Vascular dysfunction and fibrosis in stroke-prone spontaneously hypertensive rats: the aldosterone-mineralocorticoid receptor-Nox1 Axis

Aims: We questioned whether aldosterone and oxidative stress play a role in vascular damage in severe hypertension and investigated the role of Nox1 in this process. Materials and methods: We studied mesenteric arteries, aortas and vascular smooth muscle cells (VSMC) from WKY and SHRSP rats. Vascular effects of eplerenone or canrenoic acid (CA) (mineralocorticoid receptor (MR) blockers), ML171 (Nox1 inhibitor) and EHT1864 (Rac1/2 inhibitor) were assessed. Nox1-knockout mice were also studied. Vessels and VSMCs were probed for Noxs, reactive oxygen species (ROS) and pro-fibrotic/inflammatory signaling. Key findings: Blood pressure and plasma levels of aldosterone and galectin-3 were increased in SHRSP versus WKY. Acetylcholine-induced vasorelaxation was decreased (61% vs 115%) and phenylephrine-induced contraction increased in SHRSP versus WKY (Emax 132.8% vs 96.9%, p < 0.05). Eplerenone, ML171 and EHT1864 attenuated hypercontractility in SHRSP. Vascular expression of collagen, fibronectin, TGFβ, MCP-1, RANTES, MMP2, MMP9 and p66Shc was increased in SHRSP versus WKY. These changes were associated with increased ROS generation, 3-nitrotyrosine expression and Nox1 upregulation. Activation of vascular p66Shc and increased expression of Nox1 and collagen I were prevented by CA in SHRSP. Nox1 expression was increased in aldosterone-stimulated WKY VSMCs, an effect that was amplified in SHRSP VSMCs (5.2vs9.9 fold-increase). ML171 prevented aldosterone-induced VSMC Nox1-ROS production. Aldosterone increased vascular expression of fibronectin and PAI-1 in wild-type mice but not in Nox1-knockout mice. Significance: Our findings suggest that aldosterone, which is increased in SHRSP, induces vascular damage through MR-Nox1-p66Shc-mediated processes that modulate pro-fibrotic and pro-inflammatory signaling pathways

Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Foundation for the Support of Research in the State of Sao Paulo; grants 06/61523-7 and 06/54064-6)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, National Council for Scientific and Technological Development)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Foundation for the Support of Research in the State of Sao Paul

Therapeutic activity of lipoxin A4 in TiO2-induced arthritis in mice: NF-κB and Nrf2 in synovial fluid leukocytes and neuronal TRPV1 mechanisms

BackgroundLipoxin A4 (LXA4) has anti-inflammatory and pro-resolutive roles in inflammation. We evaluated the effects and mechanisms of action of LXA4 in titanium dioxide (TiO2) arthritis, a model of prosthesis-induced joint inflammation and pain.MethodsMice were stimulated with TiO2 (3mg) in the knee joint followed by LXA4 (0.1, 1, or 10ng/animal) or vehicle (ethanol 3.2% in saline) administration. Pain-like behavior, inflammation, and dosages were performed to assess the effects of LXA4in vivo.ResultsLXA4 reduced mechanical and thermal hyperalgesia, histopathological damage, edema, and recruitment of leukocytes without liver, kidney, or stomach toxicity. LXA4 reduced leukocyte migration and modulated cytokine production. These effects were explained by reduced nuclear factor kappa B (NFκB) activation in recruited macrophages. LXA4 improved antioxidant parameters [reduced glutathione (GSH) and 2,2-azino-bis 3-ethylbenzothiazoline-6-sulfonate (ABTS) levels, nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA and Nrf2 protein expression], reducing reactive oxygen species (ROS) fluorescent detection induced by TiO2 in synovial fluid leukocytes. We observed an increase of lipoxin receptor (ALX/FPR2) in transient receptor potential cation channel subfamily V member 1 (TRPV1)+ DRG nociceptive neurons upon TiO2 inflammation. LXA4 reduced TiO2‐induced TRPV1 mRNA expression and protein detection, as well TRPV1 co-staining with p-NFκB, indicating reduction of neuronal activation. LXA4 down-modulated neuronal activation and response to capsaicin (a TRPV1 agonist) and AITC [a transient receptor potential ankyrin 1 (TRPA1) agonist] of DRG neurons.ConclusionLXA4 might target recruited leukocytes and primary afferent nociceptive neurons to exert analgesic and anti-inflammatory activities in a model resembling what is observed in patients with prosthesis inflammation

Renin-angiotensin and kallikrein-kinin systems interationstudy of B1 receptor induction by angiotensin II.

Os objetivos deste trabalho foram investigar a modulação exercida pela infusão de angiotensina II (Ang II) sobre expressão de receptor B1 (RB1), mecanismos envolvidos nesta modulação, papel do RB1 na reatividade de anéis de aorta e os efeitos do antagonismo in vivo do RB1 na hipertrofia e inflamação vascular. Observamos que a Ang II causou hipertensão o que não foi afetado pelo tratamento com apocinina e com antagonista de RB1. Na aorta de ratos hipertensos observamos: indução de expressão de RB1, aumento da geração de espécies reativas de oxigênio e da atividade do NF-kB em relação ao grupo Controle e o tratamento com apocinina reduziu estes parâmetros. O agonista de RB1 causou vasodilatação dependente de endotélio e de óxido nitrito na aorta dos ANG II. A aorta dos ANG II apresentou hipertrofia, maior atividade da ERK1/2 e expressão de interleucinas, disfunção endotelial e o antagonismo de RB1 reduziu estes parâmetros, sem alterar disfunção endotelial. Nossos resultados fornecem esclarecimentos sobre a modulação de RB1 e sua funcionalidade na aorta de ratos.We investigated whether angiotensin II (Ang II) infusion modulates in vivo the kinin B1 receptor (B1R) expression, the mechanisms involved in this process, the role of the B1R activation in aorta and the effect of B1R antagonism in vivo upon vascular hypertrophy and inflammation. Ang II induced hypertension in Wistar was not affected by treatments with apocynin or B1R antagonism. The aorta of hypertensive rats presents: B1R expression, increased superoxide anion and NF-kB activity and apocynin treatment reduced those parameters. The B1R agonist promoted endothelium and NO-dependent dilation in aorta of Ang II rats. The aorta of Ang II rats presented hypertrophy, increased MAPKs activity and interleukins expression and the B1R antagonism reduced those parameters. Those data indicated that Ang II can induce B1R expression in aorta and this receptor could participate in Ang II-induced vascular hypertrophy and inflammation

Detrimental Effects of Testosterone Addition to Estrogen Therapy Involve Cytochrome P-450-Induced 20-HETE Synthesis in Aorta of Ovariectomized Spontaneously Hypertensive Rat (SHR), a Model of Postmenopausal Hypertension

Postmenopausal period has been associated to different symptoms such as hot flashes, vulvovaginal atrophy, hypoactive sexual desire disorder (HSDD) and others. Clinical studies have described postmenopausal women presenting HSDD can benefit from the association of testosterone to conventional hormonal therapy. Testosterone has been linked to development of cardiovascular diseases including hypertension and it also increases cytochrome P-450-induced 20-HETE synthesis which in turn results in vascular dysfunction. However, the effect of testosterone plus estrogen in the cardiovascular system is still very poorly studied. The aim of the present study is to evaluate the role of cytochrome P-450 pathway in a postmenopausal hypertensive female treated with testosterone plus estrogen. For that, hypertensive ovariectomized rats (OVX-SHR) were used as a model of postmenopausal hypertension and four groups were created: SHAM-operated (SHAM), ovariectomized SHR (OVX), OVX treated for 15 days with conjugated equine estrogens [(CEE) 9.6 μg/Kg/day/po] or CEE associated to testosterone [(CEE+T) 2.85 mg/kg/weekly/im]. Phenylephrine-induced contraction and generation of reactive oxygen species (ROS) were markedly increased in aortic rings from OVX-SHR compared to SHAM rats which were restored by CEE treatment. On the other hand, CEE+T abolished vascular effects by CEE and augmented both systolic and diastolic blood pressure of SHR. Treatment of aortic rings with the CYP/20-HETE synthesis inhibitor HET0016 (1 μM) reduced phenylephrine hyperreactivity and the augmented ROS generation in the CEE+T group. These results are paralleled by the increased CYP4F3 protein expression and activity in aortas of CEE+T. In conclusion, we showed that association of testosterone to estrogen therapy produces detrimental effects in cardiovascular system of ovariectomized hypertensive females via CYP4F3/20-HETE pathway. Therefore, our findings support the standpoint that the CYP/20-HETE pathway is an important therapeutic target for the prevention of cardiovascular disease in menopausal women in the presence of high levels of testosterone

Angiotensin II chronic infusion induces B1 receptor expression in aorta of rats

We investigated whether angiotensin II infusion modulates in vivo the kinin B1 receptor expression and the mechanisms involved in this process. We also evaluated the role of the B1 receptor activation in aorta. Wistar rats received 400 ng/kg per minute of angiotensin II or saline (control rats) infusion during 14 days through an osmotic minipump. To investigate the role of superoxide anion in B1 receptor expression, rats received a reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor in the drinking water during 14 days (60 mg/L of apocynin) simultaneously with angiotensin II infusion. Angiotensin II induced B1 receptor expression in the aorta and increased significantly systolic blood pressure, superoxide anion, and the nuclear factor kappa B activity. Apocynin treatment did not alter the blood pressure levels of angiotensin II rats and reduced the B1 receptor expression, superoxide anion generation, and nuclear factor kappa B activity to similar levels of the control rats. Vascular reactivity studies in isolated aorta reveal that B1 receptor agonist promoted endothelium-dependent dilation and increased the NO generation in aorta of angiotensin II rats. NO synthase inhibitor and B1 receptor antagonist inhibited the vasodilation and NO generation, which were not affected by B2 receptor antagonist or indomethacin. These results provide evidence that angiotensin II induces B1 receptor expression in aorta by superoxide anion generation, via reduced nicotinamide-adenine dinucleotide phosphate oxidase, concomitant to nuclear factor kappa B activation. We have also shown that B1 receptor agonist causes endothelium-dependent vasodilation through NO production in aortic rings, suggesting that the B1 receptor expression could be related with the vascular tonus control of angiotensin II rats.Univ São Paulo, Dept Pharmacol, Lab Hypertens, Inst Biomed Sci, BR-05508900 São Paulo, BrazilUniv São Paulo, Sch Med, Inst Heart, Vasc Biol Lab, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilWeb of Scienc

Modulation of kinin B1 receptor expression by endogenous angiotensin II in hypertensive rats

We investigated the expression and localization of B I receptor in tissues of rats submitted to a renin-dependent model of hypertension (2K-IC), and analyzed the influence of endogenous Ang II in modulating the in vivo expression of these receptors. B1 mRNA levels in the heart, kidney and thoracic aorta were quantified by real time PCR, B1 receptor protein expression was assessed by immunchistochemistry, plasma Ang II levels were analyzed by radioirnmunoassay and the effects of ATI receptor blockade were determined after losartan treatment. 2K-IC rats presented a marked increase in Ang II levels when compared to sham-operated rats. in parallel, cardiac- (but not renal and aortic) B I mRNA levels were 15-fold higher in 2K-IC than in sham rats. in 2K-IC, B1 expression was detected in the endothelium of small cardiac arteries and in cardiomyocytes. Losartan completely reverted the increased B1 mRNA levels and significantly decreased the protein expression observed in 2K-IC rats, despite reducing, but not normalizing blood pressure. We conclude that in the 2K-IC rat, induction of cardiac B I receptor might be tightly linked to ATI receptor activation. These data suggest the existence of a new site of interaction between kinins and angiotensins, and might provide important contributions for a better understanding of the pathophysiology of hypertension. (c) 2006 Elsevier B.V. All rights reserved.Univ São Paulo, Inst Biomed Sci, Dept Pharmacol, BR-05508900 São Paulo, SP, BrazilUniv Fed Minas Gerais, Dept Physiol & Biophys, Inst Biomed Sci, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, SP, BrazilWeb of Scienc

Finding density-based subspace clusters in graphs with feature vectors

Endothelial dysfunction has been implicated in portal vein obstruction, a condition responsible for major complications in chronic portal hypertension. Increased vascular tone due to disruption of endothelial function has been associated with an imbalance in the equilibrium between endothelium-derived relaxing and contracting factors. Herein, we assessed underlying mechanisms by which expression of bradykinin B-1 receptor (B1R) is induced in the endothelium and how its stimulation triggers vasoconstriction in the rat portal vein. Prolonged in vitro incubation of portal vein resulted in time- and endothelium-dependent expression of B1R and cyclooxygenase-2 (COX-2). Inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3K) significantly reduced expression of B1R through the regulation of transcription factors, activator protein-1 (AP-1) and cAMP response element-binding protein (CREB). Moreover, pharmacological studies showed that B1R-mediated portal vein contraction was reduced by COX-2, but not COX-1, inhibitors. Notably, activation of endothelial B1R increased phospholipase A(2)/COX-2-derived thromboxane A(2) (TXA(2)) levels, which in turn mediated portal vein contraction through binding to TXA(2) receptors expressed in vascular smooth muscle cells. These results provide novel molecular mechanisms involved in the regulation of B1R expression and identify a critical role for the endothelial B1R in the modulation of portal vein vascular tone. Our study suggests a potential role for B1R antagonists as therapeutic tools for diseases where portal hypertension may be involved. (C) 2012 Elsevier Inc. All rights reserved

Effect of Hyperthyroidism (Hyper) in the protein expression.

<p>Hyper reduced the expression of contractile proteins: p-MLC (A), α-actin and calponin (B). In addition, Hyper was associated with increased AT2R (C) and AngI/II (D) levels in aortas. Top: representative blots. Bottom: Densitometry values. Data are expressed as mean ± SD (n = 6) per group, *p<0.05 <i>vs.</i> control.</p