Yolanda Alvarez

Reciprocal Relationship Between Reactive Oxygen Species and Cyclooxygenase-2 and Vascular Dysfunction in Hypertension

AIMS This study evaluates a possible relationship between reactive oxygen species (ROS) and cyclooxygenase (COX)-2-derived products in conductance and resistance arteries from hypertensive animals. Angiotensin II (Ang II)-infused mice or spontaneously hypertensive rats treated with the NAD(P)H Oxidase inhibitor apocynin, the mitochondrion-targeted SOD2 mimetic Mito-TEMPO, the superoxide dismutase analog tempol, or the COX-2 inhibitor Celecoxib were used. RESULTS Apocynin, Mito-TEMPO, and Celecoxib treatments prevented Ang II-induced hypertension, the increased vasoconstrictor responses to phenylephrine, and the reduced acetylcholine relaxation. The NOX-2 inhibitor gp91ds-tat, the NOX-1 inhibitor ML171, catalase, and the COX-2 inhibitor NS398 abolished the ex vivo effect of Ang II-enhancing phenylephrine responses. Antioxidant treatments diminished the increased vascular COX-2 expression, prostanoid production, and/or participation of COX-derived contractile prostanoids and thromboxane A(2) receptor (TP) in phenylephrine responses, observed in arteries from hypertensive models. The treatment with the COX-2 inhibitor normalized the increased ROS production (O(2)·(-) and H(2)O(2)), NAD(P)H Oxidase expression (NOX-1, NOX-4, and p22phox) and activity, MnSOD expression, and the participation of ROS in vascular responses in both hypertensive models. Apocynin and Mito-TEMPO also normalized these parameters of oxidative stress. Apocynin, Mito-TEMPO, and Celecoxib improved the diminished nitric oxide (NO) production and the modulation by NO of phenylephrine responses in the Ang II model. INNOVATION This study provides mechanistic evidence of circuitous relationship between COX-2 products and ROS in hypertension. CONCLUSION The excess of ROS from NAD(P)H Oxidase and/or mitochondria and the increased vascular COX-2/TP receptor axis act in concert to induce vascular dysfunction and hypertension.

Hypertension increases the participation of vasoconstrictor prostanoids from cyclooxygenase-2 in phenylephrine responses.

Objective The present study was designed to analyse whether hypertension alters the involvement of cyclooxygenase-2-derived mediators in phenylephrine-induced vasoconstrictor responses. Methods Vascular reactivity experiments were performed in aortic segments from normotensive, Wistar–Kyoto, and spontaneously hypertensive rats (SHR); protein expression was measured by western blot and/or immunohistochemistry, and prostaglandin F2α (PGF2α), 8-isoprostane and prostacyclin release were determined by enzyme immunoassay commercial kits. Results The protein synthesis inhibitor dexamethasone (1 μmol/l), the non-selective cyclooxygenase inhibitor indomethacin (10 μmol/l), the selective cyclooxygenase-2 inhibitor NS 398 (1 μmol/l), and the thromboxane A2/prostaglandin H2 (TP) receptor antagonist SQ 29,548 (1 μmol/l), reduced the concentration–response curves to phenylephrine more in segments from hypertensive than from normotensive rats; however, the thromboxane A2 (TxA2) synthase inhibitors furegrelate (10 μmol/l) and OKY 046 (1 and 10 μmol/l) had no effect in either strain. Removing endothelium or adding dexamethasone almost abolished the NS 398 effect. Cyclooxygenase-2 protein expression, which was reduced by dexamethasone, was higher in aorta from hypertensive animals. In both strains cyclooxygenase-2 was localized mainly in endothelial cells and adventitial fibroblasts. 13,14-Dihydro-15-keto-PGF2α, 6-keto-PGF1α and 8-isoprostane levels were greater in the medium from hypertensive than from normotensive rats; NS 398 decreased levels of the three metabolites studied only in the medium from SHR. Conclusions PGF2α and 8-isoprostane seem to be involved in the response to phenylephrine in rat aorta; this involvement is greater in hypertensive rats, probably due to a higher endothelial induction of cyclooxygenase-2.

Vasodilator effects of peptides derived from egg white proteins

The aim of this work was to investigate the effect of several peptides, identified before and after simulated gastrointestinal digestion of an egg white hydrolysate, on the vascular function, in rat aorta. The sequences IVF, RADHPFL and YAEERYPIL (0.1 mM) induced vasodilatation in intact aortic rings, with the maximum percentage of dilation corresponding to RADHPFL (40.5+/-7.0%). Two of the end products of the gastrointestinal digestion, RADHP and YPI, also showed vasodilator activity with degrees of relaxation higher than 50%. However, all these peptides failed to induce relaxation in endothelium-denuded aortic rings. The relaxation induced by RADHP was concentration-dependent and it was partially blocked by the nitric oxide synthase inhibitor L-NAME (100 microM) and by the B(1) bradykinin receptor antagonist Des-HOE 140 (30 nM), thus showing that it was mediated by NO production through the activation of B(1) bradykinin receptors. These findings suggest that these peptides could reduce the vascular resistance and could be used as functional food ingredients in the prevention and treatment of hypertension.

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.

Polarization of the innate immune response by prostaglandin E2: A puzzle of receptors and signals

Eicosanoids tailor the innate immune response by supporting local inflammation and exhibiting immunomodulatory properties. Prostaglandin (PG) E2 is the most abundant eicosanoid in the inflammatory milieu due to the robust production elicited by pathogen-associated molecular patterns on cells of the innate immune system. The different functions and cell distribution of E prostanoid receptors explain the difficulty encountered thus far to delineate the actual role of PGE2 in the immune response. The biosynthesis of eicosanoids includes as the first step the Ca2+- and kinase-dependent activation of the cytosolic phospholipase A2, which releases arachidonic acid from membrane phospholipids, and later events depending on the transcriptional regulation of the enzymes of the cyclooxygenase routes, where PGE2 is the most relevant product. Acting in an autocrine/paracrine manner in macrophages, PGE2 induces a regulatory phenotype including the expression of interleukin (IL)-10, sphingosine kinase 1, and the tumor necrosis factor family molecule LIGHT. PGE2 also stabilizes the suppressive function of myeloid-derived suppressor cells, inhibits the release of IL-12 p70 by macrophages and dendritic cells, and may enhance the production of IL-23. PGE2 is a central component of the inflammasome-dependent induction of the eicosanoid storm that leads to massive loss of intravascular fluid, increases the mortality rate associated with coinfection by Candida ssp. and bacteria, and inhibits fungal phagocytosis. These effects have important consequences for the outcome of infections and the polarization of the immune response into the T helper cell types 2 and 17 and can be a clue to develop pharmacological tools to address infectious, autoimmune, and autoinflammatory diseases.

Role of NADPH oxidase and iNOS in vasoconstrictor responses of vessels from hypertensive and normotensive rats.

To analyse the influence of hypertension in the modulation induced by inducible NOS (iNOS)‐derived NO and superoxide anion (O2 •−) of vasoconstrictor responses and the sources of O2 •− implicated.

p38 MAPK contributes to angiotensin II-induced COX-2 expression in aortic fibroblasts from normotensive and hypertensive rats

Objective To investigate the effect of angiotensin II on cyclooxygenase-2 (COX-2) expression in aortic adventitial fibroblasts from normotensive [Wistar–Kyoto (WKY)] rats and spontaneously hypertensive rats (SHRs). Methods Protein expression was determined by western blot, mRNA levels by real-time PCR, transcriptional activity by luciferase assays, superoxide anion (O2•−) production by dihydroethidine fluorescence and prostaglandin E2 by enzyme immunoassay. Results Angiotensin II (0.1 μmol/l, 0.5–6 h) time dependently induced COX-2 protein expression, this effect being transient in fibroblasts from WKY rats and maintained over time in SHRs. Angiotensin II effect was abolished by valsartan (1 μmol/l), an angiotensin II type 1 receptor antagonist. Angiotensin II-induced prostaglandin E2 production was reduced by valsartan and the COX-2 inhibitor NS398 (1 μmol/l). Angiotensin II increased O2•− production more in SHR than WKY rats. This increase was reduced by apocynin (30 μmol/l) and allopurinol (10 μmol/l), respective nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidase inhibitors. However, angiotensin II-induced COX-2 expression was unaffected by apocynin, allopurinol, tempol (1 mmol/l) or catalase (1000 U/ml). Angiotensin II (2–30 min) induced p38 mitogen-activated protein kinase (MAPK) phosphorylation, transiently in WKY rats but sustained in SHRs. The p38 inhibitor SB203580 (10 μmol/l) reduced angiotensin II-induced COX-2 protein and mRNA levels. The angiotensin II effect was not prevented by inhibition of mRNA synthesis, and angiotensin II was unable to modulate COX-2 transcriptional activity. Conclusions Angiotensin II increases COX-2 expression in aortic fibroblasts through mechanisms including p38 MAPK pathway, independent of reactive oxygen species production and nonmediated by COX-2 transcriptional activity modulation. The sustained angiotensin-induced p38 MAPK activation in SHR cells might be related to the maintained COX-2 expression in this strain.

Aldosterone modulates neural vasomotor response in hypertension: role of calcitonin gene-related peptide.

OBJECTIVE We analyse the effect of aldosterone on vasomotor response induced by electrical field stimulation (EFS) in mesenteric arteries from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). RESULTS Aldosterone (0.001-1 microM) reduced vasoconstrictor response to EFS in a dose- and time-dependent manner only in SHR. Thus, the rest of experiments were performed only in SHR. Aldosterone did not affect either noradrenaline response or release. Effect of aldosterone (1 microM) on EFS response was not affected by NG-nitro-arginine-methyl esther (100 microM), and was abolished by capsaicin (0.5 microM) and the calcitonin gene-related peptide antagonist (CGRP 8-37, 0.5 microM). Calcitonin gene-related peptide (0.1 nM-0.1 microM) induced a concentration-dependent relaxation, which was enhanced by aldosterone (1 microM). Incubation with either spironolactone (1 microM), glibenclamide (10 microM), RU 486 10 microM, ODQ (10 microM) or cycloheximide (10 microM) significantly reduced the enhancement of CGRP-relaxation produced by aldosterone, while remained unmodified by SQ 22,536. CONCLUSIONS Aldosterone decreases the vasoconstrictor response to EFS in mesenteric arteries from SHR but not from WKY. This effect is mediated by an increased response to the sensory neurotransmitter CGRP, substantially, through glucocorticoid receptors activation. Furthermore, this effect is mediated by an increase of cGMP synthesis and ATP-dependent potassium channel activation.

Eicosanoids in the innate immune response: TLR and non-TLR routes.

The variable array of pattern receptor expression in different cells of the innate immune system explains the induction of distinct patterns of arachidonic acid (AA) metabolism. Peptidoglycan and mannan were strong stimuli in neutrophils, whereas the fungal extract zymosan was the most potent stimulus in monocyte-derived dendritic cells since it induced the production of PGE2, PGD2, and several cytokines including a robust IL-10 response. Zymosan activated κB-binding activity, but inhibition of NF-κB was associated with enhanced IL-10 production. In contrast, treatments acting on CREB (CRE binding protein), including PGE2, showed a direct correlation between CREB activation and IL-10 production. Therefore, in dendritic cells zymosan induces il10 transcription by a CRE-dependent mechanism that involves autocrine secretion of PGE2, thus unraveling a functional cooperation between eicosanoid production and cytokine production.

Contribution of the endothelin and renin-angiotensin systems to the vascular changes in rats chronically treated with ouabain.

Renin–angiotensin and endothelin systems are involved in the cardiovascular effects produced by treatment with ouabain. We recently demonstrated that the contractile response to phenylephrine is decreased in ouabain‐treated rats. The present study investigated whether endothelin‐1 (ET‐1) and angiotensin II (Ang II) contributes to the vascular changes observed in rats chronically treated with ouabain. Wistar rats were treated with ouabain (8.0 μg day−1, s.c. pellets for 5 weeks) alone or in combination with an endothelin type A receptor (ETA) antagonist, BMS182874 (40 mg kg−1 day−1, per gavage) or an angiotensin type 1 (AT1) receptor antagonist, losartan (15 mg kg−1 day−1, p.o.). Treatment with ouabain increased systolic blood pressure and treatment with either losartan or BMS182874 prevented the development of ouabain‐induced hypertension. The sensitivity and maximal response for phenylephrine were reduced in aortic rings from ouabain‐treated rats. Removal of the endothelium or in vitro exposure to an inhibitor of nitric oxide synthase (NOS), N‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM) increased the responses to phenylephrine, an effect that was more pronounced in aortas from ouabain‐treated rats. Endothelial NOS protein (eNOS) expression was increased after ouabain treatment. Treatment with BMS182874, but not with losartan, prevented the effects of ouabain on the reactivity of phenylephrine and in eNOS protein expression. Gene expression of pre–pro‐ET‐1 and ETA receptors was increased in aortic rings from ouabain‐treated rats. ETB receptor gene expression was not altered by ouabain treatment. In conclusion, our results suggest that endothelin and angiotensin systems play an important role in the development of ouabain‐induced hypertension. However, ET‐1, by activation of ETA receptors, but not Ang II, contributes to changes in vascular reactivity to phenylephrine induced by chronic treatment with ouabain.