AT1R gene rs389566 polymorphism contributes to MACCEs in hypertension patients.

OBJECTIVE: To investigate the possible association between AT1R gene polymorphisms and major adverse cardiovascular and cerebrovascular events (MACCEs) in hypertension patients combined with or without coronary artery disease (CAD) in Xinjiang. METHODS: 374 CAD patients and 341 non-CAD individuals were enrolled as study participants and all of them have a hypertension diagnosis. AT1R gene polymorphisms were genotyped by SNPscan™ typing assays. During the follow-up in the clinic or by telephone interview, MACCEs were recorded. Kaplan-Meier curves and Cox survival analyses were used to explore the association between AT1R gene polymorphisms and the occurrence of MACCEs. RESULTS: AT1R gene rs389566 was associated with MACCEs. The TT genotype of the AT1R gene rs389566 had a significantly higher probability of MACCEs than the AA + AT genotype (75.2% vs. 24.8%, P = 0.033). Older age (OR = 1.028, 95% CI: 1.009-1.0047, P = 0.003) and TT genotype of rs389566 (OR = 1.770, 95% CI: 1.148-2.729, P = 0.01) were risk factors of MACCEs. AT1R gene rs389566 TT genotype may be a predisposing factor for the occurrence of MACCEs in hypertensive patients. CONCLUSION: We should also pay more attention to the prevent of MACCEs in hypertension patients combined with CAD. Especially those elderly hypertensive patients carrying AT1R rs389566 TT genotype requires avoidance of unhealthy lifestyle, better management of blood pressure control and reduce the occurrence of MACCEs.

Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation.

Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca2+ homeostasis, and whose functions are modulated by G protein-coupled receptors (GPCR). In this study, we developed bioluminescent resonance energy transfer (BRET) biosensors to better study channel conformational changes following receptor activation. For this study, two intramolecular biosensors, GFP10-TRPC7-RLucII and RLucII-TRPC7-GFP10, were constructed and were assessed following the activation of various GPCRs. We first transiently expressed receptors and the biosensors in HEK293 cells, and BRET levels were measured following agonist stimulation of GPCRs. The activation of GPCRs that engage Gαq led to a Gαq-dependent BRET response of the functional TRPC7 biosensor. Focusing on the Angiotensin II type-1 receptor (AT1R), GFP10-TRPC7-RLucII was tested in rat neonatal cardiac fibroblasts, expressing endogenous AT1R and TRPC7. We detected similar BRET responses in these cells, thus validating the use of the biosensor in physiological conditions. Taken together, our results suggest that activation of Gαq-coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.

Angiotensin II type 1 receptor variants alter endosomal receptor-ß-arrestin complex stability and MAPK activation.

The angiotensin II (AngII) type 1 receptor (AT1R), a member of the G protein-coupled receptor (GPCR) family, signals through G proteins and ß-arrestins, which act as adaptors to regulate AT1R internalization and mitogen-activated protein kinase (MAPK) ERK1/2 activation. ß-arrestin-dependent ERK1/2 regulation is the subject of important studies because its spatiotemporal control remains poorly understood for many GPCRs, including AT1R. To study the link between ß-arrestin-dependent trafficking and ERK1/2 signaling, we investigated three naturally occurring AT1R variants that show distinct receptor-ß-arrestin interactions: A163T, T282M, and C289W. Using bioluminescence resonance energy transfer (BRET)-based and conformational fluorescein arsenical hairpin-BRET sensors coupled with high-resolution fluorescence microscopy, we show that all AT1R variants form complexes with ß-arrestin2 at the plasma membrane and efficiently internalize into endosomes upon AngII stimulation. However, mutant receptors imposed distinct conformations in ß-arrestin2 and differentially impacted endosomal trafficking and MAPK signaling. Notably, T282M accumulated in endosomes, but its ability to form stable complexes following internalization was reduced, markedly impairing its ability to co-traffic with ß-arrestin2. We also found that despite ß-arrestin2 overexpression, T282M's and C289W's residency with ß-arrestin2 in endosomes was greatly reduced, leading to decreased ß-arrestin-dependent ERK1/2 activation, faster recycling of receptors to the plasma membrane, and impaired AngII-mediated proliferation. Our findings reveal that naturally occurring AT1R variants alter the patterns of receptor/ß-arrestin2 trafficking and suggest conformationally dependent ß-arrestin-mediated MAPK activation as well as endosomal receptor-ß-arrestin complex stabilization in the mitogenic response of AT1R.

Angiotensin II inhibits DDAH1-nNOS signaling via AT1R and µOR dimerization to modulate blood pressure control in the central nervous system.

G protein-coupled receptors (GPCRs) are important drug targets. Blocking angiotensin II (Ang II) type 1 receptor signaling alleviates hypertension and improves outcomes in patients with heart failure. Changes in structure and trafficking of GPCR, and desensitization of GPCR signaling induce pathophysiological processes. We investigated whether Ang II, via induction of AT1R and µ-opioid receptor (µOR) dimerization in the nucleus tractus solitarius (NTS), leads to progressive hypertension. Ang II signaling increased µOR and adrenergic receptor α2A (α2A-AR) heterodimer levels and decreased expression of extracellular signal-regulated kinases 1/2T202/Y204, ribosomal protein S6 kinaseT359/S363, and nNOSS1416 phosphorylation. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression was abolished in the NTS of adult spontaneously hypertensive rats (SHRs). Endomorphin-2 was overexpressed in NTS of adult SHRs compared with that in 6-week-old Wistar-Kyoto rats (WKY). Administration of µOR agonist into the NTS of WKY increased blood pressure (BP), decreased nitric oxide (NO) production, and decreased DDAH1 activity. µOR agonist significantly reduced the activity of DDAH1 and decreased neuronal NO synthase (nNOS) phosphorylation. The AT1R II inhibitor, losartan, significantly decreased BP and abolished AT1R-induced formation of AT1R and µOR, and α2A-AR and µOR, heterodimers. Losartan also significantly increased the levels of nNOSS1416 phosphorylation and DDAH1 expression. These results show that Ang II may induce expression of endomorphin-2 and abolished DDAH1 activity by enhancing the formation of AT1R and µOR heterodimers in the NTS, leading to progressive hypertension.

Differential Contribution of Transmembrane Domains IV, V, VI, and VII to Human Angiotensin II Type 1 Receptor Homomer Formation.

G protein-coupled receptors (GPCRs) play an important role in drug therapy and represent one of the largest families of drug targets. The angiotensin II type 1 receptor (AT1R) is notable as it has a central role in the treatment of cardiovascular disease. Blockade of AT1R signaling has been shown to alleviate hypertension and improve outcomes in patients with heart failure. Despite this, it has become apparent that our initial understanding of AT1R signaling is oversimplified. There is considerable evidence to suggest that AT1R signaling is highly modified in the presence of receptor-receptor interactions, but there is very little structural data available to explain this phenomenon even with the recent elucidation of the AT1R crystal structure. The current study investigates the involvement of transmembrane domains in AT1R homomer assembly with the goal of identifying hydrophobic interfaces that contribute to receptor-receptor affinity. A recently published crystal structure of the AT1R was used to guide site-directed mutagenesis of outward-facing hydrophobic residues within the transmembrane region of the AT1R. Bioluminescence resonance energy transfer was employed to analyze how receptor mutation affects the assembly of AT1R homomers with a specific focus on hydrophobic residues. Mutations within transmembrane domains IV, V, VI, and VII had no effect on angiotensin-mediated ß-arrestin1 recruitment; however, they exhibited differential effects on the assembly of AT1R into oligomeric complexes. Our results demonstrate the importance of hydrophobic amino acids at the AT1R transmembrane interface and provide the first glimpse of the requirements for AT1R complex assembly.

Effect of the renin inhibitor aliskiren against retinal ischemia-reperfusion injury.

The purpose of this study was to investigate the effect of the renin inhibitor, aliskiren, on retinal ischemia-reperfusion injury. Retinal ischemia was induced by increasing intraocular pressure to 130 mmHg. At 7 days after ischemia, retinal damage was evaluated by measuring the retinal thickness and the number of retinal ganglion cells. Western blot was used to measure changes in the (pro)renin receptor expression. Retinal mRNA expressions of prorenin, angiotensinogen and angiotensin II type 1 receptor (AT1-R) were measured by real-time polymerase chain reaction. Rats were treated with the renin inhibitor, aliskiren. Although the number of retinal ganglion cells and the inner retinal thickness were significantly decreased at 7 days after ischemia, treatment with aliskiren significantly inhibited retinal ischemic injury. Administration of aliskiren increased mRNA expression of prorenin in the retina at 3 h after the reperfusion. The expression of the (pro)renin receptor was not changed after ischemia-reperfusion injury with or without aliskiren. Although there was an increase in the retinal expression of AT1-R at 3 h after the reperfusion, aliskiren administration suppressed this expression. A renin inhibitor attenuated subsequent ischemic damage in the rat retina via the inhibition of the prorenin-induced angiotensin generation.

Angiotensin II modulates THP-1-like macrophage phenotype and inflammatory signatures via angiotensin II type 1 receptor.

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.

Effectiveness of losartan on infrapatellar fat pad/synovial fibrosis and pain behavior in the monoiodoacetate-induced rat model of osteoarthritis pain.

Infrapatellar fat pad (IFP)/ synovial fibrosis is closely associated with the clinical symptoms of joint pain and stiffness, which contribute to locomotor restriction in osteoarthritis (OA) patients. Hence, this study was designed to gain insight on whether losartan, a selective angiotensin II type 1 receptor (AT1R) antagonist, has therapeutic benefit to reverse IFP/synovial fibrosis and secondarily to attenuate pain behavior. In male Wistar rats with monoiodoacetic acid (MIA)-induced IFP/synovial fibrosis, a possible role for increased AT1R expression in the pathogenesis of IFP/synovial fibrosis was assessed over an 8-week period. Pain behavior comprised static weight bearing and von Frey paw withdrawal thresholds (PWTs), which were assessed once or twice weekly, respectively. Groups of MIA-rats received oral losartan (30-mg/kg; n = 8 or 100-mg/kg; n = 9) or vehicle (n = 9) for 28-days according to a prevention protocol. Animals were euthanized on day 28 and various tissues (IFP/synovium, cartilage and lumbar dorsal root ganglia (DRGs)) were collected for histological, immunohistochemical and western blot analyses. Administration of once-daily losartan for 28-days dose-dependently attenuated the development of static weight bearing. This was accompanied by reduced IFP/synovial fibrosis and suppression of TGF-ß1 expression. Chronic treatment of MIA-rats with losartan had an anti-fibrotic effect and it attenuated pain behavior in this animal model.

Differential expression of the angiotensin receptors (AT1, AT2, and AT4) in the placental bed of HIV-infected preeclamptic women of African ancestry.

The Renin-Angiotensin-Aldosterone System (RAAS) is implicated in the pathophysiology of preeclampsia (PE). There is a paucity of data on uteroplacental angiotensin receptors AT1-2 and 4. We evaluated the immunoexpression of AT1R, AT2R, and AT4R within the placental bed of PE vs. normotensive (N) pregnancies stratified by HIV status. Placental bed (PB) biopsies (n = 180) were obtained from N and PE women. Both groups were stratified by HIV status and gestational age into early-and late onset-PE. Immuno-labeling of AT1R, AT2R, and AT4R was quantified using morphometric image analysis. Immunostaining of PB endothelial cells (EC) and smooth muscle cells of spiral arteries (VSMC) displayed an upregulation of AT1R expression compared to the N group (p < 0.0001). Downregulation of AT2R and AT4R expression was observed in PE vs. N group (p = 0.0042 and p < 0.0001), respectively. AT2R immunoexpression declined between HIV+ve and HIV-ve groups, while AT1R and AT4R displayed an increase. An increase in AT1R expression was noted in the EOPE-ve/+ve and LOPE-ve/+ve compared to N-ve/N+ve. In contrast, AT2R and AT4R expression decreased in EOPE-ve/+ve and LOPE-ve/+ve compared to N-ve/N+ve. We demonstrate a significant downregulation of AT2R and AT4R with a concomitant elevated AT1R immunoexpression within PB of HIV-infected PE women. In addition, a decline in AT2R and AT4R with an increase in AT1R immunoexpression in PE, EOPE, and LOPE vs. normotensive pregnancies, irrespective of HIV status. Thus highlighting differential immunoexpression of uteroplacental RAAS receptors based on pregnancy type, HIV status, and gestational age.

Soluble RAGE attenuates Ang II-induced arterial calcification via inhibiting AT1R-HMGB1-RAGE axis.

BACKGROUND AND AIMS: Arterial calcification (AC), which is an important process in the pathogenesis of atherosclerosis, is accelerated by angiotensin II (Ang II), a critical effector of the renin-angiotensin system (RAS). Receptor for advanced glycation end-product (RAGE) is an important pattern recognition receptor downstream of Ang II. Although recent studies have suggested an association between RAGE-mediated signaling and RAS in AC, the detailed mechanism, particularly in relation to Ang II, remains unclear. METHODS: Therefore, we investigated the role of RAGE-mediated signaling pathways and the therapeutic efficacy of soluble RAGE (sRAGE) in Ang II-induced AC, using both a human aortic smooth muscle cell (HAoSMC) model, and an in vivo apolipoprotein E knockout (ApoE KO) mouse model. RESULTS: According to our data, Ang II significantly increased the calcification of HAoSMCs, and the associated activation of RAGE was mediated by subsequent HMGB1 release through Angiotensin II type 1 receptor activation. Both HMGB1 neutralizing antibody and sRAGE inhibited Ang II-induced calcium deposition. Furthermore, sRAGE attenuated HMGB1 secretion and the activation of RAGE-mediated signaling. The in vivo study indicated that Ang II significantly induced calcium deposition in the aorta, and this was significantly attenuated by sRAGE. CONCLUSIONS: Our findings strongly suggest that blockade of RAGE, using sRAGE, effectively attenuates Ang II-induced arterial calcification.

Angiotensin and Endothelin Receptor Structures With Implications for Signaling Regulation and Pharmacological Targeting.

In conjunction with the endothelin (ET) type A (ETAR) and type B (ETBR) receptors, angiotensin (AT) type 1 (AT1R) and type 2 (AT2R) receptors, are peptide-binding class A G-protein-coupled receptors (GPCRs) acting in a physiologically overlapping context. Angiotensin receptors (ATRs) are involved in regulating cell proliferation, as well as cardiovascular, renal, neurological, and endothelial functions. They are important therapeutic targets for several diseases or pathological conditions, such as hypertrophy, vascular inflammation, atherosclerosis, angiogenesis, and cancer. Endothelin receptors (ETRs) are expressed primarily in blood vessels, but also in the central nervous system or epithelial cells. They regulate blood pressure and cardiovascular homeostasis. Pathogenic conditions associated with ETR dysfunctions include cancer and pulmonary hypertension. While both receptor groups are activated by their respective peptide agonists, pathogenic autoantibodies (auto-Abs) can also activate the AT1R and ETAR accompanied by respective clinical conditions. To date, the exact mechanisms and differences in binding and receptor-activation mediated by auto-Abs as opposed to endogenous ligands are not well understood. Further, several questions regarding signaling regulation in these receptors remain open. In the last decade, several receptor structures in the apo- and ligand-bound states were determined with protein X-ray crystallography using conventional synchrotrons or X-ray Free-Electron Lasers (XFEL). These inactive and active complexes provide detailed information on ligand binding, signal induction or inhibition, as well as signal transduction, which is fundamental for understanding properties of different activity states. They are also supportive in the development of pharmacological strategies against dysfunctions at the receptors or in the associated signaling axis. Here, we summarize current structural information for the AT1R, AT2R, and ETBR to provide an improved molecular understanding.

µ-Opioid Receptor-Mediated AT1R-TLR4 Crosstalk Promotes Microglial Activation to Modulate Blood Pressure Control in the Central Nervous System.

Opioids, a kind of peptide hormone involved in the development of hypertension, cause systemic and cerebral inflammation, and affects regions of the brain that are important for blood pressure (BP) control. A cause-and-effect relationship exists between hypertension and inflammation; however, the role of blood pressure in cerebral inflammation is not clear. Evidence showed that AT1R and µOR heterodimers' formation in the NTS might lead to the progression of hypertension. In this study, we investigated the formation of the µOR/AT1R heterodimer, determined its correlation with µORs level in the NTS, and explored the role of TLR4-dependent inflammation in the development of hypertension. Results showed that Ang II increased superoxide and Iba-1 (microgliosis marker: ionized calcium-binding adaptor molecule (1) levels in the NTS of spontaneously hypertensive rats (SHRs). The AT1R II inhibitor, losartan, significantly decreased BP and abolished superoxide, Iba-1, TLR4 expression induced by Ang II. Furthermore, losartan significantly increased nNsOSS1416 phosphorylation. Administration of a µOR agonist or antagonist in the NTS of WKY and SHRs increased endogenous µ-opioids, triggered the formation of µOR/AT1R heterodimers and the TLR4-dependent inflammatory pathway, and attenuated the effect of depressor nitric oxide (NO). These results imply an important link between neurotoxicity and superoxides wherein abnormal increases in NTS endogenous µ-opioids promote the interaction between Ang II and µOR, the binding of Ang II to AT1R, and the activation of microglia. In addition, the interaction between Ang II and µOR enhanced the formation of the AT1R and µOR heterodimers, and inactivated nNOS-derived NO, leading to the development of progressive hypertension.

Possible Therapeutic Interventions in COVID-19 Induced ARDS by Cotinine as an ACE-2 Promoter and AT-1R Blocker.

In these challenging times of the pandemic, as coronavirus disease 2019 (COVID-19) has taken over the planet, its complications such as acute respiratory distress syndrome (ARDS) have the potential to wipe out a large portion of our population. Whereas a serious lack of ventilators, vaccine being months away makes the condition even worse. That's why promising drug therapy is required. One of them is suggested in this article. It is the angiotensin-converting enzyme-2 (ACE-2) to which the COVID-19 virus binds and upon downregulation of which the pulmonary permeability increases and results in the filling of alveoli by proteinaceous fluids, which finally results in ARDS. ARDS can be assisted by angiotensin-II type-1 receptor (AT-1R) blocker and ACE-2 upregulator. AT-1R blocker will prevent vasoconstriction, the pro-inflammatory effect seen otherwise upon its activation. ACE-2 upregulation will ensure less formation of angiotensin II, vasodilatory effects due to the formation of angiotensin (1-7), increased breakdown of bradykinin at lung level. Overall, decreased vasoconstriction of vessels supplying lungs and decreased vasodilation of lung tissues will ensure decreased pulmonary permeability and eventually relieve ARDS. It should also be considered that all components of the renin-angiotensin-aldosterone system (RAAS) are located in the lung tissues. A drug with the least plasma protein binding is required to ensure its distribution across these lung tissues. Cotinine appears to be a promising candidate for COVID-19- induced ARDS. It acts across the board and acts as both an AT-1R blocker, and ACE-2 upregulator. It also has a weak plasma protein binding that helps to spread through the lung tissues. In this review, we summarized that cotinine, along with COVID-19 virus replication blocker anti-virals, may prove to be a promising therapy for the treatment of COVID-19 induced ARDS.

Recruitment of central angiotensin II type 1 receptor associated neurocircuits in carbon dioxide associated fear.

Individuals with fear-associated conditions such as panic disorder (PD) and posttraumatic stress disorder (PTSD) display increased emotional responses to interoceptive triggers, such as CO2 inhalation, that signal a threat to physiological homeostasis. Currently, effector systems and mechanisms underlying homeostatic modulation of fear memory are not well understood. In this regard, the renin angiotensin system (RAS), particularly the angiotensin receptor type 1 (AT1R), a primary homeostatic regulatory target, has gained attention. RAS polymorphisms have been reported in PD and PTSD, and recent studies report AT1R-mediated modulation of fear extinction. However, contribution of AT1Rs in fear evoked by the interoceptive threat of CO2 has not been investigated. Using pharmacological, behavioral, and AT1R/ACE gene transcription analyses, we assessed central AT1R recruitment in CO2-associated fear. CO2 inhalation led to significant AT1R and ACE mRNA upregulation in homeostatic regulatory regions, subfornical organ (SFO) and paraventricular nucleus (PVN), in a temporal manner. Intracerebroventricular infusion of selective AT1R antagonist, losartan, significantly attenuated freezing during CO2 inhalation, and during re-exposure to CO2 context, suggestive of AT1R modulation of contextual fear. Regional Fos mapping in losartan-treated mice post-behavior revealed significantly attenuated labeling in areas regulating defensive behavior, contextual fear, and threat responding; such as, the bed nucleus of stria terminalis, dorsal periaqueductal gray, hypothalamic nuclei, hippocampus, and prefrontal areas such as the prelimbic, infralimbic, and anterior cingulate cortices. Sub-regions of the amygdala did not show CO2-associated AT1R regulation or altered Fos labeling. Collectively, our data suggests central AT1R recruitment in modulation of fear behaviors associated with CO2 inhalation via engagement of neurocircuits regulating homeostasis and defensive behaviors. Our data provides mechanistic insights into the interoceptive regulation of fear, relevant to fear related disorders such as PD and PTSD.

Methods to Monitor the Trafficking of ß-Arrestin/G Protein-Coupled Receptor Complexes Using Enhanced Bystander BRET.

ß-Arrestins are adaptors that regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). Bioluminescence resonance energy transfer (BRET) is a sensitive and versatile method for real-time monitoring of protein-protein interactions and protein kinesis within live cells, such as the recruitment of ß-arrestins to activated receptors at the plasma membrane (PM) and the trafficking of GPCR/ß-arrestin complexes to endosomes. Trafficking of receptor/ß-arrestin complexes can be assessed by BRET through tagging ß-arrestins with the donor luciferase from Renilla reniformis (Rluc) and anchoring the acceptor green fluorescent protein from the same species (rGFP) in distinct cell compartments (e.g., PM or endosomes) to generate highly efficient bystander BRET (referred to as enhanced bystander BRET (EbBRET)) upon re-localization of ß-arrestins to these compartments following receptor activation. Here, we outline the protocol for quantitatively monitoring ß-arrestin recruitment to agonist-activated Angiotensin II type 1 receptor (AT1R) and ß2-adrenergic receptor (ß2AR) at the PM and the trafficking of receptor/ß-arrestin complexes into endosomes using EbBRET-based biosensors.