Effects of Chronic Exposure to Mercury on Angiotensin-Converting Enzyme Activity and Oxidative Stress in Normotensive and Hypertensive Rats / Efeitos da Exposição Crônica ao Mercúrio sobre a Atividade da Enzima Conversora de Angiotensina e Estresse Oxidativo em Ratos Normotensos e Hipertensos

Abstract Background: Mercury's deleterious effects are associated with increased cardiovascular risk. Objective: To determine whether chronic exposure to inorganic mercury increases the activity of angiotensin-converting enzyme and its relationship with oxidative stress in several organs and tissues. Methods: We studied male Wistar and spontaneously hypertensive rats (SHR) (3-month-old) exposed or not to HgCl2 for 30 days. At the end of treatment, we investigated the following: changes in body weight, hemodynamic parameters, angiotensin-converting enzyme (ACE) activity and oxidative stress in the heart, aorta, lung, brain and kidney in hypertensive compared to normotensive animals. A value of p < 0.05 was considered significant. Results: Chronic exposure to HgCl2 did not affect weight gain in either group. Systolic blood pressure, measured weekly, did not increase in Wistar rats but showed a small increase in SHR rats. We also observed increases in left ventricular end-diastolic pressure and ACE activity in the plasma and hearts of normotensive rats. In the SHR+Hg group, ACE activity increased in plasma but decreased in kidney, lung, heart, brain and aorta. Oxidative stress was assessed indirectly by malondialdehyde (MDA) production, which increased in Hg-treated rats in both plasma and heart. In the SHR+Hg group, MDA increased in heart and aorta and decreased in lungs and brain. Conclusion: These results suggest that chronic exposure to inorganic mercury aggravates hypertension and produces more expressive changes in ACE activity and oxidative stress in SHRs. Such exposure affects the cardiovascular system, representing a risk factor for the development of cardiovascular disorders in normotensive rats and worsening of pre-existing risks for hypertension.

Resumo Fundamento: Os efeitos deletérios do mercúrio estão associados ao risco cardiovascular aumentado. Objetivo: Determinar se a exposição crônica ao mercúrio inorgânico aumenta a atividade da enzima conversora de angiotensina e sua relação com o estresse oxidativo em vários órgãos e tecidos. Métodos: Estudamos ratos Wistar e ratos espontaneamente hipertensos (SHR) (3 meses de idade) expostos ou não a HgCl2 por 30 dias. Ao final do tratamento, investigamos: alterações de peso, parâmetros hemodinâmicos, atividade da enzima conversora de angiotensina (ECA) e estresse oxidativo no coração, aorta, pulmão, cérebro e rim de animais hipertensos comparados a animais normotensos. Um valor de p < 0,05 foi considerado significativo. Resultados: A exposição crônica ao HgCl2 não afetou o ganho de peso em nenhum dos grupos. A pressão arterial sistólica, medida semanalmente, não aumentou em ratos Wistar, mas mostrou um pequeno aumento nos ratos SHR. Também observamos aumentos na pressão diastólica final do ventrículo esquerdo e na atividade da ECA no plasma e no coração de ratos normotensos. No grupo SHR + Hg, a atividade da ECA aumentou no plasma, mas diminuiu no rim, pulmão, coração, cérebro e aorta. O estresse oxidativo foi avaliado indiretamente pela produção de MDA, que aumentou nos ratos tratados com Hg tanto no plasma quanto no coração. No grupo SHR + Hg, o MDA aumentou no coração e na aorta e diminuiu nos pulmões e no cérebro. Conclusão: Estes resultados sugerem que a exposição crônica ao mercúrio inorgânico agrava a hipertensão e produz mudanças mais expressivas na atividade da ECA e no estresse oxidativo em SHRs. Essa exposição afeta o sistema cardiovascular, representando um fator de risco para o desenvolvimento de distúrbios cardiovasculares em ratos normotensos e para piorar riscos pré-existentes para hipertensão.

Quantitative expression and localization of cysteine and aspartic proteases in human abdominal aortic aneurysms

Cysteine and aspartic proteases possess high elastolytic activity and might contribute to the degradation of the abdominal aortic aneurysm (AAA) wall. The aim of this study was to analyze, in detail, the proteases (cathepsins B, D, K, L and S, and inhibitor cystatin C) found in human AAA and healthy aortic tissue samples. The vessel walls from AAA patients (n=36) and nonaneurysmal aortae (n=10) were retrieved using conventional surgical repair and autopsy methods. Serum samples from the same AAA patients and 10 healthy volunteers were also collected. Quantitative expression analyses were performed at the mRNA level using real-time reverse transcriptase-PCR (RT-PCR). Furthermore, analyses at the protein level included western blot and immunoprecipitation analyses. Cellular sources of cysteine/aspartic proteases and cystatin C were identified by immunohistochemistry (IHC). All cysteine/aspartic proteases and cystatin C were detected in the AAA and control samples. Using quantitative RT-PCR, a significant increase in expression was observed for cathepsins B (P=0.021) and L (P=0.018), compared with the controls. Cathepsin B and cystatin C were also detected in the serum of AAA patients. Using IHC, smooth muscle cells (SMCs) and macrophages were positive for all of the tested cathepsins, as well as cystatin C; in addition, the lymphocytes were mainly positive for cathepsin B, followed by cathepsins D and S. All cysteine/aspartic proteases analyzed in our study were detected in the AAA and healthy aorta. The highest expression was found in macrophages and SMCs. Consequently, cysteine/aspartic proteases might play a substantial role in AAA.

Increased arginase II activity contributes to endothelial dysfunction through endothelial nitric oxide synthase uncoupling in aged mice

The incidence of cardiovascular disease is predicted to increase as the population ages. There is accumulating evidence that arginase upregulation is associated with impaired endothelial function. Here, we demonstrate that arginase II (ArgII) is upregulated in aortic vessels of aged mice and contributes to decreased nitric oxide (NO) generation and increased reactive oxygen species (ROS) production via endothelial nitric oxide synthase (eNOS) uncoupling. Inhibiting ArgII with small interfering RNA technique restored eNOS coupling to that observed in young mice and increased NO generation and decreased ROS production. Furthermore, enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxation responses to acetylcholine in aged vasculature were markedly improved following siRNA treatment against ArgII. These results might be associated with increased L-arginine bioavailability. Collectively, these results suggest that ArgII may be a valuable target in age-dependent vascular diseases.

The methyl ester of rosuvastatin elicited an endothelium-independent and 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent relaxant effect in rat aorta

The relaxant effect of the methyl ester of rosuvastatin was evaluated on aortic rings from male Wistar rats (250-300 g, 6 rats for each experimental group) with and without endothelium precontracted with 1.0 µM phenylephrine. The methyl ester presented a slightly greater potency than rosuvastatin in relaxing aortic rings, with log IC50 values of -6.88 and -6.07 M, respectively. Unlike rosuvastatin, the effect of its methyl ester was endothelium-independent. Pretreatment with 10 µM indomethacin did not inhibit, and pretreatment with 1 mM mevalonate only modestly inhibited the relaxant effect of the methyl ester. Nω-nitro-L-arginine methyl ester (L-NAME, 10 µM), the selective nitric oxide-2 (NO-2) inhibitor 1400 W (10 µM), tetraethylammonium (TEA, 10 mM), and cycloheximide (10 µM) partially inhibited the relaxant effect of the methyl ester on endothelium-denuded aortic rings. However, the combination of TEA plus either L-NAME or cycloheximide completely inhibited the relaxant effect. Inducible NO synthase (NOS-2) was only present in endothelium-denuded aortic rings, as demonstrated by immunoblot with methyl ester-treated rings. In conclusion, whereas rosuvastatin was associated with a relaxant effect dependent on endothelium and hydroxymethylglutaryl coenzyme A reductase in rat aorta, the methyl ester of rosuvastatin exhibited an endothelium-independent and only slightly hydroxymethylglutaryl coenzyme A reductase-dependent relaxant effect. Both NO produced by NOS-2 and K+ channels are involved in the relaxant effect of the methyl ester of rosuvastatin.

Role of NADPH oxidase in the endothelial dysfunction and oxidative stress in aorta of aged spontaneous hypertensive rats

Increased reactive oxygen species [ROS] production is implicated in the pathogenesis of arterial hypertension and the development of endothelial dysfunction. NADPH oxidase type enzyme family has been suggested to form ROS and to interfere with endotheli urn-dependent relaxation. However, the specific isoform of NADPH oxidases that may predominantly contribute to these events remains to be clarified. Here we investigated the expressional regulation of NADPH oxidase isoforms [NOX[1], NOX[2] and NOX[4]] in aorta of aged spontaneously hypertensive rats [SHR] in comparison to age matched Wistar Kyoto rats [WKY]. Moreover, we examined the effect of in vitro inhibition of NADPH oxidase by apocynin or the novel NADPH oxidase inhibitor, VAS2870 on the vascular reactivity and ROS production. Our results showed that ROS formation was largely increased in aorta of SHR as measured by dihydroethidine [DHE] fluorescence and inhibited by apocynin or VAS2870. NADPH oxidase activity, measured by lucigenin-enhanced chemiluminescence and of NOX[1] and NOX[2] protein levels were increased in aortic homogenates from SHR compared to WKY. However, NOX[4] protein expression was not significantly changed. Furthermore, the impaired acetylcholine-induced relaxation of SHR aorta was significantly improved in the presence of either apocynin or VAS2870. Collectively, our data suggest that NADPH oxidases, particularly NOX[1] and NOX[2] are relevant sources of ROS in the aorta of aged SHR thereby cause endothelial dysfunction, and VAS2870 is effective as apocynin in reversing these consequences

Mayor expresión de la enzima convertidora de angiotensina I homóloga (ECA2) en la pared arterial de ratas con niveles genéticamente elevados de angiotensina II: efectos decandesartán y fasudil / Increased expression of homologous Angiotensin I converting enzyme (ACE2) in the arterial wall of rats with genetically elevated levels of Angiotensin II: effects of candesartan and fasudil

El polimorfismo de la enzima convertidora de angiotensina I (ECA) determina mayor actividad de 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 relación entre ECA, ECA2, la estimulación del receptor de angiotensina (Ang) II y la vía transduccional Rho A/Rho kinasa no ha sido explorada. Objetivo: Determinar la actividad y expresión de ECA2 y eNOS en la aorta de ratas con niveles genéticamente elevados de ECA y Ang II y los efectos independientes de la inhibición del receptor de Ang II (RAT1) y de Rho kinasa (ROCK). Métodos: Se usaron ratas macho homocigotos de 150 grs BN y L. Para inhibir ROCK, se administró fasudil (100 mg/Kg/día por gavage) y para inhibir el RAT1 se administró candesartán (10 mg/kg/día por gavage) a ratas BN, durante 7 días. Se determinó la presión arterial sistólica (PAS), la actividad circulante de ECA y ECA2 por fluorimetría y la expresión génica de ECA2 y de eNOS por RT-PCR (en unidades de densidad óptica).Resultados como promedio(ES). Conclusión: Los mayores niveles de ECA y AngII están asociados a menor actividad circulante de ECA2 en ratas normotensas. Candesartán y fasudil aumentaron la actividad y expresión génica de ECA2 en la pared arterial de ratas BN, efecto que fue mayor al inhibir ROCK. El aumento de ECA2 se asoció a mayor expresión génica de eNOS independientemente de la vía inhibida. Estos resultados permiten plantear que óxido nítrico y ROCK pudieran ser activadores endógenos de ECA2.

Background: Angiotensin I converting enzyme (ACE) polymorphism is associated with increased ACE activity and angiotensin II (A-II) levels in Brown Norway (BN) rats and decreased ACE and A-II levels in Lewis (L) rats. The relationship of ACE, ACE 2, stimulation of A-II receptor and activity of the Rho A/ Rho kinase transductional pathway is not known. Aim: To determine the activity and expression of ACE 2 and eNOS in the aortic wall of rats with genetically elevated leves of ACE and A-II and the independent effects of A-II receptor (ART2) and Rho kinase (ROCK) inhibition. Methods: Homozygous BN and L male rats wighing 150g were used. Fasudil (100mg/kg/day via gavage) was administer to inhibit ROCK and candesartan (10 mg/Kg/day) was given to inhibit ART1, during 7 days. Systolic blood pressure (SBP, ACE and ACE2 circulating activity was measured by fluorimetry. Genetic expression of ACE2 and eNOS was determined by RT-PCR (optical density units). Results are expressed as mean +/- SEM.

Hypercholesterolemia and tissue-specific differential mRNA expression of type-1 5'-iodothyronine deiodinase under different selenium status in rats

Type-1 5'-iodothyronine deiodinase (5'-DI) is responsible for conversion of T4 to T3. Selenium (Se) is an integral part of this enzyme. Keeping in view the strong association between atherosclerosis and hypothyroidism, the present study examined the behavior of 5'-DI in liver, aorta and thyroid during hypercholesterolemia following different Se status, i.e., Se deficiency (0.02ppm), adequate (0.2ppm) and excess dose (1ppm) in SD male rats. Animals were fed a control or high-cholesterol diet (2%) for 1 and 2 months. 5'-DI activity and mRNA expression was measured by RIA and RT-PCR respectively. In liver and aorta, 5'-DI expression significantly decreased with the Se-deficient and the high-cholesterol diet. The trend was opposite in thyroid, i.e., mRNA expression increased significantly during selenium deficiency and with a high-cholesterol feeding. But with 1ppm Se supplementation, the 5'-DI expression increased in all the three tissues. The present study indicates that hypercholesterolemia along with selenium deficiency is co-responsible for differential regulation of 5'-DI enzyme in thyroidal vs. extrathyroidal tissues. Distinct regulation of 5'-DI in the thyroid reflects the clinical importance of this selenoprotein during hypercholesterolemia as this enzyme is essential for T3 production, which further has a vital role in the maintenance of lipid metabolism.

Mayor actividad de NADPH oxidasa vascular en ratas con niveles elevados de enzima convertidora de angiotensina I / Increased activity of vascular NADPH oxidase in rats with elevated levels of angiotensin I converting

Introducción: La actividad de la enzima convertidora de angiotensina I (ECA) está determinada importantemente por un polimorfismo de la misma enzima. Las ratas Brown Norway (BB) tienen mayor actividad de ECA (y niveles de angiotensina II) que ratas Lewis (LL). Debido a que Ang II induce la activación de NADPH oxidasa, se postula que el polimorfismo BB determina mayor actividad de NADPH oxidasa y mayor producción de anión superóxido (O2). Métodos: Se usaron ratas homocigotas BB y LL, que tienen niveles altos y bajos de ECA, respectivamente. La actividad enzimática de ECA plasmática se determinó por fluorimetría. La actividad enzimática de NADPH oxidasa (unidades relativas de luz por segundo, por mg de proteína; URL/seg/mg/ prot) y la producción de O2 (URL) se determinaron por quimioluminiscencia con lucigenina en aorta y miocardio.

Coexpression of cyclooxygenase-2 and matrix metalloproteinases in human aortic atherosclerotic lesions

Inflammation appears to have a major role in the development of atherosclerosis. Cyclooxygenase-2 (COX-2) is involved in the inflammatory response via the generation of prostanoids that, in turn, are involved in the production of matrix metalloproteinases (MMPs). This study aimed to investigate atherosclerosis in human aortas for in situ tissue distribution of COX-2, MMPs including MMP-9 and membrane type 1 MMP (MT1-MMP), and tissue inhibitor of metalloproteinase-2 (TIMP-2). Immunohistochemical studies were performed on atherosclerotic lesions of aortas from patients with aortic aneurysms (n = 4) and dissections (n = 3) by using antibodies to COX-2, MMP-9, MT1-MMP, and TIMP-2. Control tissues were obtained from traumatically dissected aortas (n = 2). All specimens from diseased aortas had atherosclerotic lesions ranging from fatty streak to atheromatous plaques. In control, there was no expression of COX-2, MMP-9, and MT1-MMP in all aortic layers. Immunoreactivity for COX-2 was predominantly noted in macrophages and smooth muscle cells (SMCs) of the intima including atherosclerotic plaque itself and the medial layer of the plaque base, as well as in SMCs and endothelial lining of the vasa vasorum in the adventitia. Immunoreactivity for MMP-9 and MT1-MMP was found in the same distribution as that of COX-2. Additionally, the expression of TIMP-2 increased in relation to MMP-9 expression. This study demonstrates that COX-2 is coexpressed with MMP-9 and MT1-MMP, not only by macrophages and SMCs in atherosclerotic lesions, but also in endothelial lining of the vasa vasorum of human aortas. Thus, vascular inflammatory reactions may influence extracellular matrix remodeling by coactivation of MMPs in the development of atherosclerosis and, in turn, the progression of disease.

Differential effects of isoproterenol on the activity of angiotensin-converting enzyme in the rat heart and aorta

The excessive stimulation of beta-adrenergic receptors in the heart induces myocardial hypertrophy. There are several experimental data suggesting that this hypertrophy may also depend, at least partially, on the increase of local production of angiotensin II secondary to the activation of the cardiac renin-angiotensin system. In this study we investigated the effects of isoproterenol on the activity of angiotensin-converting enzyme (ACE) in the heart and also in the aorta and plasma. Male Wistar rats weighing 250 to 305 g were treated with a dose of (ñ)-isoproterenol (0.3 mg kg-1 day-1, N = 8) sufficient to produce cardiac hypertrophy without deleterious effects on the pumping capacity of the heart. Control rats (N = 7) were treated with vehicle (corn oil). The animals were killed one week later. ACE activity was determined in vitro in the four cardiac chambers, aorta and plasma by a fluorimetric assay. A significant hypertrophy was observed in both ventricular chambers. ACE activity in the atria remained constant after isoproterenol treatment. There was a significant increase (P<0.05) of ACE activity in the right ventricle (6.9 = 0.9 to 8.2 = 0.6 nmol His-Leu g-1 min-1) and in the left ventricle (6.4 ñ 1.1 to 8.9 ñ 0.8 nmol His-Leu g-1 min-1). In the aorta, however, ACE activity decreased (P<0.01) after isoproterenol (41 = 3 to 27 = 2 nmol His-Leu g-1 min-1) while it remained unchanged in the plasma. These data suggest that ACE expression in the heart can be increased by stimulation of beta-adrenoceptors. However, this effect is not observed on other local renin-angiotensin systems, such as the aorta. Our data also suggest that the increased sympathetic discharge and the elevated plasma concentration of catecholamines may contribute to the upregulation of ACE expression in the heart after myocardial infarction and heart failure

Effect of alpha-tocopherol on isoproterenol induced myocardial infarction in rats--electrocardiographic biochemical and histological evidences.

The effect of alpha-tocopherol (6 mg/100 g body wt, orally, daily for 90 days) pretreatment in isoproterenol (20 mg/100 g body wt, subcutaneously, twice at an interval of two days at the end of the alpha-tocopherol pretreatment) induced myocardial infarction was studied in rats. Isoproterenol administered rats showed electrocardiographic changes suggestive of myocardial infarction with marked ST segment elevation, Q waves appearance and a significant increase in heart rate. In isoproterenol administered rats, a significant decrease was observed in the activities of marker enzymes such as aspartate amino transferase, alanine amino transferase, lactate dehydrogenase and creatine kinase in heart and aorta with a significant increase in their activities in serum. The levels of lipid peroxides in terms of "TBA reactants" increased significantly in serum, heart and aorta on isoproterenol administration. The histology of heart and aorta showed marked fragmentation of muscle fibres and necrotic lesions in isoproterenol administered rats. alpha-Tocopherol pretreated rats showed a near normal ECG pattern, levels of lipid peroxides, activities of marker enzymes and a near normal histology of heart and aorta on isoproterenol administration.