Examinando por Autor "Hernanz, R"

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Activation of p38 and ERK1/2 MAPK by superoxide anion participates in Angiotensin II-induced COX-2 expression in smooth muscle cells from resistance arteries
(2009-09) Hernanz, R; Beltrán, AE; Pérez-Girón, JV; Martín, A; Briones, AM; Palacios, R; Salaices, M; Alonso, MJ
Introduction: Angiotensin II (Ang II) regulates vascular smooth muscle cell (VSMC) function by activating signalling cascades that promote vasoconstriction, growth and inflammation. The mechanisms implicated in Ang II-induced pro-inflammatory actions include activation of several mitogen-activated protein kinases (MAPKs), reactive oxygen species generation and the modulation of prostaglandins production by regulating cyclooxygenase-2 (COX-2) expression. Aim: To investigate the effect of Ang II on COX-2 expression in VSMC derived from small resistance arteries and the mechanisms involved. Methods: VSMC derived from rat mesenteric resistance arteries were used. Protein expression was determined by Western Blot, mRNA levels by Q-RT-PCR and superoxide anion (O2-) production by dihydroethidine fluorescence. Results: Ang II (0.1 ¿M) time-dependently increased COX-2 protein expression (30 min - 8 h) and mRNA levels (15 min - 4 h), while COX- 1 expression remained unmodified. In addition, Ang II did not modify AT1 receptor expression. The AT1 antagonist losartan (10 ¿M), but not the AT2 antagonist PD 122319 (10 ¿M), abolished the increase in COX-2 expression induced by Ang II (2 h). COX-2 expression was also reduced by the respective NADPHox and xanthine oxidase inhibitors, apocynin (30 mM) and allopurinol (10 mM). Furthermore, Ang II (3- 30 min) increased O2- production; this effect was reduced by losartan, allopurinol and apocynin but not by PD 123319. Ang II (2-30 min) induced the phosphorylation of p38 and ERK1/2 MAPK; this effect was reduced by losartan, allopurinol and apocynin. In addition, the respective inhibitors of p38 and ERK1/2, SB 203580 (10 mM) and PD 98059 (25 mM), reduced the Ang II-induced COX-2 expression. Conclusions: The present results provide evidences that angiotensin II increases COX-2 expression in VSMC from resistance arteries, at least in part, through mechanisms that include O2- production and the subsequent activation of p38 and ERK1/2 MAPK
Atorvastatin prevents angiotensin II-induced vascular remodeling and oxidative stress
(2009-07) Briones, AM; Rodríguez-Criado, N; Hernanz, R; García-Redondo, AB; Rodrígues-Díez, RR; Alonso, MJ; Egido, J; Ruiz-Ortega, M; Salaices, M
Angiotensin II (Ang II) modulates vasomotor tone, cell growth, and extracellular matrix deposition. This study analyzed the effect of atorvastatin in the possible alterations induced by Ang II on structure and mechanics of mesenteric resistance arteries and the signaling mechanisms involved. Wistar rats were infused with Ang II (100 ng/kg per day, SC minipumps, 2 weeks) with or without atorvastatin (5 mg/kg per day). Ang II increased blood pressure and plasmatic malondialdehyde levels. Compared with controls, mesenteric resistance arteries from Ang II¿treated rats showed the following: (1) decreased lumen diameter; (2) increased wall/lumen; (3) decreased number of adventitial, smooth muscle, and endothelial cells; (4) increased stiffness; (5) increased collagen deposition; and (6) diminished fenestrae area and number in the internal elastic lamina. Atorvastatin did not alter blood pressure but reversed all of the structural and mechanical alterations of mesenteric arteries, including collagen and elastin alterations. In mesenteric resistance arteries, Ang II increased vascular O2.- production and diminished endothelial NO synthase and CuZn/superoxide dismutase but did not modify extracellular-superoxide dismutase expression. Atorvastatin improved plasmatic and vascular oxidative stress, normalized endothelial NO synthase and CuZn/superoxide dismutase expression, and increased extracellular superoxide dismutase expression, showing antioxidant properties. Atorvastatin also diminished extracellular signal¿ regulated kinase 1/2 activation caused by Ang II in these vessels, indicating an interaction with Ang II¿induced intracellular responses. In vascular smooth muscle cells, collagen type I release mediated by Ang II was reduced by different antioxidants and statins. Moreover, atorvastatin downregulated the Ang II¿induced NADPH oxidase subunit, Nox1, expression. Our results suggest that statins might exert beneficial effects on hypertension-induced vascular remodeling by improving vascular structure, extracellular matrix alterations, and vascular stiffness. These effects might be mediated by their antioxidant properties.
Losartan and tempol treatments normalize the increased response to hydrogen peroxide in resistance arteries from hypertensive rats
(2009-09) García-Redondo, AB; Briones, AM; Avendaño, MS; Hernanz, R; Alonso, MJ; Salaices, M
Objective To analyse the role of angiotensin II, via AT1 receptors, and oxidative stress in the mechanisms underlying the increased response to hydrogen peroxide (H2O2) of mesenteric resistance arteries from spontaneously hypertensive rats (SHRs). Methods Arteries from normotensive and SHRs untreated or treated with the AT1 receptor antagonist, losartan (15mg/kg per day, 12 weeks), or with the superoxide dismutase analogue, tempol (1 mmol/l, 17 days), were used. Arteries were mounted in microvascular myographs for isometric tension recording; superoxide anion (O2 S) production was evaluated by dihydroethidium fluorescence, thromboxane A2 production by enzyme immunoassay and plasma nitrite levels by the Griess method. Results H2O2 (1¿100mmol/l) induced higher contractile responses in mesenteric resistance arteries from hypertensive than normotensive rats. In SHRs, losartan and tempol treatments induced the following effects: normalized the increased H2O2 contractile responses observed; modified neither the inhibitory effects of the cyclooxygenase inhibitor, indomethacin [1-(4- chlorobenzoyl)-5-methoxy-2-methyl-1-H-indole-3-acetic acid] (1mmol/l), and the thromboxane A2/prostaglandin H2 receptor antagonist, SQ 29 548 (1mmol/l), on H2O2 contraction, nor the increase in thromboxane A2 production in response to H2O2; abolished the increased vascular O2.- production; increased both the potentiatory effect of the nitric oxide inhibitor, N(G)-nitro-L-arginine methyl ester (100mmol/l), on H2O2 responses and the acetylcholineinduced relaxation. Moreover, losartan treatment abolished the effect of the O2.- scavenger, tiron (1 mmol/l), on H2O2 responses and increased plasma nitrite levels. Conclusion Nitric oxide removal by an excessive O2.- production, probably from an upregulated renin¿ angiotensin system, participates in the increased response to H2O2 in mesenteric resistance arteries from SHRs.
Ouabain treatment increases nitric oxide bioavailability and decreases superoxide anion production in cerebral vessels
(2008-10) Hernanz, R; Briones, AM; Martín, A; Beltrán, AE; Tejerina, T; Salaices, M; Alonso, MJ
Objective Chronic administration of ouabain induces hypertension and increases the contribution of nitric oxide to vasoconstrictor responses in peripheral arteries. The aim of this study was to analyse whether ouabain treatment alters the nitric oxide bioavailability in cerebral arteries. Methods Basilar arteries from control and ouabain-treated rats (approx. 8.0mg/day, 5 weeks) were used. Vascular reactivity was analysed by isometric tension recording, protein expression by western blot, nitric oxide levels by diaminofluorescein-induced fluorescence, superoxide anion (O2.-) production by ethidium fluorescence and lucigenin chemiluminescence and plasma total antioxidant status by a commercial kit. Results The relaxations induced by bradykinin (1 nmol/l¿10mmol/l) and L-arginine (0.01¿300mmol/l) and the contractile responses induced by both N-nitro-L-arginine methyl ester (0.1¿100mmol/l) and oxyhaemoglobin (0.01¿10mmol/l) were greater in arteries from ouabain-treated than control rats. However, the relaxation to diethylamine NONOate¿nitric oxide (0.1 nmol/l¿10mmol/l) and the contractions to KCl (7.5¿120 mmol/l) and 5-hydroxytryptamine (0.01¿10mmol/l) were similar in arteries from both groups. Ouabain treatment increased basal nitric oxide levels but did not modify endothelial and neuronal nitric oxide synthase protein expression. O2.- production was lower in cerebral arteries from ouabain-treated rats; however, plasma total antioxidant status and vascular protein expression of Cu/Zn-superoxide dismutase,Mn-superoxide dismutase and extracellular superoxide dismutase were similar in both groups. Conclusion Chronic ouabain treatment increased nitric oxide basal levels in basilar arteries probably due to the decreased O2.- levels. This might be an adaptive mechanism of the cerebral vasculature to the increase in blood pressure.
Pioglitazone alters the participation of cCyclooxygenase-2 produts and reactive oxygen species on vascular reactivity of hypertensive rats
(2009-06) Hernanz, R; Martín, A; Pérez-Girón, JV; Avendaño, MS; Roque, FR; Salaices, M; Alonso, MJ
The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) is expressed in all major vascular cells, where it may play an important role in vascular disease. Thus, the PPARgamma agonists, glitazones, exert depressor action in both hypertensive subjects and various animal models, improve endothelium-dependent vasodilation, and reduce vascular contractility in response to various vasoconstrictors. In addition, glitazones have anti-inflammatory actions associated to interference with redox-sensitive transcription factors, such as NF-kappaB, involved in the transcription of several genes including COX-2. Objetive: To analyze the effect of chronic pioglitazone treatment on the vascular reactivity of mesenteric resistance arteries from spontaneously hypertensive (SHR) rats to phenylephrine as well as the role of prostanoids and reactive oxygen species in such effect. Methods: Mesenteric resistance arteries from 6- month old SHR rats untreated or treated with the PPARgamma activator pioglitazone (2.5 mg/Kg/day for 28 days) were used. Vascular reactivity was studied with wire myography and protein expression by western blot. Results: Pioglitazone did not lower blood pressure of SHR (control: 198.9±3.3; pioglitazone: 200±5.2 mmHg; P>0.05). Concentration-response curve to phenylephrine (0.1-30 microM) was similar in segments from untreated and pioglitazone-treated rats. Indomethacin (10 microM), the selective COX-2 inhibitor, NS 398 (1 microM), the TxA2 receptor antagonist, SQ 29,548 (1 microM) and the EP1 receptor antagonist, SC 19220 (10 µM) reduced the response to phenylephrine only in segments from treated rats, while the TxA2 synthase inhibitor, furegrelate (1 µM) did not modify this response in both treated and untreated rats. In addition, COX-2 expression was higher in mesenteric arteries from treated than untreated rats. Pioglitazone treatment abolished the inhibitory effect of the respective inhibitors of NADPH oxidase and xanthine oxidase inhibitor, apocynin (0.3 mM) and allopurinol (0.3 mM) on vasoconstrictor response to phenylephrine, and reduced the vascular levels of Cu/Zn- and Mn-SOD. On the other hand, the NO synthase inhibitor L-NAME (0.1 mM) potentiated the response to phenylephrine in segments from both untreated and treated rats. Conclusions: Chronic pioglitazone treatment of hypertensive rats increases mesenteric COX-2 expression, associated with increased participation of contractile prostanoids from COX-2 in vasoconstrictor responses to phenylephrine. On the other hand, the observed reduction of involvement of NADPH oxidase and xantine oxidase-derived reactive oxygen species in the contraction elicited by phenylephrine can explain the similar vasoconstrictor response to this agonist found in resistance arteries. In spite of this, the treatment reduced the expression of cytosolic and mitochondrial SOD; if pioglitazone regulates reactive oxygen species production needs to be elucidated
PPAR¿ activation improves oxidative stress and downregulates COX-2 expression in vascular cells
(2009-09) Palacios, R; Pérez-Girón, JV; Martín, A; Hernanz, R; Briones, AM; Salaices, M; Alonso, MJ
Introduction: The increased renin-angiotensin system (RAS) activity seems to contribute to the pathophysiology of hypertension by the increase in reactive oxygen species (ROS) levels and proinflammatory mediators. Endothelin-1 (ET-1) has been proposed to explain the cardiovascular damage induced by angiotensin II (AngII). Moreover, peroxisome proliferator activated receptor ¿ (PPAR¿) agonists have anti-inflammatory actions by interference with redox-sensitive transcription factors, such as NFkB or AP-1, involved in the transcription of proinflammatory genes including cyclooxygenase-2 (COX-2). Aim: To analyze if AngII contributes to the increased COX-2 levels in vascular smooth muscle cells (VSMC) from spontaneously hypertensive (SHR) rats by mechanisms dependent of ROS and ET-1 production and whether PPAR¿ activation regulates this effect. Methods: Aortic VSMC from SHR were stimulated with AngII in the absence and the presence of different drugs. mRNA levels were measured by qRT-PCR and protein expression by Western blot. Aortic segments from SHR and Wistar-Kyoto (WKY) rats untreated and treated with losartan (15 mg/Kg/day, 12 weeks) were also used. Results: COX-2 mRNA levels were greater in segments from SHR than WKY; the treatment with losartan reduced COX-2 levels in SHR. In VSMC from SHR, AngII (0.1 ¿M, 2 h) induced COX-2, ET-1 and NOX-1 mRNA levels; this effect was reduced by losartan (10 ¿M). AngII-induced COX-2 protein expression was also reduced by the NADPHox inhibitor apocynin (30 mM). The antagonist of the ETA receptor BQ 123 (1 ¿M), but not of the ETB receptor BQ 788 (1 ¿M), also reduced COX-2 and NOX-1 mRNA levels after AngII. The proteasome inhibitor lactacystin (20 ¿M) did not modify the ET-1 mRNA levels but inhibited those of NOX-1 and COX-2. AngII also increased c-jun expression; this expression was reduced by losartan but not by BQ 123. Moreover, the PPAR¿ activator pioglitazone (10 ¿M) decreased AngII-induced COX-2 and NOX-1 mRNA levels in VSMC from SHR. Conclusions: 1) The RAS activation contributes to the increased vascular COX-2 expression in hypertension. 2) AngII-induced COX-2 expression in VSMC is related with NOX-1 induction and NFkB and AP-1 activation. 3) AngII-induced ET-1 production and ETA activation contributes, at least partially, to the increased NOX-1 and COX-2 expression. 4) PPAR¿ activation inhibits AngII-induced COX-2 expression by reducing NOX-1 levels; we suggest that transrepression mechanisms on NFkB and/or AP-1 can play an important role in this inhibitory effect of PPAR¿ activation.
Toll-like receptor 4 contributes to vascular remodelling and endothelial dysfunction in angiotensin II-induced hypertension
(British Pharmacological Society, 2015-02-17) Hernanz, R; Martínez-Revelles, S; Palacios-Ramírez, R; Martín, A; Cachofeiro, V; Aguado, A; García-Redondo, L; Barrús, MT; de Batista, PR; Briones, AM; Salaices, M; Alonso, MJ
Toll-like receptor 4 (TLR4) signalling contributes to inflammatory cardiovascular diseases, but its role in hypertension and the associated vascular damage is not known. We investigated whether TLR4 activation contributed to angiotensin II (AngII)-induced hypertension and the associated vascular structural, mechanical and functional alterations. AngII was infused (1.44 mg·kg−1·day−1, s.c.) for 2 weeks in C57BL6 mice, treated with a neutralizing anti-TLR4 antibody or IgG (1 μg·day−1); systolic BP (SBP) and aortic cytokine levels were measured. Structural, mechanical and contractile properties of aortic and mesenteric arterial segments were measured with myography and histology. RT-PCR and Western blotting were used to analyse these tissues and cultured vascular smooth muscle cells (VSMC) from hypertensive rats (SHR). Aortic TLR4 mRNA levels were raised by AngII infusion. Anti-TLR4 antibody treatment of AngII-treated mice normalised: (i) increased SBP and TNF-α, IL-6 and CCL2 levels; (ii) vascular structural and mechanical changes; (iii) altered aortic phenylephrine- and ACh-induced responses; (iv) increased NOX-1 mRNA levels, superoxide anion production and NAD(P)H oxidase activity and effects of catalase, apocynin, ML-171 and Mito-TEMPO on vascular responses; and (v) reduced NO release and effects of L-NAME on phenylephrine-induced contraction. In VSMC, the MyD88 inhibitor ST-2825 reduced AngII-induced NAD(P)H oxidase activity. The TLR4 inhibitor CLI-095 reduced AngII-induced increased phospho-JNK1/2 and p65 NF-κB subunit nuclear protein expression. TLR4 up-regulation by AngII contributed to the inflammation, endothelial dysfunction, vascular remodelling and stiffness associated with hypertension by mechanisms involving oxidative stress. MyD88-dependent activation and JNK/NF-κB signalling pathways participated in these alterations.
El tratamiento con pioglitazona modifica la participación de derivados de la Ciclooxigenasa-2 y de especies reactivas de oxígeno en la respuesta a fenilefrina de arterias de resistencia de ratas hipertensas
(2009-07) Hernanz, R; Martín, A; Pérez-Girón, JV; Avendaño, MS; Roque, FR; Palacios, R; Salaices, M; Alonso, MJ
Los receptores activadores de la proliferación peroxisomal-¿ (PPAR¿) son factores de transcripción expresados en la pared vascular con actividad cardioprotectora. Además, las glitazonas, agonistas PPAR¿, tienen acciones antiinflamatorias asociadas a la interferencia con factores de transcripción redox-sensibles como NF-kappaB, implicados en la transcripción de diversos genes, incluyendo la isoforma inducible de la ciclooxigenasa (COX-2) Objetivo: Analizar el efecto del tratamiento de ratas espontáneamente hipertensas (SHR)con pioglitazona sobre la respuesta a fenilefrina de arterias de resistencia, así como el efecto del mismo sobre el papel de prostanoides y especies reactivas de oxígeno (ROS) en dicha respuesta. Métodos: Se han utilizado arterias mesentéricas de resistencia de ratas SHR de 6 meses tratadas o no con el agonista PPARgamma pioglitazona (2,5 mg/Kg/día, 28 días). La reactividad vascular se ha estudiado en un miógrafo isométrico y la expresión proteica por western blot. Resultados: El tratamiento con pioglitazona no modificó la PAS ni la contracción inducida por fenilefrina. Indometacina (10 microM), el inhibidor selectivo de COX-2 NS 398 (1 microM), el antagonista del receptor TP SQ 29,548 (1 microM) y el antagonista del receptor EP1 SC 19220 (10 µM) redujeron la respuesta a fenilefrina sólo en segmentos de ratas tratadas, mientras que el inhibidor de la TXA2 sintasa furegrelato (1 microM) no modificó la respuesta en ningún grupo. La expresión de COX-2 fue mayor en arterias mesentéricas de ratas tratadas. Además, pioglitazona abolió el efecto inhibitorio de apocinina (0.3 mM) y alopurinol (0.3 mM), inhibidores respectivos de la NADPH oxidasa y la xantina oxidasa, sobre la respuesta a fenilefrina y redujo la expresión vascular de Cu/Zn- y Mn-SOD. Conclusiones: El tratamiento crónico de ratas hipertensas con pioglitazona incrementa la expresión vascular de COX-2 y la participación de prostanoides vasoconstrictores en la respuesta a fenilefrina. A pesar de que el tratamiento reduce la expresión de las isoformas citosólica y mitocondrial de SOD, la reducción en la participación de ROS en la respuesta a fenilefrina puede explicar que la respuesta a este vasoconstrictor sea similar.