Inibidores da Na+/K+ ATPase, como a ouabaína (OUA), estão associados à gênese/manutenção da hipertensão arterial (HA). Ratos tratados com OUA ficam hipertensos e suas artérias mesentéricas de resistência (AMR) apresentam disfunção vascular, estresse oxidativo e remodelamento estrutural e mecânico. Modelos de HA dependente de volume, como o DOCA-sal, têm aumento da concentração plasmática de OUA. Interagindo com a Na+/K+ ATPase, a OUA ativa vias de proliferação e fibrose, como a via MAPK, ativada quando da fosforilação da c-Src e do EGFR. A rostafuroxina, antagonista da OUA, reduz a pressão arterial (PA) e melhora a disfunção endotelial em AMR de ratos DOCA-sal. Dados do laboratório (não publicados) mostram que a rostafuroxina reverte o remodelamento mecânico e restaura parcialmente o remodelamento estrutural das AMR de ratos DOCA-sal. Assim, o objetivo deste trabalho foi avaliar os mecanismos de sinalização celular envolvidos nas mudanças estruturais e mecânicas das AMR de ratos DOCA-sal tratados com rostafuroxina. Ratos Wistar, uninefrectomizados, foram tratados com injeções subcutâneas de DOCA (em mg/kg/semana: 20 na primeira; 12 na segunda e terceira; 6 da quarta ao fim do experimento) e ingeriram água com NaCl (1%) e KCl (0,2%). Cinco semanas após o início do tratamento, um subgrupo passou a ser tratado, concomitantemente, com rostafuroxina (gavagem, 1mg/kg/dia) por 3 semanas. Ao final do protocolo, os animais foram anestesiados, mortos, as AMR dissecadas e fixadas em paraformaldeído (4%) para as análises histológicas ou armazenadas para Western Blot. Análise estatística aplicada: ANOVA uma ou duas vias. Avaliada por meio da plestismografia de cauda, o tratamento com DOCA-sal aumentou a PA; a rostafuroxina reduziu, mas não normalizou a mesma aos valores do grupo SHAM. A razão de colágeno I/III e a expressão proteica dos mediadores fibróticos TGFβ1 e CTGF foi maior nas AMR dos DOCA, alterações restauradas após tratamento com rostafuroxina. Além do mais, houve aumento da espessura da lâmina elástica interna das AMR dos DOCA, o que foi revertido pela rostafuroxina. A expressão de endotelina-1 foi maior nas AMR tanto dos DOCA tratados ou não com rostafuroxina; a expressão do receptor ETΑ foi reduzida nas AMR de ambos os grupos. Já a expressão do receptor ETΒ foi maior nas AMR dos DOCA quando comparados aos SHAM, sendo normalizada pela rostafuroxina. A expressão de c-SrcTyr418 e EGFR, bem como das enzimas c-RAF, ERK 1/2, p-ERK 1/2 e p38MAPK foi maior nas AMR dos DOCA, sendo restauradas com a rostafuroxina. Os dados sugerem que o remodelamento das AMR do grupo DOCA pode ser atribuído à ação direta da OUA sobre a via fibrótica e sobre a elastina, interferindo na rigidez e no diâmetro interno, e também à sua ação indireta, mediada pela endotelina-1 que, agindo em receptores ETB e transativando o EGFR, ativa a via da MAPK, a qual está associada aos eventos de proliferação e fibrose celular. / Na+/K+ ATPase inhibitors, such as ouabain (OUA), are associated with genesis/maintenance of hypertension. Rats treated with OUA are hypertensive and their mesenteric resistance arteries (MRA) exhibit vascular dysfunction, oxidative stress and structural and mechanical remodeling. Volume-dependent hypertensive models, as DOCA-salt, have increased plasmatic OUA levels. Interacting with Na+/K+ ATPase, OUA activates proliferation and fibrosis pathways, such as MAPK pathway, which is activated from c-Src and EGFR phosphorylation. Rostafuroxin, an OUA antagonist, reduces arterial pressure (AP) and improves endothelial dysfunction in MRA of DOCA-salt rats. Unpublished data of our laboratory have shown that rostafuroxin reverses the mechanical and partially restores the structural remodeling of MRA of DOCA-salt rats. Thus, the aim of this study was to evaluate the cellular signaling mechanisms involved in the structural and mechanical changes in MRA of DOCA-salt rats treated with rostafuroxin. Male Wistar rats, uninephrectomized, were treated with subcutaneous injections of DOCA (mg/kg/week: 20 in the 1st; 12 in the 2nd and 3rd; 6 from 4th to the end of the experiment) and drunk water with NaCl (1%) and KCl (0,2%). After 5 weeks of treatment, a subgroup started being treated, concomitantly, with rostafuroxin (gavage, 1mg/kg/day) for 3 weeks. At the end of the protocol, animals were anesthetized, killed, the MRA dissected out and fixed in paraformaldehyde (4%) to histological analyses or stored to Western Blot. Statistical analysis applied: ANOVA one- or two-way. Evaluated by pletismography, the DOCA-salt treatment raised the AP levels; rostafuroxin reduced it, but did not normalized to the values of SHAM group. Collagen I/III ratio and protein expression of fibrosis mediators TGFβ1 and CTGF was higher in MRA of DOCA and rostafuroxin restored these parameters. In addition, an increment in the thickness of inner elastic lamina was observed in MRA of DOCA, which was reversed by rostafuroxin treatment. Endothelin-1 expression was higher in MRA of both DOCA and rostafuroxin treated rats; ETΑ receptor was less expressed in both groups. On the other hand, ETΒ was more expressed in MRA from DOCA and normalized to SHAM levels by rostafuroxin treatment. The expression of c-SrcTyr418 and EGFR, as well as of the enzymes c-RAF, ERK 1/2, p-ERK 1/2 and p38MAPK was higher in MRA of DOCA when compared to SHAM, being restores with rostafuroxin. Taken together, the present data suggest that MRA remodeling in DOCA group can be attributed to direct action of OUA on elastin and fibrotic pathway, interfering in stiffness and inner diameter, as well as to its indirect action, mediated by endothelin-1, which acting in ETB receptors and activating EGFR, activates MAPK pathway, which is associated to proliferation and fibroses events.

The application of the synthetic mineralocorticoid, deoxycorticosterone acetate (DOCA)-salt, to unilaterally nephrectomised rats induces severe hypertension due to volume-overload, and mimics human primary aldosteronism. Importantly, DOCA-salt hypertension is characterized by severe cardiac and renal lesions triggered by nuclear factor kappa B (NF-kappaB), activating protein (AP-1), and transforming growth factor beta1 (TGF-beta1) leading to end-stage organ damage. Although DOCA-salt hypertension is a low renin model, local production of angiotensin-II and aldosterone in cardiac and renal tissues stimulate TGF-beta1, fibronectin and collagen-1 causing fibrosis and hypertrophy. Since TGF-beta1 gene promoter contains binding sites for NF-kappaB and AP-1, cross-talk between TGF-beta1, NF-kappaBnand AP-1 can be envisaged. Accordingly, the activation of TGF-beta1, fibronectin, collagen, NF-kappaB and AP-1 may constitute a potent destructive force in hypertension.<p> Emerging evidence indicates that upregulation of the heme oxygenase (HO) system is cytoprotective with antioxidant, antihypertensive and antihypertrophic effects. Interestingly, the promoter region of HO-1 gene harbors consensus-binding sites for NF-kappaB and AP-1; therefore, the HO system may regulate these transcription factors to counteract tissue insults. However, the multifaceted interactions between the HO system, NF-kappaB, AP-1, TGF-beta1, fibronectin and collagen in mineralocorticoid-induced end-stage-organ damage have not been fully characterized. Similarly, the effect of the HO system on tissue angiotensin-II and aldosterone levels in mineralocorticoid-induced hypertension remains unclear. Therefore, the present study was designed to investigate the antihypertrophic effect of the HO system in cardiac and renal tissue of DOCA-salt hypertensive rats. In this study, the HO inducer, hemin, lowered blood pressure and attenuated cardiac/renal hypertrophy, whereas the HO inhibitor, chromium mesoporphyrin (CrMP), nullified the effects of hemin and exacerbated cardiac/renal injury the DOCA-salt hypertensive rats. The protective effect of hemin was associated with increased HO-1, HO activity, cyclic guanosine monophosphate (cGMP), superoxide dismutase activity, ferritin and the total antioxidant capacity in the cardiac and renal tissue. In contrast, angiotensin-II, aldosterone, 8-isoprostane, NF-kappaB and AP-1 were significantly downregulated. Furthermore, hemin therapy attenuated TGF-beta1 and extracellular matrix (ECM) proteins such as fibronectin and collagen, with corresponding reduction of cardiac histopathological lesions, including longitudinal/cross-sectional muscle fiber thickness, scarring, muscular hypertrophy, coronary arteriolar thickening and collagen deposition. Similarly, hemin attenuated structural lesions in the kidney such as glomerular hypertrophy, glomerular sclerosis, mononuclear cell infiltration, tubular cast formation, tubular dilation and renal arteriolar thickening with concomitant improvement of kidney function as evidenced by reduction of plasma creatinine, proteinuria, but enhanced creatinine clearance.<p> Collectively, these results suggest that the HO system suppressed hypertension, cardiac and renal fibrosis, and hypertrophy in the DOCA-salt hypertensive rat by downregulating transcription factors such as NF-kappaB and AP-1, reducing ECM proteins such as fibronectin and collagen, decreasing local tissue production of angiotensin-II and aldosterone, and improved renal functional capacity.

The application of the synthetic mineralocorticoid, deoxycorticosterone acetate (DOCA)-salt, to unilaterally nephrectomised rats induces severe hypertension due to volume-overload, and mimics human primary aldosteronism. Importantly, DOCA-salt hypertension is characterized by severe cardiac and renal lesions triggered by nuclear factor kappa B (NF-kappaB), activating protein (AP-1), and transforming growth factor beta1 (TGF-beta1) leading to end-stage organ damage. Although DOCA-salt hypertension is a low renin model, local production of angiotensin-II and aldosterone in cardiac and renal tissues stimulate TGF-beta1, fibronectin and collagen-1 causing fibrosis and hypertrophy. Since TGF-beta1 gene promoter contains binding sites for NF-kappaB and AP-1, cross-talk between TGF-beta1, NF-kappaBnand AP-1 can be envisaged. Accordingly, the activation of TGF-beta1, fibronectin, collagen, NF-kappaB and AP-1 may constitute a potent destructive force in hypertension.<p> Emerging evidence indicates that upregulation of the heme oxygenase (HO) system is cytoprotective with antioxidant, antihypertensive and antihypertrophic effects. Interestingly, the promoter region of HO-1 gene harbors consensus-binding sites for NF-kappaB and AP-1; therefore, the HO system may regulate these transcription factors to counteract tissue insults. However, the multifaceted interactions between the HO system, NF-kappaB, AP-1, TGF-beta1, fibronectin and collagen in mineralocorticoid-induced end-stage-organ damage have not been fully characterized. Similarly, the effect of the HO system on tissue angiotensin-II and aldosterone levels in mineralocorticoid-induced hypertension remains unclear. Therefore, the present study was designed to investigate the antihypertrophic effect of the HO system in cardiac and renal tissue of DOCA-salt hypertensive rats. In this study, the HO inducer, hemin, lowered blood pressure and attenuated cardiac/renal hypertrophy, whereas the HO inhibitor, chromium mesoporphyrin (CrMP), nullified the effects of hemin and exacerbated cardiac/renal injury the DOCA-salt hypertensive rats. The protective effect of hemin was associated with increased HO-1, HO activity, cyclic guanosine monophosphate (cGMP), superoxide dismutase activity, ferritin and the total antioxidant capacity in the cardiac and renal tissue. In contrast, angiotensin-II, aldosterone, 8-isoprostane, NF-kappaB and AP-1 were significantly downregulated. Furthermore, hemin therapy attenuated TGF-beta1 and extracellular matrix (ECM) proteins such as fibronectin and collagen, with corresponding reduction of cardiac histopathological lesions, including longitudinal/cross-sectional muscle fiber thickness, scarring, muscular hypertrophy, coronary arteriolar thickening and collagen deposition. Similarly, hemin attenuated structural lesions in the kidney such as glomerular hypertrophy, glomerular sclerosis, mononuclear cell infiltration, tubular cast formation, tubular dilation and renal arteriolar thickening with concomitant improvement of kidney function as evidenced by reduction of plasma creatinine, proteinuria, but enhanced creatinine clearance.<p> Collectively, these results suggest that the HO system suppressed hypertension, cardiac and renal fibrosis, and hypertrophy in the DOCA-salt hypertensive rat by downregulating transcription factors such as NF-kappaB and AP-1, reducing ECM proteins such as fibronectin and collagen, decreasing local tissue production of angiotensin-II and aldosterone, and improved renal functional capacity.

Cardiovascular disease is the leading cause of death globally. Chronic hypertension can lead to a gradual deterioration in the structure and performance of the cardiovascular system in a process described as cardiovascular remodelling. The ultimate response to this remodelling is heart failure. While cardiovascular remodelling is characterised by features such as cardiac and vascular hypertrophy, endothelial dysfunction and fibrosis; mechanisms leading to such pathologies are still unclear. However, oxidative stress, the damage caused by increased production of reactive oxygen species such as free radicals, or their reduced removal by anti-oxidants, appears to have a significant role. The major aim of this thesis is to investigate the involvement of reactive oxygen species in cardiovascular remodelling and to further investigate the potentials of three anti-oxidant approaches (a combination of alpha-lipoic acid and vitamin E, apocynin and a red-wine component, resveratrol) in preventing or reversing cardiovascular remodelling. These studies were conducted in two well-established rat models of cardiovascular remodelling including the deoxycorticosterone acetate (DOCA)-salt hypertensive rat and the ageing spontaneously hypertensive rat (SHR). Additionally, this thesis also characterises the effects of ovariectomy on the blood pressure and survival rate of female SHR. The cardiovascular structure and function in the animals have been defined using in vivo echocardiography, ex vivo isolated Langendorff heart perfusion, isolated thoracic aortic rings, histological analysis of the myocardial extracellular matrix and inflammation along with terminal organ weight measurements. Reactive oxygen species were assessed through the measurement of plasma malondialdehyde (MDA) while the anti-oxidant capacities have been assessed through measurements of plasma total anti-oxidant status (TAS) and plasma glutathione peroxidase (GPx) activity. DOCA-salt treated rats exhibited hypertension, oxidative stress and cardiovascular remodelling, evidenced by their increased left ventricular weights, excess collagen deposition in the heart and increased values for diastolic stiffness, increased plasma MDA concentrations along with impaired contraction and relaxation of the vessels. Treatment with a combination of alpha-lipoic acid and vitamin E or apocynin significantly inhibited the increases in blood pressure, left ventricular weights, cardiac stiffness, interstitial collagen deposition along with improvements in the vascular responses. The treatment of ageing male SHRs with resveratrol, although without any decrease in blood pressure, was shown to reduce left ventricular hypertrophy, fibrosis, diastolic stiffness and improve functional performance of the heart. The level of oxidative stress was also lowered in the male SHRs treated with resveratrol as evidenced by decreased plasma MDA and increased TAS. Female SHRs had a higher blood pressure and diastolic stiffness compared to their age-matched WKYs. Similar to male SHRs, treatment with resveratrol did not affect blood pressure, but attenuated the increased diastolic stiffness, in female SHRs. Furthermore, the ovariectomised SHRs were proven to be extremely hypertensive and had a significantly poorer survival rate. Overall, these studies demonstrated great potential for the alpha-lipoic acid and vitamin E combination and apocynin in the treatment of cardiovascular remodelling. However, further clinical and experimental research is essential to confirm the complete cardiovascular health benefits of resveratrol or red wine intake.

Orientador: Carlos Alan Candido Dias Junior / Resumo: Durante a gestação, diversas adaptações fisiológicas ocorrem para suportar o feto em desenvolvimento. Em algumas mulheres, falhas nesses mecanismos podem causar desordens hipertensivas da gestação. A pré-eclâmpsia (PE) é uma desordem hipertensiva da gestação caracterizada por um de novo aumento na pressão arterial (> 140x90 mmHg), após a 20ª semana gestacional. É normalmente acompanhada de proteinúria e acomete 5-7% das gestantes. Atualmente é amplamente aceito que a fisiopatologia da PE envolve dois diferentes estágios: o primeiro, relacionado à má placentação e consequente isquemia/hipóxia placentária e liberação de fatores bioativos, e o segundo, relacionado à promoção de danos às células endoteliais. A disfunção endotelial é caracterizada por um aumento na liberação de substâncias vasoconstritoras, como a endotelina, e concomitante redução na liberação de substâncias vasodilatadoras, como o óxido nítrico (NO). Recentes estudos apontam para a importância do sulfeto de hidrogênio (H2S), uma molécula que apresenta diversas semelhanças com o NO, como a natureza gasosa e a produção por enzimas de localização endotelial. No sistema cardiovascular, o H2S exerce várias funções, como modulação da angiogênese e efeitos vasodilatadores, entretanto, poucos trabalhos avaliaram seu papel na hipertensão gestacional. Para a realização dos trabalhos, ratas Wistar prenhes foram utilizadas. Inicialmente a hipertensão gestacional foi induzida com o uso do inibidor da NO sintase (NOS), Nω-nit... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Throughout gestation, several physiological adaptations occur to support the growing fetus. In some women, fails in these mechanisms may cause gestational hypertensive disorders. Preeclampsia (PE) is a gestational hypertensive disorder characterized by a de novo increase in arterial blood pressure (> 140x90 mmHg) after the 20th gestational week. It is normally accompanied by proteinuria and affects 5-7% of pregnant women. It is currently accepted that the physiopathology of PE involves two different stages: the first one is related to a bad placentation process, consequent placental ischemia/hypoxia and release of bioactive factors; and the second stage is related to the occurrence of endothelial cell damage. Endothelial dysfunction is characterized by an increased release of vasoconstrictors substances, such as endothelin, and a concurrent reduction in the release of vasodilators substances, such as nitric oxide (NO). Recent studies point to the importance of hydrogen sulfide (H2S), a molecule that shows several similarities with NO, such as its gaseous nature and the enzymatic production in endothelium. In cardiovascular system, H2S exerts numerous functions, such as angiogenesis modulation and vasodilators effects, however only a few studies evaluated H2S role in gestational hypertension. For the accomplishment of the works, pregnant Wistar rats were used. Initially, gestational hypertension was induced with the use of NO synthase (NOS) inhibitor, Nω-nitro-L-arginina-metil... (Complete abstract click electronic access below) / Doutor

Intravenous injection of substance P (SP) stimulates sympathetic ganglia to evoke a greater increase in renal sympathetic nerve activity, heart rate (HR) and blood pressure (BP) in hypertensive than normotensive rats due to upregulation of the NK1 receptor. These experiments were designed to determine the cellular basis for the enhanced ganglionic responsiveness to NK1 agonists in spontaneously hypertensive rats (SHR) in comparison to their normotensive counterparts, Wistar-Kyoto rats (WKY). Studies were also conducted to determine whether the increased ganglion responsiveness to SP in SHR is causally related to the increased BP or is a unique characteristic of this model of essential hypertension. Nerve recordings were made from the external carotid branch of the superior cervical ganglion (SCG) in pentobarbital anesthetized rats. Animals were treated with the ganglion blocking agent chlorisondamine (10.5 μmol/kg) and pre- and postganglionic SCG nerves were cut. SP (1.0 to 100 nmol/kg) evoked a greater increase in postganglionic nerve firing from the SCG of SHR vs. WKY. Intracellular microelectrode recordings were made from isolated SCG. Membrane properties were similar between strains. Picospritzer application of the NK1 agonist GR-73632 (100 μM, 1 s) caused slow depolarization and increased neuron excitability. Depolarization amplitude and duration were similar between strains, however, a greater percentage of neurons were depolarized by the NK1 agonist in SHR. To determine if the ganglion sensitivity to SP was correlated with blood pressure WKY were made hypertensive by unilateral nephrectomy and deoxycorticosterone acetate (DOCA)/salt treatment. Tail cuff BP was the same in treated WKY and untreated SHR. Increases in sympathetic nerve activity, HR and BP in response to SP (1.0 to 100 nmol/kg) were the same in treated and untreated WKY rats. In conclusion, SHR are more responsive to ganglion stimulation by NK1 agonists due to a greater number of responsive cells within their SCG rather than an enhanced responsiveness of individual neurons. The increased sympathetic nerve responsiveness to SP is an inherent characteristic and not an adaptive response of sympathetic ganglion neurons to hypertension. This enhanced action of SP at sympathetic ganglia may contribute to the elevated sympathetic outflow observed in this model of hypertension.

No description available.

Tem sido demonstrado que na hipertensão arterial ocorre aumento de ouabaína plasmática, um inibidor da Na+K+-ATPase. Em 1998, Ferrari et al. desenvolveram uma molécula denominada de rostafuroxina, a qual é capaz de antagonizar os efeitos da ouabaína por deslocar a ligação desse glicosídeo com a Na+K+-ATPase. Dentro desse contexto, parece razoável sugerir que um anti-hipertensivo capaz de antagonizar os efeitos da ouabaína possa representar uma nova ferramenta farmacológica para o tratamento da hipertensão arterial. Baseado em tais premissas, o presente estudo avaliou o papel da ouabaína por meio do tratamento com rostafuroxina sobre: a pressão arterial, a reatividade vascular em artérias de resistência e a atividade simpática de ratos DOCA-sal. Os resultados demonstraram que os animais DOCA-sal tratados com rostafuroxina apresentaram redução da pressão arterial sistólica e da hiperatividade simpática e melhora na função vascular. Assim, sugere-se que a ouabaína seja um possível alvo para o tratamento da hipertensão arterial dependente de volume. / It has been shown that some types of hypertension have increased plasma levels of ouabain, a factor inhibitor of Na+K+-ATPase. In 1998, Ferrari et al. developed a molecule called rostafuroxin that antagonizes the effects of ouabain. In this context, it seems reasonable to suggest that an antihypertensive capable of antagonizing the effects of ouabain might be a new and specific pharmacological tool for the treatment of hypertension. In so doing, the present study aimed to evaluate the role of endogenous ouabain by treatment with rostafuroxin on blood pressure, vascular reactivity in resistance arteries and sympathetic activity of DOCA-salt rats. Our data have shown that the treatment with rostafuroxin decreased the systolic blood pressure and sympathetic activity and improve the vascular function of the DOCA-salt rats. Thus, it is suggested that ouabain is a putative target for the treatment of volume-dependent hypertension.

Tem sido demonstrado que na hipertensão arterial ocorre aumento de ouabaína plasmática, um inibidor da Na+K+-ATPase. Em 1998, Ferrari et al. desenvolveram uma molécula denominada de rostafuroxina, a qual é capaz de antagonizar os efeitos da ouabaína por deslocar a ligação desse glicosídeo com a Na+K+-ATPase. Dentro desse contexto, parece razoável sugerir que um anti-hipertensivo capaz de antagonizar os efeitos da ouabaína possa representar uma nova ferramenta farmacológica para o tratamento da hipertensão arterial. Baseado em tais premissas, o presente estudo avaliou o papel da ouabaína por meio do tratamento com rostafuroxina sobre: a pressão arterial, a reatividade vascular em artérias de resistência e a atividade simpática de ratos DOCA-sal. Os resultados demonstraram que os animais DOCA-sal tratados com rostafuroxina apresentaram redução da pressão arterial sistólica e da hiperatividade simpática e melhora na função vascular. Assim, sugere-se que a ouabaína seja um possível alvo para o tratamento da hipertensão arterial dependente de volume. / It has been shown that some types of hypertension have increased plasma levels of ouabain, a factor inhibitor of Na+K+-ATPase. In 1998, Ferrari et al. developed a molecule called rostafuroxin that antagonizes the effects of ouabain. In this context, it seems reasonable to suggest that an antihypertensive capable of antagonizing the effects of ouabain might be a new and specific pharmacological tool for the treatment of hypertension. In so doing, the present study aimed to evaluate the role of endogenous ouabain by treatment with rostafuroxin on blood pressure, vascular reactivity in resistance arteries and sympathetic activity of DOCA-salt rats. Our data have shown that the treatment with rostafuroxin decreased the systolic blood pressure and sympathetic activity and improve the vascular function of the DOCA-salt rats. Thus, it is suggested that ouabain is a putative target for the treatment of volume-dependent hypertension.