ABSTRACT
Antecedentes: La ECA2 ha mostrado ser un regulador esencial de la funcionalidad cardíaca. En un modelo experimental de insuficiencia cardíaca (IC) con Fier, modelo de coartación de aorta (COA), se encontró activación de la vía Rho-kinasa. La inhibición de esta vía con fasudil no mejoró el remodelado cardíaco ni la disfunción sistólica. Se desconoce en este modelo, si el deterioro de la función cardíaca y activación de la vía rho-kinasa se asocia con una disminución de la ECA2 cardíaca y si la inhibición de Rho-kinasa tiene un efecto sobre la expresión de ECA2. Objetivo: Nuestro objetivo es determinar si en la falla cardaca experimental por coartación aórtica, los niveles proteicos de ECA2 en el miocardio se asocian a disfunción sistólica y cual es su interacción con la actividad de ROCK en el miocardio. Métodos: Ratones C57BL6J machos de 7-8 semanas se randomizaron en 3 grupos experimentales. Grupo COA por anudación de la aorta + vehículo; Grupo COA + Fasudil (100 mg/Kg día) por bomba osmótica desde la semana 5 post-cirugía; y grupo control o Sham. Se determinaron las dimensiones y función cardíaca por ecocardiografía. Posterior a la eutanasia, se determinaron los niveles de ECA2 del VI por Western-blot y actividad de la Rho-kinasa Resultados: En los grupos COA+vehículo y COA-FAS hubo deterioro de la función cardíaca, reflejada por la reducción de la FE (47,9 ± 1,53 y 45,5 ± 2,10, p < 0,05, respectivamente) versus SHAM (68,6 ± 1,19). Además, aumentaron las dimensiones cardíacas y hubo desarrollo de hipertrofia (0,53 ± 0,02 / 0,53 ± 0,01, p < 0,05) medida por aumento de la masa cardíaca relativa respecto del grupo SHAM (0,40 ± 0,01). En los grupos COA+vehículo y COA-FAS se encontró una disminución significativa del 35% en la expresión de ECA2 cardíaca respecto al grupo control. Conclusiones: La disfunción sistólica por coartación aórtica se asocia con aumento de la actividad de Rho-kinasa y significativa disminución de la expresión de ECA2. La inhibición de Rho-kinasa no mejoró el remodelado cardíaco, la disfunción sistólica y tampoco modificó los niveles de ECA2 cardíaca.
Background: ACE2 has been described as an essential regulator of cardiac function. In an experimental model of heart failure (HF) and heart failure reduced ejection fraction (HFrEF), the aortic coarctation (COA) model, activation of the Rho-kinase pathway of cardiac remodeling was found. Inhibition of this pathway did not improve cardiac remodeling or systolic ventricular dysfunction. It is unknown in this model whether the impairment of cardiac function and activation of the rho-kinase pathway is associated with a decrease in ACE2 and whether rho-kinase inhibition has an effect on ACE2 expression. Objective: To determine if in experimental heart failure due to aortic coarctation, ACE2 protein levels in the myocardium are associated with systolic dysfunction and what is its interaction with ROCK activity in the myocardium. Methods: Male C57BL6J mice aged 7-8 weeks were divided into 3 groups and anesthetized: One group underwent COA+ vehicle; A second group COA + Fasudil (100 mg/Kg/d) by osmotic pump from week 5 post-surgery and; the third group, control(SHAM). Echocardiograms were performed to determine cardiac dimensions and systolic function. Rats were then euthanized. Ventricular expression of ACE2, activity of the Rho-kinase pathway by MYPT-1 phosphorylation, relative cardiac mass, area and perimeter of cardiomyocytes were determined by Western blot. Results: In both COA+vehicle and COA+FAS groups there was deterioration of cardiac function, reflected in the reduction of EF (47.9 ± 1.53 and 45.5 ± 2.10, p < 0.05, respectively) versus the SHAM group (68.6 ± 1.19). In addition, cardiac dimensions and hypertrophy increased (0.53 ± 0.02 / 0.53 ± 0.01, p < 0.05) due to increased relative cardiac mass compared to the SHAM group (0.40 ± 0.01). In the COA+vehicle and COA+FAS groups a significant decrease of 35% in cardiac ACE2 expression was found compared to the control group. Conclusions: Systolic dysfunction due to aortic coarctation is associated with increased Rhokinase activity and a significant decrease in ACE2 expression. Rho-kinase inhibition did not improve cardiac remodeling, systolic dysfunction, nor did it change cardiac ACE2 levels.
Subject(s)
Animals , Mice , Angiotensin-Converting Enzyme 2 , Heart Failure/enzymology , Aortic Coarctation , Blotting, Western , Hypertrophy, Left Ventricular , Ventricular Dysfunction, Left , Disease Models, Animal , Mice, Inbred C57BLABSTRACT
Background: Reverse remodeling is a clinically relevant endpoint in heart failure with reduced ejection fraction (HFrEF). Rho-kinase (ROCK) signaling cascade activation correlates with cardiac remodeling and left ventricular (LV) systolic dysfunction in HFrEF patients. Cardiac resynchronization therapy (CRT) is effective in HFrEF, especially when there is a left bundle block, as this treatment may stimulate reverse remodeling, thereby improving quality of life and prolonging survival for patients with this severe condition. Here, we evaluate the hypothesis that ROCK activation is reduced after effective CRT in HFrEF. Methods: ROCK activation in circulating leukocytes was evaluated in 28 HFrHF patients, using Western blot (myosin light chain phosphatase subunit 1 phosphorylation, MYPT1p/t), before and three months after initiation of CRT. LV systolic function and remodeling were assessed by echocardiography. Results: Three months after CRT, LV ejection fraction increased an average of 14.5% (p < 0.001) in 13 patients (responders), while no change was observed in 15 patients (non-responders). End-systolic diameter decreased 16% (p < 0.001) in responders, with no change in non-responders. ROCK activation in PBMCs decreased 66% in responders (p < 0.05) but increased 10% in non-responders (NS). LV end-diastolic diameter was also 5.2 mm larger in non-responders vs. responders (p = 0.058). LV ejection fraction, systolic diameter, and ROCK activation levels were similar in both groups at baseline. Conclusion: In HFrEF patients, 3 months of effective CRT induced reverse myocardial remodeling, and ROCK activation was significantly decreased in circulating leukocytes. Thus, decreased ROCK activation in circulating leukocytes may reflect reverse cardiac remodeling in patients with heart failure.
ABSTRACT
BACKGROUND: The intracellular ROCK signaling pathway is an important modulator of blood pressure and of cardiovascular and renal remodeling when Rho-kinase activity is increased. Besides, in preclinical models of diabetes, ROCK activation has also a role in abnormal glucose metabolism as well as in subsequent vascular and myocardial dysfunction. In humans, there are a few data assessing ROCK activation in patients with type 2 diabetes mellitus (T2D) and no studies assessing upstream/downstream components of the ROCK pathway. We assessed here levels of ROCK activation and some of the RhoA/ROCK cascade molecules in peripheral blood mononuclear cells (PBMCs) in T2D patients under current treatment. METHODS: Cross-sectional observational study comparing 28 T2D patients under current antidiabetic treatment with 31 consecutive healthy subjects, matched by age and gender. Circulating levels of malondialdehyde, angiotensin II and inflammatory cytokines IL-6 and IL-8 were determined in all subjects. ROCK activation in PMBCs, upstream and downstream cascade proteins, and levels of the proinflammatory molecules VCAM, ICAM-1 and IL-8 were determined in their PMBCs by Western blot. RESULTS: Compared to healthy controls, ROCK activation in T2D patients measured by 2 direct ROCK targets in PBMCs was increased by 420 and 570% (p < 0001) and it correlated significantly with serum glucose levels. p38 MAPK phosphorylation (downstream from ROCK) and JAK-2 (upstream from ROCK) were significantly higher in the T2D patients by 580% and 220%, respectively. In T2D patients, significantly increased PBMC levels of the proinflammatory molecules VCAM-1, ICAM-1 and IL-8 were observed compared to control subjects (by 180%, 360% and 260%, respectively). Circulating levels of Ang II and MDA were significantly higher in T2D patients by 29 and 63%, respectively. CONCLUSIONS: T2D patients under treatment with glucose-lowering drugs, antihypertensive treatment as well as with statins have significantly increased ROCK activation in their circulating leukocytes along with higher phosphorylation of downstream cascade proteins despite pharmacologic treatment, along with increased plasma angiotensin II and MDA levels. ROCK inhibition might have an additional role in the prevention and treatment of T2D.
Subject(s)
Diabetes Mellitus, Type 2/enzymology , Leukocytes, Mononuclear/enzymology , rho-Associated Kinases/blood , Aged , Angiotensin II/blood , Antihypertensive Agents/therapeutic use , Biomarkers/blood , Case-Control Studies , Cross-Sectional Studies , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/diagnosis , Diabetes Mellitus, Type 2/drug therapy , Enzyme Activation , Female , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Hypoglycemic Agents/therapeutic use , Intercellular Adhesion Molecule-1/blood , Interleukin-8/blood , Janus Kinase 2/blood , Male , Malondialdehyde/blood , Middle Aged , Signal Transduction , Vascular Cell Adhesion Molecule-1/blood , p38 Mitogen-Activated Protein Kinases/bloodABSTRACT
INTRODUCCIÓN: Los ratones SR-B1 KO/ApoER6 1h/h que son alimentados con una dieta rica en grasas saturadas, desarrollan enfermedad coronaria aterosclerótica severa, complicaciones isquémicas e insuficiencia cardíaca, con alta mortalidad. Los estudios con este modelo se han enfocado fundamentalmente en la enfermedad coronaria y menos en el remodelado cardíaco. El OBJETIVO del trabajo ha sido caracterizar el remodelado miocárdico, evaluar la evolución temporal de la función ventricular izquierda y la sobrevida asociada a enfermedad cardíaca por ateromatosis. MÉTODO: Ratones homocigotos SR-B1 KO/ApoER6 1h/h fueron alimentados por 8 semanas con dieta aterogénica o dieta normal y se comparó la sobrevida en ambos grupos. A las 4 semanas se realizó un ecocardiograma bidimensional. En los ratones eutanasiados se evaluó en la pared cardíaca fibrosis miocárdica y tamaño de los cardiomiocitos por morfometría, apoptosis con técnica de TUNEL e infiltración por células inflamatorias mononucleares (ED1) por inmunohistoquímica. RESULTADOS: En el grupo que recibió dieta aterogénica la sobrevida se redujo en 46,7% (p < 0.001), debido a muerte súbita y a falla cardíaca progresiva. En este grupo, a las 4 semanas se observó dilatación de cavidades izquierdas y disminución de la fracción de eyección del ventrículo izquierdo en comparación con el grupo control (79,3 ± 1,3% vs 66 ± 3,7%, p<0,01). También se observó aumento de la masa cardíaca relativa de 2.1 veces (p<0,001) y del peso pulmonar relativo en 80% (p<0,001), sin cambios en las dimensiones de los cardiomiocitos. En el miocardio de los ratones que recibieron dieta aterogénica hubo un aumento de la fibrosis cardíaca de 7.9 veces (p < 0.01) y del número de cardiomiocitos apoptóticos en 55.9 veces (p < 0.01), junto a un aumento del número de células inflamatorias mononucleares ED1. CONCLUSIONES: En el modelo de falla cardíaca severa de etiología isquémica con alta mortalidad en el ratón homocigoto SR-B1 KO/ApoER6 1h/h sometido a una dieta aterogénica, con falla cardíaca izquierda por disfunción sistólica, el remodelado patológico del miocardio está dado fundamentalmente por apoptosis y fibrosis. También se observa un aumento discreto de macrófagos en la pared cardíaca. Es posible que el edema parietal también pueda ser un mecanismo de remodelado relevante en este modelo.
Abstract: SR-B1 KO/ApoER6 1h/h mice fed a high saturated fat diet develop severe coronary atheromatosis, and cardiac failure with a high mortality rate. Cardiac remodeling under these conditions has not been well studied. AIM: To evaluate the time course of left ventricular function, cardiac remodeling and survival associated to the administration of an atherogenic diet. METHOD: Homozygote SR-B1 KO/ApoER6 1h/h mice received an atherogenic diet for 8 weeks. Mice receiving a normal diet served as controls. Survival rate, myocardial fibrosis, cardiomyocyte size, apoptosis and infiltration by inflammatory or mononuclear cells were compared between groups. A TUNEL technique was used to evaluate apoptosis. RESULTS: A 46.7% survival reduction compared to controls was observed in the experimental group (p<0.01), due to left ventricular and atrial dilatation associated to a decrease in ejection fraction (79,3 ± 1,3% vs 66 ± 3,7%, p<0,01, respectively). Also, an increased cardiac weight, 2.6 times greater was observed in the experimental group, compared to controls. Mice receiving the atherogenic diet showed an 80% increased lung weight. There was no evident change in cardiomyocytes, but there was more (7.9 times) cardiac fibrosis (p<0.01) and 55.9 times more apoptotic cells. (p<0.01), along with a greater number of inflammatory cells and ED1 mononuclear cells. CONCLUSION: Mice receiving an atherogenic diet develop heart failure and reduced survival rate. This is associated with cardiac remodeling with underlying apoptosis an ventricular wall fibrosis. It is posible that wall edema might contribute to the observed cardiac remodeling.
Subject(s)
Animals , Mice , Ventricular Remodeling , Diet, Atherogenic , Heart Failure/etiology , Hyperlipidemias/pathology , Ischemia/etiology , Fibrosis , Survival Analysis , Ventricular Function, Left , Apoptosis , Mice, Knockout , Ventricular Dysfunction , Disease Models, Animal , Heart Failure/physiopathology , Heart Failure/mortality , Heart Failure/pathology , Ischemia/physiopathology , Ischemia/mortality , Ischemia/pathologyABSTRACT
BACKGROUND: Increased Rho-kinase activity in circulating leucocytes is observed in heart failure with reduced ejection fraction (HFrEF). However, there is little information in HFrEF regarding other Rho-kinase pathway components an on the relationship between Rho-kinase and apoptosis. Here, Rho-kinase activation levels and phosphorylation of major downstream molecules and apoptosis levels were measured for the first time both in HFrEF patients and healthy individuals. METHODS: Cross-sectional study comparing HFrEF patients (n = 20) and healthy controls (n = 19). Rho-kinase activity in circulating leucocytes (peripheral blood mononuclear cells, PBMCs) was determined by myosin light chain phosphatase 1 (MYPT1) and ezrin-radixin-moesin (ERM) phosphorylation. Rho-kinase cascade proteins phosphorylation p38-MAPK, myosin light chain-2, JAK and JNK were also analysed along with apoptosis. RESULTS: MYPT1 and ERM phosphorylation were significantly elevated in HFrEF patients, (3.9- and 4.8-fold higher than in controls, respectively). JAK phosphorylation was significantly increased by 300% over controls. Phosphorylation of downstream molecules p38-MAPK and myosin light chain-2 was significantly higher by 360% and 490%, respectively, while JNK phosphorylation was reduced by 60%. Catecholamine and angiotensin II levels were significantly higher in HFrEF patients, while angiotensin-(1-9) levels were lower. Apoptosis in circulating leucocytes was significantly increased in HFrEF patients by 2.8-fold compared with controls and significantly correlated with Rho-kinase activation. CONCLUSION: Rho-kinase pathway is activated in PMBCs from HFrEF patients despite optimal treatment, and it is closely associated with neurohormonal activation and with apoptosis. ROCK cascade inhibition might induce clinical benefits in HFrEF patients, and its assessment in PMBCs could be useful to evaluate reverse remodelling and disease regression.
Subject(s)
Apoptosis , Heart Failure/blood , Heart Failure/physiopathology , Leukocytes, Mononuclear/enzymology , Leukocytes, Mononuclear/pathology , Signal Transduction , Stroke Volume , rho-Associated Kinases/metabolism , Angiotensins/blood , Animals , Antigens, CD/metabolism , Catecholamines/blood , Cytokines/blood , Cytoskeletal Proteins/metabolism , Disease Models, Animal , Enzyme Activation , Female , Heart Failure/diagnostic imaging , Humans , Janus Kinase 2/metabolism , Male , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Middle Aged , Myocardium/pathology , Myosin-Light-Chain Phosphatase/metabolism , Natriuretic Peptide, Brain/blood , Phosphorylation , Rats , Systole , Ventricular RemodelingABSTRACT
RESUMEN: Introducción: La vía clásica del sistema renina-angiotensina (SRA) está activado en pacientes con hipertensión arterial pulmonar (HAP). Previamente, hemos encontrado que en la disfunción ventricular post infarto al miocardio experimental la activación del eje clásico del SRA, dado por la enzima convertidora de angiotensina I (ECA) y angiotensina (Ang ) II se correlaciona negativamente con el eje paralelo del SRA dado por la ECA homóloga (ECA2) y el péptido vasoactivo y cardioprotector Ang-(1-9). Resultados preclínicos muestran la eficacia de la administración de Ang-(1-9) en el tratamiento del remodelamiento cardiovascular patológico. Hasta la fecha no existen antecedentes de los niveles circulantes de Ang-(1-9) en pacientes con hipertensión arterial pulmonar comparados con sujetos sanos. Objetivo: Determinar los niveles circulantes del péptido vasoactivo y cardiprotector Ang-(1-9) en pacientes con HAP y compararlos con sujetos sanos pareados por edad y sexo. Métodos: Estudio comparativo transversal en pacientes con HAP (grupo I, OMS) con presión de arteria pulmonar media (mPAP) ≥25 mmHg bajo tratamiento con furosemida (40%), espironolactona (53%), Acenocumarol/Warfarina (47%), Bosentan/Ambrisentan (27%), Sildenafil (80%), iloprost (7%) y digoxina (13%). Los sujetos controles correspondieron a sujetos asintomáticos sanos sin enfermedad cardiovascular, cardiopatía estructural ni pulmonar (n=14). En todos los pacientes se determinó mPAP, proBNP, resistencia vascular pulmonar (RVP, WU), presión capilar pulmonar (PCP, mmHg), gasto cardíaco (L/min), capacidad funcional por test de caminata 6 minutos (TC6M), cambio del área fraccional del ventrículo derecho VD (FAC, %). Se utilizó prueba t de Student y programa estadístico SPSS10.0. Un valor de p < 0,05 fue considerado como estadísticamente significativo. Resultados: Los pacientes ingresados al estudio mostraron: etiología de la HAP, idiopática (86,7%), VIH (13,3%), capacidad funcional I (6,2%), II (68,3) y III (25%) y promedio mPAP 51,3±1,9. Pacientes con HAP (grupo I, OMS) versus sujetos sanos mostraron disminución significativa de FAC, actividad plasmática de la ECA2 y niveles circulantes de Ang-(1-9). En la vía clásica del RAAS pacientes con HAP mostraron mayor actividad plasmática de ECA y niveles circulantes e Ag II. Correlaciones significativas se encontraron entre niveles de Ang-(1-9) y mPAP (r = -0.701, p < 0,001) y Ang-(1-9) vs FAC (r = 0.549, p < 0,01). Conclusiones: En pacientes con HAP (grupo I, OMS), los niveles circulantes de Ang-(1-9) están significativamente disminuidos y se asocian inversamente con la PAP, severidad del remodelamiento y disfunción del ventrículo derecho. El uso terapéutico de Ang-(1-9) como agente vasodilatador y cardioprotector podría ser relevante y potencialmente útil, desde una perspectiva clínica, en la HAP. Ang-(1-9) podría reducir la PAP y mejorar el remodelamiento vascular y del ventrículo derecho en la HAP. Por lo tanto, este péptido podría ser útil como blanco terapéutico en la HAP.
ABSTRACTS: Classic renin-angiotensis pathway (RAP) is activated in patients with pulmonary artery hypertension (PAH). We have previously shown that in patients with post myocardial infarction systolic dysfunction the activation of RAP mediated by angiotensin converting enzyme (ACE) and angiotensin II (Ang II) is inversely correlated with the parallel RAP axis mediated by homologous ACE (ACE2) and by the vasoactive and cardioprotective peptide Ang-(1-9). Pre clinical studies show that administration of Ang-(1-9) leads to a favorable ventricular remodelling. At present there is no information regarding levels of Ang-(1-9) in PAH patients compared to healthy subjects. Methods: 16 PAH patients (WHO group 1), with mean PA pressure > 25mmHg being treated with furosemide (40%), Bosentan/Ambrisentan (27%), Sildenafil (80%), iloprost (7%) were compared with healthy subjects (n=14). mPAP, pro BNP, pulmonary vascular resistance (Wu), pulmonary capillary pressure (PCP mmHg), cardiac output (L/min), functional capacity (6 min walking test) (6mWT), and changes in right ventricular fractional area (RV FA), were measured in all subjects. Results: In HAP subjects, the eiotology of PAH was unknown in 87%, or HIV (13%). Functional class was I (6.2 %), II (68.3%) or III (25%). Mean PAP was 51.3±1.9. Compared to healthy subjects, PAH patients had significantly lower RV FA, ACE2 and Ang-(19) levels. Also they had greater ACE plasma activity and AngII circulating levels. Significant correlations were found between Ang-(1-9) and mPAP (-0.701, p < 0,001) and between Ang-(1-9) and RV FA (r = 0.549, p < 0,01). Conclusion: group I PAH subjects, circulating levels of Ang-(1-9) are significantly lower than in healthy subjects and are inversely related to PAP, severity of ventricular remodeling and right ventricular dysfunction. The use of Ang-(1-9) as a vasodilator and cardioprotector agent could be clinically useful in PAH subjects.
Subject(s)
Humans , Male , Female , Middle Aged , Angiotensins/blood , Pulmonary Arterial Hypertension/blood , Peptides , Cross-Sectional StudiesABSTRACT
Resumen: Los modelos experimentales de falla cardíaca con fracción de eyección disminuida en murinos son pocos. Uno de estos modelos es el de coartación de la aorta torácica en el arco aórtico (COA) en ratones. Un aspecto importante en su desarrollo es la evaluación precoz del procedimiento y su relación con la función sistólica posterior. En este sentido, las velocidades de flujo carotídeo y la relación entre ambos flujos (derecho, pre-coartación; izquierdo post coartación) pueden permitir evaluar tempranamente la precisión del procedimiento y relacionarse más tardíamente con la función sistólica VI. Nuestro objetivo fue comparar precozmente (semana 2 post operatoria) las velocidades de flujo en ambas carótidas (Doppler continuo) y tardíamente (semana 5 postoperatoria) la función sistólica VI (Ecocardiograma de superficie) en ratones seudocoartados o sham (n= 6) vs ratones COA (n = 12). Se confirmó una diferencia estadísticamente significativa en la relación de velocidades de flujo entre ambas carótidas medida precozmente entre los ratones sham y COA (1,1 ± 0,1 vs 2,5 ± 0,5, p< 0,001), lo que se correlacionó con un deterioro significativo de la función sistólica del ventrículo izquierdo evaluada a las 5 semanas en los ratones COA. Conclusión: En este modelo preclínico de falla cardíaca por sobrecarga de presión con fracción de eyección VI disminuida en ratón, el aumento precoz de la velocidad de flujo en la arteria carótida derecha (pre-coartación en el modelo COA) y sobre todo de la relación entre las velocidades de flujo carotídeo entre ambas carótidas se asocia a deterioro importante de la función sistólica VI cinco semanas después de efectuada la COA, lo que permite predecir la efectividad del procedimiento en este modelo experimental.
Abstract: There are few experimental models of heart failure with reduced ejection fraction in murines. One of these models is transverse aortic coarctation (TAC) in mice. However, an important challenge in its development is the early evaluation of the procedure and its relationship with late systolic LV function. In this sense, carotid flow velocities and the relationship between both (right, precoarctation, left post-coarctation) may allow early evaluation of the accuracy of the procedure and be related to late LV systolic function. The aim was to compare early (week 2 post-operative) flow velocities determined in both carotid arteries (by continuous Doppler) with late (week 5 postoperative) LV systolic function (by echocardiogram) in sham (n= 6) vs. TAC (n: 12) mice. We confirmed a statistically significant difference in the early ratio of carotid flow velocities (left/right common carotid velocity ratio) between sham and TAC mice (1.1 ± 0.1 vs 2.5 ± 0.5, p< 0.001) and this correlated well with a deteriorated left ventricular function in the TAC mice after 5 weeks. In this preclinical model of cardiac failure due to pressure overload with reduced LV ejection fraction in the mouse, the early increase in right carotid flow velocity (precoarctation) and especially the relationship between precoarctation/postcoarctation carotid flow velocities is associated with significant impairment of LV systolic function five weeks after the TAC, which allows to predict the effectiveness of the procedure in this experimental model.
Subject(s)
Animals , Mice , Aortic Coarctation/physiopathology , Ventricular Dysfunction, Left/physiopathology , Heart Failure, Systolic/physiopathology , Aortic Coarctation/surgery , Regional Blood Flow , Stroke Volume , Blood Flow Velocity , Echocardiography/methods , Carotid Arteries/physiopathology , Disease Models, Animal , Heart Failure, Systolic/surgery , Mice, Inbred C57BLABSTRACT
Hypertension-induced cardiovascular and renal damage can be mediated by activation of the renin-angiotensin-aldosterone system. There are different factors beyond renin-angiotensin-aldosterone system involved in hypertension and renal damage. Inflammation has emerged as an important mediator of hypertension and cardiovascular and kidney damage. Angiotensin-(1-9), a peptide of the renin-angiotensin system, counter-regulates both the physiological and pathological actions of angiotensin II. Recent data has shown that angiotensin-(1-9) protects the heart and blood vessels from adverse cardiovascular remodeling in experimental models of hypertension and/or heart failure and reduces cardiac fibrosis in stroke-prone, spontaneously hypertensive rats. These effects are mediated by the angiotensin II type 2 receptor (AT2R). However, it remains unknown whether angiotensin-(1-9) also has an anti-inflammatory effect. In the present study, we investigate whether angiotensin-(1-9) reduces inflammation and fibrosis in the heart, arteries, and kidney in a DOCA-salt hypertensive model and explore the mechanisms underlying the amelioration of end-organ damage. DOCA-salt hypertensive rats received: a) vehicle, b) angiotensin-(1-9), c) PD123319 (AT2R blocker), d) angiotensin-(1-9) plus A779 (a Mas receptor blocker) or e) angiotensin-(1-9) plus PD123319, and sham rats were used as a control. Our results showed that angiotensin-(1-9) decreased hypertension and increased vasodilation in DOCA-salt hypertensive rats. These actions were partially inhibited by PD123319. Moreover, angiotensin-(1-9) decreased diuresis, fibrosis, and inflammation. These beneficial effects were not mediated by Mas or AT2R blockers. We concluded that angiotensin-(1-9) protects against volume overload-induced hypertensive cardiovascular and kidney damage by decreasing inflammation in the heart, aortic wall, and kidney, through mechanisms independent of the Mas or AT2R.
Subject(s)
Angiotensins/pharmacology , Hypertension/chemically induced , Inflammation/chemically induced , Kidney Diseases/chemically induced , Renin/metabolism , Actins/genetics , Actins/metabolism , Animals , Aorta/cytology , Aorta/drug effects , Aorta/pathology , Desoxycorticosterone/toxicity , Hypertension/drug therapy , Inflammation/drug therapy , Kidney Diseases/drug therapy , Male , Rats , Rats, Sprague-DawleyABSTRACT
Rho-kinase has relevant functions in blood pressure modulation and cardiovascular remodeling. Rho-kinase activity is determined in circulating leukocytes measuring phosphorylation of its target myosin phosphatase target subunit 1 (MYPT1), but its relationship with Rho-kinase activity in the myocardium and in vasculature in hypertension has not been evaluated.The aim was to determine the degree of association between Rho-kinase cascade activation in circulating leukocytes with cardiac and aortic Rho-kinase pathway activation in a model of hypertension and to analyze it with a cause-effect perspective.Hypertensive deoxycorticosterone (DOCA)-salt rats received the Rho-kinase antagonist fasudil (DOCA-Fas, 100 mg/kg/day, 3 weeks). Results were compared with an untreated DOCA-salt and a sham group.Rho-kinase inhibition reduced significantly blood pressure, cardiac hypertrophy, myocardial collagen and macrophage infiltration, but not aortic wall hypertrophy. Fasudil decreased significantly Rho-kinase activity in peripheral blood mononucleated cells (PBMC), myocardium and aortic wall to similar levels as in the sham group. A significant correlation was found between PBMC Rho-kinase activity and cardiac remodeling, specifically with hypertrophy (r = 0.51, P≤0.01), myocardial collagen (r = 0.40, P≤0.05) and ED1 immunostaining (r = 0.48, P≤0.01). In the untreated hypertensive group, increased levels (P<0.05) of the proinflammatory molecules p65 NF-κB, vascular cell adhesion molecule 1 and interleukin-6 antibody in the myocardium, aortic wall and PBMC were observed and were reduced with fasudil (P<0.05).In conclusion, in this hypertension model, Rho-kinase and its pathway activation determined in circulating leukocytes reflect the activation of this pathway in the myocardium and in the aortic wall and are significantly related to myocardial remodeling (hypertrophy, fibrosis and inflammation).
Subject(s)
Atrial Remodeling/physiology , Hypertension/metabolism , Leukocytes, Mononuclear/enzymology , rho-Associated Kinases/metabolism , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , Atrial Remodeling/drug effects , Blood Pressure/drug effects , Blood Pressure/physiology , Cardiomegaly/physiopathology , Cardiomegaly/prevention & control , Cells, Cultured , Enzyme Activation/drug effects , Hypertension/blood , Hypertension/physiopathology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Male , Protein Kinase Inhibitors/pharmacology , Rats, Inbred SHR , Rats, Sprague-Dawley , rho-Associated Kinases/antagonists & inhibitorsABSTRACT
Resumen: Objetivo: Determinar algunos mecanismos moleculares por los cuales la activación de ROCK cardíaca post infarto del miocardio (IAM) participa en el remodelado y en deterioro de la función sistólica. Métodos: Determinación simultánea de niveles de proteínas blanco de ROCK cardíaca, de función sistólica in vivo del ventrículo izquierdo (VI) y de fibrosis e hipertrofia cardíaca en ratas con IAM en condiciones de inhibición de ROCK con fasudil. Resultados : Siete días post IAM la masa ventricular relativa aumentó significativamente en un 30% en el grupo MI y se redujo con fasudil. La disfunción sistólica VI mejoró significativamente con fasudil mientras que la activación de ROCK cardíaca se redujo a niveles del grupo control. El inhibidor de ROCK también redujo significativamente los niveles cardíacos elevados de las isoformas ROCK1 y ROCK2, de MHC-β y del colágeno miocárdico. En el grupo con IAM aumentaron significativamente los niveles de fosforilación de ERK 42 y ERK 44 (en 2 veces y en 63%, respectivamente), mientras que en el grupo IAM tratado con fasudil estos niveles fueron similares a los del grupo control. El IAM aumentó significativamente los niveles fosforilados del factor de transcripción GATA-4, que se normalizaron con el inhibidor de ROCK. Conclusiones: La disfunción sistólica post IAM se asoció fuertemente con la activación del ROCK cardíaca y con la fosforilación de proteínas río abajo de ROCK que promueven remodelado cardíaco como β-MHC y la vía ERK / GATA-4.
Abstracts: Objective: to determine some molecular mechanisms by which cardiac ROCK activation after myocardial infarction (MI) intervene in cardiac systolic function decline and remodeling. Methods: simultaneous measurement of different cardiac ROCK target proteins levels, in vivo left ventricular (LV) systolic function, myocardial fibrosis, and hypertrophy in rats with MI under ROCK inhibition with fasudil were performed. Results: seven days after MI the relative ventricular mass increased significantly by 30% in the MI groupand was reduced with fasudil. LV systolic dysfunction improved significantly with fasudil whereas at the same time cardiac ROCK activation was reduced to sham levels. The ROCK inhibitor also reduced increased cardiac levels of both ROCK1 and ROCK2 isoforms, β-MHC levels and myocardial collagen volume fraction decline. MI significantly increased phosphorylation levels of ERK 42 and ERK 44 by 2-fold and 63% respectively whereas in the fasudil-treated MI group these levels were similar to those in the sham group. MI significantly increased phosphorylated levels of the transcription factor GATA-4 which were normalyzed by the ROCK inhibitor. Conclusion: LV systolic dysfunction after MI was strongly associated to cardiac ROCK activation and subsequent phosphorylation of ROCK target proteins that promote ventricular remodeling, such as β-MHC and the ERK/GATA-4 pathway. ROCK inhibition with fasudil significantly improved systolic function, diminished myocardial fibrosis, and normalized β-MHC and ERK/GATA-4 phosphorylation levels.
Subject(s)
Animals , Rats , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , Protein Kinase Inhibitors/pharmacology , rho-Associated Kinases/antagonists & inhibitors , Myocardial Infarction/drug therapy , Organ Size/drug effects , Phosphorylation , Blotting, Western , Ventricular Function, Left/drug effects , Rats, Sprague-Dawley , Cardiomegaly/drug therapy , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Ventricular Remodeling/drug effects , Disease Models, Animal , Myocardial Infarction/enzymologyABSTRACT
Previamente hemos demostrado que la eficacia de enalapril, candesartán y de fasudil -inhibidor de la vía RhoA/ROCK,- en el tratamiento del remodelado cardíaco en la hipertensión (HTA) e infarto al miocardio está mediada por aumento en los niveles circulantes del péptido vasoactivo angiotensina [Ang]-(1-9). Sin embargo, no hay información disponible si el antagonista del receptor de mineralocorticoide, espironolactona (espiro) disminuye el remodelado cardíaco aumentando los niveles circulantes de Ang-(1-9). El objetivo de este trabajo fue determinar si espironolac-tona disminuye el remodelado cardíaco aumentando los niveles circulantes de Ang-(1-9) en la hipertensión arterial experimental. Métodos. Estudio comparativo de 3 grupos experimentales. Se utilizaron ratas Sprague Dawley macho (150 ± 10 grs) unifrectomizadas tratadas con desoxi-corticosterona (DOCA, 60mg/Kg 2 veces sem, im) por 6 semanas. Como controles (Sham) se usaron ratas unifrectomizadas. A partir de la 3° semana las ratas DOCA con HTA> 140 mmHg fueron randomizadas a recibir vehículo o espiro (100 mg/kg día, gavage) por 3 sem. Al finalizar el tratamiento se determinó la presión arterial sistólica (PAS), masa corporal, peso del corazón (PC) y masa cardíaca relativa al largo de la tibia (MCR, mg ventrículos/LT*100). El grado de hipertrofia car-diomiocitaria se determinó midiendo el área y perímetro de los cardiomiocitos y la fibrosis por el contenido de colágeno en cortes teñidos con rojo picrosirio. Resultados (promedio ± ES): Conclusión: Espironolactona disminuye la PAS y aumenta los niveles circulantes de Ang-(1-9). Este aumento en los niveles circulantes de Ang-(1-9) se asocia con una disminución significativa de la hipertrofia y la fibrosis cardiaca hipertensiva. Este nuevo efecto de espironolactona en los niveles circulantes de Ang-(1-9), - péptido vasoactivo de la vía paralela del sistema renina-angiotensina-aldosterona,- podría contribuir al efecto antihipertensivo y disminución del daño cardiaco en la hipertensión y remodelamiento cardiovascular y renal patológico. Estos hallazgos pueden tener relevancia terapéutica en términos que Ang-(1-9) podría disminuir el daño cardiovascular patológico.
We have previously demonstrated that the efficacy of enalapril, candesartan and fasudil,- RhoA / ROCK inhibitor-, in the treatment of cardiac remodeling in hypertension (HT) and myocardial infarction is mediated by an increase in circulating levels of the vasoactive peptide angiotensin (Ang) -(1-9). However, it is not known whether the mineralocorticoid receptor antagonist, spironolactone (spiro) decreases cardiac remodeling by increasing the circulating levels of Ang- (1-9). The aim of this study was to determine whether spironolactone decreases cardiac remodeling by increasing circulating levels of Ang-(1-9) in experimental hypertension. Methods. Comparative study of 3 experimental groups. Unifirectomized male Sprague Dawley rats (150 ± 10 grams) were treated with deoxycorticos-terone (DOCA, 60 mg / kg 2 times a week, im) for 6 weeks. Unifirectomized rats were used as controls (Sham). At 3rd week after surgery, DOCA rats with HTA> 140 mmHg were randomized to receive vehicle or spironolactone (Spiro, 100 mg / kg day, gavage) for 3 weeks. At the end of treatment, systolic blood pressure (SBP), body mass (BM), heart weight (HW) and relative cardiac mass to the tibia length (MCR, mg ventricles / LT * 100) were determined. The degree of cardiomyocyte hypertrophy was determined by measuring the area and perimeter of cardiomyocytes and fibrosis by collagen content in sections stained with picrosirius red. Results (mean ± ES): Conclusion: Spironolactone decreases systolic blood pressure and increases circulating levels of Ang-(1-9). This increase in circulating levels of Ang- (1-9) was associated with a significant decrease in hypertrophy and hypertensive cardiac fibrosis. This new effect of spironolactone on the circulating levels of Ang- (1-9) - vasoactive peptide of the parallel pathway of the re-nin-angiotensin-aldosterone system - could contribute to the antihypertensive effect and decrease of cardiac damage in HT and cardiovascular remodeling and renal disease. These findings may have therapeutic relevance supporting that Ang-(1-9) may decreases pathologic cardiovascular damage.
Subject(s)
Animals , Male , Rats , Spironolactone/pharmacology , Angiotensins/drug effects , Ventricular Remodeling/drug effects , Hypertension/drug therapy , Rats, Sprague-Dawley , Disease Models, Animal , Mineralocorticoid Receptor Antagonists/pharmacologyABSTRACT
OBJECTIVE: The objective of this study was to determine the molecular mechanisms by which cardiac Rho-associated coiled-coil containing protein kinase (ROCK) activation after myocardial infarction (MI) does intervene in cardiac systolic function decline and remodeling. METHODS: Simultaneous measurement of different cardiac ROCK target proteins levels, in vivo left ventricular (LV) systolic function, myocardial fibrosis and hypertrophy in rats with MI under ROCK inhibition with fasudil. RESULTS: Seven days after MI, the ventricular mass increased significantly by 5.6% in the MI group and was reduced with fasudil. LV systolic dysfunction improved significantly with fasudil whereas cardiac ROCK activation was reduced to sham levels. The ROCK inhibitor also reduced increased cardiac levels of both ROCK1 and ROCK2 isoforms, cardiomyocyte ROCK2 fluorescence levels and ß-myosin heavy chain (MHC) levels in addition to myocardial collagen volume fraction decline. Compared with sham rats, troponin phosphorylation levels after MI were similar and ROCK inhibition reduced them. MI significantly increased phosphorylation levels of extracellular-signal-regulated kinase (ERK) 42 and ERK 44 by twofold and 63%, respectively, whereas in the fasudil-treated MI group these levels were similar to those in the sham group. MI significantly increased phosphorylated levels of the transcription factor GATA-4 and the ROCK inhibitor normalized them. CONCLUSIONS: LV systolic dysfunction after MI was strongly associated with cardiac ROCK activation and subsequent phosphorylation of ROCK target proteins that promote ventricular remodeling such as ß-MHC and the ERK/GATA-4 pathway. ROCK inhibition with fasudil significantly improved systolic function, diminished myocardial fibrosis and normalized ß-MHC and ERK/GATA-4 phosphorylation levels.
Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , Myocardial Infarction/drug therapy , Ventricular Remodeling/drug effects , rho-Associated Kinases/antagonists & inhibitors , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , Disease Models, Animal , Extracellular Signal-Regulated MAP Kinases/metabolism , GATA4 Transcription Factor/metabolism , Male , Myocardial Infarction/physiopathology , Myosin Heavy Chains/metabolism , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Sprague-Dawley , Ventricular Dysfunction, Left/drug therapy , Ventricular Dysfunction, Left/physiopathology , rho-Associated Kinases/metabolismABSTRACT
Introducción: Angiotensina (Ang)-(1-9) posee propiedades anti-hipertensivas y efecto protector a nivel cardiovascular en ratas hipertensas. Sin embargo, se desconoce si estos efectos están asociados a un mecanismo de desbalance de sodio a nivel renal. Objetivo: Determinar si el efecto anti-hipertensivo de Ang-(1-9) está asociado a un mecanismo diurético-na-triurético. Método: Ratas macho Sprague Dawley (200 ± 10g) fueron aleatorizadas para recibir Ang II (400 ng/kgmin) vía bomba osmótica. Como control se utilizaron ratas con operación sham (n=18). Después de 2 semanas desde la instalación de bomba, las ratas Sham e hipertensas fueron randomizadas para recibir vehículo (n=10), Ang-(1-9) (602 ng/kg/min, n=17) o una co-administración de Ang-(1-9) y A779 (100 ng kg-1min-1, n=7 bloqueador del receptor MAS) por 2 semanas. Resultados: Se determinó la presión arterial sistólica (PAS), masa ventricular relativa (MVR), área y perímetro de los cardiomiocitos (AC y PC) y la fracción volumétrica de colágeno total (FVCT). Para evaluar la diuresis y natriuresis se utilizaron ratas normotensas que fueron randomizadas para recibir vehículo (n=8) o Ang-(1-9) (600 ngKg-1min-1, n=8) por 6 días. Se observó un incremento significativo(p<0.05) de PAS (33%), MVR (17%), AC (64%), PC (20%), FVCT (46%). La administración crónica de Ang-(1-9) disminuyó PAS (20%), MVR (13 %), AC (35%), PC (20%) y FVCT (20%). Estos efectos no fueron mediados por el receptor MAS. Al comparar las ratas normotensas tratadas con vehículo o Ang-(1-9), se observó un aumento significativo de la diuresis y natriuresis en los días 2 y 3 en los animales con infusión de Ang-(1-9). Conclusión: Ang-(1-9) reduce la hipertensión y el remodelamiento cardíaco en ratas hipertensas. En animales normotensos se demostró que el tratamiento con Ang-(1-9)-induce diuresis y natriuresis. Este es el primer reporte que señala que el efecto de Ang-(1-9) está asociado a una regulación del sodio a nivel renal.
Background: Angiotensin-(1-9) has anti-hypertensive properties and protective cardiovascular effect in hypertensive rats. However, it is unknown whether its effects are related to a kidney mechanism to balance sodium. Aim: To determine if the anti-hypertensive effect of Ang-(1-9) is associated to a diuretic-natriuretic mechanism. Method: Sprague Dawley male rats (200±10 grs) were randomized to receive Angiotensin II by osmotic pump (400 ng/kg/min). Sham operated rats were utilized as control (n=18). Two weeks after pump setting, Sham rats with hypertension were randomized to receive placebo (n=10), Ang-(1-9)(602 ng/kg/min, n=17) or Ang-(1-9) plus A779 (Ang-(1-7) Receptor Mas blocker, 100ng/kg-1min-1, n=7) co-administration for two weeks. Arterial systolic pressure (PAS), ventricular relative mass (MVR), cardiomyocytes area and perimeter (AC and PC) and total collagen volume fraction (FVCT) were measured. Normotensive rats were utilized to evaluate diuresis and natriuresis which were randomized to receive placebo (n=8) or Ang-(1-9) (600ng/kg-1/min-1, n=8) for six days. Results: It was observed a significant rise (p<0.05) of PAS (33%), MVR (17%), AC (64%), PC (26%), FVCT (46%) was observed. Chronic administration of Ang-(1-9) reduced PAS (20%), MVR (13%), AC (35%), PC (20%) and FCVT (20%). All those effects were not mediated by Mas receptor. A significant raise was observed of diuresis and natriuresis at the second and third day of treatment in rats receiving Ang-(1-9) in comparison with normotensive rats treated with placebo. Conclusion: Ang-(1-9) reduces hypertension and cardiac remodeling in hypertensive rats. Ang-(1-9) induces natriuresis and diuresis in normotensive rats. This is the first report showing that Ang-(1-9) is associated to sodium balance in the kidney.
Subject(s)
Animals , Rats , Angiotensin II/pharmacology , Diuresis/drug effects , Natriuresis/drug effects , Antihypertensive Agents/pharmacology , Rats, Sprague-Dawley , Heart/drug effectsABSTRACT
Antecedentes: Angiotensina [Ang-(1-9)] disminuye la presión arterial (PA) y el remodel amiento cardíaco en la hipertensión arterial experimental independiente de renina. No hay antecedentes sobre el efecto de Ang-(1-9) en la progresión de daño renal de ratas hipertensas por expansión de volumen (con renina baja). Objetivo: Determinar la participación de Ang-(1-9) en la progresión del daño renal en ratas hipertensas (DOCA-sal). Métodos: Se utilizaron ratas Sprague Dawley macho de 150 +/- 10 g uninefrectomizadas tratadas con DOCA (60mg/Kg/2 veces sem, im) por 4 semanas. Como controles (Sham) se usaron ratas uninefrectomizadas. Desde la 2a semana las ratas DOCA, con presión arterial sistólica (PAS) > 140 mmHg, recibieron vehículo o Ang-(1-9) [602 ng/Kg min] por 2 semanas (minibomba Alzet). Se evaluó la respuesta inflamatoria y el daño renal profibrótico por la presencia de macrófagos infiltrativos y de mio-fibroblastos intersticiales. Se determinó, además, la presión arterial sistólica (PAS), masa corporal (MC), masa del riñón derecho (MR) y masa renal relativa (MRR). Resultados: Se observó una disminución del daño renal en las ratas DOCA-sal, cuando recibieron Ang-(1-9), respecto a aquellas que no la recibieron, evidenciado por una significativa disminución de macrófagos infiltrativos y miofibroblastos en el intersticio renal. El bloqueo de los receptores Mas y AT2, no tuvieron efectos adicionales. Conclusion: En este modelo experimental, Ang-(1-9) disminuyó la hipertensión y redujo significativamente la infiltración de macrófagos y la aparición de miofibroblastos en el intersticio renal. Estos resultados son la primera evidencia de que Ang-(1-9) reduce la fibrosis túbulo-intersticial renal y el daño renal hipertensivo.
Angiotensin [Ang] - (1-9) decreases blood pressure (BP) and cardiac remodeling in renin independent hypertension. There are not studies about the effect of Ang- (1-9) in the progression of hypertensive renal damage by volume overload (low-renin). The aim of this study was to determine the effect of Ang- (1-9) on renal damage in volume overload hypertensive rats by (DOCA-salt rats). Methods: Male Sprague Dawley rats, 150 +/- 10 g, were uni-nephrectomized and treated with DOCA (60mg/Kg) 2 times per week, for 4 weeks. Uninephrec-tomized rats were used as controls (Sham). From the 2nd week on DOCA rats with systolic blood pressure (SBP)> 140 mmHg received vehicle or Ang- (1-9) [602 ng / kg min] for 2 weeks (Alzet minipump). Inflammatory and profibrotic renal damage was evaluated by the presence of infiltrating macrophages and interstitial myofibroblasts. Results: Compared to rats receiving vehicle renal damage in DOCA-salt rats decreased when they received Ang- (1-9), as evidenced by a significant decrease in infiltrating macrophages and myofibroblasts in the renal interstitium. Mas and AT2 receptor blockade had no additional effect. The SBP, body mass (BM), mass of the right kidney (MR) and relative renal mass (MRR) were also determined. Conclusion: in this experimental model, Ang-(1-9) decreased hypertension and significantly reduced macrophage infiltration and the appearance of myofibroblasts in the renal interstitium. These results are the first evidence that Ang-(1-9) reduces renal tubulointerstitial fibrosis and hypertensive renal damage.
Subject(s)
Male , Animals , Rats , Angiotensin I/administration & dosage , Peptide Fragments/administration & dosage , Hypertension/drug therapy , Kidney , Kidney/pathology , Angiotensin I/pharmacology , Body Composition , Fibroblasts , Peptide Fragments/pharmacology , Macrophages , Arterial Pressure , Rats, Sprague-Dawley , Renin/bloodABSTRACT
BACKGROUND: Little is known about the biological effects of angiotensin-(1-9), but available evidence shows that angiotensin-(1-9) has beneficial effects in preventing/ameliorating cardiovascular remodeling. OBJECTIVE: In this study, we evaluated whether angiotensin-(1-9) decreases hypertension and reverses experimental cardiovascular damage in the rat. METHODS AND RESULTS: Angiotensin-(1-9) (600â ng/kg per min for 2 weeks) reduced already-established hypertension in rats with early high blood pressure induced by angiotensin II infusion or renal artery clipping. Angiotensin-(1-9) also improved cardiac (assessed by echocardiography) and endothelial function in small-diameter mesenteric arteries, cardiac and aortic wall hypertrophy, fibrosis, oxidative stress, collagen and transforming growth factor type ßâ-â1 protein expression (assessed by western blot). The beneficial effect of angiotensin-(1-9) was blunted by coadministration of the angiotensin type 2(AT2) receptor blocker PD123319 (36 âng/kg per min) but not by coadministration of the Mas receptor blocker A779 (100 âng/kg per min). Angiotensin-(1-9) treatment also decreased circulating levels of Ang II, angiotensin-converting enzyme activity and oxidative stress in aorta and left ventricle. Whereas, Ang-(1-9) increased endothelial nitric oxide synthase mRNA levels in aorta as well as plasma nitrate levels. CONCLUSION: Angiotensin-(1-9) reduces hypertension, ameliorates structural alterations (hypertrophy and fibrosis), oxidative stress in the heart and aorta and improves cardiac and endothelial function in hypertensive rats. These effects were mediated by the AT2 receptor but not by the angiotensin-(1-7)/Mas receptor axis.
Subject(s)
Angiotensin II/chemistry , Angiotensin I/chemistry , Angiotensin-Converting Enzyme Inhibitors/chemistry , Cardiovascular Diseases/drug therapy , Hypertension/drug therapy , Peptide Fragments/chemistry , Angiotensin II Type 1 Receptor Blockers/chemistry , Animals , Aorta/pathology , Blood Pressure/drug effects , Cardiovascular Diseases/prevention & control , Disease Models, Animal , Echocardiography , Endothelium, Vascular/pathology , Heart Ventricles , Hemodynamics , Hypertension/physiopathology , Imidazoles/chemistry , Male , Oxidative Stress , Pyridines/chemistry , Rats , Rats, Sprague-DawleyABSTRACT
Introducción: El polimorfismo del gen de la enzima convertidora de angiotensina I (ECA) determina mayor actividad de la ECA y mayores niveles de angioten-sina (Ang) II. Un polimorfismo similar ha sido descrito en humanos. La ECA2, a través de Ang-(1-9) más que Ang-(1-7), contrarresta los efectos deletéreos de Ang II. Se desconoce si el polimorfismo de la ECA frente a un estímulo hipertensivo modifica el eje ECA2/Ang-(1-9) y determina mayor remodelamiento de la pared aórtica de ratas hipertensas. Objetivo: Determinar el efecto del polimorfismo del gen de la ECA en la actividad del eje ECA2/Ang-(1-9) y su efecto en el remodelamiento de la pared aórtica secundaria a la hipertensión arterial (HTA) experimental. Métodos: Se usaron ratas macho homocigotas de 150 gr BN y LL. Se indujo HTA por el procedimiento Goldblatt (GB, 2 K-1clip). Ratas pseudo-operadas se usaron como controles (Sham). A las 6 semanas post cirugía se determinaron en la aorta las actividades de ECA y ECA2, los niveles de Ang II/Ang-(1-9), colágeno tipo I, células positivas para el marcador de inflamación ED-1, área y grosor de la túnica media (ATM, GTM). Resultados: El polimorfismo de la ECA con mayores niveles de ECA y Ang II determinó una mayor disminución de la actividad de ECA2, menores niveles de Ang-(1-9) y mayor remodelamiento de la pared aórtica tanto en animales normotensos como hipertensos. Conclusión: El polimorfismo de la ECA con mayor actividad de ECA y AngII determina una interregu-lación de los ejes ECA/AngII y ECA2/Ang-(1-9) lo que se asocia a mayor remodelamiento de la pared aórtica. Fondecyt 1100874.
background: The angiotensin converting enzyme (ACE) gene polymorphism determines increased ACE activity and angiotensin (Ang) II levels in Brown Norway rats (BN), compared to Lewis rats (LL). Similar polymorphism has been described in humans. ACE2 through Ang-(1-9) rather than Ang-(1-7) counteracts the deleterious effects of Ang II. It is unknown whether the ACE polymorphism counteracts the ECA2/Ang1-9 axis and determines increased remodeling of the aortic wall in hypertensive rats. Objective: To determine the effects of ACE gene polymorphism in the ECA2/Ang1-9 axis activity and its impact on the aortic wall remodeling secondary to hypertension (HT). Methods: Male homozygous rats BN and LL were used. Hypertension was induced by the Goldblatt procedure (GB, 2 K-1clip). Pseudo-operated rats were used as controls (Sham). At 6 weeks after surgery, we determined the body weight (BW) and systolic blood pressure (SBP). In aorta, we determined the ACE and ACE2 activities, Ang II/Ang1-9 levels, protein expression of collagen type I, positive cells for ED-1 inflammatory cells and medial thickness (MT) and area (MA) of aortic wall. Results: ACE polymorphism with higher levels of ACE and Ang II determined a significant decrease of ACE2 activity, Ang-(1-9) levels and aortic wall remodeling in normotensives and hypertensives rats. Conclusion: ACE polymorphism with increased ACE activity and AngII levels determines a significant inter-regulation between ACE/AngII and ACE2/Ang-(1-9) axis which is associated with increased remodeling of the aortic wall. Fondecyt 1100874.
Subject(s)
Rats , Angiotensin II/genetics , Aorta/pathology , Hypertension/genetics , Peptidyl-Dipeptidase A/genetics , Polymorphism, Genetic , Rats, Inbred BNABSTRACT
Resumen: La enzima convertidora de angiotensina I (ECA2) a través de Angiotensina (Ang)-(1-9) más que Ang-(1-7) contrarresta los efectos deletéreos de ECA y Ang II. Se desconoce si Ang-(1-9) es efectiva en el tratamiento del remodelamiento cardiovascular (RMCV) hipertensivo, en ratas con polimorfismo del gen de la ECA. Objetivo: Determinar el efecto de Ang-(1-9) en el tratamiento del RMCV hipertensivo en ratas con niveles genéticamente determinados de ECA y Ang II. Métodos: Ratas normotensas homocigotas, Lewis (LL) y Brown Norway (BN), se les indujo HTA a través del modelo Goldblatt (GB, 2 riñones-1 pinzado). Después de 4 semanas, las ratas hipertensas se rando-mizaron para recibir Ang-(1-9) (602 ng/Kg min) o una coadministración de Ang-(1-9)+A779 (100 ng/Kg min, antagonista del receptor MAS de Ang-(1-7)) durante 14 días mediante una minibomba. Como controles se usaron ratas sometidas a operación ficticia (Sham). Se determinó masa corporal (MC), presión arterial sistólica (PAS), masa ventricular (MV), área de cardiomiocitos (AC), área y grosor de la túnica media (ATM, GTM), fracción volumétrica de colágeno total (FVCT) en el ventrículo izquierdo (VI), niveles proteicos de colágeno tipo I (Col I) en la aorta (Ao) y la infiltración de macrófagos en Ao y VI, por medio de su molécula especifica ED1 (ED1-Ao, ED1-VI). Resultados: La administración de Ang-(1-9) disminuyó significativamente PAS, MV, AC, FVCT, Col I, ATM, GTM, ED1-Ao (-) y ED1-VI, en las ratas hipertensas LL y BN respecto a las ratas GB sin tratamiento, respectivamente. Este efecto no fue inhibido por el antagonista A779. El polimorfismo de la ECA no modificó la respuesta al tratamiento. Conclusión: Ang-(1-9) redujo eficazmente la HTA y el RMCV secundario, independiente al polimorfismo en el gen de la ECA. Este efecto posiblemente es directo ya que no fue mediado por Ang-(1-7). Fondecyt 1100874.
Background: The angiotensin I converting enzyme 2 (ACE2) counteracts the deleterious effects of ACE and Ang II through angiotensin (Ang) -(1-9) rather than Ang-(1-7). In addition, it is not clear whether Ang-(1-9) is effective in the reversal of hypertensive cardiovascular remodeling (CVRM) in rats with ACE gene polymorphism. Objective: To determine the effect of Ang-(1-9) in the prevention of hypertensive CVRM in rats with genetically determined levels of ACE and Ang II. Methods: In normotensive homozygous Lewis (LL) and Brown Norway (BN) rats hypertension was induced by the Goldblatt 2 kidney-1 pinch model. After 4 weeks, rats were randomized to receive Ang- (1-9) (602 ng / Kg min) or the co administration of Ang- (19) + A779 (100 ng / kg min, a MAS receptor antagonist of Ang- (1-7)) for 14 days. Sham operated rats were used as controls. We determined body mass (BM), systolic blood pressure (SBP), ventricular mass (VM), cardiomyocyte area (CA), area and thickness of the aortic media (ATM, TTM), LV total collagen volume fraction (FVCT), type I collagen protein levels (Col I) in the aorta (Ao) and macrophage infiltration in LV and Ao, through its specific molecule ED1 (ED1-Ao, ED1-VI). Results: Continuous administration of Ang- (1-9) significantly decreased SBP, VM, CA, TCVF, Col I, TTM, and ED1 in the aorta and left ventricle of hypertensive rats. This effect was not inhibited by the antagonist A779. ACE polymorphism did not modify the response to treatment. Conclusion: Ang- (1-9) effectively reduced hypertension induced CVRM independent of ACE gene polymorphism. This effect was not mediated by Ang- (1-7).
Subject(s)
Animals , Rats , Hypertension/chemically induced , Peptidyl-Dipeptidase A/administration & dosage , Peptidyl-Dipeptidase A/genetics , Cardiovascular System/pathology , Polymorphism, Genetic , Cardiovascular System/enzymologyABSTRACT
Antecedentes: La sobreexpresion génica de la enzima convertidora de angiotensina I homologa (ECA2) se asocia con prevención de la hipertrofia y fibrosis cardiaca dependiente de angiotensina (Ang) II. Sin embargo se desconoce si su sobreexpresion reduce la hipertensión arterial (HTA) y revierte el consecuente remodelado miocárdico (RM) dependiente de Ang II. Objetivo: Determinar si la sobreexpresion adenoviral (Ad) del gen de la ECA2 en el miocardio disminuye la HTA y RM experimental en ratas con niveles genéticamente determinados de ECA y Ang II. Métodos: Ratas homocigotas normotensas Lewis (LL) y Brown Borway (BN), con menores y mayores niveles circulantes de ECA y Ang II, respectivamente, se hicieron hipertensas por el procedimiento Goldblatt (GB). Como controles se usaron ratas seudo-operadas (sham). A la semana 5 post cirugía y con HTA establecida > 140 mmHg, las ratas se randomizaron a inyección intramiocárdica con un AdECA2 o Ad proteína fluorescente verde (GFP) como controles de infección. A la semana post infección adenoviral, los ratas se sacrificaron y se determinaron peso corporal (PC, g), masa cardiaca (MC, mg), presión arterial sistólica (RAS, mmHg), área (AC, um2) y perímetro (PERC, um) de cardiomiocitos y contenido de colágeno ( por ciento) miocárdico (CM), sub-endocárdico (CS)ytotal(CT). Resultados: La HTA aumentó significativamente la MC, MCR, AC, PERC como también el CM, CS y CT en las ratas GB vs Sham, sin diferencias en el PC ni por efecto del polimorfismo de la ECA. La sobreexpresion de ECA2 disminuyó significativamente la RAS (15 por ciento y 27 por ciento), AC (25 por ciento y 25 por ciento ) y PERC (17 por ciento y 18 por ciento) en las ratas LL y BN vs ratas hipertensas, respectivamente. Estos resultados se asociaron a una disminución significativa del CS (LL = 37 por ciento, BN = 39 por ciento), CM (LL = 54 por ciento) y CT (LL = 42 por ciento, BN = 22 por ciento) respecto a las ratas GB. Conclusión: En ratas con HTA...
Objective Background. Over expression of the gene for homologous angiotensin I converting enzyme (ACE) is associated with prevention of angiotensin II dependent cardiac hypertrophy and fibrosis. Whether this over expression is able to reduce hypertension and revert cardiac remodeling is unknown. Aim: To determine whether adenoviral ACE2 gene over expression in the myocardium decreases experimental hypertension and cardiac remodeling in rats with genetically determined levels of ACE and ANGII. Methods: Homozygous normotensive Lewis (LL) and Brown Norway (BN) rats with decreased or increased levéis of ACE and Ang II, respectively, were made hy-pertensive using the Goldblatt procedure. Sham opera-ted rats were used as control s. 5 weeks after surgery, when systolic blood pressure reached > 140 mmHg, rats were randomized to receive intramyocardial injec-tion of either AdACE2 or green fluorescent Ad protein (GFP) as infection controlling agents. One week after adenoviral infection, rats were sacrificed. Body weight (BW, Gr), relative cardiac mass (RCM, mG), systolic blood pressure (SBP, mmHg), cardiomyocite área (MA um2) and perimeter (MPER, uM), and myocardial co-llagen content, both subendocardial (SC, percent) and total (TC percent) were measured. Results: Hypertension was associated to significant in-creases in total and RCM, MA, MPER as well as SC and TC in Goldblatt vs normal rats. This was independent of BW and not affected by ACE polymorphism. ACE II over expression significantly decreased SBP (27 vs 15 percent), MA (25 vs 25 percent) and MPER (18 vs 17 percent) hy-pertensive vs normal rats, respectively. Conclusion: Over expression of ACE2 decreased hypertension, hypertrophy and fibrosis in rats with experimental hypertension and different levels of ACE and Ang II. (Fondecyt 1070662).
Subject(s)
Male , Animals , Rats , Heart , Hypertension/drug therapy , Peptidyl-Dipeptidase A/administration & dosage , Ventricular Remodeling , Body Weight , Cardiomegaly/prevention & control , Fibrosis , Endomyocardial Fibrosis/prevention & control , Gene Expression , Genetic Therapy , Myocytes, Cardiac , Myocardium/pathology , Arterial Pressure , Rats, Inbred Strains , Organ SizeABSTRACT
Antecedentes: Recientemente hemos propuesto en un modelo experimental de infarto al miocardio una significativa interregulación entre los niveles de la enzima convertidora de angiotensina I (ECA) y su homóloga (ECA-2), junto con que angiotensina (Ang)-(1-9) más que Ang-(1-7) actuaría como uncontrarregulador de Ang II. Sin embargo tal relación no se ha investigado en el remodelado aórtico hipertensivo. Objetivo: Determinar la expresión de ECA y ECA-2, los niveles de Angs I, II, (1-7) y (1-9) y los parámetros de remodelado de la pared aórtica de ratas hipertensas. Métodos: Ratas normotensas Lewis (n=18) fueron randomizadas a hipertensión (HTA) por sobrecarga de presión (modelo Goldblatt, GB, 2 riñones-1 pinzado, n=9). Ratas pseudo-operadas se usaron como controles (S, n=9). A las 6 semanas post cirugía, se determinó la masa cardíaca relativa (MCR) y la presión arterial sistólica (PAS). En la aorta torácica se determinó el grosor de la túnica media (GTM), área de la TM (ATM), niveles de mRNA de ECA y ECA-2, factor de crecimiento transformante tipo beta (TGF-beta), inhibidor del activador de plasminógeno (PAI-1) y de la proteína quimioatractante de monocitos (MCP-1) por RT-PCR. La actividad y niveles proteicos de ECA y ECA-2 por fluorimetría y Western blot y los niveles de Angs I, II, (1-7) y (1-9) por HPLC y radioinmunoensayo. Resultados: La MCR y la PAS aumentaron significativamente (p<0,05) en el grupo GB respecto a su control S. Las ratas hipertensas mostraron un aumento significativo (p<0.05) del GTM (18 por ciento), ATM (31 por ciento), niveles de mRNA de ECA (164 por ciento), TGF-beta (105 por ciento), PAI-1(51 por ciento), MCP-1 (53 por ciento) junto con mayor actividad (89 por ciento), niveles proteicos de ECA (130 por ciento) y Ang II (48 por ciento). Esos efectos se asociaron a una significativa disminución del mRNA, los niveles proteicos y actividad...
Background: In experimental models of myocardial infarction we have recently proposed a significantinter-regulation between levels of Angiotensin I converting enzyme (ACE) and its homologous, ACE-2; in addition, we have proposed that Angiotensin 1-9 (Ang-(1-9)) rather than Ang-(1-7) counter regulates Ang II. These relations have not been investigated in hypertensive aortic wall remodeling. Aim: To measure de expression of ACE and ACE-2, the aortic wall levels of Ang I, Ang II, Ang-(1-7) and Ang-(1-9), along with parameters of aortic wall remodeling in hypertensive rats. Methods: 18 Lewis rats were randomized to Goldblatt (2 kidneys, 1 clamped) induced hypertension (n=9) or sham operation (controls, n=9). Six weeks after surgery, relative cardiac mass (RCM), systolic blood pressure (SBP), medial layer aortic wall thickness (MLT) and ML area (MLA) were measured. The aortic wall levels of ACE and ACE-2, tissue growth factor beta (TGF- beta), plasminogen activator inhibitor (PAI-1) and monocyte chemoattractant protein (MCP-1) were determined by RT-PCR. Activity and protein levels of ACE and ACE-2 were measured by fluorometry and Western Blot and ANG I, Ang II, Ang-(1-7) and Ang-(1-9) levels were determined using HPLC and radioimmunoassay. Results: RCM and SBP increased significantly in hypertensive as opossed to sham operated rats...
Subject(s)
Animals , Rats , Angiotensin I/physiology , Angiotensin II/physiology , Aorta, Thoracic/enzymology , Hypertension , Peptidyl-Dipeptidase A/physiology , Renin-Angiotensin SystemABSTRACT
Introducción: El polimorfismo de la enzima convertidora de angiotensina I (ECA) determina mayor actividadde ECA y niveles de angiotensina (Ang) II en ratas Brown Norway (BN) y menor actividad de ECA y niveles de Ang II en ratas Lewis (L). La interregulación entre ECA, ECA2 y su relación con remodelamiento aórtico hipertensivo no ha sido explorada Objetivo: Determinar la expresión de ECA y ECA2 y los parámetros de remodelamiento vascular hipertensivo en la aorta de ratas con niveles genéticamente determinados de ECA. Métodos: A ratas macho homocigotas de 150 grs BN y LL, se les indujo HTA por 6 semanas por el procedimiento Goldblatt (GB, 2 K-1clip). Ratas pseudo-operadas se usaron como controles (sham). Se determinó la presión arterial sistólica (PAS), el grosor de la túnica media (GTM), área de la TM (ATM), expresión génica deECA, ECA2 ,TGF-beta, PAI-1 y MCP-1 por RT-PCR y también proteica de ECA y ECA2 por Western Blot. Resultados: La masa cardiaca relativa y la PAS aumentaron significativamente en los grupos GB respecto a sus controles Sham, sin diferencias por efecto del polimorfismo de la ECA. En condiciones de normotensión las ratas BN mostraron que la pared aórtica expresa mayores niveles génicos y proteicos de la ECA(60% y 134%, respectivamente) y menores de ECA2 (74% y 73%, respectivamente) respecto de las ratas L(p<0.05). Estos resultados se asociaron con mayores GTM y ATM como en los niveles de mRNA de TGF-beta y, PAI-1 en las aortas de ratas BN respecto de las ratas L (p<0,05). En respuesta a un estrés hipertensivo las ratas con mayores niveles de ECA y menores niveles de ECA2 mostraron mayor GTM (p<0,05, respecto de GB-L), sin diferencias en los otros parámetros evaluados...
Background: Angiotensin I converting enzyme (ACE) polymorphism determines increased ACE and Ang IIlevels in Brown Norway rats (BN) and decreased ACE and Ang II levels in Lewis (L) rats. The interactionbetween ACE and ACE2 in relation to aortic remodeling associated to hypertension has not been explored. Aim: to determine the expression of ACE and ACE2 along with parameters of remodeling in hypertensive rats with genetically determined levels of ACE. Methods: BN and L rats weighing 150 g were made hypertensive by the Goldblatt procedure (GB, 2K-1 clip). Sham operated rats were used as controls. Systolic blood pressure (SBP), media thickness (MT), and MT area were measured. RT-PCR was used to determine the genetic expression of ACE, ACE2, TGF-beta, PAI-1 and MCP-1. Western Blot was used to measure the protein fraction of ACE and ACE2 Results: Relative cardiac mass and SBP increases significantly in GB rats compared to controls; ACE polymorphism did not influence this effect. The aortic wall of normotensive BN rats expressed increased genic and protein levels of ACE (60% and 134%, respectively) and decreased levels of ACE2 (74% and 73%, respectively) compared to L rats (p<0.05). These findings were associated to increased MT and MT area as well as increased mRNA for TGF-beta and PAI 1 in BN rats compared to L rats (p<0.05). In response to hypertensive stress, rats with increased ACE and decreased ACE2 levels developed increased MT compared to GB-L rats; other parameters of remodeling were not affected...