RESUMO
Endothelial dysfunction (ED) is a key factor for the development of cardiovascular diseases. Due to its chronic, life-threatening nature, ED only can be studied experimentally in animal models. Therefore, this work was aimed to characterize a murine model of ED induced by a daily intraperitoneal administration of angiotensin II (AGII) for 10 weeks. Oxidative stress, inflammation, vascular remodeling, hypertension, and damage to various target organs were evaluated in treated animals. The results indicated that a chronic intraperitoneal administration of AGII increases the production of systemic soluble VCAM, ROS and ICAM-1 expression, and the production of TNFα, IL1ß, IL17A, IL4, TGFß, and IL10 in the kidney, as well as blood pressure levels; it also promotes vascular remodeling and induces non-alcoholic fatty liver disease, glomerulosclerosis, and proliferative retinopathy. Therefore, the model herein proposed can be a representative model for ED; additionally, it is easy to implement, safe, rapid, and inexpensive.
Assuntos
Angiotensina II/administração & dosagem , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Doenças Vasculares/metabolismo , Angiotensina II/toxicidade , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Infusões Parenterais , Molécula 1 de Adesão Intercelular/metabolismo , Interleucinas/metabolismo , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Doenças Vasculares/etiologia , Doenças Vasculares/patologia , Remodelação VascularRESUMO
Cardiac fibroblasts (CF) play an important role in the healing process and in pathological remodeling of cardiac tissue. As sentinel cells in the heart, they respond to inflammatory stimuli, expressing cytokines and cell adhesion proteins, which ultimately lead to increased recruitment of monocytes and enhancement of the inflammatory response. Angiotensin II (Ang II) triggers an inflammatory response, leading to cardiac tissue remodeling. On the other hand, RvD1 has been shown to contribute to the resolution of inflammation; however, its role in Ang II-treated CF has not been addressed until now. The present research aimed to study the effect of RvD1 on cytokine levels, cell adhesion proteins expression in a model of Ang II-triggered inflammatory response. CF from adult Sprague Dawley rats were used to study mRNA and protein levels of MCP-1, IL-6, TNF-a, IL-10, ICAM-1 and VCAM-1; and adhesion of spleen mononuclear cells to CF after Ang II stimulation. Our results show that Ang II increased IL-6, MCP-1 and TNF-a mRNA levels, but only increased IL-6 and MCP-1 protein levels. These effects were blocked by Losartan, but not by PD123369. Moreover, RvD1 was able to prevent all Ang II effects in CF. Additionally, RvD1 reduced the intracellular Ca2+ increase triggered by Ang II, indicating that RvD1 acts in an early manner to block Ang II signaling. Conclusion: our findings confirm the pro-resolutive effects of inflammation by RvD1, which at the cardiovascular level, could contribute to repair damaged cardiac tissue.
Assuntos
Angiotensina II/toxicidade , Adesão Celular/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Ácidos Docosa-Hexaenoicos/farmacologia , Monócitos/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Adesão Celular/fisiologia , Células Cultivadas , Citocinas/biossíntese , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica , Masculino , Monócitos/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
ABSTRACT: Disruption of protein quality control occurs with aging and cardiovascular pathologies including arterial stiffness and hypertension. Angiotensin II (Ang II) is believed to induce endoplasmic reticulum stress in vascular smooth muscle cells (VSMCs), thus contributing to vascular remodeling and dysfunction. However, whether Ang II increases formation of protein aggregates and mediates proteotoxicity in VSMCs remain obscure. Accordingly, this study aimed to establish a quantitative method of protein aggregate detection induced by Ang II and to investigate their potential involvement in inflammatory and senescence responses. Proteostat staining showed increased aggregate numbers per cell on Ang II exposure. Immunoblot analysis further showed an increase in preamyloid oligomer presence in a detergent insoluble protein fraction purified from VSMCs stimulated with Ang II. Moreover, these responses were attenuated by treatment with chemical chaperone, 4-phenylbutyrate. 4-phenylbutyrate further blocked Ang II-induced senescence associated ß-galactosidase activity and THP-1 monocyte adhesion in VSMCs. These data suggest that Ang II induces proteotoxicity in VSMCs which likely contributes to aging and inflammation in the vasculature.
Assuntos
Angiotensina II/toxicidade , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Agregados Proteicos , Animais , Adesão Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Masculino , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Agregação Patológica de Proteínas , Ratos Sprague-Dawley , Células THP-1RESUMO
BACKGROUND: Angiotensin II (Ang II) contributes to the progression of renal diseases associated with proteinuria and glomerulosclerosis mainly by inducing podocyte apoptosis. In the present study, we investigated whether the chronic effects of Ang II via AT1 receptor (AT1R) would result in endoplasmic reticulum (ER) stress/PKC-delta/p38 MAPK stimulation, and consequently podocyte apoptosis. METHODS: Wistar rats were treated with Ang II (200 ng·kg-1·min-1, 42 days) and or losartan (10 mg·kg-1·day-1, 14 days). Immortalized mouse podocyte were treated with 1 µM Ang II and/or losartan (1 µM) or SB203580 (0.1 µM) (AT1 receptor antagonist and p38 MAPK inhibitor) for 24 h. Kidney sections and cultured podocytes were used to evaluate protein expression by immunofluorescence and immunoblotting. Apoptosis was evaluated by flow cytometry and intracellular pH (pHi) was analyzed using microscopy combined with the fluorescent probe BCECF/AM. RESULTS: Compared with controls, Ang II via AT1R increased chaperone GRP 78/Bip protein expression in rat glomeruli (p < 0.001) as well as in podocyte culture (p < 0.01); increased phosphorylated eIf2-α (p < 0.05), PKC-delta (p < 0.01) and p38 MAPK (p < 0.001) protein expression. Furthermore, Ang II induced p38 MAPK-mediated late apoptosis and increased the Bax/Bcl-2 ratio (p < 0.001). Simultaneously, Ang II via AT1R induced p38 MAPK-NHE1-mediated increase of pHi recovery rate after acid loading. CONCLUSION: Together, our results indicate that Ang II-induced podocyte apoptosis is associated with AT1R/ER stress/PKC-delta/p38 MAPK axis and enhanced NHE1-mediated pHi recovery rate.
Assuntos
Angiotensina II/toxicidade , Estresse do Retículo Endoplasmático/fisiologia , Podócitos/metabolismo , Proteína Quinase C-delta/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Transformada , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Camundongos , Podócitos/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
Inflammation and oxidative stress play major roles in endothelial dysfunction, and are key factors in the progression of cardiovascular diseases. The aim of this study was to evaluate in vitro the effect of three subfractions (SFs) from the Cucumis sativus aqueous fraction to reduce inflammatory factors and oxidative stress induced by angiotensin II (Ang II) in human microvascular endothelial cells-1 (HMEC-1) cells. The cells were cultured with different concentrations of Ang II and 0.08 or 10 µg/mL of SF1, SF2, or SF3, or 10 µmol of losartan as a control. IL-6 (Interleukin 6) concentration was quantified. To identify the most effective SF combinations, HMEC-1 cells were cultured as described above in the presence of four combinations of SF1 and SF3. Then, the effects of the most effective combination on the expression of adhesion molecules, the production of reactive oxygen species (ROS), and the bioavailability of nitric oxide (NO) were evaluated. Finally, a mass spectrometry analysis was performed. Both SF1 and SF3 subfractions decreased the induction of IL-6 by Ang II, and C4 (SF1 and SF3, 10 µg/mL each) was the most effective combination to inhibit the production of IL-6. Additionally, C4 prevented the expression of adhesion molecules, reduced the production of ROS, and increased the bioavailability of NO. Glycine, arginine, asparagine, lysine, and aspartic acid were the main components of both subfractions. These results demonstrate that C4 has anti-inflammatory and antioxidant effects.
Assuntos
Aminoácidos/farmacologia , Angiotensina II/toxicidade , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Cucumis sativus , Células Endoteliais/efeitos dos fármacos , Inflamação/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Aminoácidos/isolamento & purificação , Anti-Inflamatórios/isolamento & purificação , Antioxidantes/isolamento & purificação , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Cucumis sativus/química , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Óxido Nítrico/metabolismo , Fitoterapia , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Espécies Reativas de Oxigênio/metabolismoRESUMO
The renin-angiotensin system (RAS) plays a pivotal role in the pathogenesis of cardiovascular diseases. New members of this system have been characterized and shown to have biologically relevant actions. Alamandine and its receptor MrgD are recently identified components of RAS. In the cardiovascular system, alamandine actions included vasodilation, antihypertensive, and antifibrosis effects. Currently, the actions of alamandine on cardiomyocytes are unknown. Here our goal was twofold: 1) to unravel the signaling molecules activated by the alamandine/MrgD axis in cardiomyocytes; and 2) to evaluate the ability of this axis to prevent angiotensin II (ANG II)-induced hypertrophy. In cardiomyocytes from C57BL/6 mice, alamandine treatment induced an increase in nitric oxide (NO) production, which was blocked by d-Pro7-ANG-(1-7), a MrgD antagonist. This NO rise correlated with increased phosphorylation of AMPK. Alamandine-induced NO production was preserved in Mas-/- myocytes and lost in MrgD-/- cells. Binding of fluorescent-labeled alamandine was observed in wild-type cells, but it was dramatically reduced in MrgD-/- myocytes. We also assessed the consequences of prolonged alamandine exposure to cultured neonatal rat cardiomyocytes (NRCMs) treated with ANG II. Treatment of NRCMs with alamandine prevented ANG II-induced hypertrophy. Moreover, the antihypertrophic actions of alamandine were mediated via MrgD and NO, since they could be prevented by d-Pro7-ANG-(1-7) or inhibitors of NO synthase or AMPK. ß-Alanine, a MrgD agonist, recapitulated alamandine's cardioprotective effects in cardiomyocytes. Our data show that alamandine via MrgD induces AMPK/NO signaling to counterregulate ANG II-induced hypertrophy. These findings highlight the therapeutic potential of the alamandine/MrgD axis in the heart.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Angiotensina II/toxicidade , Cardiomegalia/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Proteínas do Tecido Nervoso/agonistas , Óxido Nítrico/metabolismo , Oligopeptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/enzimologia , Cardiomegalia/patologia , Células Cultivadas , Ativação Enzimática , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Proteínas do Tecido Nervoso/metabolismo , Oligopeptídeos/metabolismo , Fosforilação , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/agonistas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ratos Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The in vivo effect of continuous angiotensin II (Ang II) infusion on arterial blood pressure, vascular hypertrophy and α1 -adrenoceptors (α1 -ARs) expression was explored. Alzet(®) minipumps filled with Ang II (200 ng kg(-1) min(-1) ) were subcutaneously implanted in male Wistar rats (3 months-old). Groups of rats were also treated with losartan, an AT1 R antagonist, or with BMY 7378, a selective α1D -AR antagonist. Blood pressure was measured by tail-cuff; after 2 or 4 weeks of treatment, vessels were isolated for functional and structural analyses. Angiotensin II increased systolic blood pressure. Phenylephrine-induced contraction in aorta was greater (40% higher) in Ang II-treated rats than in the controls, and similar effect occurred with KCl 80 mm. Responses in tail arteries were not significantly different among the different groups. Angiotensin II decreased α1D -ARs without modifying the other α1 -ARs and induced an increase in media thickness (hypertrophy) in aorta, while no structural change occurred in tail artery. Losartan prevented and reversed hypertension and hypertrophy, while BMY 7378 prevented and reversed the aorta's hypertrophic response, without preventing or reversing hypertension. Findings indicate that Ang II-induced aortic hypertrophic response involves Ang II-AT1 Rs and α1D -ARs. Angiotensin II-induced α1D -AR-mediated vascular remodeling occurs independently of hypertension. Findings identify a α1D -AR-mediated process whereby Ang II influences aortic hypertrophy independently of blood pressure elevation.
Assuntos
Angiotensina II/toxicidade , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Músculo Liso Vascular/fisiologia , Receptores Adrenérgicos alfa 1/fisiologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Relação Dose-Resposta a Droga , Hipertrofia/induzido quimicamente , Hipertrofia/metabolismo , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ratos , Ratos WistarRESUMO
The kallikrein-kinin and renin-angiotensin systems interact at multiple levels. In the present study, we tested the hypothesis that the B1 kinin receptor (B1R) contributes to vascular hypertrophy in angiotensin II (ANG II)-induced hypertension, through a mechanism involving reactive oxygen species (ROS) generation and extracellular signal-regulated kinase (ERK1/2) activation. Male Wistar rats were infused with vehicle (control rats), 400 ng/Kg/min ANG II (ANG II rats) or 400 ng/Kg/min ANG II plus B1 receptor antagonist, 350 ng/Kg/min des-Arg(9)-Leu(8)-bradykinin (ANGII+DAL rats), via osmotic mini-pumps (14 days) or received ANG II plus losartan (10 mg/Kg, 14 days, gavage - ANG II+LOS rats). After 14 days, ANG II rats exhibited increased systolic arterial pressure [(mmHg) 184 ± 5.9 vs 115 ± 2.3], aortic hypertrophy; increased ROS generation [2-hydroxyethidium/dihydroethidium (EOH/DHE): 21.8 ± 2.7 vs 6.0 ± 1.8] and ERK1/2 phosphorylation (% of control: 218.3 ± 29.4 vs 100 ± 0.25]. B1R expression was increased in aortas from ANG II and ANG II+DAL rats than in aortas from the ANG II+LOS and control groups. B1R antagonism reduced aorta hypertrophy, prevented ROS generation (EOH/DHE: 9.17 ± 3.1) and ERK1/2 phosphorylation (137 ± 20.7%) in ANG II rats. Cultured aortic vascular smooth muscle cells (VSMC) stimulated with low concentrations (0.1 nM) of ANG II plus B1R agonist exhibited increased ROS generation, ERK1/2 phosphorylation, proliferating-cell nuclear antigen expression and [H3]leucine incorporation. At this concentration, neither ANG II nor the B1R agonist produced any effects when tested individually. The ANG II/B1R agonist synergism was inhibited by losartan (AT1 blocker, 10 µM), B1R antagonist (10 µM) and Tiron (superoxide anion scavenger, 10 mM). These data suggest that B1R activation contributes to ANG II-induced aortic hypertrophy. This is associated with activation of redox-regulated ERK1/2 pathway that controls aortic smooth muscle cells growth. Our findings highlight an important cross-talk between the DABK and ANG II in the vascular system and contribute to a better understanding of the mechanisms involved in vascular remodeling in hypertension.
Assuntos
Hipertensão/patologia , Sistema Calicreína-Cinina/fisiologia , Sistema Renina-Angiotensina/fisiologia , Angiotensina II/toxicidade , Animais , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/uso terapêutico , Aorta/metabolismo , Aorta/patologia , Pressão Sanguínea/efeitos dos fármacos , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Células Cultivadas , Sinergismo Farmacológico , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Hipertrofia/metabolismo , Sistema Calicreína-Cinina/efeitos dos fármacos , Losartan/farmacologia , Losartan/uso terapêutico , Masculino , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptor B1 da Bradicinina/agonistas , Receptor B1 da Bradicinina/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
The renin-angiotensin system is critically involved in regulating arterial blood pressure (BP). Inappropriate angiotensin type-1 receptor activation by angiotensin-II (Ang-II) is related to increased arterial BP. Mg has a role in BP; it can affect cardiac electrical activity, myocardial contractility, and vascular tone. To evaluate the relationship between high BP induced by a high sodium (Na) diet and Mg, and other mineral balances, two experimental rat models of salt-sensitive, induced-hypertension were used: Ang-II infused and Dahl salt-sensitive (SS) rats. We found that: 1) Ang-II infusion progressively increased BP, which was accompanied by hypomagnesuria and signs of secondary hyperaldosteronism; 2) an additive effect between Ang-II and a high Na load may have an effect on strontium (Sr), zinc (Zn) and copper (Cu) balances; 3) Dahl SS rats fed a high Na diet had a slow pressor response, accompanied by altered Mg, Na, potassium (K), and phosphate (P) balances; and 4) losartan prevented BP increases induced by Ang II-NaCl, but did not modify mineral balances. In Dahl SS rats, losartan attenuated high BP and ameliorated magnesemia, Na and K balances. Mg metabolism maybe considered a possible defect in this strain of rat that may contribute to hypertension.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Losartan/uso terapêutico , Magnésio/fisiologia , Sistema Renina-Angiotensina/fisiologia , Angiotensina II/fisiologia , Angiotensina II/toxicidade , Animais , Pressão Sanguínea/fisiologia , Peso Corporal/efeitos dos fármacos , Diurese/efeitos dos fármacos , Ingestão de Líquidos/efeitos dos fármacos , Hiperaldosteronismo/etiologia , Hipertensão/etiologia , Hipertensão/genética , Hipertensão/fisiopatologia , Magnésio/sangue , Masculino , Minerais/sangue , Minerais/urina , Polidipsia/induzido quimicamente , Distribuição Aleatória , Ratos , Ratos Endogâmicos Dahl , Ratos Sprague-Dawley , Cloreto de Sódio na Dieta/efeitos adversosRESUMO
The renin-angiotensin (Ang) system plays a pivotal role in the pathogenesis of cardiovascular disease, with Ang II being the major effector of this system. Multiple lines of evidence have shown that Ang-(1-7) exerts cardioprotective effects in the heart by counterregulating Ang II actions. The questions that remain are how and where Ang-(1-7) exerts its effects. By using a combination of molecular biology, confocal microscopy, and a transgenic rat model with increased levels of circulating Ang-(1-7) (TGR[A1-7]3292), we evaluated the signaling pathways involved in Ang-(1-7) cardioprotection against Ang II-induced pathological remodeling in ventricular cardiomyocytes. Rats were infused with Ang II for 2 weeks. We found that ventricular myocytes from TGR(A1-7)3292 rats are protected from Ang II pathological remodeling characterized by Ca(2+) signaling dysfunction, hypertrophic fetal gene expression, glycogen synthase kinase 3beta inactivation, and nuclear factor of activated T-cells nuclear accumulation. Moreover, cardiomyocytes from TGR(A1-7)3292 rats infused with Ang II presented increased expression levels of neuronal NO synthase. To provide a signaling pathway involved in the beneficial effects of Ang-(1-7), we treated neonatal cardiomyocytes with Ang-(1-7) and Ang II for 36 hours. Treatment of cardiomyocytes with Ang-(1-7) prevented Ang II-induced hypertrophy by modulating calcineurin/nuclear factor of activated T-cell signaling cascade. Importantly, antihypertrophic effects of Ang-(1-7) on Ang II-treated cardiomyocytes were prevented by N(G)-nitro-l-arginine methyl ester and 1H-1,2,4oxadiazolo4,2-aquinoxalin-1-one, suggesting that these effects are mediated by NO/cGMP. Taken together, these data reveal a key role for NO/cGMP as a mediator of Ang-(1-7) beneficial effects in cardiac cells.