RESUMO
Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.
Assuntos
Endotélio Vascular/fisiopatologia , Hipertensão/fisiopatologia , Estresse Oxidativo , Peptidil Dipeptidase A/metabolismo , Vasodilatação/fisiologia , Enzima de Conversão de Angiotensina 2 , Animais , Aorta Torácica/citologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ativação Enzimática , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/enzimologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Peptidil Dipeptidase A/efeitos dos fármacos , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Vasodilatação/efeitos dos fármacos , Xantonas/farmacologiaRESUMO
We evaluated the hypothesis that activation of endogenous angiotensin-converting enzyme (ACE) 2 would improve cardiac dysfunction induced by diabetes. Ten days after diabetes induction (streptozotocin, 50 mg/kg, i.v.), male Wistar rats were treated with the ACE2 activator 1-[[2-(dimethylamino)ethyl]amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl)sulfonyl]oxy]-9H-xanthen-9-one (XNT, 1 mg/kg/day, gavage) or saline (control) for 30 days. Echocardiography was performed to analyze the cardiac function and kinetic fluorogenic assays were used to determine cardiac ACE and ACE2 activities. Cardiac ACE2, ACE, Mas receptor, AT(1) receptor, AT(2) receptor and collagen types I and III mRNA and ACE2, ACE, Mas, AT(1) receptor, AT(2) receptor, ERK1/2, Akt, AMPK-α and AMPK-ß(1) protein were measured by qRT-PCR and western blotting techniques, respectively. Histological sections of hearts were analyzed to evaluate the presence of hypertrophy and fibrosis. Diabetic animals presented hyperglycemia and diastolic dysfunction along with cardiac hypertrophy and fibrosis. XNT treatment prevented further increase in glycemia and improved the cardiac function, as well as the hypertrophy and fibrosis. These effects were associated with increases in cardiac ACE2/ACE ratios (activity: ~26%; mRNA: ~113%; and protein: ~188%) and with a decrease in AT(1) receptor expression. Additionally, XNT inhibited ERK1/2 phosphorylation and prevented changes in AMPK-α and AMPK-ß(1) expressions. XNT treatment did not induce any significant change in AT(2) receptor and Akt expression. These results indicate that activation of intrinsic cardiac ACE2 by oral XNT treatment protects the heart against diabetes-induced dysfunction through mechanisms involving ACE, ACE2, ERK1/2, AMPK-α and AMPK-ß(1) modulations.
Assuntos
Cardiomegalia/tratamento farmacológico , Coração/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , Disfunção Ventricular/tratamento farmacológico , Xantonas/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Western Blotting , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Cardiotônicos/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/fisiopatologia , Avaliação Pré-Clínica de Medicamentos , Ecocardiografia , Ativação Enzimática , Coração/fisiopatologia , Hiperglicemia/induzido quimicamente , Hiperglicemia/metabolismo , Hiperglicemia/fisiopatologia , Sistema de Sinalização das MAP Quinases , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Disfunção Ventricular/induzido quimicamente , Disfunção Ventricular/fisiopatologiaRESUMO
Our previous studies have indicated that chronic treatment with 1-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one (XNT), an angiotensin-converting enzyme 2 (ACE2) activator, reverses hypertension-induced cardiac and renal fibrosis in spontaneously hypertensive rats (SHRs). Furthermore, XNT prevented pulmonary vascular remodelling and right ventricular hypertrophy and fibrosis in a rat model of monocrotaline-induced pulmonary hypertension. The aim of this study was to determine the mechanisms underlying the protective effects of XNT against cardiac fibrosis. Hydroxyproline assay was used to measure cardiac collagen content in control and XNT-treated (200 ng kg(-1) min(-1) for 28 days) SHRs. Cardiac ACE2 activity and protein levels were determined using the fluorogenic peptide assay and Western blot analysis, respectively. Extracellular signal-regulated kinases (ERKs; p44 and p42) and angiotensin II type 1 (AT(1)) receptor levels were quantified by Western blotting. Cardiac ACE2 protein levels were â¼15% lower in SHRs compared with Wistar-Kyoto control animals (ACE2/glyceraldehyde 3-phosphate dehydrogenase ratio: Wistar-Kyoto, 1.00 ± 0.02 versus SHR, 0.87 ± 0.01). However, treatment of SHRs with XNT completely restored the decreased cardiac ACE2 levels. Also, chronic infusion of XNT significantly increased cardiac ACE2 activity in SHRs. This increase in ACE2 activity was associated with decreased cardiac collagen content. Furthermore, the antifibrotic effect of XNT correlated with increased cardiac angiotensin-(1-7) immunostaining, though no change in cardiac AT(1) protein levels was observed. The beneficial effects of XNT were also accompanied by a reduction in ERK phosphorylation (phospho-ERK/total ERK ratio: Wistar-Kyoto, 1.00 ± 0.04; control SHR, 1.46 ± 0.25; treated SHR, 0.86 ± 0.02). Our observations demonstrate that XNT activates cardiac ACE2 and inhibits fibrosis. These effects are associated with increases in angiotensin-(1-7) and inhibition of cardiac ERK signalling.