Role of COX-2-derived PGE2 on vascular stiffness and function in hypertension.

BACKGROUND AND PURPOSE: Prostanoids derived from COX-2 and EP receptors are involved in vascular remodelling in different cardiovascular pathologies. This study evaluates the contribution of COX-2 and EP1 receptors to vascular remodelling and function in hypertension. EXPERIMENTAL APPROACH: Spontaneously hypertensive rats (SHR) and angiotensin II (AngII)-infused (1.44 mg · kg(-1) · day(-1), 2 weeks) mice were treated with the COX-2 inhibitor celecoxib (25 mg · kg(-1) · day(-1) i.p) or with the EP1 receptor antagonist SC19220 (10 mg · kg(-1) · day(-1) i.p.). COX-2(-/-) mice with or without AngII infusion were also used. KEY RESULTS: Celecoxib and SC19220 treatment did not modify the altered lumen diameter and wall : lumen ratio in mesenteric resistance arteries from SHR-infused and/or AngII-infused animals. However, both treatments and COX-2 deficiency decreased the augmented vascular stiffness in vessels from hypertensive animals. This was accompanied by diminished vascular collagen deposition, normalization of altered elastin structure and decreased connective tissue growth factor and plasminogen activator inhibitor-1 gene expression. COX-2 deficiency and SC19220 treatment diminished the increased vasoconstrictor responses and endothelial dysfunction induced by AngII infusion. Hypertensive animals showed increased mPGES-1 expression and PGE2 production in vascular tissue, normalized by celecoxib. Celecoxib treatment also decreased AngII-induced macrophage infiltration and TNF-α expression. Macrophage conditioned media (MCM) increased COX-2 and collagen type I expression in vascular smooth muscle cells; the latter was reduced by celecoxib treatment. CONCLUSIONS AND IMPLICATIONS: COX-2 and EP1 receptors participate in the increased extracellular matrix deposition and vascular stiffness, the impaired vascular function and inflammation in hypertension. Targeting PGE2 receptors might have benefits in hypertension-associated vascular damage.

HuR mediates the synergistic effects of angiotensin II and IL-1ß on vascular COX-2 expression and cell migration.

BACKGROUND AND PURPOSE: Angiotensin II (AngII) and IL-1ß are involved in cardiovascular diseases through the induction of inflammatory pathways. HuR is an adenylate- and uridylate-rich element (ARE)-binding protein involved in the mRNA stabilization of many genes. This study investigated the contribution of HuR to the increased expression of COX-2 induced by AngII and IL-1ß and its consequences on VSMC migration and remodelling. EXPERIMENTAL APPROACH: Rat and human VSMCs were stimulated with AngII (0.1 µM) and/or IL-1ß (10 ng · mL(-1)). Mice were infused with AngII or subjected to carotid artery ligation. mRNA and protein levels were assayed by quantitative PCR, Western blot, immunohistochemistry and immunofluorescence. Cell migration was measured by wound healing and transwell assays. KEY RESULTS: In VSMCs, AngII potentiated COX-2 and tenascin-C expressions and cell migration induced by IL-1ß. This effect of AngII on IL-1ß-induced COX-2 expression was accompanied by increased COX-2 3' untranslated region reporter activity and mRNA stability, mediated through cytoplasmic HuR translocation and COX-2 mRNA binding. These effects were blocked by ERK1/2 and HuR inhibitors. VSMC migration was reduced by blockade of ERK1/2, HuR, COX-2, TXAS, TP and EP receptors. HuR, COX-2, mPGES-1 and TXAS expressions were increased in AngII-infused mouse aortas and in carotid-ligated arteries. AngII-induced tenascin-C expression and vascular remodelling were abolished by celecoxib and by mPGES-1 deletion. CONCLUSIONS AND IMPLICATIONS: The synergistic induction of COX-2 by AngII and IL-1ß in VSMCs involves HuR through an ERK1/2-dependent mechanism. The HuR/COX-2 axis participates in cell migration and vascular damage. HuR might be a novel target to modulate vascular remodelling.

Toll-like receptor 4 contributes to vascular remodelling and endothelial dysfunction in angiotensin II-induced hypertension.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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). KEY RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: 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.

The role of cyclooxygenase (COX)-2 derived prostanoids on vasoconstrictor responses to phenylephrine is increased by exposure to low mercury concentration.

We have previously demonstrated that chronic exposure to low-dose of mercury induced endothelial dysfunction and increased vasoconstrictor responses. The aim of this work was to investigate if mercury exposure alters contractile prostanoids production from cyclooxygenase-2 (COX-2) and its contribution to phenylephrine responses. For this, aortic segments from 3-month old Wistar rats daily treated with HgCl(2) (1(st) dose 4.6 microg/kg, subsequent dose 0.07 microg/kg/day, i.m.) or vehicle for 30 days were used. Mercury treatment did not affect systolic blood pressure but increased phenylephrine-induced vasoconstriction. The non selective COX inhibitor, indomethacin (10 micromol/l) reduced the response to phenylephrine more in aortic segments from mercury-treated than control rats. The selective COX-2 inhibitor NS 398 (1 micromol/l), the thromboxane A(2)/prostaglandin H(2) receptor (TP) antagonist SQ 29,548 (1 micromol/l), the TXA(2) synthase inhibitor furegrelate (1 micromol/l), the EP(1) receptor antagonist SC 19220 (1 micromol/l) and the AT(1) receptor antagonist losartan (10 micromol/l) reduced phenylephrine response only in vessels from mercury-treated rats. TXA(2) and PGE(2) levels were greater in the incubation medium of vessels from treated than untreated rats; NS 398 decreased these levels only in the mercury group. COX-2 protein was localized in adventitial and endothelial cells. Aortic COX-2 mRNA expression and plasma angiotensin converting enzyme activity were greater in mercury-treated rats. These results suggest that treatment with low doses of mercury increases the release of COX-2-derived vasoconstrictor prostanoids and its participation in phenylephrine responses. The increased activation of the renin-angiotensin system after mercury treatment might be associated to this increased COX-2 activity.

Characterization of microbial trophic structures of two anaerobic bioreactors processing sulfate-rich waste streams.

A multi-compartment anaerobic bioreactor, designated the anaerobic migrating blanket reactor (AMBR), did not perform well in terms of chemical oxygen demand (COD) removal after an increase in sulfate load, compared to a conventional upflow anaerobic sludge blanket (UASB) reactor. The trophic structures of the bioreactors were characterized by analyzing the electron flows, formation and consumption of fermentation intermediates and terminal product (methane and hydrogen sulfide) formation. Critical performance parameters were linked to operational perturbations such as increase in sulfate load and changes in flow reversal schemes in the AMBR. Both of these manipulations affected the microbial communities, which were monitored by terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the bacterial and archaeal domains. The less stable AMBR did not produce granular biomass, and in response to increased sulfate concentrations, experienced a reversal in the distribution of hydrogenotrophic methanogens that correlated with a shift in electron flow from butyrate to propionate. As this shift occurred, bacterial populations such as butyrate-producing clostridia, became predominant, thus leading to reactor imbalance. The stable UASB reactor developed and retained granules and maintained a relatively stable archaeal community. Sulfate perturbation led to the selection of a novel bacterial group (Thermotogaceae), which was most likely well adapted to the increasingly sulfidogenic conditions in the bioreactor.

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.

Low mercury concentrations cause oxidative stress and endothelial dysfunction in conductance and resistance arteries.

Increased cardiovascular risk after mercury exposure has been described, but the underlying mechanisms are not well explored. We analyzed the effects of chronic exposure to low mercury concentrations on endothelium-dependent responses in aorta and mesenteric resistance arteries (MRA). Wistar rats were treated with mercury chloride (1st dose 4.6 microg/kg, subsequent dose 0.07 microg.kg(-1).day(-1) im, 30 days) or vehicle. Blood levels at the end of treatment were 7.97 +/- 0.59 ng/ml. Mercury treatment: 1) did not affect systolic blood pressure; 2) increased phenylephrine-induced vasoconstriction; 3) reduced acetylcholine-induced vasodilatation; and 4) reduced in aorta and abolished in MRA the increased phenylephrine responses induced by either endothelium removal or the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 100 microM). Superoxide dismutase (SOD, 150 U/ml) and the NADPH oxidase inhibitor apocynin (0.3 mM) decreased the phenylephrine-induced contraction in aorta more in mercury-treated rats than controls. In MRA, SOD did not affect phenylephrine responses; however, when coincubated with l-NAME, the l-NAME effect on phenylephrine response was restored in mercury-treated rats. Both apocynin and SOD restored the impaired acetylcholine-induced vasodilatation in vessels from treated rats. Endothelial NOS expression did not change in aorta but was increased in MRA from mercury-treated rats. Vascular O2(-) production, plasmatic malondialdehyde levels, and total antioxidant status increased with the mercury treatment. In conclusion, chronic exposure to low concentrations of mercury promotes endothelial dysfunction as a result of the decreased NO bioavailability induced by increases in oxidative stress. These findings offer further evidence that mercury, even at low concentrations, is an environmental risk factor for cardiovascular disease.

Chronic ouabain treatment increases the contribution of nitric oxide to endotheliumdependent relaxation

The aim of this study was to analyze the contribution of nitric oxide, prostacyclinand endothelium-dependent hyperpolarizing factor to endothelium-dependentvasodilation induced by acetylcholine in rat aorta from control and ouabain-inducedhypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omeganitro-L-arginine methyl esther (L-NAME) inhibited the vasodilator response toacetylcholine in segments from both groups but to a greater extent in segments fromouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higherin segments from ouabain-treated rats. Preincubation with the prostacyclin synthesisinhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacininhibited the vasodilator response to acetylcholine in aortic segments from bothgroups. The Ca2+-dependent potassium channel blocker charybdotoxin inhibited thevasodilator response to acetylcholine only in segments from control rats. Theseresults indicate that hypertension induced by chronic ouabain treatment is accompaniedby increased endothelial nitric oxide participation and impaired endotheliumdependenthyperpolarizing factor contribution in acetylcholine-induced relaxation.These effects might explain the lack of effect of ouabain treatment on acetylcholineresponses in rat aorta (AU)

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

BACKGROUND AND PURPOSE: To analyse the influence of hypertension in the modulation induced by inducible NOS (iNOS)-derived NO and superoxide anion (O(2) (*-)) of vasoconstrictor responses and the sources of O(2) (*-) implicated. EXPERIMENTAL APPROACH: Vascular reactivity experiments were performed in segments of aorta from normotensive, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR); protein and mRNA expressions were respectively measured by western blot and quantitative reverse transcription-polymerase chain reaction and O(2) (*-) production was evaluated by ethidium fluorescence. KEY RESULTS: The contractile responses to phenylephrine (1 nM-30 microM) and 5-hydroxytryptamine (0.1-100 microM) were greater in aortic segments from SHR than WKY. The selective iNOS inhibitor, 1400W (10 microM), increased the phenylephrine contraction only in WKY segments; however, iNOS protein and mRNA expressions were greater in aorta from SHR than WKY. Superoxide dismutase (SOD, 150 U ml(-1)) reduced phenylephrine and 5-hydroxytryptamine responses only in aorta from SHR; the NAD(P)H oxidase inhibitor apocynin (0.3 mM) decreased phenylephrine and 5-hydroxytryptamine responses more in vessels from SHR than WKY. Co-incubation with SOD plus 1400W potentiated the phenylephrine and 5-hydroxytryptamine responses more in segments from SHR than WKY. O(2) (*-) production was greater in aorta from SHR than WKY; apocynin abolished this difference. CONCLUSIONS AND IMPLICATIONS: Increased O(2) (*-) formation from NADP(H) oxidase in vessels from hypertensive rats contributes to the vasoconstrictor responses and counteract the increase of NO from iNOS and the consequent modulation of these responses.

Alteraciones electrocargiográficas en la hipercaliemia / Electrocardiographic changes in hyperkaliaemia

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Ageing affects nitric oxide synthase, cyclooxygenase and oxidative stress enzymes expression differently in mesenteric resistance arteries.

1 Our aim was to study the role of nitric oxide (NO) and arachidonic acid pathways in the alpha(1)-adrenoceptor-mediated vasoconstriction in mesenteric resistance arteries from 3--4 and 22 to 23-month-old Sprague-Dawley rats. 2 The expression of NO synthase (NOS), cyclooxygenase (COX) isoforms, soluble guanylate cyclase, superoxide dismutase and the NAD(P)H oxidase subunits p22(phox) and p 47(phox) were determined. 3 The N(G)-nitro-l-arginine methyl ester, a non-selective NOS inhibitor, shifted to the left but indomethacin and NS 398, non-selective and selective COX-2 inhibitors, shifted to the right the concentration-response curve for the vasoconstriction by phenylephrine in both age groups. 4 Ageing up-regulated endothelial NOS and p22(phox) expression but did not modify COX, soluble guanylate cyclase, superoxide dismutase and p 47(phox) expression. 5 These data suggest that the observed enhancement of eNOS protein expression could constitute a compensatory mechanism to counter-regulate a chronic loss of NO possibly through increased superoxide anion production from NAD(P)H oxidase induced by age.

Increased anandamide induced relaxation in mesenteric arteries of cirrhotic rats: role of cannabinoid and vanilloid receptors.

BACKGROUND AND AIMS: Anandamide is an endocannabinoid that evokes hypotension by interaction with peripheral cannabinoid CB1 receptors and with the perivascular transient receptor potential vanilloid type 1 protein (TRPV1). As anandamide has been implicated in the vasodilated state in advanced cirrhosis, the study investigated whether the mesenteric bed from cirrhotic rats has an altered and selective vasodilator response to anandamide. METHODS: We assessed vascular sensitivity to anandamide, mRNA and protein expression of cannabinoid CB1 receptor and TRPV1 receptor, and the topographical distribution of cannabinoid CB1 receptors in resistance mesenteric arteries of cirrhotic and control rats. RESULTS: Mesenteric vessels of cirrhotic animals displayed greater sensitivity to anandamide than control vessels. This vasodilator response was reverted by CB1 or TRPV1 receptor blockade, but not after endothelium denudation or nitric oxide inhibition. Anandamide had no effect on distal femoral arteries. CB1 and TRPV1 receptor protein was higher in cirrhotic than in control vessels. Neither CB1 mRNA nor protein was detected in femoral arteries. Immunochemistry showed that CB1 receptors were mainly in the adventitia and in the endothelial monolayer, with higher expression observed in vessels of cirrhotic rats than in controls. CONCLUSIONS: These results indicate that anandamide is a selective splanchnic vasodilator in cirrhosis which predominantly acts via interaction with two different types of receptors, CB1 and TRPV1 receptors, which are mainly located in perivascular sensory nerve terminals of the mesenteric resistance arteries of these animals.

Influence of hypertension on nitric oxide synthase expression and vascular effects of lipopolysaccharide in rat mesenteric arteries.

1. Experiments were designed to investigate the effects of the inducible nitric oxide synthase (iNOS) stimulator, lipopolysaccharide (LPS), on noradrenaline (NA) responses and on NOS activity and its expression in intact mesenteric resistance arteries (MRAs) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. 2. In MRAs from WKY, LPS (10 microg ml(-1); 1-5 h) reduced the vasoconstrictor responses to NA (0.1 - 30 microM) in the presence, but not in the absence of L-arginine (L-Arg, 10 microM). However, in SHR arteries, LPS induced an incubation-time dependent reduction of NA responses in the absence, as well as the presence, of L-Arg. The LPS inhibitory effect was reduced by the non-specific NOS inhibitor L-N(G)-nitroarginine methyl ester (L-NAME, 100 microM) and the selective iNOS inhibitor, aminoguanidine (100 microM). 3. L-NAME alone similarly shifted the concentration-response curve to NA leftward in arteries from both strains, while aminoguanidine had no effect. L-Arg shifted the curve to NA rightward only in SHR MRAs. 4. Basal activity of both iNOS and constitutive NOS (conversion of [(3)H]-L-Arg to [(3)H]-L-citrulline) was similar in arteries from both strains. After 5 h incubation with LPS, only iNOS activity in arteries from SHR was increased. 5. Basal iNOS protein expression was undetectable; basal endothelial (eNOS) protein expression was similar in arteries from both strains, while neuronal (nNOS) was greater in arteries from SHR. LPS induced iNOS protein expression, that was higher in arteries from SHR than in those from WKY. 6. These results indicate that NO production, via iNOS induction, is greater than in those from MRAs from SHR to WKY.

Estimating microbial population counts by 'most probable number' using Microsoft Excel.

A computer-assisted method for determining population counts using the 'most probable number' (MPN) was developed. The Microsoft Excel spreadsheet and its Solver tool were used to generate MPNs, error estimates and confidence limits. Our method was flexible, allowing the use of unbalanced replication schemes and varying replication numbers and inoculation volumes. Furthermore, it required no programming skills and generated fast results, which were comparable to those of standard MPN tables and MPN software.

Role of iNOS in the vasodilator responses induced by L-arginine in the middle cerebral artery from normotensive and hypertensive rats.

1. The substrate of nitric oxide synthase (NOS), L-arginine (L-Arg, 0.01 microM - 1 mM), induced endothelium-independent relaxations in segments of middle cerebral arteries (MCAs) from normotensive Wistar-Kyoto (WKY) and hypertensive rats (SHR) precontracted with prostaglandin F2alpha (PGF2alpha). These relaxations were higher in SHR than WKY arteries. 2. L-N(G)-nitroarginine methyl ester (L-NAME) and 2-amine-5,6-dihydro-6-methyl-4H-1,3-tiazine (AMT), unspecific and inducible NOS (iNOS) inhibitors, respectively, reduced those relaxations, specially in SHR. 3. Four- and seven-hours incubation with dexamethasone reduced the relaxations in MCAs from WKY and SHR, respectively. 4. Polymyxin B and calphostin C, protein kinase C (PKC) inhibitors, reduced the L-Arg-induced relaxation. 5. Lipopolysaccharide (LPS, 7 h incubation) unaltered and inhibited these relaxations in WKY and SHR segments, respectively. LPS antagonized the effect polymyxin B in WKY and potentiated L-Arg-induced relaxations in SHR in the presence of polymyxin B. 6. The contraction induced by PGF2alpha was greater in SHR than WKY arteries. This contraction was potentiated by dexamethasone and polymyxin B although the effect of polymyxin B was higher in SHR segments. LPS reduced that contraction and antagonized dexamethasone- and polymyxin B-induced potentiation, these effects being greater in arteries from SHR. 7. These results suggest that in MCAs: (1) the induction of iNOS participates in the L-Arg relaxation and modulates the contraction to PGF2alpha; (2) that induction is partially mediated by a PKC-dependent mechanism; and (3) the involvement of iNOS in such responses is greater in the hypertensive strain.

Analysis of DNA Polymorphism and Virulence in Philippine Strains of Xanthomonas oryzae pv. oryzicola.

Molecular tools were used to analyze the genetic diversity and population structure of Xanthomonas oryzae pv. oryzicola, the bacterial leaf streak pathogen of rice in the Philippines. Representative pathogen strains were selected and used to assess resistance in rice germplasm. A partial genomic library of X. oryzae pv. oryzicola was constructed, and a 459-bp clone containing the repetitive DNA element R41 was selected as a probe for restriction fragment length polymorphism (RFLP) analysis and sequenced. R41 shared 44% sequence homology with the putative transposase gene of IS1112, an insertion element cloned from X. oryzae pv. oryzae. Using R41 as a probe for RFLP analysis, 26 band profiles were discerned in a collection of 123 strains of X. oryzae pv. oryzicola. Analysis of PstI digestion patterns of DNA from the same collection resolved 36 haplotypes. Several clusters of strains were detected after grouping of data based on either pR41 as a probe or Pst1 digestion patterns. However, based on bootstrap analysis, the clusters were not robust. Genetic diversity was high for the entire collection as well as within spatially and temporally defined subsets of strains. Even a set of strains collected from a single site at a single time was highly diverse. Strains representing the different DNA types were inoculated to a set of diverserice cultivars. Consistent rice varietal groupings were obtained from disease reaction data, but there was no correlation between pathogen isolate cluster and host reaction across inoculation trials. Isozyme group I of rice, representing tropical japonica and javanica germplasm, is a promising source of resistance to bacterial leaf streak.