 2-Ang (1-7), similar to olmesartan treatment, while no obvious feedback activation of circulating or local renin-angiotensin system (RAS) was only observed in vaccine group. In rat mesangial cells, the anti-ATR-001 antibody inhibited high glucose-induced transforming growth factor-β1 (TGF)-β1/Smad3 signal pathway. Additionally, no significant immune-mediated damage was detected in vaccinated animals. In conclusion, the ATRQβ-001 vaccine ameliorated streptozotocin-induced diabetic renal injury via modulating two RAS axes and inhibiting TGF-β1/Smad3 signal pathway, providing a novel, safe, and promising method to treat diabetic nephropathy.

The role of autoimmunity in neurodegeneration has been increasingly suggested. The renin-angiotensin system (RAS) autoantibodies play a major role in several peripheral inflammatory processes. Dysregulation of brain RAS has been involved in neuroinflammation and neurodegeneration. We aimed to know whether angiotensin type-1 receptor (AT1) autoantibodies (AT1 agonists) and angiotensin-converting enzyme 2 (ACE2) autoantibodies (ACE2 antagonists) may be involved in Parkinson's disease (PD) progression and constitute a new therapeutical target. Both AT1 and ACE2 serum autoantibodies were higher in a group of 117 PD patients than in a group of 106 controls. Serum AT1 autoantibodies correlated with several cytokines, particularly Tumor Necrosis Factor Ligand Superfamily Member 14 (TNFSF14, LIGHT), and 27-hydroxycholesterol levels. Serum ACE2 autoantibodies correlated with AT1 autoantibodies. Both autoantibodies were found in cerebrospinal fluid (CSF) of four PD patients with CSF samples. Consistent with the observations in patients, experimental dopaminergic degeneration, induced by 6-hydroxydopamine, increased levels of autoantibodies in serum and CSF in rats, as well as LIGHT levels and transglutaminase activity in rat substantia nigra. In cultures, administration of AT1 autoantibodies enhanced dopaminergic neuron degeneration and increased levels of neuroinflammation markers, which was inhibited by the AT1 antagonist candesartan. The results suggest dysregulation of RAS autoantibodies as a new mechanism that can contribute to PD progression. Therapeutical strategies blocking the production, or the effects of these autoantibodies may be useful for PD treatment, and the results further support repurposing AT1 blockers (ARBs) as treatment against PD progression.

The aims of this study were to assess the renal expression of angiotensin II type 1 receptor (AT1R), angiotensin II type 2 receptor (AT2R), and MAS receptor in human type 2 diabetic nephropathy (DN).

We recently characterized an autocrine renin angiotensin system (RAS) in canine heart. Activation of Angiotensin II Type 1 Receptors (AT1Rs) induced electrical remodeling, including inhibition of the transient outward potassium current Ito, prolongation of the action potential (AP), increased calcium entry and increased contractility. Electrical properties of the mouse heart are very different from those of dog heart, but if a similar system existed in mouse, it could be uniquely studied through genetic manipulations. To investigate the presence of a RAS in mouse, we measured APs and Ito in isolated myocytes. Application of angiotensin II (A2) for 2 or more hours reduced Ito magnitude, without affecting voltage dependence, and prolonged APs in a dose-dependent manner. Based on dose-inhibition curves, the fast and slow components of Ito (Ito,fast and IK,slow) appeared to be coherently regulated by [A2], with 50% inhibition at an A2 concentration of about 400 nM. This very high K0.5 is inconsistent with systemic A2 effects, but is consistent with an autocrine RAS in mouse heart. Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on Ito and AP duration, suggesting these effects depend on intracellular trafficking. Application of the biased agonist SII ([Sar1-Ile4-Ile8]A2), which stimulates receptor internalization without G protein activation, caused Ito reduction and AP prolongation similar to A2-induced changes. These data demonstrate AT1R mediated regulation of Ito in mouse heart. Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.

Cardiac hypertrophy (CH) is an independent risk factor for cardiovascular diseases (CVDs). Mitigating or preventing CH is the most effective strategy for the treatment of CVDs. DanHong injection (DH) is a Chinese herbal medicine preparation (CHMP) widely used in clinical treatment of several CVDs in China. However, the direct targets and cellular mechanisms for these protective effects remain unclear. This study was designed to illustrate the direct targets of DH in protecting against CH and investigate CH molecular pathogenesis. A hypertrophic cell model was induced by endothelin-1 (ET-1) on human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). Real time cellular analysis (RTCA) cardio system and high content analysis (HCA) were used to detect the changes in contractile function, morphology and protein level of hypertrophic hiPS-CMs. Agonist and antagonist assay on receptors were performed using calcium mobilization high-throughput screening (HTS). DH significantly attenuated CH by modulating myocardial contractility, suppressing cell area enlargement and down-regulating ET-1-induced brain natriuretic peptide (BNP), actinin alpha 2 (ACTN2) and cardiac muscle troponin T (TNNT2) protein expression (P < 0.05). Endothelin receptor type B (ETBR) and angiotensin II receptor type 1 (AT1R) were DH direct targets, with IC50 value of 25.67 μL/mL and 1.10 μL/mL, respectively. Proteomics analysis showed that proteins involved in cell cycle inhibition, RNA processing, mitochondrial translation and cytoskeleton are significant regulated by DH treatment. These data revealed that ETBR and AT1R are DH direct targets on protecting against CH, providing a strategy to explore direct targets of CHMPs.

Emerging evidences suggest that the angiotensin receptor type 1 (AT1R) contributes heavily to the pathogenesis of atherosclerotic cerebral infarction (ACI). Herein, we examined a potential link between AT1R gene polymorphisms and ACI risk among a Southern Han Chinese population.

The heterodimerization of angiotensin II receptors (AT1R and AT2R) with adiponectin receptor AdipoR1 and AdipoR2 may instigate high glucose (HG)-induced renal tubulointerstitial injury. This study examined the effect of telmisartan on diabetic nephropathy (DN) and its underlying mechanism.

In the recent years, a role of the immune system in Huntington's disease (HD) is increasingly recognized. Here we investigate the presence of T cell activating auto-antibodies against angiotensin II type 1 receptors (AT1R) in all stages of the disease as compared to healthy controls and patients suffering from multiple sclerosis (MS) as a prototype neurologic autoimmune disease.

The mechanisms for increased risk of chronic kidney disease (CKD) in obesity remain unclear. The renin-angiotensin system is implicated in the pathogenesis of both adiposity and CKD. We investigated whether the angiotensin type 1 (AT(1)) receptor, composed of dominant AT(1a) and less expressed AT(1b) in wild-type (WT) mice, modulates development and progression of kidney injury in a high-fat diet (HFD)-induced obesity model. WT mice had increased body weight, body fat, and insulin levels and decreased adiponectin levels after 24 wk of a high-fat diet. Identically fed AT(1a) knockout (AT1aKO) mice gained weight similarly to WT mice, but had lower body fat and higher plasma cholesterol. Both obese AT1aKO and obese WT mice had increased visceral fat and kidney macrophage infiltration, with more proinflammatory M1 macrophage markers as well as increased mesangial expansion and tubular vacuolization, compared with lean mice. These abnormalities were heightened in the obese AT1aKO mice, with downregulated M2 macrophage markers and increased macrophage AT(1b) receptor. Treatment with an AT(1) receptor blocker, which affects both AT(1a) and AT(1b), abolished renal macrophage infiltration with inhibition of renal M1 and upregulation of M2 macrophage markers in obese WT mice. Our data suggest obesity accelerates kidney injury, linked to augmented inflammation in adipose and kidney tissues and a proinflammatory shift in macrophage and M1/M2 balance.

Hypertension is a leading risk factor for the development and progression of diabetic retinopathy and contributes to a variety of other retinal diseases in the absence of diabetes mellitus. Inhibition of the renin-angiotensin system has been shown to provide beneficial effects against diabetic retinopathy, both in the absence and presence of hypertension, suggesting that angiotensin II (Ang II) and the Ang II type 1 receptor may contribute to retinal vascular dysfunction. We investigated the effects of the Ang II type 1 receptor antagonist candesartan on retinal vascular permeability (RVP) in normotensive rats with streptozotocin-induced diabetes mellitus and in rats with Ang II-induced hypertension. We showed that candesartan treatment decreased diabetes mellitus- and Ang II-stimulated RVP by 58% (P<0.05) and 79% (P<0.05), respectively, compared with untreated controls, suggesting that activation of the Ang II type 1 receptor contributes to blood-retinal barrier dysfunction. We found that plasma kallikrein levels are increased in the retina of rats with Ang II-stimulated hypertension and that intravitreal injection of either plasma kallikrein or bradykinin is sufficient to increase RVP. We showed that a novel small molecule inhibitor of plasma kallikrein, 1-benzyl-1H-pyrazole-4-carboxylic acid 4-carbamimidoyl-benzylamide, delivered systemically via a subcutaneous pump, decreased Ang II-stimulated RVP by 70% (P<0.05) and ameliorates Ang II-induced hypertension, measured from the carotid artery by telemetry, but did not reduce Ang II-induced retinal leukostasis. These findings demonstrate that activation of the Ang II type 1 receptor increases RVP and suggest that systemic plasma kallikrein inhibition may provide a new therapeutic approach for ameliorating blood-retinal barrier dysfunction induced by hypertension.