Roberto Palacios

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.

Spironolactone Prevents Endothelial Nitric Oxide Synthase Uncoupling and Vascular Dysfunction Induced by β-Adrenergic Overstimulation: Role of Perivascular Adipose Tissue.

Sustained stimulation of &bgr;-adrenoceptors (&bgr;-ARs) and activation of renin–angiotensin–aldosterone system are common features of cardiovascular diseases with rising sympathetic activation, including essential hypertension, myocardial infarction, and heart failure. In this study, we investigated the role of AT1 receptor and mineralocorticoid receptor (MR) in the vascular alterations caused by &bgr;-AR overstimulation. &bgr;-AR overstimulation with associated cardiac hypertrophy and increased vasoconstrictor response to phenylephrine in aorta were modeled in rats by 7-day isoproterenol treatment. The increased vasoconstrictor response to phenylephrine in this model was blunted by the MR antagonist spironolactone, but not by the AT1 receptor antagonist losartan, despite the blunting of cardiac hypertrophy with both drugs. Spironolactone, but not losartan, restored NO bioavailability in association with lower endothelial nitric oxide synthase–derived superoxide production, increased endothelial nitric oxide synthase dimerization, and aortic HSP90 upregulation. MR genomic and nongenomic functions were activated in aortas from isoproterenol-treated rats. Isoproterenol did not modify plasma levels of MR ligands aldosterone and corticosterone but rather increased perivascular adipose tissue–derived corticosterone in association with increased expression of 11&bgr;-hydroxysteroid dehydrogenase type 1. The anticontractile effect of aortic perivascular adipose tissue was impaired by &bgr;-AR overstimulation and restored by MR blockade. These results suggest that activation of vascular MR signaling contributes to the vascular dysfunction induced by &bgr;-AR overstimulation associated with endothelial nitric oxide synthase uncoupling. These findings reveal an additional explanation for the protective effects of MR antagonists in cardiovascular disorders with sympathetic activation.

Aluminum exposure at human dietary levels promotes vascular dysfunction and increases blood pressure in rats: A concerted action of NAD(P)H oxidase and COX-2

Aluminum (Al) is a non-essential metal and a significant environmental contaminant and is associated with a number of human diseases including cardiovascular disease. We investigated the effects of Al exposure at doses similar to human dietary levels on the cardiovascular system over a 60day period. Wistar male rats were divided into two major groups and received orally: 1) Low aluminum level - rats were subdivided and treated for 60days as follows: a) Untreated - ultrapure water; b) AlCl3 at a dose of 8.3mg/kg bw for 60days, representing human Al exposure by diet; and 2) High aluminum level - rats were subdivided and treated for 42days as follows: C) Untreated - ultrapure water; d) AlCl3 at 100mg/kg bw for 42days, representing a high level of human exposure to Al. Effects on systolic blood pressure (SBP) and vascular function of aortic and mesenteric resistance arteries (MRA) were studied. Endothelium and smooth muscle integrity were evaluated by concentration-response curves to acetylcholine (ACh) and sodium nitroprusside. Vasoconstrictor responses to phenylephrine (Phe) in the presence and absence of endothelium and in the presence of the NOS inhibitor L-NAME, the potassium channels blocker TEA, the NAD(P)H oxidase inhibitor apocynin, superoxide dismutase (SOD), the non-selective COX inhibitor indomethacin and the selective COX-2 inhibitor NS 398 were analyzed. Vascular reactive oxygen species (ROS), lipid peroxidation and total antioxidant capacity, were measured. The mRNA expressions of eNOS, NAD(P)H oxidase 1 and 2, SOD1, COX-2 and thromboxane A2 receptor (TXA-2 R) were also investigated. Al exposure at human dietary levels impaired the cardiovascular system and these effects were almost the same as Al exposure at much higher levels. Al increased SBP, decreased ACh-induced relaxation, increased response to Phe, decreased endothelial modulation of vasoconstrictor responses, the bioavailability of nitric oxide (NO), the involvement of potassium channels on vascular responses, as well as increased ROS production from NAD(P)H oxidase and contractile prostanoids mainly from COX-2 in both aorta and mesenteric arteries. Al exposure increased vascular ROS production and lipid peroxidation as well as altered the antioxidant status in aorta and MRA. Al decreased vascular eNOS and SOD1 mRNA levels and increased the NAD(P)H oxidase 1, COX-2 and TXA-2 R mRNA levels. Our results point to an excess of ROS mainly from NAD(P)H oxidase after Al exposure and the increased vascular prostanoids from COX-2 acting in concert to decrease NO bioavailability, thus inducing vascular dysfunction and increasing blood pressure. Therefore, 60-day chronic exposure to Al, which reflects common human dietary Al intake, appears to pose a risk for the cardiovascular system.

Spironolactone Prevents Endothelial Nitric Oxide Synthase Uncoupling and Vascular Dysfunction Induced by β-Adrenergic Overstimulation

Sustained stimulation of &bgr;-adrenoceptors (&bgr;-ARs) and activation of renin–angiotensin–aldosterone system are common features of cardiovascular diseases with rising sympathetic activation, including essential hypertension, myocardial infarction, and heart failure. In this study, we investigated the role of AT1 receptor and mineralocorticoid receptor (MR) in the vascular alterations caused by &bgr;-AR overstimulation. &bgr;-AR overstimulation with associated cardiac hypertrophy and increased vasoconstrictor response to phenylephrine in aorta were modeled in rats by 7-day isoproterenol treatment. The increased vasoconstrictor response to phenylephrine in this model was blunted by the MR antagonist spironolactone, but not by the AT1 receptor antagonist losartan, despite the blunting of cardiac hypertrophy with both drugs. Spironolactone, but not losartan, restored NO bioavailability in association with lower endothelial nitric oxide synthase–derived superoxide production, increased endothelial nitric oxide synthase dimerization, and aortic HSP90 upregulation. MR genomic and nongenomic functions were activated in aortas from isoproterenol-treated rats. Isoproterenol did not modify plasma levels of MR ligands aldosterone and corticosterone but rather increased perivascular adipose tissue–derived corticosterone in association with increased expression of 11&bgr;-hydroxysteroid dehydrogenase type 1. The anticontractile effect of aortic perivascular adipose tissue was impaired by &bgr;-AR overstimulation and restored by MR blockade. These results suggest that activation of vascular MR signaling contributes to the vascular dysfunction induced by &bgr;-AR overstimulation associated with endothelial nitric oxide synthase uncoupling. These findings reveal an additional explanation for the protective effects of MR antagonists in cardiovascular disorders with sympathetic activation.

Spironolactone Prevents Endothelial Nitric Oxide Synthase Uncoupling and Vascular Dysfunction Induced by β-Adrenergic OverstimulationNovelty and Significance

Sustained stimulation of &bgr;-adrenoceptors (&bgr;-ARs) and activation of renin–angiotensin–aldosterone system are common features of cardiovascular diseases with rising sympathetic activation, including essential hypertension, myocardial infarction, and heart failure. In this study, we investigated the role of AT1 receptor and mineralocorticoid receptor (MR) in the vascular alterations caused by &bgr;-AR overstimulation. &bgr;-AR overstimulation with associated cardiac hypertrophy and increased vasoconstrictor response to phenylephrine in aorta were modeled in rats by 7-day isoproterenol treatment. The increased vasoconstrictor response to phenylephrine in this model was blunted by the MR antagonist spironolactone, but not by the AT1 receptor antagonist losartan, despite the blunting of cardiac hypertrophy with both drugs. Spironolactone, but not losartan, restored NO bioavailability in association with lower endothelial nitric oxide synthase–derived superoxide production, increased endothelial nitric oxide synthase dimerization, and aortic HSP90 upregulation. MR genomic and nongenomic functions were activated in aortas from isoproterenol-treated rats. Isoproterenol did not modify plasma levels of MR ligands aldosterone and corticosterone but rather increased perivascular adipose tissue–derived corticosterone in association with increased expression of 11&bgr;-hydroxysteroid dehydrogenase type 1. The anticontractile effect of aortic perivascular adipose tissue was impaired by &bgr;-AR overstimulation and restored by MR blockade. These results suggest that activation of vascular MR signaling contributes to the vascular dysfunction induced by &bgr;-AR overstimulation associated with endothelial nitric oxide synthase uncoupling. These findings reveal an additional explanation for the protective effects of MR antagonists in cardiovascular disorders with sympathetic activation.

Spironolactone Prevents Endothelial Nitric Oxide Synthase Uncoupling and Vascular Dysfunction Induced by β-Adrenergic OverstimulationNovelty and Significance: Role of Perivascular Adipose Tissue

Sustained stimulation of &bgr;-adrenoceptors (&bgr;-ARs) and activation of renin–angiotensin–aldosterone system are common features of cardiovascular diseases with rising sympathetic activation, including essential hypertension, myocardial infarction, and heart failure. In this study, we investigated the role of AT1 receptor and mineralocorticoid receptor (MR) in the vascular alterations caused by &bgr;-AR overstimulation. &bgr;-AR overstimulation with associated cardiac hypertrophy and increased vasoconstrictor response to phenylephrine in aorta were modeled in rats by 7-day isoproterenol treatment. The increased vasoconstrictor response to phenylephrine in this model was blunted by the MR antagonist spironolactone, but not by the AT1 receptor antagonist losartan, despite the blunting of cardiac hypertrophy with both drugs. Spironolactone, but not losartan, restored NO bioavailability in association with lower endothelial nitric oxide synthase–derived superoxide production, increased endothelial nitric oxide synthase dimerization, and aortic HSP90 upregulation. MR genomic and nongenomic functions were activated in aortas from isoproterenol-treated rats. Isoproterenol did not modify plasma levels of MR ligands aldosterone and corticosterone but rather increased perivascular adipose tissue–derived corticosterone in association with increased expression of 11&bgr;-hydroxysteroid dehydrogenase type 1. The anticontractile effect of aortic perivascular adipose tissue was impaired by &bgr;-AR overstimulation and restored by MR blockade. These results suggest that activation of vascular MR signaling contributes to the vascular dysfunction induced by &bgr;-AR overstimulation associated with endothelial nitric oxide synthase uncoupling. These findings reveal an additional explanation for the protective effects of MR antagonists in cardiovascular disorders with sympathetic activation.

ANGIOTENSIN-II-INDUCED ENDOTHELIN-1 PRODUCTION CONTRIBUTES TO THE INCREASED COX-2 EXPRESSION IN HYPERTENSIVE RATS. ROLE OF PPAR: PP.24.478

The increased angiotensin-II levels seem to contribute to the increased reactive oxygen species (ROS) and proinflammatory markers production in hypertension. Endothelin-1 has been proposed to explain the cardiovascular damage induced by angiotensin-II. Peroxisome proliferator activated receptor-γ (PPARγ) agonists have anti-inflammatory actions by interference with NFκB signalling pathway. Aim: To analyze the involvement of ROS and andothelin-1 on angiotensin-II-induced cyclooxygenase-2 expression and the role of PPARγ activation on this effect. Methods: Aortic vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and aortic segments from SHR and Wistar-Kyoto (WKY) rats were used to quantify mRNA and protein levels by qRT-PCR and Western blot, respectively. Results: cyclooxygenase-2 mRNA levels were greater in SHR than WKY; losartan (15 mg/Kg/day, 12 weeks) reduced those levels in SHR. In VSMC from SHR, angiotensin-II (0.1 μM, 2 h) induced cyclooxygenase-2, endothelin-1 and NOX-1 transcription; this effect was reduced by losartan (10 μM). Angiotensin-II-induced cyclooxygenase-2 expression was reduced by apocynin (30 μM). BQ123 (1 μM) reduced cyclooxygenase-2 and NOX-1 transcription. Lactacystin (20 μM) only inhibited NOX-1 and cyclooxygenase-2 mRNA levels. Pioglitazone (10 μM) decreased angiotensin-II-induced cyclooxygenase-2 and NOX-1 mRNA levels and p65-NFκB subunit nuclear translocation. Conclusions: Angiotensin-II contributes to the increased vascular cyclooxygenase-2 expression in hypertension. Angiotensin-II-induced cyclooxygenase-2 expression in SHR is related with NOX-1 induction and NFκB activation. Angiotensin-II-induced endothelin-1 production contributes to the increased NOX-1 and cyclooxygenase-2 expression by ETA receptors activation. PPARγ activation inhibits angiotensin-II-induced cyclooxygenase-2 by decreasing NOX-1 expression and probably activating transrepression mechanisms on NFκB.