Augmented oxidative stress and preserved vasoconstriction induced by hydrogen peroxide in coronary arteries in obesity: role of COX-2.

BACKGROUND AND PURPOSE: Oxidative stress plays a key role in the vascular and metabolic abnormalities associated with obesity. Herein, we assessed whether obesity can increase coronary vasoconstriction induced by hydrogen peroxide (H2 O2 ) and the signalling pathways involving COX-2 and superoxide (O2.- ) generation. EXPERIMENTAL APPROACH: Contractile responses to H2 O2 and O2.- generation were measured in coronary arteries from genetically obese Zucker rats (OZR) and compared to lean Zucker rats (LZR). KEY RESULTS: Both basal and H2 O2 -stimulated O2.- production were enhanced in coronary arteries from OZR, but H2 O2 -induced vasoconstriction was unchanged. The selective COX-2 inhibitor NS398 significantly reduced H2 O2 -induced contractions in endothelium-denuded arteries from LZR and OZR, but only in endothelium-intact arteries from LZR. PGI2 (IP) receptor antagonism modestly reduced the vasoconstrictor action of H2 O2 while antagonism of the PGE2 receptor 4 (EP4 ) enhanced H2 O2 contractions in arteries from OZR but not LZR. Basal release of COX-2-derived PGE2 was higher in coronary arteries from OZR where the selective agonist of EP4 receptors TCS 2519 evoked potent relaxations. COX-2 was up-regulated after acute exposure to H2 O2 in coronary endothelium and vascular smooth muscle (VSM) and inhibition of COX-2 markedly reduced H2 O2 -elicited O2.- generation in coronary arteries and myocardium. Expression of Nox subunits in VSM and NADPH-stimulated O2.- generation was enhanced and contributed to H2 O2 vasoconstriction in arteries from obese rats. CONCLUSION AND IMPLICATIONS: COX-2 contributes to cardiac oxidative stress and to the endothelium-independent O2.- -mediated coronary vasoconstriction induced by H2 O2 in obesity, which is offset by the release of COX-2-derived endothelial PGE2 acting on EP4 vasodilator receptors.

Cerebrovascular endothelial dysfunction induced by mercury exposure at low concentrations.

Mercury (Hg) has many harmful vascular effects by increasing oxidative stress, inflammation and vascular/endothelial dysfunction, all of which may contribute to cerebrovascular diseases development. We aimed to explore the effects of chronic low-mercury concentration on vascular function in cerebral arteries and the mechanisms involved. Basilar arteries from control (vehicle-saline solution, im) and mercury chloride (HgCl2)-treated rats for 30 days (first dose 4.6µg/kg, subsequent dose 0.07µg/kg/day, im, to cover daily loss) were used. Vascular reactivity, protein expression, nitric oxide (NO) levels and superoxide anion (O2(-)) production were analyzed. HgCl2 exposure increased serotonin contraction and reduced the endothelium-dependent vasodilatation to bradykinin. After NO synthase inhibition, serotonin responses were enhanced more in control than in mercury-treated rats while bradykinin-induced relaxation was abolished. NO levels were greater in control than Hg-treated rats. Tiron and indomethacin reduced vasoconstriction and increased the bradykinin-induced relaxation only in HgCl2-treated rats. Vascular O2(-) production was greater in mercury-treated when compared to control rats. Protein expressions of endothelial NO synthase, copper/zinc (Cu/Zn), Manganese (Mn) and extracellular-superoxide dismutases were similar in cerebral arteries from both groups. Results suggest that Hg treatment increases cerebrovascular reactivity by reducing endothelial negative modulation and NO bioavailability; this effect seems to be dependent on increased reactive oxygen species and prostanoids generation. These findings show, for the first time, that brain vasculature are also affected by chronic mercury exposure and offer further evidence that even at small concentration, HgCl2 is hazardous and might be an environmental risk factor accounting for cerebral vasospasm development.

Reactive oxygen species and vascular remodeling in cardiovascular diseases / Especies reactivas de oxígeno y remodelado vascular en enfermedades cardiovasculares

Reactive oxygen species (ROS) are reactive derivatives of O2 metabolism produced by all types of vascular cells. ROS play an important role in both physiological and pathological situations by acting as intracellular signaling molecules which regulate vascular function and structure. Accordingly, oxidative stress is implicated among other processes in inflammation, hypertrophy, migration, growth/apoptosis and extracellular matrix protein turnover which are important processes involved in vascular remodeling in cardiovascular diseases. In the cardiovascular system, the major source of ROS is the NADPH oxidase family of enzymes composed by seven members where NOX-1 and NOX-4 are the main isoforms in vascular smooth muscle cells. This review highlights the importance of NOX-derived ROS in vascular biology and focuses on the potential role of oxidative stress in vascular remodeling

Las especies reactivas de oxígeno son derivados reactivos del metabolismo del O2 producido por todos los tipos celulares a nivel vascular. Las especies reactivas de oxígeno juegan un papel importante en situaciones tanto fisiológicas como patológicas mediante su actuación como moléculas de señalización intracelular que regulan la función y estructura vascular. De esta manera, el estrés oxidativo está implicado, entre otros procesos, en la inflamación, hipertrofia, migración, proliferación/apoptosis y reciclaje de proteínas de matriz extracelular, los cuales son procesos importantes implicados en el remodelado vascular durante enfermedades cardiovasculares. En el sistema cardiovascular, la mayor fuente de especies reactivas de oxígeno es la familia de enzimas NADPH oxidase formadas por siete miembros donde NOX-1 y NOX-4 son las principales isoformas en células musculares lisas vasculares. Esta revisión destaca la importancia de las especies reactivas de oxígeno derivadas de NOX en la biología vascular y se centra en el papel potencial del estrés oxidativo en el remodelado vascular

MAPK pathway activation by chronic lead-exposure increases vascular reactivity through oxidative stress/cyclooxygenase-2-dependent pathways.

Chronic exposure to low lead concentration produces hypertension; however, the underlying mechanisms remain unclear. We analyzed the role of oxidative stress, cyclooxygenase-2-dependent pathways and MAPK in the vascular alterations induced by chronic lead exposure. Aortas from lead-treated Wistar rats (1st dose: 10 µg/100g; subsequent doses: 0.125µg/100g, intramuscular, 30days) and cultured aortic vascular smooth muscle cells (VSMCs) from Sprague Dawley rats stimulated with lead (20µg/dL) were used. Lead blood levels of treated rats attained 21.7±2.38µg/dL. Lead exposure increased systolic blood pressure and aortic ring contractile response to phenylephrine, reduced acetylcholine-induced relaxation and did not affect sodium nitroprusside relaxation. Endothelium removal and L-NAME left-shifted the response to phenylephrine more in untreated than in lead-treated rats. Apocynin and indomethacin decreased more the response to phenylephrine in treated than in untreated rats. Aortic protein expression of gp91(phox), Cu/Zn-SOD, Mn-SOD and COX-2 increased after lead exposure. In cultured VSMCs lead 1) increased superoxide anion production, NADPH oxidase activity and gene and/or protein levels of NOX-1, NOX-4, Mn-SOD, EC-SOD and COX-2 and 2) activated ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized superoxide anion production, NADPH oxidase activity and mRNA levels of NOX-1, NOX-4 and COX-2. Blockade of the ERK1/2 and p38 signaling pathways abolished lead-induced NOX-1, NOX-4 and COX-2 expression. Results show that lead activation of the MAPK signaling pathways activates inflammatory proteins such as NADPH oxidase and COX-2, suggesting a reciprocal interplay and contribution to vascular dysfunction as an underlying mechanisms for lead-induced hypertension.

The C-terminal module IV of connective tissue growth factor, through EGFR/Nox1 signaling, activates the NF-κB pathway and proinflammatory factors in vascular smooth muscle cells.

AIMS: Connective tissue growth factor (CTGF/CCN2) is a developmental gene upregulated in pathological conditions, including cardiovascular diseases, whose product is a matricellular protein that can be degraded to biologically active fragments. Among them, the C-terminal module IV [CCN2(IV)] regulates many cellular functions, but there are no data about redox process. Therefore, we investigated whether CCN2(IV) through redox signaling regulates vascular responses. RESULTS: CCN2(IV) increased superoxide anion (O2(•-)) production in murine aorta (ex vivo and in vivo) and in cultured vascular smooth muscle cells (VSMCs). In isolated murine aorta, CCN2(IV), via O2(•-), increased phenylephrine-induced vascular contraction. CCN2(IV) in vivo regulated several redox-related processes in mice aorta, including increased nonphagocytic NAD(P)H oxidases (Nox)1 activity, protein nitrosylation, endothelial dysfunction, and activation of the nuclear factor-κB (NF-κB) pathway and its related proinflammatory factors. The role of Nox1 in CCN2(IV)-mediated vascular responses in vivo was investigated by gene silencing. The administration of a Nox1 morpholino diminished aortic O2(•-) production, endothelial dysfunction, NF-κB activation, and overexpression of proinflammatory genes in CCN2(IV)-injected mice. The link CCN2(IV)/Nox1/NF-κB/inflammation was confirmed in cultured VSMCs. Epidermal growth factor receptor (EGFR) is a known CCN2 receptor. In VSMCs, CCN2(IV) activates EGFR signaling. Moreover, EGFR kinase inhibition blocked vascular responses in CCN2(IV)-injected mice. INNOVATION AND CONCLUSION: CCN2(IV) is a novel prooxidant factor that in VSMCs induces O2(•-) production via EGFR/Nox1 activation. Our in vivo data demonstrate that CCN2(IV) through EGFR/Nox1 signaling pathway induces endothelial dysfunction and activation of the NF-κB inflammatory pathway. Therefore, CCN2(IV) could be considered a potential therapeutic target for redox-related cardiovascular diseases.

Lysyl oxidase (LOX) in vascular remodelling. Insight from a new animal model.

Lysyl oxidase (LOX) is an extracellular matrix-modifying enzyme that seems to play a critical role in vascular remodelling. However, the lack of viable LOX-deficient animal models has been an obstacle to deep in LOX biology. In this study we have developed a transgenic mouse model that over-expresses LOX in vascular smooth muscle cells (VSMC) to clarify whether LOX could regulate VSMC phenotype and vascular remodelling. The SM22α proximal promoter drove the expression of a transgene containing the human LOX cDNA. Two stable transgenic lines, phenotypically indistinguishable, were generated by conventional methods (TgLOX). Transgene expression followed the expected SMC-specific pattern. In TgLOX mice, real-time PCR and immunohistochemistry evidenced a strong expression of LOX in the media from aorta and carotid arteries, coincident with a higher proportion of mature collagen. VSMC isolated from TgLOX mice expressed high levels of LOX pro-enzyme, which was properly secreted and processed into mature and bioactive LOX. Interestingly, cell proliferation was significantly reduced in cells from TgLOX mice. Transgenic VSMC also exhibited low levels of Myh10 (marker of SMC phenotypic switching), PCNA (marker of cell proliferation) and MCP-1, and a weak activation of Akt and ERK1/2 in response to mitogenic stimuli. Accordingly, neointimal thickening induced by carotid artery ligation was attenuated in TgLOX mice that also displayed a reduction in PCNA and MCP-1 immunostaining. Our results give evidence that LOX plays a critical role in vascular remodelling. We have developed a new animal model to study the role of LOX in vascular biology.

New 5-unsubstituted dihydropyridines with improved CaV1.3 selectivity as potential neuroprotective agents against ischemic injury.

C5-unsubstituted-C6-aryl-1,4-dihydropyridines were prepared by a CAN-catalyzed multicomponent reaction from chalcones, ß-dicarbonyl compounds, and ammonium acetate. These compounds were able to block Ca(2+) entry after a depolarizing stimulus and showed an improved Cav1.3/Cav1.2 selectivity in comparison with nifedipine. Furthermore, they were able to protect neuroblastoma cells against Ca(2+) overload and oxidative stress models. Their selectivity ratio makes them highly interesting for the treatment of neurological disorders where Ca(2+) dyshomeostasis and high levels of oxidative stress have been demonstrated. Furthermore, their low potency toward the cardiovascular channel subtype makes them safer by reducing their probable side effects, in comparison to classical 1,4-dihydropyridines. Some compounds afforded good protective profile in a postincubation model that simulates the real clinical situation of ictus patients, offering a therapeutic window of opportunity of great interest for patient recovery after a brain ischemic episode. Good activities were also found in acute ischemia/reperfusion models of oxygen and glucose deprivation.

Leptin induces cardiac fibrosis through galectin-3, mTOR and oxidative stress: potential role in obesity.

OBJECTIVE: Leptin acts as a cardiac profibrotic factor. However, the mechanisms underlying this effect are unclear. Therefore, we sought to elucidate the mediators involved in this process and the potential role of leptin in cardiac fibrosis associated with obesity. METHODS: Male Wistar rats were fed either a high-fat diet (HFD; 33.5% fat), or a standard diet (3.5% fat) for 6 weeks. RESULTS: HFD animals show cardiac hypertrophy, fibrosis and an increase in O2- production as evaluated by dihydroethidium. Echocardiographic parameters of cardiac structure and systolic function were similar in both groups. Cardiac levels of leptin, collagen I, galectin-3 and transforming growth factor ß (TGF-ß) were higher in HFD than in controls. In cardiac myofibroblasts, leptin (10-100 ng/ml) increased O2-, collagen I, galectin-3, TGF-ß and connective tissue growth factor production (CTGF). These effects were prevented by the presence of either melatonin (10 mmol/l) or the inhibitor of mTOR, rapamycin (10 mmol/l). Blockage of galectin-3 activity by N-acetyllactosamine (LacNac 10 mmol/l) reduced both collagen I and O2(*-) production induced by leptin. The p70S6 kinase activation/phosphorylation, the downstream mediator of mTOR, induced by leptin was not modified by melatonin. Leptin reduced the metalloproteinase (MMP) 2 activity and the presence of melatonin, rapamycin or LacNac were unable to prevent it. CONCLUSION: The data suggest that leptin locally produced in the heart could participate in the fibrosis observed in HFD by affecting collagen turnover. Collagen synthesis induced by leptin seems to be mediated by the production of galectin-3, TGF-ß and CTGF through oxidative stress increased by activation of mTOR pathway.

Differential structural and functional changes in penile and coronary arteries from obese Zucker rats.

Erectile dysfunction frequently coexists with coronary artery disease and has been proposed as a potential marker for silent coronary artery disease in type 2 diabetes. In the present study, we comparatively assessed the structural and functional changes of both penile arteries (PAs) and coronary arteries (CAs) from a prediabetic animal model. PAs and CAs from 17- to 18-wk-old obese Zucker rats (OZRs) and from their control counterparts [lean Zucker rats (LZRs)] were mounted in microvascular myographs to evaluate vascular function, and stained arteries were subjected to morphometric analysis. Endothelial nitric oxide (NO) synthase (eNOS) protein expression was also assessed. The internal diameter was reduced and the wall-to-lumen ratio was increased in PAs from OZRs, but structure was preserved in CAs. ACh-elicited relaxations were severely impaired in PAs but not in CAs from OZRs, although eNOS expression was unaltered. Contractions to norepinephrine and 5-HT were significantly enhanced in both PAs and CAs, respectively, from OZRs. Blockade of NOS abolished endothelium-dependent relaxations in PAs and CAs and potentiated norepinephrine and 5-HT contractions in arteries from LZRs but not from OZRs. The vasodilator response to the phosphodiesterase 5 inhibitor sildenafil was reduced in both PAs and CAs from OZRs. Pretreatment with SOD reduced the enhanced vasoconstriction in both PAs and CAs from OZRs but did not restore ACh-induced relaxations in PAs. In conclusion, the present results demonstrate vascular inward remodeling in PAs and a differential impairment of endothelial relaxant responses in PAs and CAs from insulin-resistant OZRs. Enhanced superoxide production and reduced basal NO activity seem to underlie the augmented vasoconstriction in both PAs and CAs. The severity of the structural and functional abnormalities in PAs might anticipate the vascular dysfunction of the more preserved coronary vascular bed.

Hypertension increases contractile responses to hydrogen peroxide in resistance arteries through increased thromboxane A2, Ca2+, and superoxide anion levels.

This study investigated the mechanisms underlying the response to hydrogen peroxide (H(2)O(2)) in mesenteric resistance arteries from spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto (WKY) rats. Arteries were mounted in microvascular myographs for isometric tension recording and for simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)), superoxide anion (O(2)(.)) production was evaluated by dihydroethidium fluorescence and confocal microscopy, and thromboxane A(2) (TXA(2)) production was evaluated by enzyme immunoassay. H(2)O(2) (1-100 microM) induced biphasic responses characterized by a transient endothelium-dependent contraction followed by relaxation. Simultaneous measurements of tension and Ca(2+) showed a greater effect of H(2)O(2) in arteries from hypertensive than normotensive rats. The cyclooxygenase (cox) inhibitor, indomethacin [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-H-indole-3-acetic acid] (1 microM); the COX-1 inhibitor, SC-58560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole] (1 microM); the thromboxane (TXA(2)) synthase inhibitor, furegrelate [5-(3-pyridinylmethyl)-2-benzofurancarboxylic acid, sodium salt] (10 microM); and the TXA(2)/prostaglandin H(2) receptor antagonist, SQ 29,548 ([1S-[1.alpha.,2.alpha.(Z),3.alpha.,4.alpha.]]-7-[3-[[2-[(phenylamino) carbonyl] hydrazino] methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid)) (1 microM) abolished H(2)O(2) contraction in arteries from WKY rats but only reduced it in SHRs. The O(2)(.) scavenger, tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt) (1 mM), and the NADPH oxidase inhibitor, apocynin (4'-hydroxy-3'-methoxyacetophenone) (0.3 mM), decreased H(2)O(2) contraction in arteries from SHRs but not in WKY rats. H(2)O(2) induced TXA(2) and O(2)(.) production that was greater in SHRs than in WKY rats. The TXA(2) analog, U46619 [9,11-di-deoxy-11 alpha,9 alpha-epoxymethano prostaglandin F(2 alpha) (0.1 nM-1 microM)], also increased O(2)(.) production in SHR vessels. H(2)O(2)-induced TXA(2) production was decreased by SC-58560. H(2)O(2)-induced O(2)(.) production was decreased by tiron, apocynin, and SQ 29,548. In conclusion, the enhanced H(2)O(2) contraction in resistance arteries from SHRs seems to be mediated by increased TXA(2) release from COX-1 followed by elevations in vascular smooth muscle [Ca(2+)](i) levels and O(2)(.) production. This reveals a new mechanism of oxidative stress-induced vascular damage in hypertension.