RESUMO
The peptide hormone angiotensin II regulates blood pressure mainly through the type 1 angiotensin II receptor AT1 R and its downstream signaling proteins Gq and ß-arrestin. AT1 R blockers, clinically used as antihypertensive drugs, inhibit both signaling pathways, whereas AT1 R ß-arrestin-biased agonists have shown great potential for the treatment of acute heart failure. Here, we present a cryo-electron microscopy (cryo-EM) structure of the human AT1 R in complex with a balanced agonist, Sar1 -AngII, and Gq protein at 2.9 Å resolution. This structure, together with extensive functional assays and computational modeling, reveals the molecular mechanisms for AT1 R signaling modulation and suggests that a major hydrogen bond network (MHN) inside the receptor serves as a key regulator of AT1 R signal transduction from the ligand-binding pocket to both Gq and ß-arrestin pathways. Specifically, we found that the MHN mutations N1113.35 A and N2947.45 A induce biased signaling to Gq and ß-arrestin, respectively. These insights should facilitate AT1 R structure-based drug discovery for the treatment of cardiovascular diseases.
Assuntos
Angiotensina II , Transdução de Sinais , Humanos , Microscopia Crioeletrônica , Transdução de Sinais/fisiologia , beta-Arrestinas/metabolismo , Angiotensina II/química , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Receptores de Angiotensina/metabolismoRESUMO
We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or -mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.
Assuntos
Núcleo Celular , Receptores ErbB , Receptor Tipo 1 de Angiotensina , Receptores ErbB/metabolismo , Humanos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Núcleo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Membrana Celular/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Ligação Proteica , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genéticaRESUMO
BACKGROUND AND AIMS: Anti-hypertensive agents are one of the most frequently used drugs worldwide. However, no blood pressure-lowering strategy is superior to placebo with respect to survival in diabetic hypertensive patients. Previous findings show that Wnt co-receptors LDL receptor-related proteins 5 and 6 (LRP5/6) can directly bind to several G protein-coupled receptors (GPCRs). Because angiotensin II type 1 receptor (AT1R) is the most important GPCR in regulating hypertension, this study examines the possible mechanistic association between LRP5/6 and their binding protein Dickkopf-1 (DKK1) and activation of the AT1R and further hypothesizes that the LRP5/6-GPCR interaction may affect hypertension and potentiate cardiac impairment in the setting of diabetes. METHODS: The roles of serum DKK1 and DKK1-LRP5/6 signalling in diabetic injuries were investigated in human and diabetic mice. RESULTS: Blood pressure up-regulation positively correlated with serum DKK1 elevations in humans. Notably, LRP5/6 physically and functionally interacted with AT1R. The loss of membrane LRP5/6 caused by injection of a recombinant DKK1 protein or conditional LRP5/6 deletions resulted in AT1R activation and hypertension, as well as ß-arrestin1 activation and cardiac impairment, possibly because of multiple GPCR alterations. Importantly, unlike commonly used anti-hypertensive agents, administration of the anti-DKK1 neutralizing antibody effectively prevented diabetic cardiac impairment in mice. CONCLUSIONS: These findings establish a novel DKK1-LRP5/6-GPCR pathway in inducing diabetic injuries and may resolve the long-standing conundrum as to why elevated blood DKK1 has deleterious effects. Thus, monitoring and therapeutic elimination of blood DKK1 may be a promising strategy to attenuate diabetic injuries.
Assuntos
Diabetes Mellitus Experimental , Cardiomiopatias Diabéticas , Hipertensão , Receptores de LDL , Animais , Humanos , Camundongos , Anti-Hipertensivos , Cardiomiopatias Diabéticas/prevenção & controle , Hipertensão/prevenção & controle , Receptores de LDL/antagonistas & inibidoresRESUMO
The renin-angiotensin system plays a crucial role in the regulation of blood pressure. Activation of the angiotensin II (Ang II)-Ang II type 1 receptor (AT1R) signaling pathway contributes to the pathogenesis of hypertension and subsequent organ damage. AT1R-associated protein (ATRAP) has been identified as an endogenous inhibitory protein of the AT1R pathological activation. We have shown that mouse Atrap (Atrap) represses various Ang II-AT1R-mediated pathologies, including hypertension in mice. The expression of human ATRAP (ATRAP)/Atrap can be altered in various pathological states in humans and mice, such as Ang II stimulation and serum starvation. However, the regulatory mechanisms of ATRAP/Atrap are not yet fully elucidated. miRNAs are 21 to 23 nucleotides of small RNAs that post-transcriptionally repress gene expression. Single miRNA can act on hundreds of target mRNAs, and numerous miRNAs have been identified as the Ang II-AT1R signaling-associated disease phenotype modulator, but nothing is known about the regulation of ATRAP/Atrap. In the present study, we identified miR-125a-5p/miR-125b-5p as the evolutionarily conserved miRNAs that potentially act on ATRAP/Atrap mRNA. Further analysis revealed that miR-125a-5p/miR-125b-5p can directly repress both ATRAP and Atrap. In addition, the inhibition of miR-125a-5p/miR-125b-5p resulted in the suppression of the Ang II-AT1R signaling in mouse distal convoluted tubule cells. Taken together, miR-125a-5p/miR-125b-5p activates Ang II-AT1R signaling by the suppression of ATRAP/Atrap. Our results provide new insights into the potential approaches for achieving the organ-protective effects by the repression of the miR-125 family associated with the enhancement of ATRAP/Atrap expression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Hipertensão , MicroRNAs , Receptor Tipo 1 de Angiotensina , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Hipertensão/metabolismo , Túbulos Renais Distais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismoRESUMO
Amphetamine (AMPH) exposure induces behavioural and neurochemical sensitization observed in rodents as hyperlocomotion and increased dopamine release in response to a subsequent dose. Brain Angiotensin II modulates dopaminergic neurotransmission through its AT1 receptors (AT1-R), positively regulating striatal dopamine synthesis and release. This work aims to evaluate the AT1-R role in the development and maintenance of AMPH-induced sensitization. Also, the AT1-R involvement in striatal dopamine reuptake was analysed. The sensitization protocol consisted of daily AMPH administration for 5 days and tested 21 days after withdrawal. An AT1-R antagonist, candesartan, was administered before or after AMPH exposure to evaluate the participation of AT1-R in the development and maintenance of sensitization, respectively. Sensitization was evaluated by locomotor activity and c-Fos immunostaining. Changes in dopamine reuptake kinetics were evaluated 1 day after AT1-R blockade withdrawal treatment, with or without the addition of AMPH in vitro. The social interaction test was performed as another behavioural output. Repeated AMPH exposure induced behavioural and neurochemical sensitization, which was prevented and reversed by candesartan. The AT1-R blockade increased the dopamine reuptake kinetics. Neither the AMPH administration nor the AT1-R blockade altered the performance of social interaction. Our results highlight the AT1-R's crucial role in AMPH sensitization. The enhancement of dopamine reuptake kinetics induced by the AT1-R blockade might attenuate the neuroadaptive changes that lead to AMPH sensitization and its self-perpetuation. Therefore, AT1-R is a prominent candidate as a target for pharmacological treatment of pathologies related to dopamine imbalance, including drug addiction and schizophrenia.
Assuntos
Anfetamina , Bloqueadores do Receptor Tipo 1 de Angiotensina II , Angiotensina II , Benzimidazóis , Compostos de Bifenilo , Corpo Estriado , Dopamina , Animais , Anfetamina/farmacologia , Masculino , Dopamina/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Angiotensina II/farmacologia , Compostos de Bifenilo/farmacologia , Benzimidazóis/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Ratos Wistar , Ratos , Receptor Tipo 1 de Angiotensina/metabolismo , Tetrazóis/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Interação Social/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
BACKGROUND: Extracellular matrix (ECM) stiffness is closely related to the progress of diabetic cardiomyopathy (DCM) and the response of treatment of DCM to anti-diabetic drugs. Dapagliflozin (Dapa) has been proven to have cardio-protective efficacy for diabetes and listed as the first-line drug to treat heart failure. But the regulatory relationship between ECM stiffness and treatment efficacy of Dapa remains elusive. MATERIALS AND METHODS: This work investigated the effect of ECM stiffness on DCM progression and Dapa efficacy using both in vivo DCM rat model and in vitro myocardial cell model with high glucose injury. First, through DCM rat models with various levels of myocardial injury and administration with Dapa treatment for four weeks, the levels of myocardial injury, myocardial oxidative stress, expressions of AT1R (a mechanical signal protein) and the stiffness of myocardial tissues were obtained. Then for mimicking the stiffness of myocardial tissues at early and late stages of DCM, we constructed cell models through culturing H9c2 myocardial cells on the polyacrylamide gels with two stiffness and exposed to a high glucose level and without/with Dapa intervention. The cell viability, reactive oxygen species (ROS) levels and expressions of mechanical signal sensitive proteins were obtained. RESULTS: The DCM progression is accompanied by the increased myocardial tissue stiffness, which can synergistically exacerbate myocardial cell injury with high glucose. Dapa can improve the ECM stiffness-induced DCM progression and its efficacy on DCM is more pronounced on the soft ECM, which is related to the regulation pathway of AT1R-FAK-NOX2. Besides, Dapa can inhibit the expression of the ECM-induced integrin ß1, but without significant impact on piezo 1. CONCLUSIONS: Our study found the regulation and effect of biomechanics in the DCM progression and on the Dapa efficacy on DCM, providing the new insights for the DCM treatment. Additionally, our work showed the better clinical prognosis of DCM under early Dapa intervention.
Assuntos
Compostos Benzidrílicos , Cardiomiopatias Diabéticas , Matriz Extracelular , Glucosídeos , Miócitos Cardíacos , Estresse Oxidativo , Ratos Sprague-Dawley , Inibidores do Transportador 2 de Sódio-Glicose , Animais , Cardiomiopatias Diabéticas/fisiopatologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/tratamento farmacológico , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/patologia , Glucosídeos/farmacologia , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/patologia , Compostos Benzidrílicos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Masculino , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Espécies Reativas de Oxigênio/metabolismo , Ratos , Quinase 1 de Adesão Focal/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/complicaçõesRESUMO
While orthosteric ligands of the angiotensin II (AngII) type 1 receptor (AT1R) are available for clinical and research applications, allosteric ligands are not known for this important G protein-coupled receptor (GPCR). Allosteric ligands are useful tools to modulate receptor pharmacology and subtype selectivity. Here, we report AT1R allosteric ligands for a potential application to block autoimmune antibodies. The epitope of autoantibodies for AT1R is outside the orthosteric pocket in the extracellular loop 2. A molecular dynamics simulation study of AT1R structure reveals the presence of a druggable allosteric pocket encompassing the autoantibody epitope. Small molecule binders were then identified for this pocket using structure-based high-throughput virtual screening. The top 18 hits obtained inhibited the binding of antibody to AT1R and modulated agonist-induced calcium response of AT1R. Two compounds out of 18 studied in detail exerted a negative allosteric modulator effect on the functions of the natural agonist AngII. They blocked antibody-enhanced calcium response and reactive oxygen species production in vascular smooth muscle cells as well as AngII-induced constriction of blood vessels, demonstrating their efficacy in vivo. Our study thus demonstrates the feasibility of discovering inhibitors of the disease-causing autoantibodies for GPCRs. Specifically, for AT1R, we anticipate development of more potent allosteric drug candidates for intervention in autoimmune maladies such as preeclampsia, bilateral adrenal hyperplasia, and the rejection of organ transplants.
Assuntos
Autoanticorpos , Desenho de Fármacos , Receptor Tipo 1 de Angiotensina/agonistas , Angiotensina II , Animais , Especificidade de Anticorpos , Cálcio/metabolismo , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G , Ligantes , Camundongos , Simulação de Dinâmica Molecular , Ligação Proteica , Coelhos , Receptores Opioides , Vasoconstrição/efeitos dos fármacosRESUMO
Cardiovascular outcome in Marfan syndrome (MFS) patients most prominently depends on aortic aneurysm progression with subsequent aortic dissection. Angiotensin II receptor blockers (ARBs) prevent aneurysm formation in MFS mouse models. In patients, ARBs only slow down aortic dilation. Downstream signalling from the angiotensin II type 1 receptor (AT1R) is mediated by G proteins and ß-arrestin recruitment. AT1R also interacts with the monocyte chemoattractant protein-1 (MCP-1) receptor, resulting in inflammation. In this study, we explore the targeting of ß-arrestin signalling in MFS mice by administering TRV027. Furthermore, because high doses of the ARB losartan, which has been proven beneficial in MFS, cannot be achieved in humans, we investigate a potential additive effect by combining lower concentrations of losartan (25 mg/kg/day and 5 mg/kg/day) with barbadin, a ß-arrestin blocker, and DMX20, a C-C chemokine receptor type 2 (CCR2) blocker. A high dose of losartan (50 mg/kg/day) slowed down aneurysm progression compared to untreated MFS mice (1.73 ± 0.12 vs. 1.96 ± 0.08 mm, p = 0.0033). TRV027, the combination of barbadin with losartan (25 mg/kg/day), and DMX-200 (90 mg/kg/day) with a low dose of losartan (5 mg/kg/day) did not show a significant beneficial effect. Our results confirm that while losartan effectively halts aneurysm formation in Fbn1C1041G/+ MFS mice, neither TRV027 alone nor any of the other compounds combined with lower doses of losartan demonstrate a notable impact on aneurysm advancement. It appears that complete blockade of AT1R function, achieved by administrating a high dosage of losartan, may be necessary for inhibiting aneurysm progression in MFS.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II , Aneurisma Aórtico , Losartan , Síndrome de Marfan , Transdução de Sinais , Camundongos , Síndrome de Marfan/tratamento farmacológico , Síndrome de Marfan/patologia , Modelos Animais de Doenças , Aneurisma Aórtico/tratamento farmacológico , Aneurisma Aórtico/prevenção & controle , Oligopeptídeos/administração & dosagem , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/patologia , Pirimidinas/administração & dosagem , Combinação de Medicamentos , Losartan/administração & dosagem , Antagonistas de Receptores de Angiotensina/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Humanos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/administração & dosagemRESUMO
HLA donor-specific antibodies (DSAs) pre and post transplant increase the risk of antibody-mediated rejection (AMR) and lead to poor graft survival. Increasing data exist to support the involvement of non-HLA antibodies in triggering an immunological response. The development of non-HLA antibodies specific for AT1R is associated with poor clinical outcomes in orthotopic heart transplant recipients. This case presents an investigation of non-HLA antibodies in a 56-year-old female heart transplant recipient diagnosed with AMR in the absence of DSAs.
Assuntos
Transplante de Coração , Transplante de Rim , Feminino , Humanos , Pessoa de Meia-Idade , Autoanticorpos , Antígenos HLA , Rejeição de Enxerto , Transplante de Coração/efeitos adversosRESUMO
Medical procedures, such as radiation therapy, are a vital element in treating many cancers, significantly contributing to improved survival rates. However, a common long-term complication of such exposure is radiation-induced skin fibrosis (RISF), a complex condition that poses substantial physical and psychological challenges. Notably, about 50% of patients undergoing radiation therapy may achieve long-term remission, resulting in a significant number of survivors managing the aftereffects of their treatment. This article delves into the intricate relationship between RISF, reactive oxygen species (ROS), and angiotensin II (Ang II) signaling. It proposes the underlying mechanisms and examines potential treatments for mitigating skin fibrosis. The primary goal is to offer essential insights in order to better care for and improve the quality of life of cancer survivors who face the risk of developing RISF.
Assuntos
Angiotensina II , Fibrose , Espécies Reativas de Oxigênio , Pele , Humanos , Angiotensina II/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pele/efeitos da radiação , Pele/patologia , Animais , Lesões por Radiação/etiologia , Radioterapia/efeitos adversos , Transdução de SinaisRESUMO
The success of Angiotensin II receptor blockers, specifically Angiotensin II type 1 receptor (AT1R) antagonists as antihypertensive drug emphasizes the involvement of AT1R in Essential hypertension. The structural insights and mutational studies of Ang II-AT1R have brought about the vision to design Ang II analogs that selectively activate the pathways with beneficial and cardioprotective effects such as cell survival and hinder the deleterious effects such as hypertrophy and cell death. AT1R belongs to G-protein coupled receptors and is regulated by G-protein coupled receptor kinases (GRKs) that either uncouples Gq protein for receptor desensitization or phosphorylate C-terminus to recruit ß-arrestin for internalization of the receptor. The interaction of GRKs with ligand activated AT1R induces conformational changes and signal either Gq dependent or Gq independent pathways. These interactions might explain the complex regulatory mechanisms and offer promising ideas for hypertension therapeutics. This article reviews the functional role of AT1R, organization of GRK genes and regulation of AT1R by GRKs that play significant role in desensitization and internalization of the receptors.
Assuntos
Hipertensão , Receptor Tipo 1 de Angiotensina , Humanos , beta-Arrestinas/metabolismo , beta-Arrestinas/farmacologia , Pressão Sanguínea , Hipertensão/genética , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Doença da Artéria Coronariana , Hipertensão , Receptor Tipo 1 de Angiotensina , Idoso , Humanos , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/genética , Genótipo , Hipertensão/diagnóstico , Hipertensão/epidemiologia , Hipertensão/genética , Polimorfismo Genético , Receptor Tipo 1 de Angiotensina/genética , Fatores de RiscoRESUMO
The aetiological mechanism of preeclampsia (PE) is unclear exactly, so we attempted to investigate the association between susceptibility to preeclampsia and renin-angiotensin-aldosterone system (RAAS) gene polymorphisms to explore the aetiology in terms of genetics. A systematic search was performed in electronic databases to identify relevant studies. Eventually 73 studies were enrolled, odds ratios were generated by 5 genetic models. In overall analysis, significant associations were detected for AGT M235T, AT1R A1166C and CYP11B2 C344T whereas negative correlation was shown for AGT T174M. As stratified by race and geography, AGT 235T allele and AT1R 1166C allele increased preeclampsia risk and AGT T174M was justified uncorrelated with preeclampsia. Our meta-analysis illustrated that AGT 235T allele and AT1R 1166C allele increased and CYP11B2 344T allele decreased preeclampsia risk while AGT T174M polymorphism did not change preeclampsia risk. Hence, pregnant women carrying high-risk genotypes need strengthened management to prevent and early identification of preeclampsia.
Assuntos
Pré-Eclâmpsia , Sistema Renina-Angiotensina , Feminino , Humanos , Gravidez , Sistema Renina-Angiotensina/genética , Pré-Eclâmpsia/genética , Citocromo P-450 CYP11B2/genética , Angiotensinogênio/genética , Polimorfismo Genético , Genótipo , Predisposição Genética para DoençaRESUMO
Animal models and clinical studies suggest an influence of angiotensin II (AngII) on the pathogenesis of liver diseases via the renin-angiotensin system. AngII application increases portal blood pressure, reduces bile flow, and increases permeability of liver tight junctions. Establishing the subcellular localization of angiotensin II receptor type 1 (AT1R), the main AngII receptor, helps to understand the effects of AngII on the liver. We localized AT1R in situ in human and porcine liver and porcine gallbladder by immunohistochemistry. In order to do so, we characterized commercial anti-AT1R antibodies regarding their capability to recognize heterologous human AT1R in immunocytochemistry and on western blots, and to detect AT1R using overlap studies and AT1R-specific blocking peptides. In hepatocytes and canals of Hering, AT1R displayed a tram-track-like distribution, while in cholangiocytes AT1R appeared in a honeycomb-like pattern; i.e., in liver epithelia, AT1R showed an equivalent distribution to that in the apical junctional network, which seals bile canaliculi and bile ducts along the blood-bile barrier. In intrahepatic blood vessels, AT1R was most prominent in the tunica media. We confirmed AT1R localization in situ to the plasma membrane domain, particularly between tight and adherens junctions in both human and porcine hepatocytes, cholangiocytes, and gallbladder epithelial cells using different anti-AT1R antibodies. Localization of AT1R at the junctional complex could explain previously reported AngII effects and predestines AT1R as a transmitter of tight junction permeability.
Assuntos
Bile , Receptor Tipo 1 de Angiotensina , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Bile/metabolismo , Western Blotting , Humanos , Peptídeos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina , SuínosRESUMO
Hypertension is controlled via the alteration of microRNAs (miRNAs), their therapeutic targets angiotensin II type I receptor (AT1R) and cross talk of signaling pathways. The stimulation of the Ang II/AT1R pathway by deregulation of miRNAs, has also been linked to cardiac remodeling as well as the pathophysiology of high blood pressure. As miRNAs have been associated to ACE2/Apelin and Mitogen-activated protein kinases (MAPK) signaling, it has revealed an utmost protective impact over hypertension and cardiovascular system. The ACE2-coupled intermodulation between RAAS, Apelin system, MAPK signaling pathways, and miRNAs reveal the practicalities of high blood pressure. The research of miRNAs may ultimately lead to the expansion of an innovative treatment strategy for hypertension, which indicates the need to explore them further at the molecular level. Therefore, here we have focused on the mechanistic importance of miRNAs in hypertension, ACE2/Apelin signaling as well as their biological functions, with a focus on interplay and crosstalk between ACE2/Apelin signaling, miRNAs, and hypertension, and the progress in miRNA-based diagnostic techniques with the goal of facilitating the development of new hypertension-controlling therapeutics.
Assuntos
Hipertensão , MicroRNAs , Enzima de Conversão de Angiotensina 2 , Apelina , Humanos , Hipertensão/genética , MicroRNAs/genética , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Transdução de SinaisRESUMO
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 â¼ 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.
Assuntos
Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina , Angiotensina II/química , Angiotensina II/farmacologia , Animais , Humanos , Imidazóis , Ligantes , Camundongos , Ratos , Receptor Tipo 2 de Angiotensina/agonistas , Sulfonamidas , TiofenosRESUMO
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.
Assuntos
Benzimidazóis , Receptor Tipo 2 de Angiotensina , Benzimidazóis/farmacologia , Humanos , Imidazóis , Ligantes , Receptor Tipo 2 de Angiotensina/agonistas , Sulfonamidas , Tiazóis , TiofenosRESUMO
Pulmonary arterial hypertension (PAH) is a progressive disease with a complex aetiology and high mortality. Functional and structural changes in the small pulmonary arteries lead to elevated pulmonary arterial pressure, resulting in right heart failure. The pathobiology of PAH is not fully understood, and novel treatment targets in PAH are desperately needed. The renin-angiotensin system is critical for maintaining homeostasis of the cardiovascular system. The system consists of the angiotensin converting enzyme (ACE)-angiotensin (Ang) II-angiotensin type 1 receptor (AT1R) axis and the ACE2-Ang-(1-7)-Mas receptor axis. The former, the ACE-Ang II-AT1R axis, is involved in vasoconstrictive and hypertensive actions along with cardiac and vascular remodelling. The latter, the ACE2-Ang-(1-7)-Mas axis, generally mediates counterbalancing effects against those mediated by the ACE-Ang II-AT1R axis. Based on established functions, the ACE2-Ang-(1-7)-Mas axis may represent a novel target for the treatment of PAH. This review focuses on recent advances in pulmonary circulation science and the role of the ACE2-Ang-(1-7)-Mas axis in PAH.
Assuntos
Peptidil Dipeptidase A , Hipertensão Arterial Pulmonar , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2 , Humanos , Fragmentos de Peptídeos , Peptidil Dipeptidase A/metabolismo , Hipertensão Arterial Pulmonar/tratamento farmacológico , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sistema Renina-AngiotensinaRESUMO
Progress in the study of Covid-19 disease in rodents has been hampered by the lack of angiotensin-converting enzyme 2 (ACE2; virus entry route to the target cell) affinities for the virus spike proteins across species. Therefore, we sought to determine whether a modified protocol of lipopolysaccharide (LPS)-induced acute respiratory distress syndrome in rats can mimic both cell signalling pathways as well as severe disease phenotypes of Covid-19 disease. Rats were injected via intratracheal (IT) instillation with either 15 mg/kg of LPS (model group) or saline (control group) before being killed after 3 days. A severe acute respiratory syndrome (SARS)-like effect was observed in the model group as demonstrated by the development of a "cytokine storm" (>2.7 fold increase in blood levels of IL-6, IL-17A, GM-CSF, and TNF-α), high blood ferritin, demonstrable coagulopathy, including elevated D-dimer (approximately 10-fold increase), PAI-1, PT, and APTT (p < 0.0001). In addition, LPS increased the expression of lung angiotensin II type I receptor (AT1R)-JAK-STAT axis (>4 fold increase). Chest imaging revealed bilateral small patchy opacities of the lungs. Severe lung injury was noted by the presence of both, alveolar collapse and haemorrhage, desquamation of epithelial cells in the airway lumen, infiltration of inflammatory cells (CD45+ leukocytes), widespread thickening of the interalveolar septa, and ultrastructural alterations similar to Covid-19. Thus, these findings demonstrate that IT injection of 15 mg/kg LPS into rats, induced an AT1R/JAK/STAT-mediated cytokine storm with resultant pneumonia and coagulopathy that was commensurate with moderate and severe Covid-19 disease noted in humans.
Assuntos
Lesão Pulmonar Aguda/etiologia , Transtornos da Coagulação Sanguínea/etiologia , COVID-19/patologia , Síndrome da Liberação de Citocina/etiologia , Hemorragia/etiologia , Lipopolissacarídeos/efeitos adversos , Pneumopatias/etiologia , Receptor Tipo 1 de Angiotensina/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Lesão Pulmonar Aguda/patologia , Animais , Transtornos da Coagulação Sanguínea/patologia , COVID-19/etiologia , Síndrome da Liberação de Citocina/patologia , Modelos Animais de Doenças , Hemorragia/patologia , Janus Quinases , Pneumopatias/patologia , Masculino , Ratos , Ratos WistarRESUMO
Angiotensin II receptor type 1 (AT1R) and endothelin-1 receptor type A (ETAR) are G-protein-coupled receptors (GPCRs) expressed on the surface of a great variety of cells: immune cells, vascular smooth cells, endothelial cells, and fibroblasts express ETAR and AT1R, which are activated by endothelin 1 (ET1) and angiotensin II (AngII), respectively. Certain autoantibodies are specific for these receptors and can regulate their function, thus being known as functional autoantibodies. The function of these antibodies is similar to that of natural ligands, and it involves not only vasoconstriction, but also the secretion of proinflammatory cytokines (such as interleukin-6 (IL6), IL8 and TNF-α), collagen production by fibroblasts, and reactive oxygen species (ROS) release by fibroblasts and neutrophils. The role of autoantibodies against AT1R and ETAR (AT1R-AAs and ETAR-AAs, respectively) is well described in the pathogenesis of many medical conditions (e.g., systemic sclerosis (SSc) and SSc-associated pulmonary hypertension, cystic fibrosis, and allograft dysfunction), but their implications in cardiovascular diseases are still unclear. This review summarizes the current evidence regarding the effects of AT1R-AAs and ETAR-AAs in cardiovascular pathologies, highlighting their roles in heart transplantation and mechanical circulatory support, preeclampsia, and acute coronary syndromes.