Raquel Hernanz

Atorvastatin Prevents Angiotensin II–Induced Vascular Remodeling and Oxidative Stress

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 reduces the increased participation of cyclooxygenase-2-derived products in vascular responses of hypertensive rats.

This study analyzes the role of angiotensin II (Ang II), via AT1 receptors, in the involvement of cyclooxygenase (COX)-2-derived prostanoids in phenylephrine responses in normotensive rats (Wistar Kyoto; WKY) and spontaneously hypertensive rats (SHR). Aorta from rats untreated or treated for 12 weeks with losartan (15 mg/kg · day) or hydralazine plus hydrochlorothiazide (44 and 9.4 mg/kg · day, respectively) and vascular smooth muscle cells (VSMC) from SHR were used. Vascular reactivity was analyzed by isometric recording; COX-2 expression by Western blot and reverse transcription-polymerase chain reaction; prostaglandin (PG)I2, PGF2α, 8-isoprostane, and total antioxidant status (TAS) by commercial kits; superoxide anion (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document}) by lucigenin chemiluminescence; and plasmatic malondialdehyde (MDA) by thiobarbituric acid assay. The COX-2 inhibitor N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398) at 1 μM reduced phenylephrine responses more in SHR than in WKY rats. COX-2 protein and mRNA expressions, PGF2α, PGI2, 8-isoprostane, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} production, and MDA levels were higher in SHR, but TAS was similar in both strains. Losartan, but not hydralazine-hydrochlorothiazide treatment, reduced COX-2 expression and the effect of NS-398 on phenylephrine responses in SHR. Losartan also increased TAS and reduced PGF2α, PGI2, 8-isoprostane, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} production and MDA levels in SHR. Ang II (0.1 μM) induced COX-2 expression in VSMC from SHR that was reduced by 30 μM apocynin and 100 μM allopurinol, NADPH oxidase, and xanthine oxidase inhibitors, respectively. In conclusion, AT1 receptor activation by Ang II could be involved in the increased participation of COX-2-derived contractile prostanoids in vasoconstriction to phenylephrine with hypertension, probably through COX-2 expression regulation. The increased oxidative stress seems to be one of the mechanisms involved.

Peroxisome proliferator-activated receptor-γ activation reduces cyclooxygenase-2 expression in vascular smooth muscle cells from hypertensive rats by interfering with oxidative stress.

Aims: Hypertension is associated with increased plasma inflammatory markers such as cytokines and increased vascular cyclooxygenase-2 (COX-2) expression. The ability of peroxisome proliferator-activated receptor-&ggr; (PPAR&ggr;) agonists to reduce oxidative stress seems to contribute to their anti-inflammatory properties. This study analyzes the effect of pioglitazone, a PPAR&ggr; agonist, on interleukin-1&bgr;-induced COX-2 expression and the role of reactive oxygen species (ROS) on this effect. Methods and results: Vascular smooth muscle cells from hypertensive rats stimulated with interleukin-1&bgr; (10 ng/ml, 24 h) were used. Interleukin-1&bgr; increased: 1) COX-2 protein and mRNA levels; 2) protein and mRNA levels of the NADPH oxidase subunit NOX-1, NADPH oxidase activity and ROS production; and 3) phosphorylation of inhibitor of nuclear factor kappa B (I&kgr;B) kinase (IKK) nuclear expression of the p65 nuclear factor kappa B (NF-&kgr;B) subunit and cell proliferation, all of which were reduced by apocynin (30 &mgr;mol/l). Interleukin-1&bgr;-induced COX-2 expression was reduced by apocynin, tempol (10 &mgr;mol/l), catalase (1000 U/ml) and lactacystin (5 &mgr;mol/l). Moreover, H2O2 (50 &mgr;mol/l, 90 min) induced COX-2 expression, which was reduced by lactacystin. Pioglitazone (10 &mgr;mol/l) reduced the effects of interleukin-1&bgr; on: 1) COX-2 protein and mRNA levels; 2) NOX-1 protein and mRNA levels, NADPH oxidase activity and ROS production; and 3) p-IKK, p65 expressions and cell proliferation. Pioglitazone also reduced the H2O2-induced COX-2 expression and increased Cu/Zn and Mn-superoxide dismutase protein expression. PPAR&ggr; small interfering RNA (5 nmol/l) further increased interleukin-1&bgr;-induced COX-2 and NOX-1 mRNA levels. In addition, pioglitazone increased the interleukin-1&bgr;-induced PPAR&ggr; mRNA levels. Conclusion: PPAR&ggr; activation with pioglitazone reduces interleukin-1&bgr;-induced COX-2 expression by interference with the redox-sensitive transcription factor NF-&kgr;B.

Mechanisms involved in the early increase of serotonin contraction evoked by endotoxin in rat middle cerebral arteries

The present study investigated the mechanisms involved in the increased 5‐hydroxytryptamine (5‐HT) vasoconstriction observed in rat middle cerebral arteries exposed in vitro to lipopolysaccharide (LPS, 10 μg ml−1) for 1–5 h. Functional, immunohistochemical and Western blot analysis and superoxide anion measurements by ethidium fluorescence were performed. LPS exposure increased 5‐HT (10 μM) vasoconstriction only during the first 4 h. In contrast to control tissue, indomethacin (10 μM), the COX‐2 inhibitor NS 398 (10 μM), the TXA2/PGH2 receptor antagonist SQ 29,548 (1 μM) and the TXA2 synthase inhibitor furegrelate (1 μM) reduced 5‐HT contraction of LPS‐treated arteries from hour one. The iNOS inhibitor aminoguanidine (0.1 mM) increased 5‐HT contraction from hour three of LPS incubation. The superoxide anion scavenger superoxide dismutase (SOD, 100 U ml−1) and the H2O2 scavenger catalase (1000 U ml−1), as well as the respective inhibitors of NAD(P)H oxidase and xanthine oxidase, apocynin (0.3 mM) and allopurinol (0.3 mM), reduced 5‐HT contraction after LPS incubation. LPS induced an increase in superoxide anion levels that was abolished by PEG‐SOD. Subthreshold concentrations of the TXA2 analogue U 46619, xanthine/xanthine oxidase and H2O2 potentiated, whereas those of sodium nitroprusside inhibited, the 5‐HT contraction. COX‐2 expression was increased at 1 and 5 h of LPS incubation, while that of iNOS, Cu/Zn‐SOD and Mn‐SOD was only increased after 5 h. All the three vascular layers expressed COX‐2 and Cu/Zn‐SOD. iNOS expression was detected in the endothelium and adventitia after LPS. In conclusion, increased production of TXA2 from COX‐2, superoxide anion and H2O2 enhanced vasoconstriction to 5‐HT during the first few hours of LPS exposure; iNOS and SOD expression counteracted that increase at 5 h. These changes can contribute to the disturbance of cerebral blood flow in endotoxic shock.

Hypertension alters the participation of contractile prostanoids and superoxide anions in lipopolysaccharide effects on small mesenteric arteries

The involvement of cyclooxygenase-2 (COX-2)-derived products and superoxide anion in the effect of lipopolysaccharide in noradrenaline (NA)-induced contraction was investigated in small mesenteric arteries (SMA) from normotensive, Wistar Kyoto (WKY), and spontaneously hypertensive (SHR) rats. In WKY, lipopolysaccharide (10 microg/ml, 1 and 5 h) only inhibited the NA response (0.1-30 microM) in the presence of dexamethasone (1 microM), indomethacin (10 microM), the selective COX-2 inhibitor, NS 398 (10 microM), and the TXA(2)/PGH(2) receptor antagonist, SQ 29,548 (10 microM) but not of superoxide dismutase (SOD, 100 U/ml). In SHR, lipopolysaccharide inhibited the NA response by itself; this inhibition was potentiated by dexamethasone, indomethacin, NS 398, SQ 29,548 and SOD. The effect of lipopolysaccharide plus indomethacin, NS 398 or SQ 29,548 was higher in SMA from WKY than SHR only after 1 h lipopolysaccharide incubation. N(G)-nitro-L-arginine methyl ester (100 microM) and endothelium removal abolished the indomethacin-induced potentiatory effect of lipopolysaccharide in both strains. Endothelium removal also abolished the SOD potentiatory effect in SMA from SHR. Lipopolysaccharide increases COX-2 expression to a similar level in both strains and iNOS expression in a greater extent in SHR; these increases were reduced by dexamethasone. These results indicate: 1) lipopolysaccharide induces the endothelial production of contractile prostanoids from COX-2 in SMA, probably to compensate the increase in NO from iNOS; 2) the production of prostanoids in the presence of lipopolysaccharide seems to be greater in normotensive than hypertensive rats only after lipopolysaccharide short incubation times; 3) endothelial production of O(2)(.-) contributes to counteract depression of NA contraction caused by lipopolysaccharide only in SHR.

Alterations of the nitric oxide pathway in cerebral arteries from spontaneously hypertensive rats

Hypertension-associated alterations of the nitric oxide (NO) pathway were analyzed in middle cerebral arteries (MCA) from normotensive (WKY) and hypertensive (SHR) rats. The vasoconstrictor response to prostaglandin F2&agr; (PGF2&agr;, 30 and 100 &mgr;M) was smaller in MCA from SHR than from WKY. Endothelium-dependent relaxations to bradykinin (1 n M–10 &mgr;M) or acetylcholine (10 &mgr;M) were similar in MCA from both strains, whereas the endothelium-independent response to sodium nitroprusside (1 n M–0.1 m M) was smaller in MCA from SHR. L-arginine (L-Arg, 10 &mgr;M) similarly inhibited the vasoconstrictor responses in both strains; however, the inhibitory effect of 100 &mgr;M of L-Arg was greater in MCA from SHR. Nω-nitro-L-arginine methyl ester (L-NAME, 100 &mgr;M), but not aminoguanidine (100 &mgr;M) or 7-nitroindazole (10 &mgr;M), increased basal tone, potentiated the PGF2&agr;-induced vasoconstrictor responses and reduced the bradykinin-elicited relaxation in a similar way in MCA from WKY and SHR. Nω-nitro-L-arginine methyl ester also antagonized the inhibitory effect of 10 &mgr;M of L-Arg. Incubation for 5 h with lipopolysaccharide (10 &mgr;g/ml) similarly reduced the response to PGF2&agr; in MCA from WKY and SHR; this reduction was antagonized by dexamethasone (1 &mgr;M). Cerebral arteries expressed endothelial (eNOS) and neuronal (nNOS) NO synthase similarly in both strains, but inducible NOS (iNOS) expression was more evident in SHR. Lipopolysaccharide increased iNOS expression in both strains to a similar level. The basal constitutive NOS (cNOS) and iNOS activities were similar in arteries from WKY and SHR. Lipopolysaccharide increased iNOS activity only in arteries from SHR. These results indicate that hypertension did not impair endothelial NO production by NOS activation but induced an up-regulation of basal iNOS expression.

Losartan and tempol treatments normalize the increased response to hydrogen peroxide in resistance arteries from hypertensive rats

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 (15 mg/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•−) production was evaluated by dihydroethidium fluorescence, thromboxane A2 production by enzyme immunoassay and plasma nitrite levels by the Griess method. Results H2O2 (1–100 μmol/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] (1 μmol/l), and the thromboxane A2/prostaglandin H2 receptor antagonist, SQ 29 548 (1 μmol/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 (100 μmol/l), on H2O2 responses and the acetylcholine-induced 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.

Chronic ouabain treatment increases the contribution of nitric oxide to endothelium dependent relaxation

The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega-nitro-l-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higher in segments from ouabain-treated rats. Preincubation with the prostacyclin synthesis inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments from both groups. The Ca2+-dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only in segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompanied by increased endothelial nitric oxide participation and impaired endothelium-dependent hyperpolarizing factor contribution in acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment on acetylcholine responses in rat aorta.ResumenSe analiza en este trabajo la contribución del óxido nítrico, la prostaciclina y el factor hiperpolarizante dependiente de endotelio (EDHF) en la vasodilatación inducida por acetilcolina en aorta de ratas controles y con hipertensión inducida por ouabaína. La preincubación con el inhibidor de la óxido nítrico sintasa N-omega-nitro-L-arginina metil éster (L-NAME) inhibió la respuesta vasodilatadora a acetilcolina en segmentos de ambos grupos experimentales, pero en mayor medida en los de ratas tratadas con ouabaína. La liberación de óxido nítrico basal e inducida por acetilcolina fue mayor en segmentos de animales tratados con ouabaína. La preincubación con tranilcipromina, inhibidor de la prostaciclina sintasa, y con indometacina, inhibidor de la ciclooxigenasa, inhibió la respuesta vasodilatadora a acetilcolina en segmentos de aorta de ambos grupos experimentales. El bloqueante de los canales de potasio dependientes de Ca2+, charibdotoxina, inhibió la respuesta vasodilatadora a acetilcolina sólo en segmentos de animales control. Estos resultados indican que la hipertensión inducida por el tratamiento crónico con ouabaína se acompaña de aumento en la participación del óxido nítrico endotelial y de disminución del efecto del EDHF mediado por canales de potasio dependientes de Ca2+ en la relajación inducida por acetilcolina. Estos efectos opuestos podrían explicar el hecho de que el tratamiento con ouabaína no modifique la respuesta a la acetilcolina.

University of Wisconsin solution increases hyperpolarizing mechanisms in response to bradykinin.

BACKGROUND The aim of this study was to determine the effect of University of Wisconsin solution (UWS) incubation on bradykinin-induced vasodilation. METHODS Porcine coronary arteries were incubated in Krebs-Henseleit solution (KHS) or UWS at 4 degrees C for 20 hours. Endothelium-dependent relaxation to bradykinin and endothelium-independent relaxation to nitric oxide were tested after U46619 or KCl pre-contraction. Nitric oxide synthase activity and protein expression was determined by [3H]-L-citrulline formation and western blot analysis, respectively. RESULTS The relaxation to bradykinin (0.1 to 300 nmol/liter) after U46619 (30 to 300 nmol/liter) pre-contraction was similar with both KHS and UWS pre-incubation; however, it was reduced after KCl pre-contraction (15 to 20 mmol/liter), this reduction being greater after UWS incubation. The inhibitory effect of N(G)-nitro-L-arginine methylester (0.1 mmol/liter) on bradykinin-induced relaxation was lower in UWS- than KHS-incubated segments after U46619 pre-contraction, but similar after KCl pre-contraction; however, the inhibitory effect of 0.5 mmol/liter ouabain was unaffected. Tetraethylammonium (5 mmol/liter) reduced the response to bradykinin more strongly after UWS pre-incubation. UWS did not modify relaxation to nitric oxide (0.1 to 30 micromol/liter) in pre-incubated UWS or KHS segments. UWS failed to modify both total nitric oxide synthase activity and endothelial nitric oxide synthase expression. CONCLUSIONS UWS incubation decreased nitric oxide participation and increased the hyperpolarizing mechanisms produced by bradykinin.