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BACKGROUND: Circulating extracellular vesicles (EVs) carry protected cargoes of nucleic acids, proteins, and metabolites. Here we identified and validated the surface proteins and enzymatic activity of chymase, angiotensin converting enzymes 1 (ACE) and 2 (ACE2), and neprilysin (NEP) in EVs isolated from the blood and urine of primary hypertensive patients. METHODS: Peripheral venous blood and spot urine from 34 hypertensive patients were processed to isolate plasma and urinary EVs. Immuno-gold labeling and transmission electron microscopy validated the presence of the exosomal marker protein CD63 on the surface of plasma and urinary EVs. Flow cytometry characterized plasma and urinary EVs for CD63, CD9, and CD81 surface markers. In addition, exosomal CD63, TSG101, and Alix were analyzed in urine by Western blotting. Urinary EVs did not express the endoplasmic reticulum protein calnexin and Golgi protein GM130. Chymase, ACE, ACE2, and NEP activities on 125I substrates â angiotensin-(1-12) [Ang-(1â12)] and angiotensin II (Ang II) â [1 nmol/L each] were quantified by HPLC. Data were analyzed based on whether the patient's blood pressure was controlled (Group I: <140/80 mm Hg) or not controlled (Group II: ≥ 140/80 mm Hg). RESULTS: Chymase activity on Ang-(1â12) was significantly higher in plasma and urinary EVs than in ACE, ACE2, and NEP. In addition, chymase activity in urine EVs was more than 3-fold higher than in plasma EVs. Chymase activity increased in plasma and urine EVs retrieved from Group II patients. No comparable differences were found in the enzymatic activities of ACE, ACE2, and NEP urinary EVs between Group I and Group II. CONCLUSION: These studies reveal a differential enzymatic activity of renin angiotensin system enzymes in plasma and urine EVs isolated from hypertensive patients. Demonstrating a comparatively high chymase enzymatic activity in EVs expands a previously documented finding of increased plasma Ang-(1â12) in hypertensive patients.
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Angiotensina II , Hipotensão , Humanos , Hipotensão/tratamento farmacológico , Angiotensina II/administração & dosagem , Relação Dose-Resposta a Droga , Vasoconstritores/administração & dosagem , Vasoconstritores/uso terapêutico , Procedimentos Cirúrgicos Operatórios/efeitos adversos , Masculino , FemininoRESUMO
The renin-angiotensin system is the most important peptide hormone system in the regulation of cardiovascular homeostasis. Its classical arm consists of the enzymes, renin, and angiotensin-converting enzyme, generating angiotensin II from angiotensinogen, which activates its AT1 receptor, thereby increasing blood pressure, retaining salt and water, and inducing cardiovascular hypertrophy and fibrosis. However, angiotensin II can also activate a second receptor, the AT2 receptor. Moreover, the removal of the C-terminal phenylalanine from angiotensin II by ACE2 (angiotensin-converting enzyme 2) yields angiotensin-(1-7), and this peptide interacts with its receptor Mas. When the aminoterminal Asp of angiotensin-(1-7) is decarboxylated, alamandine is generated, which activates the Mas-related G-protein-coupled receptor D, MrgD (Mas-related G-protein-coupled receptor type D). Since Mas, MrgD, and the AT2 receptor have opposing effects to the classical AT1 receptor, they and the enzymes and peptides activating them are called the alternative or protective arm of the renin-angiotensin system. This review will cover the historical aspects and the current standing of this recent addition to the biology of the renin-angiotensin system.
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Angiotensina II , Sistema Renina-Angiotensina , Angiotensina I/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos , Peptidil Dipeptidase A/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Renina , Sistema Renina-Angiotensina/fisiologia , HumanosRESUMO
BACKGROUND: Extracellular vesicles (EVs) have emerged as a promising liquid biopsy for various diseases. For the first time, using plasma and urinary EVs, we assessed the activity of renin-angiotensin system (RAS), a central regulator of renal, cardiac, and vascular physiology, in patients with control (Group I) or uncontrolled (Group II) primary hypertension. METHODS: EVs were isolated from 34 patients with history of hypertension, and characterized for size and concentration by nanoparticle tracking analyses, exosomal biomarkers by immunogold labeling coupled with transmission electron microscopy, flow cytometry and immunoblotting. EVs were analyzed for the hydrolytic activity of chymase, angiotensin converting enzyme (ACE), ACE2, and neprilysin (NEP) by HPLC. RESULTS: Plasma and urinary EVs were enriched for small EVs and expressed exosomal markers (CD63, CD9, and CD81). The size of urinary EVs (but not plasma EVs) was significantly larger in Group II compared to Group I. Differential activity of RAS enzymes was observed, with significantly higher chymase activity compared to ACE, ACE2, and NEP in plasma EVs. Similarly, urinary EVs exhibited higher chymase and NEP activity compared to ACE and ACE2 activity. Importantly, compared to Group I, significantly higher chymase activity was observed in urinary EVs (p = 0.03) from Group II, while no significant difference in activity was observed for other RAS enzymes. CONCLUSIONS: Bioactive RAS enzymes are present in plasma and urinary EVs. Detecting chymase in plasma and urinary EVs uncovers a novel mechanism of angiotensin II-forming enzyme and could also mediate cell-cell communication and modulate signaling pathways in recipient cells.
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INTRODUCTION: Non-angiotensin converting enzyme mechanisms of angiotensin II production remain underappreciated in part due to the success of current therapies to ameliorate the impact of primary hypertension and atherosclerotic diseases of the heart and the blood vessels. This review scrutinize the current literature to highlight chymase role as a critical participant in the pathogenesis of cardiovascular disease and heart failure. AREAS COVERED: We review the contemporaneous understanding of circulating and tissue biotransformation mechanisms of the angiotensins focusing on the role of chymase as an alternate tissue generating pathway for angiotensin II pathological mechanisms of action. EXPERT OPINION: While robust literature documents the singularity of chymase as an angiotensin II-forming enzyme, particularly when angiotensin converting enzyme is inhibited, this knowledge has not been fully recognized to clinical medicine. This review discusses the limitations of clinical trials' that explored the benefits of chymase inhibition in accounting for the failure to duplicate in humans what has been demonstrated in experimental animals.
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Doenças Cardiovasculares , Insuficiência Cardíaca , Animais , Humanos , Quimases/metabolismo , Quimases/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Angiotensina II/metabolismo , Angiotensina II/uso terapêuticoRESUMO
Aims: Upregulation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) contributes to the pathogenesis of cardiovascular disease, including hypertension. Transgenic rats expressing the human angiotensinogen gene [TGR (hAGT)L1623] are a new novel humanized model of hypertension that associates with declines in cardiac contractile function and ß-adrenergic receptor (AR) reserve. The molecular mechanisms are unclear. We tested the hypothesis that in TGR (hAGT)L1623 rats, left ventricular (LV) myocyte CaMKIIδ and ß3-AR are upregulated, but ß1-AR is down-regulated, which are important causes of cardiac dysfunction and ß-AR desensitization. Main methods: We compared LV myocyte CaMKIIδ, CaMKIIδ phosphorylation (at Thr287) (pCaMKIIδ), and ß1-and ß3-AR expressions and determined myocyte functional and [Ca2+]I transient ([Ca2+]iT) responses to ß-AR stimulation with and without pretreatment of myocytes using an inhibitor of CaMKII, KN-93 (10-6 M, 30 min) in male Sprague Dawley (SD; N = 10) control and TGR (hAGT)L1623 (N = 10) adult rats. Key findings: Hypertension in TGR (hAGT)L1623 rats was accompanied by significantly increased LV myocyte ß3-AR protein levels and reduced ß1-AR protein levels. CaMKIIδ phosphorylation (at Thr287), pCaMKIIδ was significantly increased by 35%. These changes were followed by significantly reduced basal cell contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca2+]iT. Isoproterenol (10-8 M) produced significantly smaller increases in dL/dtmax, dR/dtmax, and [Ca2+]iT. Moreover, only in TGR (hAGT)L1623 rats, pretreatment of LV myocytes with KN-93 (10-6 M, 30 min) fully restored normal basal and isoproterenol-stimulated myocyte contraction, relaxation, and [Ca2+]iT. Significance: LV myocyte CaMKIIδ overactivation with associated contrast changes in ß3-AR and ß1-AR may be the key molecular mechanism for the abnormal contractile phenotype and ß-AR desensitization in this humanized model of hypertension.
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Introduction: Chymase is a highly destructive serine protease rapidly neutralized in the circulation by protease inhibitors. Here we test whether pericardial fluid (PCF) chymase activation and other inflammatory biomarkers determine intensive care unit length of stay, and explore mechanisms of chymase delivery by extracellular vesicles to the heart. Methods: PCF was collected from adult patients (17 on-pump; 13 off-pump) 4â h after cardiac surgery. Extracellular vesicles (EVs) containing chymase were injected into Sprague-Dawley rats to test for their ability to deliver chymase to the heart. Results: The mean intensive care unit (ICU) stay and mean total length of stay was 2.17 ± 3.8 days and 6.41 ± 1.3 days respectively. Chymase activity and 32 inflammatory markers did not differ in on-pump vs. off-pump cardiac surgery. Society of Thoracic Surgeons Predicted Risk of Morbidity and Mortality Score (STS-PROM), 4-hour post-surgery PCF chymase activity and C-X-C motif chemokine ligand 6 (CXCL6) were all independent predictors of ICU and total hospital length of stay by univariate analysis. Mass spectrometry of baseline PCF shows the presence of serine protease inhibitors that neutralize chymase activity. The compartmentalization of chymase within and on the surface of PCF EVs was visualized by immunogold labeling and transmission electron microscopy. A chymase inhibitor prevented EV chymase activity (0.28â fmol/mg/min vs. 14.14â fmol/mg/min). Intravenous injection of PCF EVs obtained 24â h after surgery into Sprague Dawley rats shows diffuse human chymase uptake in the heart with extensive cardiomyocyte damage 4â h after injection. Discussion: Early postoperative PCF chymase activation underscores its potential role in cardiac damage soon after on- or off-pump cardiac surgery. In addition, chymase in extracellular vesicles provides a protected delivery mechanism from neutralization by circulating serine protease inhibitors.
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The natriuretic peptide system (NPS) and renin-angiotensin-aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date supports this notion. This study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro. Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in vivo to determine the influence of ANGII on ANP actions. The underlying mechanisms were further explored via in vitro approaches. In humans, ANGII demonstrated an inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and the interaction term between ANGII and natriuretic peptides increased the predictive accuracy of the base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed a positive association between cGMP and ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at a physiological dose attenuated cGMP generation mediated by ANP infusion. In vitro, we found the suppressive effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), as this suppression can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using surface plasmon resonance (SPR), we showed ANGII has low binding affinity to the guanylyl cyclase A (GC-A) receptor compared to ANP or BNP. Our study reveals ANGII is a natural suppressor for the cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights the importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular protection.
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Angiotensina II , Guanilato Ciclase , Humanos , Ratos , Animais , Guanilato Ciclase/metabolismo , Angiotensina II/farmacologia , Fator Natriurético Atrial/farmacologia , Fator Natriurético Atrial/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Peptídeo Natriurético Encefálico , GMP Cíclico/metabolismo , Peptídeos NatriuréticosRESUMO
Obesity is a major risk factor for multiple chronic diseases. Anthropometric and imaging approaches are primarily used to assess adiposity, and there is a dearth of techniques to determine the changes in adipose tissue (AT) at the molecular level. Extracellular vesicles (EVs) have emerged as a novel and less invasive source of biomarkers for various pathologies. Furthermore, the possibility of enriching cell or tissue-specific EVs from the biofluids based on their unique surface markers has led to classifying these vesicles as "liquid biopsies", offering valuable molecular information on hard-to-access tissues. Here, we isolated small EVs from AT (sEVAT) of lean and diet-induced obese (DIO) mice, identified unique surface proteins on sEVAT by surface shaving followed by mass spectrometry, and developed a signature of five unique proteins. Using this signature, we pulled out sEVAT from the blood of mice and validated the specificity of isolated sEVAT by measuring the expression of adiponectin, 38 adipokines on an array, and several adipose tissue-related miRNAs. Furthermore, we provided evidence of sEV applicability in disease prediction by characterizing sEVAT from the blood of lean and DIO mice. Interestingly, sEVAT-DIO cargo showed a stronger pro-inflammatory effect on THP1 monocytes compared to sEVAT-Lean and a significant increase in obesity-associated miRNA expression. Equally important, sEVAT cargo revealed an obesity-associated aberrant amino acid metabolism that was subsequently validated in the corresponding AT. Lastly, we show a significant increase in inflammation-related molecules in sEVAT isolated from the blood of nondiabetic obese (>30 kg/m2) individuals. Overall, the present study offers a less-invasive approach to characterize AT.
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Tecido Adiposo , Vesículas Extracelulares , Tecido Adiposo/química , Biópsia Líquida , Vesículas Extracelulares/química , Obesidade , Humanos , Animais , Camundongos , BiomarcadoresRESUMO
Purpose: Angiotensin-(1-12) [Ang-(1-12)] serves as a primary substrate to generate angiotensin II (Ang II) by angiotensin-converting enzyme and/or chymase suggests it may be an unrecognized source of Ang II-mediated microvascular complication in hypertension-mediated retinopathy. We investigated Ang-(1-12) expression and internalization in adult retinal pigment epithelial-19 (ARPE-19) cultured cells. We performed the internalization of Ang-(1-12) in ARPE-19 cells in the presence of a highly specific monoclonal antibody (mAb) developed against the C-terminal end of the Ang-(1-12) sequence. Methods: All experiments were performed in confluent ARPE-19 cells (passage 28-35). We employed high-performance liquid chromatography to purify radiolabeled, 125I-Ang-(1-12) and immuno-neutralization with Ang-(1-12) mAb to demonstrate Ang-(1-12)'s internalization in ARPE-19 cells. Internalization was also demonstrated by immunofluorescence (IF) method. Results: These procedures revealed internalization of an intact 125I-Ang-(1-12) in ARPE-19 cells. A significant reduction (â¼53%, P < 0.0001) in 125I-Ang-(1-12) internalization was detected in APRE-19 cells in the presence of the mAb. IF staining experiments further confirms internalization of Ang-(1-12) into the cells from the extracellular culture medium. No endogenous expression was detected in the ARPE-19 cells. An increased intensity of IF staining was detected in cells exposed to 1.0 µM Ang-(1-12) compared with 0.1 µM. Furthermore, we found hydrolysis of Ang-(1-12) into Ang II by ARPE-19 cells' plasma membranes. Conclusions: Intact Ang-(1-12) peptide is internalized from the extracellular spaces in ARPE-19 cells and metabolized into Ang II. The finding that a selective mAb blocks cellular internalization of Ang-(1-12) suggests alternate therapeutic approaches to prevent/reduce the RPE cells Ang II burden.
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Angiotensina II , Radioisótopos do Iodo , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Pigmentos da Retina , Células CultivadasRESUMO
Background: Natriuretic peptide system (NPS) and renin angiotensin aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date support this notion. Objectives: This study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro for translational insights. Methods: Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in rat model to determine influence of ANGII on ANP actions. Multiple engineered HEK293 cells and surface plasmon resonance (SPR) technology were leveraged for mechanistic exploration. Results: In humans, ANGII showed inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and interaction term between ANGII and natriuretic peptide increased predicting accuracy of base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed positive association between cGMP with ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at physiological dose attenuated blood pressure reduction and cGMP generation triggered by ANP infusion. In vitro, we showed that the suppression effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), which can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using SPR, we showed ANGII has low affinity for particulate guanylyl cyclase A (GC-A) receptor binding compared to ANP or BNP. Conclusions: Our study reveals ANGII as a natural suppressor for cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular disease.
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PURPOSE OF REVIEW: To address contemporary hypertension challenges, a critical reexamination of therapeutic accomplishments using angiotensin converting enzyme inhibitors and angiotensin II receptor blockers, and a greater appreciation of evidence-based shortcomings from randomized clinical trials are fundamental in accelerating future progress. RECENT FINDINGS: Medications targeting angiotensin II mechanism of action are essential for managing primary hypertension, type 2 diabetes, heart failure, and chronic kidney disease. While the ability of angiotensin converting enzyme inhibitors and angiotensin II receptor blockers to control blood pressure is undisputed, practitioners, hypertension specialists, and researchers hold low awareness of these drugs' limitations in preventing or reducing the risk of cardiovascular events. Biases in interpreting gained knowledge from data obtained in randomized clinical trials include a pervasive emphasis on using relative risk reduction over absolute risk reduction. Furthermore, recommendations for clinical practice in international hypertension guidelines fail to address the significance of a residual risk several orders of magnitude greater than the benefits. We analyze the limitations of the clinical trials that have led to current recommended treatment guidelines. We define and quantify the magnitude of the residual risk in published hypertension trials and explore how activation of alternate compensatory bioprocessing components within the renin angiotensin system bypass the ability of angiotensin converting enzyme inhibitors and angiotensin II receptor blockers to achieve a significant reduction in total and cardiovascular deaths. We complete this presentation by outlining the current incipient but promising potential of immunotherapy to block angiotensin II pathology alone or possibly in combination with other antihypertensive drugs. A full appreciation of the magnitude of the residual risk associated with current renin angiotensin system-based therapies constitutes a vital underpinning for seeking new molecular approaches to halt or even reverse the cardiovascular complications of primary hypertension and encourage investigating a new generation of ACE inhibitors and ARBs with increased capacity to reach the intracellular compartments at which Ang II can be generated.
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Diabetes Mellitus Tipo 2 , Hipertensão , Humanos , Sistema Renina-Angiotensina/fisiologia , Antagonistas de Receptores de Angiotensina/uso terapêutico , Antagonistas de Receptores de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Angiotensina II/farmacologia , Diabetes Mellitus Tipo 2/complicações , Anti-Hipertensivos/uso terapêutico , Anti-Hipertensivos/farmacologia , ReninaRESUMO
A large body of evidence implicates the renin-angiotensin system in the pathogenesis of cardiovascular disease. However, not everyone understands that the magnitude of the risk reduction achieved in clinical trials with angiotensin-converting enzyme inhibitors and angiotensin receptor blockers is only a fraction of the residual risk for cardiovascular events and death. This paper addresses limitations of current therapeutic approaches based on renin-angiotensin system blockade for hypertension and cardiovascular disease by illustrating the complex biochemical physiology and mechanism of classical and alternate angiotensin peptide formation. Emerging evidence of alternate mechanisms that bypass both renin and angiotensin-converting enzyme to produce the angiotensins in tissues and cells is not currently universally recognized. Currently available treatment would benefit from further insights to help fully meet the aims of patient care, and the challenge is to delve more deeply into the renin-angiotensin system cascade, with the aim of enhancing therapeutics for renin-angiotensin system inhibition. This article provides a reappraisal of the renin-angiotensin-aldosterone cascade, highlighting newly elucidated intermediary components and interplay, and their consequent implications and relevance for understanding the long-term contribution of angiotensin II in cardiovascular diseases and their therapy.
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We engineered a monoclonal antibody (mAb) against the human C-terminus of angiotensin-(1-12) [h-Ang-(1-12)] and performed a biochemical characterization in concert with direct in vivo and ex vivo (carotid artery strips) assessments of h-Ang-(1-12) vasoconstrictor activity in 78 (36 females) transgenic rats expressing the human angiotensinogen gene [TGR(hAGT)L1623] and 26 (10 female) Sprague Dawley (SD) controls. The mAb shows high specificity in neutralizing angiotensin II formation from h-Ang-(1-12) and did not cross-react with human and rat angiotensins. Changes in arterial pressure and heart rate in Inactin® hydrate anesthetized rats were measured before and after h-Ang-(1-12) injections [dose range: 75-300 pmol/kg i.v.] prior to and 30-60 minutes after administration of the h-Ang-(1-12) mAb. Neutralization of circulating Ang-(1-12) inhibited the pressor action of h-Ang-(1-12), prevented Ang-(1-12) constrictor responses in carotid artery rings in both SD and TGR(hAGT)L1623 rats, and caused a fall in the arterial pressure of male and female transgenic rats. The Ang-(1-12) mAb did not affect the response of comparable dose-related pressor responses to Ang II, pre-immune IgG, or the rat sequence of Ang-(1-12). This h-Ang-(1-12) mAb can effectively suppress the pressor actions of the substrate in the circulation of hypertensive rats or in carotid artery strips from both SD and transgenic rats. The demonstration that this Ang-(1-12) mAb by itself, induced a fall in arterial pressure in transgenic hypertensive rats supports further exploring the potential abilities of Ang-(1-12) mAb in the treatment of hypertension.
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Angiotensinogênio , Hipertensão , Angiotensina I/farmacologia , Angiotensina II/farmacologia , Angiotensinogênio/genética , Angiotensinogênio/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Pressão Sanguínea , Feminino , Humanos , Masculino , Fragmentos de Peptídeos/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
AIMS: G protein-coupled estrogen receptor 30 (GPR30) activation by its agonist, G1, exhibits beneficial actions in female with heart failure (HF). Recent evidence indicates its cardiovascular benefits may also include male as well. However, whether and how GPR30 activation may limit HF progression and have a salutary role in males is unknown. We hypothesized that chronic G1 treatment improves LV and cardiomyocyte function, [Ca2+]i regulation and ß-adrenergic reserve, thus limiting HF progression in male. MAIN METHODS: We compared left ventricle (LV) and myocyte function, [Ca2+]i transient ([Ca2+]iT) and ß-AR modulation in control male mice (12/group) and isoproterenol-induced HF (150 mg/kg s.c. for 2 days). Two weeks after isoproterenol injection, HF mice received placebo, or G1 (150 µg/kg/day s.c. mini-pump) for 2 weeks. KEY FINDINGS: Isoproterenol-treated mice exhibited HF with preserved ejection fraction (HFpEF) at 2-weeks and progressed to HF with reduced EF (HFrEF) at 4-weeks, manifested by significantly increased LV time constant of relaxation (τ), decreased EF and mitral flow (dV/dtmax), which were accompanied by reduced myocyte contraction (dL/dtmax), relaxation (dR/dtmax) and [Ca2+]iT. Acute isoproterenol-superfusion caused significantly smaller increases in dL/dtmax, dR/dtmax and [Ca2+]iT. G1 treatment in HF increased basal and isoproterenol-stimulated increases in EF and LV contractility of EES. Importantly, G1 improved basal and isoproterenol-stimulated dL/dtmax, dR/dtmax and [Ca2+]iT to control levels and restored normal cardiac ß-AR subtypes modulation. SIGNIFICANCE: Chronic G1 treatment restores normal myocyte basal and ß-AR-stimulated contraction, relaxation, and [Ca2+]iT, thereby reversing LV dysfunction and playing a rescue role in a male mouse model of HF.
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Insuficiência Cardíaca/tratamento farmacológico , Ventrículos do Coração/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Quinolinas/uso terapêutico , Animais , Modelos Animais de Doenças , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/fisiopatologia , Isoproterenol , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Quinolinas/farmacologiaRESUMO
G protein-coupled estrogen receptor (GPER) activation by G1 attenuates diastolic dysfunction from estrogen loss, which may be partly due to suppression of angiotensin II pathological actions. We aimed to determine the independent effects of 8 weeks of G1 (100 µg/kg/d, subcutaneous pellet), ACE-inhibition (ACEi; lisinopril 10 mg/kg, drinking water), or combination therapy versus vehicle in the ovariectomized (OVX) spontaneously hypertensive rat (SHR) on cardiac function and morphometrics (echocardiography), serum equilibrium of angiotensins (mass spectroscopy) and cardiac components of the RAS (Western blotting). G1 alone and when combined with ACEi enhanced myocardial relaxation (é: 30 and 17%) and diastolic wall strain (DWS: 76 and 68%) while reducing relative wall thickness (RWT: 20 and 33%) and filling pressures (E/é: 30 and 37%). Cardiac expression levels of Mas receptor (Mas-R) and ACE2 also increased in the presence of G1. Strong antihypertensive effects of lisinopril monotherapy were associated with reductions in RWT, collagen deposition and E/é without overtly altering é or DWS. Chronic ACEi also increased cardiac levels of Mas-R and AT1-R and tilted the circulating RAS toward the formation of Ang-(1-7), which was amplified in the presence of G1. In vitro studies further revealed that an inhibitor to prolyl endopeptidase (PEP), but not to neprilysin, significantly reduced serum Ang-(1-7) levels in G1-treated rats, suggesting that G1 might be increasing Ang-(1-7) formation via PEP. We conclude that activating GPER with G1 augments components of the cardiac RAS and improves diastolic function without lowering blood pressure, and that lisinopril-induced blood pressure control and cardiac alterations in OVX SHR are permissive in facilitating G1 to augment Ang-(1-7) in serum, thereby strengthening its cardioprotective benefits.
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Angiotensina I/fisiologia , Ciclopentanos/farmacologia , Diástole/efeitos dos fármacos , Lisinopril/farmacologia , Fragmentos de Peptídeos/fisiologia , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Angiotensina I/sangue , Animais , Feminino , Ovariectomia , Fragmentos de Peptídeos/sangue , Ratos , Ratos Endogâmicos SHR , Receptores Acoplados a Proteínas G/fisiologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologiaRESUMO
The dodecapeptide angiotensin-(1-12) [Ang-(1-12)] functions as an intracrine/paracrine substrate for local production of angiotensin II. We developed a reliable and specific radioimmunoassay (RIA) method for the measurement of Ang-(1-12) in human plasma and urine using an affinity purified antibody fraction directed towards the C-terminus of the human Ang-(1-12) sequence. The RIA method was applied to quantify the Ang-(1-12) in plasma and urine collected from thirty-four human subjects (29 treated with antihypertensive medicines and 5 untreated patients). Plasma Ang-(1-12) level was significantly higher (P < 0.05) in patients with systolic blood pressure ≥140 mm Hg (n = 10) compared to the group with systolic blood pressure <140 mm Hg (n = 24). No significant difference (P = 0.22) was found in spot urine between the groups. Our study also shows that the polyclonal antibody neutralizes the cleavage sites of the human Ang-(1-12) from recombinant human chymase (rhChymase) and serum angiotensin converting enzyme (ACE) mediated Ang II generating hydrolysis. Overall, this newly developed RIA method is reliable and applicable to accurately quantify the Ang-(1-12) level in clinical samples (plasma and urine). Further, our in vitro neutralization study suggests that the anti-Ang-(1-12)-antibody might be used as an in vivo therapeutic agent for preventing Ang-(1-12)/Ang II-mediated hypertension and organ damage.
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Angiotensinogênio/sangue , Angiotensinogênio/urina , Hipertensão/genética , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/urina , Radioimunoensaio/métodos , Sistema Renina-Angiotensina/genética , Idoso , Angiotensina II/sangue , Angiotensina II/genética , Angiotensina II/urina , Angiotensinogênio/genética , Anticorpos/química , Anticorpos/isolamento & purificação , Anti-Hipertensivos/uso terapêutico , Pressão Sanguínea/genética , Estudos de Casos e Controles , Quimases/sangue , Quimases/genética , Feminino , Regulação da Expressão Gênica , Humanos , Hipertensão/sangue , Hipertensão/tratamento farmacológico , Hipertensão/urina , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/genética , Radioimunoensaio/normas , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Transdução de Sinais , Equilíbrio Hidroeletrolítico/genéticaRESUMO
We comment on the publication of a paper in which Brazilian investigators evaluate the anticontractile response of perivascular adipose tissue (PVAT) in experimental heart failure (HF) induced in rats by occlusion of a coronary artery.
Assuntos
Insuficiência Cardíaca , Doenças Vasculares , Tecido Adiposo/metabolismo , Animais , Insuficiência Cardíaca/metabolismo , Ratos , Sistema Renina-Angiotensina , Doenças Vasculares/metabolismo , VasoconstriçãoRESUMO
PURPOSE OF REVIEW: Hospitalizations for COVID-19 dramatically increase with age. This is likely because of increases in fragility across biological repair systems and a weakened immune system, including loss of the cardiorenal protective arm of the renin--angiotensin system (RAS), composed of angiotensin-converting enzyme-2 (ACE2)/angiotensin-(1--7) [Ang-(1--7)] and its actions through the Mas receptor. The purpose of this review is to explore how cardiac ACE2 changes with age, cardiac diseases, comorbid conditions and pharmaceutical regimens in order to shed light on a potential hormonal unbalance facilitating SARs-CoV-2 vulnerabilities in older adults. RECENT FINDINGS: Increased ACE2 gene expression has been reported in human hearts with myocardial infarction, cardiac remodeling and heart failure. We also found ACE2 mRNA in atrial appendage tissue from cardiac surgical patients to be positively associated with age, elevated by certain comorbid conditions (e.g. COPD and previous stroke) and increased in conjunction with patients' chronic use of antithrombotic agents and thiazide diuretics but not drugs that block the renin--angiotensin system. SUMMARY: Cardiac ACE2 may have bifunctional roles in COVID-19 as ACE2 not only mediates cellular susceptibility to SARS-CoV-2 infection but also protects the heart via the ACE2/Ang-(1--7) pathway. Linking tissue ACE2 from cardiac surgery patients to their comorbid conditions and medical regimens provides a unique latform to address the influence that altered expression of the ACE2/Ang-(1-7)/Mas receptor axis might have on SARs-CoV-2 vulnerability in older adults.