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Obesity can lead to the loss of the anticontractile properties of perivascular adipose tissue (PVAT). Given that cafeteria (CAF) diet reflects the variety of highly calorie and easily accessible foods in Western societies, contributing to obesity and metabolic disorders, we sought to investigate the impact of CAF diet on PVAT vasoactive profile and the involvement of renin-angiotensin system, oxidative stress, and cyclooxygenase pathway.Male Balb/c mice received standard or CAF diet for 4 weeks. Oral glucose tolerance and insulin sensitivity tests were performed, and fasting serum glucose, cholesterol and triglyceride parameters were determined. Vascular reactivity, fluorescence and immunofluorescence analyzes were carried out in intact thoracic aorta in the presence or absence of PVAT.CAF diet was effective in inducing obesity and metabolic disorders, as demonstrated by increased body weight gain and adiposity index, hyperlipidemia, hyperglycemia, glucose intolerance and insulin insensitivity. Importantly, CAF diet led to a significant decrease in aortic contractility which was restored in the presence of PVAT, exhibiting therefore a contractile profile. The contractile effect of PVAT was associated with the activation of ATThese findings suggest that the contractile profile of PVAT involving the renin-angiotensin system activation, reactive oxygen species and cyclooxygenase-1 metabolites may be a protective compensatory adaptive response during early stage of CAF diet-induced obesity as an attempt to restore the impaired vascular contraction observed in the absence of PVAT, contributing to the maintenance of vascular tone.

Vascular dysfunction induced by angiotensin-II can result from direct effects on vascular and inflammatory cells and indirect hemodynamic effects. Using isolated and functional cultured aortas, we aimed to identify the effects of angiotensin-II on cyclooxygenase (COX) and inducible nitric oxide synthase (iNOS) and evaluate their impact on vascular reactivity. Aortic rings from mice were incubated overnight in culture medium containing angiotensin-II (100 nmol/L) or vehicle to induce vascular disfunction. Vascular reactivity of cultured arteries was evaluated in a bath chamber. Immunofluorescence staining for COX-1 and COX-2 was performed. Nitric oxide (NO) formation was approached by the levels of nitrite, a NO end product, and using a fluorescent probe (DAF). Oxidative and nitrosative stress were determined by DHE fluorescence and nitrotyrosine staining, respectively. Arteries cultured with angiotensin-II showed impairment of endothelium-dependent relaxation, which was reversed by the ATsub1/subreceptor antagonist. Inhibition of COX and iNOS restored vascular relaxation, suggesting a common pathway in which angiotensin-II triggers COX and iNOS, leading to vasoconstrictor receptors activation. Moreover, using selective antagonists, TP and EP were identified as the receptors involved in this response. Endothelium-dependent contractions of angiotensin-II-cultured aortas were blunted by ibuprofen, and increased COX-2 immunostaining was found in the arteries, indicating endothelium release of vasoconstrictor prostanoids. Angiotensin-II induced increased reactive oxygen species and NO production. An iNOS inhibitor prevented NO enhancement and nitrotyrosine accumulation in arteries stimulated with angiotensin-II. These results confirm that angiotensin-II causes vascular inflammation that culminates in endothelial dysfunction in an iNOS and COX codependent manner.

Perivascular adipose tissue (PVAT) has recently entered in the realm of cardiovascular diseases as a putative target for intervention. Notwithstanding its relevance, there is still a long way before the role of PVAT in physiology and pathology is fully understood. The general idea that PVAT anti-contractile effect is beneficial and its pro-contractile effect is harmful is being questioned by several reports. The role of some PVAT important products or systems such as nitric oxide (NO), reactive oxygen species (ROS), and RAS may vary depending on the context, disease, place of production, etc., which adds doubts on how mediators of PVAT anti- and pro-contractile effects are called to action and their final result. This short review will address some points regarding NO, ROS, and RAS in the beneficial and harmful roles of PVAT.

ObjectiveDengue fever is one of the most important arboviral diseases in the world, and its severe forms are characterised by a broad spectrum of systemic and cardiovascular hallmarks. However, much remains to be elucidated regarding the pathogenesis triggered by Dengue virus (DENV) in the heart. Herein, we evaluated the cardiac outcomes unleashed by DENV infection and the possible mechanisms associated with these effects.MethodsA model of an adapted DENV-3 strain was used to infect male BALB/c mice to assess haemodynamic measurements and the functional, electrophysiological, inflammatory and oxidative parameters in the heart.ResultsDENV-3 infection resulted in increased systemic inflammation and vascular permeability with consequent reduction of systolic blood pressure and increase in heart rate. These changes were accompanied by a decrease in the cardiac output and stroke volume, with a reduction trend in the left ventricular end-systolic and end-diastolic diameters and volumes. Also, there was a reduction trend in the calcium current density in the ventricular cardiomyocytes of DENV-3 infected mice. Indeed, DENV-3 infection led to leucocyte infiltration and production of inflammatory mediators in the heart, causing pericarditis and myocarditis. Moreover, increased reactive oxygen species generation and lipoperoxidation were also verified in the cardiac tissue of DENV-3 infected mice.ConclusionsDENV-3 infection induced a marked cardiac dysfunction, which may be associated with inflammation, oxidative stress and electrophysiological changes in the heart. These findings provide new cardiac insights into the mechanisms involved in the pathogenesis triggered by DENV, contributing to the research of new therapeutic targets for clinical practice.

Endothelial dysfunction and consequent vasoconstriction are a common condition in patients with hypertension and other cardiovascular diseases. Endothelial cells produce and release vasodilator substances that play a pivotal role in normal vascular tone. The mechanisms underlying endothelial dysfunction are multifactorial. However, enhanced reactive oxygen species (ROS) production and consequent vasoconstriction instead of endothelium-derived relaxant generation and consequent vasodilatation contribute to this dysfunction considerably. The main targets of the drugs that are currently used to treat vascular diseases concerning enzyme activities and protein functions that are impaired by endothelial nitric oxide synthase (eNOS) uncoupling and ROS production. Nitric oxide (NO) bioavailability can decrease due to deficient NO production by eNOS and/or NO release to vascular smooth muscle cells, which impairs endothelial function. Considering the NO cellular mechanisms, tackling the issue of eNOS uncoupling could avoid endothelial dysfunction: provision of the enzyme cofactor tetrahydrobiopterin (BH4) should elicit NO release from NO donors, to activate soluble guanylyl cyclase. This should increase cyclic guanosine-monophosphate (cGMP) generation and inhibit phosphodiesterases (especially PDE5) that selectively degrade cGMP. Consequently, protein kinase-G should be activated, and K+ channels should be phosphorylated and activated, which is crucial for cell membrane hyperpolarization and vasodilation and/or inhibition of ROS production. The present review summarizes the current concepts about the vascular cellular mechanisms that underlie endothelial dysfunction and which could be the target of drugs for the treatment of patients with cardiovascular disease.

Aims To determine the role of reactive oxygen species (ROS) on sodium nitroprusside (SNP)-induced tolerance. Additionally, we evaluated the role of ROS on NF-κB activation and pro-inflammatory cytokines production during SNP-induced tolerance. Main methods To induce in vitro tolerance, endothelium-intact or -denuded aortic rings isolated from male Balb-c mice were incubated for 15, 30, 45 or 60 min with SNP (10 nmol/L). Key findings Tolerance to SNP was observed after incubation of endothelium-denuded, but not endothelium-intact aortas for 60 min with this inorganic nitrate. Pre-incubation of denuded rings with tiron (superoxide anion (O2−) scavenger), and the NADPH oxidase inhibitors apocynin and atorvastatin reversed SNP-induced tolerance. l -NAME (non-selective NOS inhibitor) and l -arginine (NOS substrate) also prevented SNP-induced tolerance. Similarly, ibuprofen (non-selective cyclooxygenase (COX) inhibitor), nimesulide (selective COX-2 inhibitor), AH6809 (prostaglandin PGF2α receptor antagonist) or SQ29584 [PGH2/thromboxane TXA2 receptor antagonist] reversed SNP-induced tolerance. Increased ROS generation was detected in tolerant arteries and both tiron and atorvastatin reversed this response. Tiron prevented tolerance-induced increase on O2– and hydrogen peroxide (H2O2) levels. The increase onp65/NF-κB expression and TNF-α production in tolerant arteries was prevented by tiron. The major new finding of our study is that SNP-induced tolerance is mediated by NADPH-oxidase derived ROS and vasoconstrictor prostanoids derived from COX-2, which are capable of reducing the vasorelaxation induced by SNP. Additionally, we found that ROS mediate the activation of NF-κB and the production of TNF-α in tolerant arteries. Significance These findings identify putative molecular mechanisms whereby SNP induces tolerance in the vasculature.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior The gastrointestinal (GI) tract harbors commensal microorganisms as well as invasive bacteria, toxins and other pathogens and, therefore, plays a pivotal barrier and immunological role against pathogenic agents. The vagus nerve is an important regulator of the GI tract-associated immune system, having profound effects on inflammatory responses. Among GI tract organs, the liver is a key site of immune surveillance, as it has a large population of resident macrophages and receives the blood drained from the guts through the hepatic portal circulation. Although it is widely accepted that the hepatic tissue is a major target for vagus nerve fibers, the role of this neural circuit in liver immune functions is still poorly understood. Herein we used in vivo imaging techniques, including confocal microscopy and scintigraphy, to show that vagus nerve stimulation increases the phagocytosis activity by resident macrophages in the liver, even on the absence of an immune challenge. The activation of this neural circuit in a non-lethal model of sepsis optimized the removal of bacteria in the liver and resulted in the production of anti-inflammatory and pro-regenerative cytokines. Our findings provide new insights into the neural regulation of the immune system in the liver.

Xanthenones were synthesized via one-pot tricomponent reaction, under solvent-free conditions, using aldehydes, phenolic and cyclic 1,3-dicarbonyl compounds. Natural organic acids (NOAs), compounds present in many living metabolisms, were used as potential green catalysts. NOA are considered to be more eco-friendly and user-friendly alternative to traditional methodologies. Optimization studies showed that oxalic acid was the best NOA catalyst for such reaction furnishing the xanthenones with up to 93% of yield. Theoretical calculations were performed to evaluate this reaction mechanism and regioselectivity. The results showed that the regiospecificity of this three-component reaction is kinetically and thermodynamically controlled by the addition of β-naphthol C2, instead of C10, to the aldehyde. Our results also disclosed two xanthenones as novel calcium channels blockers. Eco-friendly reaction conditions, easy workup procedure, short reaction times and good yields are some of the advantages of our methodology.

Infection with Plasmodium falciparum may result in severe disease affecting various organs, including liver, spleen, and brain, resulting in high morbidity and mortality. Plasmodium berghei Anka infection of mice recapitulates many features of severe human malaria. The aryl hydrocarbon receptor (AhR) is an intracellular receptor activated by ligands important in the modulation of the inflammatory response. We found that AhR-knockout (KO) mice infected with P. berghei Anka displayed increased parasitemia, earlier mortality, enhanced leukocyte-endothelial cell interactions in the brain microvasculature, and increased inflammation in brain (interleukin-17 [IL-17] and IL-6) and liver (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]) compared to infected wild-type (WT) mice. Infected AhR-KO mice also displayed a reduction in cytokines required for host resistance, including TNF-α, IL-1β, and IFN-γ, in the brain and spleen. Infection of AhR-KO mice resulted in an increase in T regulatory cells and transforming growth factor β, IL-6, and IL-17 in the brain. AhR modulated the basal expression of SOCS3 in spleen and brain, and P. berghei Anka infection resulted in enhanced expression of SOCS3 in brain, which was absent in infected AhR-KO mice. These data suggest that AhR-mediated control of SOCS3 expression is probably involved in the phenotype seen in infected AhR-KO mice. This is, to our knowledge, the first demonstration of a role for AhR in the pathogenesis of malaria.

Several attempts have been made toward the production of oxide (NO)- releasing agents based on metallic complexes. However, many of these agents have been shown to undergo hydroxide electrophilic attack, thus generating the corresponding nitro species. Entrapped nitrosyl ruthenium species can be stabilized in an oil-in-water (o/w) emulsion and be further used for local NO delivery. This study describes the transdermal permeation of cis and trans- (RuCl(bpy)2NO)(PF6)2 complexes as NO-donor agents from a topically applied emulsion, with NO release rates of 0.042 ± 0.002 and 0.035 ± 0.003 � mol/cm 2 .h, respectively. In vitro skin permeation studies performed with the cis and trans- (RuCl(bpy)2(NO))(PF6)2 complexes in o/w emulsion showed that they tend to accumulate in the stratum corneum (SC) and viable skin, with spontaneous NO release occurring in the viable skin only. NO release was detected by using an NO- sensor when the complexes were in contact with the sonicated skin. The vascular responses to the NO released from the cis and trans-(RuCl(bpy)2(NO))(PF6)2 complexes in o/w emulsion were also evaluated by vasodilation experiments.

The present investigation was designed to investigate the effect of the diterpene ent-pimara-8(14),15-dien-19-oic acid (pimaradienoic acid, PA) on smooth muscle extracellular Ca2+ influx. To this end, the effect of PA on phenylephrine- and KCl-induced increases in cytosolic calcium concentration ([Ca2+]c), measured by the variation in the ratio of fluorescence intensities (R340/380 nm) of Fura-2, was analysed. Whether bolus injection of PA could induce hypotensive responses in conscious normotensive rats was also evaluated. PA inhibited the contraction induced by phenylephrine (0.03 or 10 μmol L−1) and KCl (30 or 90 μmol L−1) in endothelium-denuded rat aortic rings in a concentration dependent manner. Pre-treatment with PA (10, 100, 200 μmol L−1) attenuated the contraction induced by CaCl2 (0.5 nmol L−1 or 2.5 μmol L−1) in denuded rat aorta exposed to Ca2+-free medium containing phenylephrine (0.1 μmol L−1) or KCl (30 μmol L−1). Interestingly, the inhibitory effect displayed by PA on CaCl2-induced contraction was more pronounced when KCl was used as the stimulant. Phenylephrine- and KCl-induced increases in [Ca2+]c were inhibited by PA. Similarly, verapamil, a Ca2+-channel blocker, also inhibited the increase in [Ca2+]c induced by either phenylephrine or KCl. Finally, bolus injection of PA (1–15 mg kg−1) produced a dose-dependent decrease in mean arterial pressure in conscious normotensive rats. The results provide the first direct evidence that PA reduces vascular contractility by reducing extracellular Ca2+ influx through smooth muscle cellular membrane, a mechanism that could mediate the hypotensive response induced by this diterpene in normotensive rats.

Background and purpose: Epidemiological data suggest that the risk of ethanol-associated cardiovascular disease is greater in men than in women. This study investigates the mechanisms underlying gender-specific vascular effects elicited by chronic ethanol consumption in rats. Experimental approach: Vascular reactivity experiments using standard muscle bath procedures were performed on isolated thoracic aortae from rats. mRNA and protein for inducible NO synthase (iNOS) and for endothelial NOS (eNOS) was assessed by RT-PCR or western blotting, respectively. Key results: In male rats, chronic ethanol consumption enhanced phenylephrine-induced contraction in both endothelium-intact and denuded aortic rings. However, in female rats, chronic ethanol consumption enhanced phenylephrine-induced contraction only in endothelium denuded aortic rings. After pre-incubation of endothelium-intact rings with L-NAME, both male and female ethanol-treated rats showed larger phenylephrine-induced contractions in aortic rings, compared to the control group. Acetylcholine-induced relaxation was not affected by ethanol consumption. The effects of ethanol on responses to phenylephrine were similar in ovariectomized (OVX) and intact (non-OVX) female rats. In the presence of aminoguanidine, but not 7-nitroindazole, the contractions to phenylephrine in rings from ethanol-treated female rats were greater than that found in control tissues in the presence of the inhibitors. mRNA levels for eNOS and iNOS were not altered by ethanol consumption. Ethanol intake reduced eNOS protein levels and increased iNOS protein levels in aorta from female rats. Conclusions and implications: Gender differences in the vascular effects elicited by chronic ethanol consumption were not related to ovarian hormones but seemed to involve the upregulation of iNOS. British Journal of Pharmacology (2008) 153, 468–479; doi:10.1038/sj.bjp.0707589; published online 26 November 2007

The aim of the investigation was to study the possible effects of in vivo infusion of nitric oxide (NO) blockers upon the in vitro endothelium-dependent femoral reactivity. The experimental model tested herein was the inferior canine hindlimb global ischemia induced by infrarenal abdominal aortic cross-clamping followed by reperfusion. The NO blockers employed in the tests were N(G)-nitro-l-arginine methyl ester (L-NAME), aminoguanidine (AMG), and methylene blue (MB), which were infused immediately after the anesthesia induction. The research protocol was standardized in two main experimental groups, control and ischemia/reperfusion (I/R) injury, randomized in eight subgroups including controls and NO blockers. The femoral artery vascular reactivity was studied in vitro with the aid of a setup consisting of eight organ chambers, where segments of 4-5 mm were suspended and connected to force transducers in the presence of indomethacin to block the cyclooxygenase pathway. The NO-release pathway was evaluated by using specific pharmacological agonists in the in vitro experiments. The L-NAME in vivo infusion led to in vitro endothelium dysfunction in both groups and was associated with high mortality in the animals submitted to I/R. AMG and MB, two clinically used drugs, did not cause in vitro endothelium dysfunction in either of the two groups, which gives evidence that these drugs are not deleterious in the milieu of I/R injury. Nitrite/nitrate plasma levels were not significant except for the L-NAME groups, which presented significant NO decrease.

We studied the mechanisms involved in the relaxation induced by nitric oxide (NO) donors, ruthenium complex ([Ru(terpy)(bdq)NO(+)](3+)-TERPY) and sodium nitroprusside (SNP) in denuded rat aorta. Both NO donors induced vascular relaxation independent of the agonist used in the pre-contraction. [Ru(terpy)(bdq)NO(+)](3+) and SNP activated guanylyl cyclase (GC) and K(+) channels. The production of cGMP induced by [Ru(terpy)(bdq)NO(+)](3+) - was higher than that obtained with SNP. The combination of GC inhibitor with K(+)channels blocker almost abolished the relaxation induced by the NO donors. The extracellular NO scavenger oxyhemoglobin reduced the potency without changing the maximum effect (Emax) of both NO donors. By using specific NO species scavengers, hydroxocobalamin and l-cysteine, we have identified the contribution of free radical NO (NO()) and nytroxil anion (NO(-)), respectively, to the rat aorta relaxation induced by both NO donors. The selective scavengers for NO() and NO(-) reduced the potency but not the Emax of [Ru(terpy)(bdq)NO(+)](3+). However, the NO(-) scavenger had no effect on the relaxation induced by SNP and NO() scavenger reduced only the potency to SNP. The inhibition of sarcoplasmic reticulum Ca(2+)-ATPase reduced only the potency of SNP without effect on the relaxation induced by [Ru(terpy)(bdq)NO(+)](3+). Our results demonstrate that both NO donors induce relaxation by activating the GC and K(+) channels. The NO() is the unique NO specie involved in the SNP-relaxation. On the other hand, the relaxant effect of [Ru(terpy)(bdq)NO(+)](3+) involves both NO() and NO(-), that produce higher concentration of cGMP. The inhibition of sarcoplasmic reticulum Ca(2+)-ATPase reduces the relaxation induced by SNP but it did not alter the relaxation induced by [Ru(terpy)(bdq)NO(+)](3+).

The photochemical and pharmacological studies of the novel [Ru(L)(tpy)NO]3+ L = bpy (2,2′-bipyridine), NH · NHq (quinonediimine) and NH2.NH2cat (o-phenylenediamine) were investigated in aqueous medium. The synthesized nitrosyl ruthenium complexes showed nitric oxide (NO) release under light irradiation at 355 nm for [Ru(L)(tpy)NO]3+ complex with quantum yield of 0.14 ± 0.02, 0.47 ± 0.03 and 0.46 ± 0.02 mol Einstein−1 for L = bpy, NH · NHq and NH2 · NH2cat, respectively, and 0.0065 ± 0.001 mol Einstein−1 for light irradiation at 532 nm for [Ru(NH · NHq)(tpy)NO]3+ complex. The photochemical pathway at 355 nm light irradiation was described as a multi-step mechanism, although at 532 nm it was better attributed to a photo-induced electron transfer. The vasorelaxation induced by NO release produced by light irradiation in visible region from physiological solution of [Ru(NH · NHq)(tpy)NO]3+ complex was evaluated and compared with sodium nitroprusside (SNP). The results showed very similar vasodilator power between both species.

Preliminary pharmacological studies of various nitric oxide (NO) photo-releasing agents are reported based on the flash-photolysis studies of the nitro ruthenium complexes cis-[RuII(NO2)L(bpy)2]+ (bpy = 2,2′-bipyridine and L = pyridine, 4-picoline and pyrazine) and [RuII(NO2)(bpy)(terpy)]+ (terpy = terpyridine) in physiological medium. The net photoreactions under these conditions are two primary photoproducts, in (I) there is RuII–NO2 photoaquation, where the photoproducts are RuII–H2O plus NO 2 - and (II) homolytic dissociation of NO from a coordinated nitrito to derive the RuII–OH2 specie and NO. Based on photochemical processes, the nitro ruthenium complexes were incorporated in water in oil (W/O) microemulsion and used in the vasorelaxation induced experiment. Denuded rat aortas were contracted with KCl and nitro ruthenium complexes in microemulsion were added. Perfusion pressures were recorded while arteries were irradiated at 355 nm The time to reach maximum relaxation was longer for [RuII(NO2)(bpy)(terpy)]+ complex (ca. 50 min, n = 6) than for cis-[Ru(NO2)L(bpy)2]+ with L = py and 4-pic complex (ca. 28 min, n = 6) and cis-[Ru(NO2)(bpy)2 (pz)]2+ complex (ca. 24 min, n = 5).

1. We studied the effect of the new compound trans-[RuCl([15]aneN(4))NO](2+) (15-ane) in denuded aortic rings of two kidney (2K) normotensive and two kidney, one clip (2K-1C) hypertensive rats. 2. The compound 15-ane releases nitric oxide (NO) when reduced by a catecholamine (noradrenaline). 3. Oxyhemoglobin (HbO(2)), an NO scavenger, completely abolished the effect of 15-ane in both 2K and 2K-1C rats, indicating that the relaxation is really due to NO release. 4. We tested the hypothesis that an impairment of K(+) channels plays an important role in the vasodilation induced by 15-ane. 5. The selective inhibitor of soluble guanylyl-cyclase, namely 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ; 1 micromol/L) reduced the relaxation induced by 15-ane. In 2K-1C rat aortic rings, ODQ reduced the maximum effect (E(max)) of 15-ane, whereas in 2K rat aortic rings ODQ reduced E(max) and pD(2) values to 15-ane. 6. The selective K(+) channel blockers glibenclamide (blocks K(ATP); 3 micromol/L), 4-aminopyridine (blocks K(V); 1 mmol/L) and the small conductance K(Ca) channel blocker apamin (1 micromol/L) reduced E(max) and pD(2) values for 15-ane-induced relaxation responses of aortas from 2K rats. However, iberiotoxin, a blocker of large conductance K(Ca) channels, reduced only the E(max) to 15-ane. None of these K(+) channel blockers had any effect on the relaxation induced by 15-ane of aortas from 2K-1C rats. 7. These data indicate that an impaired functional activity of K(+) channels contributes to the deficient relaxation induced by the NO donor 15-ane in renal hypertensive 2K-1C rat aortas.

The purpose of the present study was to investigate the effects of chronic methionine treatment on vascular smooth muscle contractility for angiotensin II (Ang II). Methionine at 0.1, 1 and 2 g/kg body weight was administered daily in the drinking water for 2, 4, 8 and 16 weeks. Rat carotid rings from control and treated animals were placed in an organ chamber containing Krebs solution. Concentration-response curves for Ang II and potassium chloride (KCl) were determined. Methionine-rich diet enhanced the plasmatic homocysteine concentration, and the magnitude of the contractile response to Ang II was increased in carotid rings from treated animals after 8 and 16 weeks. However, the treatment did not alter KCl-induced contraction. In another set of experiments, the rings were incubated with indomethacin and curves for Ang II were obtained. Exposure of the rings to indomethacin inhibited the enhancement in the contractile response to Ang II. The present findings show that chronic methionine treatment enhances homocysteine plasmatic concentration leading to an enhanced Ang-II-induced contraction, which appears to be related to the release of vasoconstrictor prostanoid(s).

ent -Pimara-8(14),15-dien-19-oic acid (PA) isolated from Viguiera arenaria (Asteraceae; Heliantheae) inhibited rat carotid rings contraction induced by phenylephrine (10 −8 mol/l) or potassium chloride (45 mmol/l) at concentration ranging from 5 to 20 μg/ml. This inhibitory effect was not reversible after the removal of this compound from the medium bath.

This study aimed to investigate the modulation of nitric oxide/reactive oxygen species in sodium nitroprusside relaxation in mice aorta. Sodium nitroprusside induced relaxation in endothelium-intact (e+) and endothelium-denuded (e-) aortas with greater potency in e+ than in e-. The nitric oxide synthase inhibitor did not alter the sodium nitroprusside relaxation in both e+ and e- aortas. However, the superoxide anion scavenger abolished the difference in sodium nitroprusside potency between e+ and e-. Sodium nitroprusside reduced dihydroethidium-derived fluorescent products in both groups; however, the difference between intact and denuded mice aorta remains. The glutathione levels and basal antioxidant activity of superoxide dismutase were reduced in e- aorta when compared with e+, and these values were not altered by sodium nitroprusside. Confirming these results, the levels of lipid peroxidation in e+ were significantly lower when compared to e-, and these values were not altered by sodium nitroprusside. The sodium nitroprusside potency in the presence of a nonselective COX inhibitor or the EP/DP prostaglandin receptor antagonist in endothelium denuded was similar to that in intact mice aorta. Based on these results, we performed the COX-1 and COX-2 mRNA level studies, and in denuded mice aorta, there was an upregulation in COX-1 mRNA levels. Taken together, our findings show that in the absence of endothelium, there is an enhancement of superoxide levels, leading to GSH consumption and higher levels of lipid peroxidation, showing an intense redox status. Furthermore, in denuded mice aorta, there was an upregulation of COX-1 mRNA expression, leading to vasoconstrictor prostanoids synthesis. The interaction of vasoconstrictor prostanoids with its receptors EP/DP negatively modulates the vascular relaxation induced by SNP in denuded mice aorta.

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F(1α) (6-keto-PGF(1α)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1α) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS.

Made available in DSpace on 2013-09-27T14:53:45Z (GMT). No. of bitstreams: 1 WOS000291623600023.pdf: 848109 bytes, checksum: 66c40f67ba3999c3c029f2cdc0feafbc (MD5) Previous issue date: 2011-06-25 Made available in DSpace on 2013-09-30T18:08:03Z (GMT). No. of bitstreams: 1 WOS000291623600023.pdf: 848109 bytes, checksum: 66c40f67ba3999c3c029f2cdc0feafbc (MD5) Previous issue date: 2011-06-25 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:25:36Z No. of bitstreams: 1 WOS000291623600023.pdf: 848109 bytes, checksum: 66c40f67ba3999c3c029f2cdc0feafbc (MD5) Made available in DSpace on 2014-05-20T13:25:36Z (GMT). No. of bitstreams: 1 WOS000291623600023.pdf: 848109 bytes, checksum: 66c40f67ba3999c3c029f2cdc0feafbc (MD5) Previous issue date: 2011-06-25 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The present work investigates the mechanisms involved in the vasorelaxant effect of ent-16 alpha-methoxykauran-19-oic acid (KA-OCH(3)), a semi-synthetic derivative obtained from the kaurane-type diterpene ent-kaur-16-en-19-oic acid (kaurenoic acid). Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats using standard muscle bath procedures. The cytosolic calcium concentration ([Ca(2+)]c) was measured by confocal microscopy using the fluorescent probe Fluo-3 AM. Blood pressure measurements were performed in conscious rats. KA-OCH(3) (10,50 and 100 mu mol/l) inhibited phenylephrine-induced contraction in either endothelium-intact or endothelium-denuded rat aortic rings. KA-OCH(3) also reduced CaCl(2)-induced contraction in a Ca(2+)-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 mu mol/l). KA-OCH(3) (0.1-300 mu mol/l) concentration-dependently relaxed endothelium-intact and endothelium-denuded aortas pre-contracted with either phenylephrine or KCl, to a greater extent than kaurenoic acid. Moreover, a Ca(2+) mobilisation study showed that KA-OCH(3) (100 mu mol/l) inhibited the increase in Ca(2+) concentration in smooth muscle and endothelial cells induced by phenylephrine or KCl. Pre-incubation of intact or denuded aortic rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mu mol/l), 7-nitroindazole (100 mu mol/l), wortmannin (0.5 mu mol/l) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ 1 mu mol/l) produced a rightward displacement of the KA-OCH(3) concentration-response curve. Intravenous administration of KA-OCH(3) (1-10 mg/kg) reduced mean arterial blood pressure in normotensive rats. Collectively, our results show that KA-OCH(3) induces vascular relaxation and hypotension. The mechanisms underlying the cardiovascular actions of KA-OCH(3) involve blockade of Ca(2+) influx and activation of the NO-cGMP pathway. (C) 2011 Elsevier B.V. All rights reserved. Univ São Paulo, Escola Enfermagem Ribeirao Preto, Coll Nursing Ribeirao Preto, Dept Psychiat Nursing & Human Sci,Lab Pharmacol, BR-14040902 Ribeirao Preto, SP, Brazil Univ São Paulo, Fac Med Ribeirao Preto, Dept Pharmacol, BR-14040902 Ribeirao Preto, SP, Brazil São Paulo State Univ, UNESP, Sch Pharmaceut Sci, Dept Nat Act Principles & Toxicol, Araraquara, SP, Brazil Universidade Federal de Minas Gerais (UFMG), Inst Biol Sci, Dept Pharmacol, Belo Horizonte, MG, Brazil Univ Franca, Franca, SP, Brazil Univ São Paulo, Fac Pharmaceut Sci Ribeirao Preto, Pharmacol Lab, Dept Chem & Phys, BR-14040902 Ribeirao Preto, SP, Brazil São Paulo State Univ, UNESP, Sch Pharmaceut Sci, Dept Nat Act Principles & Toxicol, Araraquara, SP, Brazil FAPESP: 09/52629-4

Pimarane-type diterpenes were described to exert antispasmodic and relaxant activities. Based on this observation we hypothesized that the diterpene ent-8(14),15-pimaradien-3beta-ol (PA-3beta-ol) induced vascular relaxation. With this purpose, the present work investigates the mechanisms involved in the vasorelaxant effect of the pimarane-type diterpene PA-3beta-ol. Vascular reactivity experiments, using standard muscle bath procedures, were performed in isolated aortic rings from male Wistar rats. Cytosolic calcium concentration ([Ca(2+)]c) was measured by confocal microscopy using the fluorescent probe Fluo-3AM. PA-3beta-ol (10, 50 and 100 micromol/l) inhibited phenylephrine and KCl-induced contraction in either endothelium-intact or denuded rat aortic rings. PA-3beta-ol also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 micromol/l). PA-3beta-ol (1-300 micromol/l) concentration dependently relaxed phenylephrine-pre-contracted rings with intact or denuded endothelium. The diterpene also relaxed KCl-pre-contracted rings with intact or denuded endothelium. Moreover, Ca(2+) mobilization study showed that PA-3beta-ol (100 micromol/l) and verapamil (1 micromol/l) inhibited the increase in Ca(2+)-concentration in smooth muscle and endothelial cells induced by phenylephrine (10 micromol/l) or KCl (60 mmol/l). Pre-incubation of intact or denuded aortic rings with N(G)-nitro-l-arginine methyl ester (L-NAME, 100 micromol/l) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 micromol/l) produced a rightward displacement of the PA-3beta-ol concentration-response curves. On the other hand, 7-nitroindazole (100 micromol/l), 1400 W (1 micromol/l), indomethacin (10 micromol/l) and tetraethylammonium (1 mmol/l) did not affect PA-3beta-ol-induced relaxation. Collectively, our results provide evidence that the effects elicited by PA-3beta-ol involve extracellular Ca(2+) influx blockade. Its effects are also partly mediated by the activation of NO-cGMP pathway.

The carotid artery has a pivotal role in the body since it supplies the head and neck with oxygenated blood. Alterations in the functional and structural integrity of these vessels can decrease blood flow to the brain. For this reason, it is important to understand how the carotid artery responds to various stimuli. The organ bath is a traditional experimental set-up that has been used extensively to investigate the (patho)physiology and pharmacology of in vitro tissue preparations including the rat carotid artery. Molecular biology developed from related fields such as biochemistry, genetics and biophysics is now considered an important tool for understanding physiological pathways in a variety of tissues. Several local and systemic factors regulate carotid reactivity, including vaso-active peptides, such as endothelin 1 (ET-1), angiotensin II (Ang II) and bradykinin (BK). These vaso-active peptides play a fundamental role in controlling the functional and structural integrity of the arterial wall and may be important in physiological processes and in pathological mechanisms underlying vascular diseases. In the rat carotid, these peptides induce vasoconstriction or relaxation by the release of endothelium-derived relaxing factors, such as nitric oxide and prostacyclin. Identification of such signal transduction processes is essential for understanding the mechanisms that regulate vascular smooth muscle cell function, both physiologically and pathophysiologically. The present review discusses the mechanisms of action, distribution of ET-1, Ang II and BK and their receptors in the rat carotid. With this purpose, data obtained in functional studies using classical pharmacological approaches as well as data obtained in molecular biology experiments are discussed.

Nitrosyl ruthenium complexes have been characterized as nitric oxide (NO) donors that induce relaxation in the denuded rat aorta. There are some differences in their vascular relaxation mechanisms compared with sodium nitroprusside. This study investigates whether the endothelium could interfere with the [Ru(terpy)(bdq)NO](3+)-TERPY-induced vascular relaxation, by analyzing the maximal relaxation (Emax) and potency (pD(2)) of TERPY. Vascular reactivity experiments showed that the endothelium negatively modulates (pD(2): 6.17+/-0.07) the TERPY relaxation in intact rat aortic rings compared with the denuded rat aorta (pD(2): 6.65+/-0.07). This effect is abolished by a non-selective NO-synthase (NOS) inhibitor L-NAME (pD(2): 6.46+/-0.10), by the superoxide anion (O(2)(-)) scavenger TIRON (pD(2): 6.49+/-0.08), and by an NOS cofactor BH(4) (pD(2): 6.80+/-0.10). The selective dye for O(2)(-) (DHE) shows that TERPY enhances O(2)(-) concentration in isolated endothelial cells (intensity of fluorescence (IF):11258.00+/-317.75) compared with the basal concentration (IF: 7760.67+/-381.50), and this enhancement is blocked by L-NAME (IF: 8892.33+/-1074.41). Similar results were observed in vascular smooth muscle cells (concentration of superoxide after TERPY: 2.63+/-0.17% and after TERPY+L-NAME: -4.63+/-0.14%). Considering that TERPY could induce uncoupling NOS, thus producing O(2)(-), we have also investigated the involvement of prostanoids in the negative modulation of the endothelium. The non-selective cyclooxygenase (COX) inhibitor indomethacin and the selective tromboxane (TXA(2)) receptor antagonist SQ29548 reduce the effect of the endothelium on TERPY relaxation (pD(2) INDO: 6.80+/-0.17 and SQ29548: 6.85+/-0.15, respectively). However, a selective prostaglandin F(2alpha) receptor antagonist (AH6809) does not change the endothelium effect. Moreover, TERPY enhances the concentration of TXA(2) stable metabolite (TXB(2)), but this effect is blocked by L-NAME and TIRON. The present findings indicate that TERPY induces uncoupling of eNOS, enhancing O(2)(-) concentration. This enhancement in O(2)(-) concentration induces COX activation, producing TXA(2), which negatively modulates the rat aorta relaxation induced by the NO donor TERPY.

Sodium nitroprusside (SNP) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle (VSM). Endothelium modulates the vascular tone through the release of vasoactive agents, such as NO. The aim of this study was to investigate the contribution of the endothelium on SNP vasorelaxation, NO release and Ca2+ mobilization. Vascular reactivity experiments showed that endothelium potentiates the SNP-relaxation in rat aortic rings and this effect was abolished by l-NAME. SNP-relaxation in intact endothelium aorta was inhibited by NOS inhibitors for the constitutive isoforms (cNOS). Furthermore, endogenous NO is involved on the SNP-effect and this endogenous NO is released by cNOS. Moreover, Ca2+ mobilization study shows that l-NAME inhibited the reduction of Ca2+-concentration in VSM cells and reduced the increase in Ca2+-concentration in endothelial cells induced by SNP. This enhancement in Ca2+-concentration in the endothelial cells is due to a voltage-dependent Ca2+ channels activation. The present findings indicate that the relaxation and [Ca2+]i decrease induced by SNP in VSM cells is potentiated by endothelial production of NO by cNOS-activation in rat aorta.

This work aimed to functionally characterize the mechanisms underlying the relaxation induced by bradykinin (BK) in the rat carotid artery. Vascular reactivity experiments, using standard muscle bath procedures, showed that BK (0.1 nmol/L-3 mumol/L) induced relaxation of phenylephrine-pre-contracted rings in a concentration-dependent manner. Endothelial removal strongly attenuated BK-induced relaxation. HOE-140, the selective antagonist of bradykinin B(2) receptors concentration-dependently reduced the relaxation induced by BK. Pre-incubation of endothelium-intact rings with L-NAME (100 micromol/L), a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME (100 micromol/L), a selective inhibitor of the eNOS or 7-nitroindazole (100 micromol/L), the selective inhibitor of nNOS, reduced BK-induced relaxation. Conversely, 1400 W (10 nmol/L), a selective inhibitor of iNOS, did not alter the relaxation induced by BK. Surprisingly, indomethacin (10 micromol/L) a non-selective inhibitor of cyclooxygenase (COX) increased BK-induced relaxation in endothelium-intact but not denuded rings. Neither SQ29548 (3 micromol/L), a competitive antagonist of PGH(2)/TXA(2) receptors nor AH6809 (10 micromol/L), an antagonist of PGF(2alpha) receptors significantly altered the relaxation induced by BK in endothelium-intact rings. The combination of SQ29548 and AH6809 increased BK-induced relaxation. The present study shows that the vasorelaxant action displayed by BK in the rat carotid is mediated by endothelial B(2) receptors and the activation of the NO pathway. The major finding of this work is that it demonstrated functionally that endothelial-derived vasoconstrictor prostanoids (probably PGH(2), TXA(2) and PGF(2alpha)) counteract the vasorelaxant action displayed by BK.

Irradiation of trans-[RuCl(cyclam)(NO)](2+), cyclam is 1,4,8,11-tetraazacyclotetradecane, at pHs 1-7.4, with near UV light results in the release of NO and formation of trans-[Ru(III)Cl(OH)(cyclam)](+) with pH dependent quantum yields (from approximately 0.01 to 0.16 mol Einstein(-1)) lower than that for trans-[RuCl([15]aneN(4))(NO)](2+), [15]aneN(4) is 1,4,8,12-tetaazacyclopentadecane, (0.61 mol Einstein(-1)). After irradiation with 355 nm light, the trans-[RuCl([15]aneN(4))(NO)](2+) induces relaxation of the aortic ring, whereas the trans-[RuCl(cyclam)(NO)](2+) complex does not. The relaxation observed with trans-[RuCl([15]aneN(4))(NO)](2+) is consistent with a larger quantum yield of release of NO from this complex.

Nitric oxide (NO) plays an important role in the control of vascular tone. NO donors have therapeutic use and the most used NO donors, nitroglycerin and sodium nitroprusside have problems in their use. Thus, new NO donors have been synthesized to minimize these undesirable effects. Nytrosil ruthenium complexes have been studied as a new class of NO donors. trans-[RuCl([15]aneN(4))NO](2+), induces vasorelaxation only in presence of reducing agent. In this study, we characterized the mechanisms of vasorelaxation of trans-[RuCl([15]aneN(4))NO](2+) in denuded rat aorta and identified which NO forms are involved in this relaxation. We also evaluated the effect of this NO donor in decreasing the cytosolic Ca(2+) concentration ([Ca(2+)]c) of the vascular smooth muscle cells. Vasorelaxation to trans-[RuCl([15]aneN(4))NO](2+) (E(max): 101.8 +/- 2.3%, pEC(50): 5.03 +/- 0.15) was almost abolished in the presence of the NO* scavenger hydroxocobalamin (E(max): 4.0 +/- 0.4%; P < 0.001) and it was partially inhibited by the NO(-) scavenger L-cysteine (E(max): 79.9 +/- 6.9%, pEC(50): 4.41 +/- 0.06; P < 0.05). The guanylyl cyclase inhibitor ODQ reduced the E(max) (57.7 +/- 4.0%, P < 0.001) and pEC(50) (4.21 +/- 0.42, P < 0.01) and the combination of ODQ and TEA abolished the response to trans-[RuCl([15]aneN(4))NO](2+). The blockade of voltage-dependent (K(v)), ATP-sensitive (K(ATP)), and Ca(2+)-activated (K(Ca) K(+) channels reduced the vasorelaxation induced by trans-[RuCl([15]aneN(4))NO](2+). This compound significantly reduced [Ca(2+)]c (from 100% to 85.9 +/- 3.5%, n = 4). In conclusion, our data demonstrate that this NO donor induces vascular relaxation involving NO* and NO(-) species, that is associated to a decrease in [Ca(2+)]c. The mechanisms of vasorelaxation involve guanylyl cyclase activation, cGMP production and K(+) channels activation.

The vasorelaxation induced by a nitrosyl macrocyclic ruthenium complex, proposed as a new nitric oxide (NO) carrier, was studied in rat isolated aorta. The compound trans -[RuCl([15]aneN 4 )NO] 2+ was characterized by elemental analysis, UV–visible spectrum, and infrared spectrum. Based on the electrochemical process, the reduction of the compound was followed by NO release, which was also observed using norepinephrine as a reducing agent and NO released was analyzed by a sensor. Vasorelaxation induced by this NO donor was studied and compared to those obtained with sodium nitroprusside (SNP). The relaxation induced by the compound was concentration-dependent in denuded rat aortas and occurred only in pre-contracted arteries with norepinephrine. The macrocyclic compound induced relaxation with a similar efficacy as SNP, although the potency of SNP was slightly greater. The time to reach maximum relaxation (595 s) was longer than that of SNP (195 s). Relaxation was completely abolished by oxyhemoglobin, a known NO scavenger.

Nitric oxide has been pointed out as the main agent involved in the vasodilatation, which is the major symptom of septic shock. However, there must be another mediator contributing to the circulatory failure observed in sepsis. This study aimed to investigate the endothelium-dependent relaxation induced by acetylcholine and the factors involved in this relaxation, using aortic rings isolated from rats submitted to cecal ligation and perforation (CLP), 2 h after induction of sepsis, which characterizes the hyperdynamic phase of sepsis. Under inhibition of constitutive NO-synthases (cNOS), the relaxation induced by acetylcholine was greater in the aortic rings of rats submitted to CLP compared with sham-operated rat aortic rings. The cyclooxygenase inhibitor indomethacin normalized this response, and the concentration of the stable metabolite of prostacyclin in the aorta of CLP rats increased in basal conditions and after stimulation with acetylcholine. Acetylcholine-induced NO production was lower in the endothelial cells from the aorta of CLP rats compared with sham rat aorta, but the protein expression of the cNOS was not altered. Moreover, iNOS protein expression could not be detected. Therefore, prostacyclin, and not only nitric oxide, is a mediator of the vasorelaxation induced by acetylcholine in aortas from rats submitted to CLP.

Aims To examine the vasodilatation induce by the NO donors, [Ru(terpy)(bdq)NO] 3+ (TERPY) and sodium nitroprusside (SNP), and to compare their effects in aortic rings from hypertensive 2K-1C and normotensive 2K rats. Main methods Vascular reactivity was performed in aortic rings pre-contracted with phenylephrine (Phe 100 nM). We have analyzed the maximal relaxation (Emax) and potency (pD 2 ) of NO donors. Key findings Potency of SNP was greater than TERPY in both arterial groups. The vasodilatation induced by TERPY was greater in 2K than in 2K-1C, and it was inhibited by sGC inhibitor ODQ in 2K and in 2K-1C aortic rings. ODQ did not alter the efficacy to SNP, but it reduced its potency in 2K and 2K-1C. The blockade of K + channels reduced the potency of TERPY only in aortic rings of 2K. On the other hand, the potency of SNP was reduced in both 2K and 2K-1C. The combination of ODQ and TEA reduced the relaxation induced by TERPY and SNP in 2K and reduced the efficacy to SNP in 2K-1C aortic rings but it had no additional effect on the TERPY relaxation in 2K-1C aortas. The production of cGMP induced by TERPY was greater than that produced by SNP, which was similarly increased in 2K and 2K-1C. Sarcoplasmic reticulum Ca-ATPase inhibition only impaired the relaxation induced by SNP in 2K aortic rings. Significance Taken together, our results provide evidences that in this model of hypertension, impaired K + channels activation by TERPY and SERCA activation by SNP may contribute to decreased vasodilatation.

O vírus Chikungunya (CHIKV) é um arbovírus reemergente, cuja infecção causa doença febril associada a fortes dores nas articulações, mialgia e comprometimento cardiovascular. Embora a resposta imune ao CHIKV seja bem descrita, demonstrando a participação de diversas células do sistema imune, um melhor entendimento dos mecanismos de interação vírus-hospedeiro que afetam o sistema vascular pode levar a intervenções terapêuticas mais eficazes. Há evidências que a Chikungunya está associada a sintomas de bradicardia e hipotensão, além de outros efeitos cardiovasculares, incluindo a geração de espécies reativas de oxigênio (EROs) e produção de citocinas pró e anti-inflamatórias. Considerando que a resposta inflamatória e o equilíbrio redox são capazes de interferir na homeostase vascular, foi testada a hipótese que a infecção por CHIKV compromete a função arterial por ações diretas em células vasculares e por mecanismos que envolvem estresse oxidativo. Foram utilizados camundongos C57BL/6J, machos, com seis semanas. Os camundongos foram divididos em dois grupos experimentais: 1) infectados com CHIKV e 2) Mock-veículo. Os camundongos foram infundidos com CHIKV (1,0 x 106 PFU) ou meio de cultura pela via intracaudal. A função vascular foi avaliada em anéis de aorta torácica usando um miógrafo para registrar a tensão isométrica. Células endoteliais (EA.hy926) foram infectadas com CHIKV (MOI 1.0). A produção de ROS foi avaliada por quimiluminescência com lucigenina e a expressão de proteínas, por western blot. Diferenças significativamente estatísticas foram consideradas quando p

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico Doenças cardiovasculares (DCVs) são consideradas as principais causas de morte no mundo. Trabalhos recentes demonstram que há um aumento na incidência de DCVs em pacientes que sobrevivem a um quadro de infecção aguda como a sepse, entre o primeiro e o quinto ano após a infecção. A partir disso, o objetivo desse estudo foi avaliar a função cardiovascular dos animais sobreviventes à sepse. Para tal, em camundongos Balb/c machos foi realizada a ligação e perfuração do ceco para indução da sepse. Após 15 dias, foram avaliados o eletrocardiograma e ecocardiograma, experimentos de Langendorff, contratilidade de cardiomiócitos e reatividade vascular na aorta torácica. Nossos resultados demonstraram um aumento significativo do volume sistólico final associado a uma redução da fração de ejeção e do débito cardíaco no grupo sobrevivente à sepse. Além disso, nesse mesmo grupo, os resultados do eletrocardiograma demonstraram um prolongamento no tempo para iniciar a repolarização ventricular. Os experimentos de Langendorff confirmaram o prejuízo da função cardíaca basal nos sobreviventes à sepse, e esse prejuízo foi mantido quando submetido ao estresse farmacológico induzido por isoprenalina. Nos experimentos de reatividade vascular, o efeito contrátil induzido por fenilefrina em aortas torácicas dos animais sobreviventes à sepse foi significativamente maior quando comparado ao grupo controle. Esse aumento na vasoconstrição foi relacionado à ativação de receptores AT1, ânion superóxido e derivados da COX. Com isso, camundongos sobreviventes à sepse apresentaram aumento da contratilidade vascular e prejuízo da função cardíaca, onde o tratamento com propranolol foi capaz de impedir a hiperreatividade vascular aórtica, bem como reverteu alguns pontos do prejuízo cardíaco. Cardiovascular diseases (CVDs) are considered the leading cause of death in the world. Recent studies have shown an increase in the incidence of CVDs in patients with acute infection, such as sepsis. Sepsis survivors show an increase in the development of CVDs between 1 and 5 years after the previous infection. Thus, the aim of this study was to evaluate the cardiovascular parameters in animals surviving sepsis. For that, in male Balb/c mice was performed cecal ligation and a puncture to induce sepsis. After 15 days, electrocardiogram and echocardiogram, Langendorff experiments, cardiomyocytes contraction, and vascular reactivity in thoracic aorta were performed. Our results showed an increase in systolic volume, ejection fraction and cardiac output in the sepsissurvivor group (SSG). Besides that, the electrocardiogram showed an increase in time to initiate ventricular repolarization in SSG. Langendorff’s experiments confirmed the impairment of cardiac function in the SSG group, and this impairment was not reversed in a stressful condition induced by isoprenaline. About vasomotor tonus, the contractile effect induced by phenylephrine in thoracic aortas from SSG was significantly higher when compared to the control group. This effect was associated with the activation ofAT1 receptors, anions superoxide and COX derived prostanoids. Taken together, sepsis-surviving mice showed vascular contractility enhancement and impairment of cardiac function. Propranolol treatment was able to reverse the increased vascular resistance triggered by sepsis and some points of cardiac function.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A obesidade está entre as causas mais importantes de doenças cardiovasculares. O tecido adiposo perivascular (PVAT) exerce importante influência sobre os vasos sanguíneos, podendo se relacionar a progressão ou regressão de doenças cardiovasculares relacionadas à obesidade. Sabendo-se que a dieta de cafeteria (dieta CAF) reflete a variedade de alimentos altamente calóricos e de fácil acesso nas sociedades ocidentais, contribuindo para o desenvolvimento da obesidade e desordens metabólicas, o objetivo desse trabalho foi avaliar o impacto da dieta CAF no sistema cardiovascular, bem como sua influência no perfil vasoativo do PVAT. Os resultados encontrados mostraram que a dieta CAF induziu aumento do peso corporal e do índice de adiposidade, hiperlipidemia, hiperglicemia, intolerância à glicose e resistência à insulina. A análise do eletrocardiograma e da função cardíaca demonstrou que a dieta CAF promoveu atraso na condução atrioventricular e aumento na contratilidade/relaxamento basal do ventrículo esquerdo. O mesmo resultado foi observado a nível celular, em que a dieta CAF exacerbou a resposta de contração/relaxamento de cardiomiócitos. Entretanto, frente ao estresse farmacológico com isoprenalina, a contratilidade/relaxamento do ventrículo esquerdo no grupo CAF demonstrou ser menos responsiva. A dieta CAF também induziu redução da contratilidade vascular induzida por fenilefrina (PE) na ausência do PVAT, relacionada ao aumento nos níveis séricos de leptina e à ativação de seus receptores, ativação das enzimas PI3K e óxido nítrico sintase endotelial, e produção de óxido nítrico e peróxido de hidrogênio. Por outro lado, a presença do PVAT aumentou a vasoconstrição induzida por PE no grupo CAF, sugerindo um perfil vasoconstritor do PVAT na tentativa de restabelecer a contratilidade vascular, relacionado a ativação dos receptores AT1, ativação da enzima NADPH oxidase e produção de ânions superóxido, e ativação da enzima ciclooxigenase 1 e dos receptores de tromboxano A2 e prostaglandina F2α. Em conjunto, os resultados encontrados demonstraram que a obesidade desencadeada pela dieta CAF predispõe alterações cardíacas e vasculares, com alterações no perfil vasoativo do PVAT. Contudo, o tratamento dietético alternando a dieta CAF para a dieta padrão para roedores se mostrou efetivo para reverter essas alterações. Obesity is among the most important causes of cardiovascular disease. The perivascular adipose tissue (PVAT) is closely related to the cardiovascular system playing an important role in vascular homeostasis, which may be associated with the progression or regression of cardiovascular diseases related to obesity. Knowing that the cafeteria diet (CAF diet) reflects the variety of highly caloric and easily accessible foods in Western societies, contributing to the development of obesity and metabolic disorders, the aim of this study was to evaluate the impact of CAF diet on the cardiovascular system, as well as its influence on the PVAT vasoactive profile. The results found showed that the CAF diet induced an increase in the body weight and adiposity index, hyperlipidemia, hyperglycemia, glucose intolerance and insulin resistance. The analysis of the electrocardiogram and cardiac function showed that the CAF diet caused a delay in atrioventricular conduction and an increase in baseline left ventricular contractility/relaxation. The same result was observed at the cellular level, in which the CAF diet exacerbated the contraction/relaxation response of cardiomyocytes. However, under a pharmacological stress induced by isoprenaline, left ventricular contractility/relaxation in the CAF group proved to be less responsive. The CAF diet also induced a reduction in vascular contractility induced by phenylephrine (PE) in the absence of PVAT related to the increase in serum leptin levels and the activation of its receptors, activation of PI3K enzymes and nitric oxide endothelial synthase, and production of nitric oxide and hydrogen peroxide. On the other hand, the presence of PVAT increased PE-induced vasoconstriction in the CAF group, suggesting a vasoconstrictor profile of PVAT in an attempt to reestablish the vascular contractility related to the activation of AT1 receptors, activation of NADPH oxidase and production of superoxide anions, and activation of the cyclooxygenase 1 and the thromboxane A2 and prostaglandin F2α receptors. Taken together, our findings showed that obesity triggered by CAF diet predisposes to cardiac and vascular alterations, with alterations in the vasoactive profile of PVAT. However, dietary treatment alternating the CAF diet to the standard diet proved to be effective in reversing these changes.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior O tecido adiposo perivascular (PVAT) reveste a camada adventícia e envolve a maioria dos vasos sanguíneos. O PVAT libera moléculas biologicamente ativas que regulam o tônus vascular. Sabendo da importância desse tecido no controle do tônus vascular e que a hipotensão sistêmica, gerada em um quadro de sepse, é a principal causa de morte, o objetivo desse trabalho foi estudar os efeitos vasoativos e inflamatórios do PVAT na hiporreatividade aórtica induzida por sepse aguda. Para tanto, utilizou-se camundongos Balb/c machos (8 semanas), que foram divididos em dois grupos: sham e séptico. No grupo séptico, a sepse foi induzida por ligadura e perfuração transversal do ceco com agulha 26G. No grupo sham, a etapa de perfuração transversal do ceco não foi realizada. 6 horas após a indução da sepse, o papel do PVAT no tônus vascular foi avaliado em aortas torácicas dos grupos sham e séptico. Nossos resultados demonstram que apesar dos grupos sham e séptico apresentarem 100% de sobrevida, o grupo séptico apresentou leucopenia marcada por neutropenia, hipotermia, hipotensão arterial e alta pontuação de escore clínico quando comparado ao grupo sham. A vasoconstrição induzida por fenilefrina foi significativamente reduzida no grupo séptico em comparação ao grupo sham na ausência de PVAT. Entretanto, na presença do PVAT, o prejuízo na contratilidade vascular observado no grupo séptico foi reestabelecido. Ou seja, o PVAT apresentou um perfil vasoconstritor, se contrapondo a hiporreatividade causada pela sepse. Entre os mecanismos envolvidos nesse processo, sugerimos uma regulação recíproca entre espécies reativas de oxigênio, neste caso, ânion superóxido, gerado provavelmente a partir do complexo NADPH oxidase, e que por uma cascata de autoperpetuação, a COX-2 e seus derivados vasocontráteis, como tromboxano A2 e prostaglandina F2α também atuam “alimentando” essa via. Portanto sugerimos que o perfil contrátil apresentado pelo PVAT na sepse aguda (6h) se deva principalmente à participação de ânions superóxido, além de derivados da COX-2 e que ambos “autoalimentam” o ciclo favorecendo a contração observada. Perivascular adipose tissue (PVAT) lines the adventitial layer and surrounds most blood vessels. PVAT releases biologically active molecules that regulate vascular tone. Knowing the importance of this tissue in the control of vascular tone and that the systemic hypotension, generated in a case of sepsis, is the main cause of death, this study aimed to study the vasoactive and inflammatory effects of PVAT on aortic hyporeactivity induced by acute sepsis. For this purpose, male Balb / c mice (8 weeks) were used, which were divided into two groups: sham and septic. In the septic group, sepsis was induced by ligation and transverse perforation of the cecum with a 26G needle. In the sham group, the transverse perforation step of the cecum was not performed. 6 hours after sepsis induction, the role of PVAT in vascular tone was assessed in thoracic aortas of the sham and septic groups. Our results demonstrate that although the sham and septic groups have 100% survival, the septic group presented leukopenia marked by neutropenia, hypothermia, arterial hypotension, and a high clinical score when compared to the sham group. Phenylephrine-induced vasoconstriction was significantly reduced in the septic group compared to the sham group in the absence of PVAT. However, in the presence of PVAT, the impairment of vascular contractility observed in the septic group was reestablished. In other words, PVAT presented a vasoconstrictor profile, in contrast to the hyporeactivity caused by sepsis. Among the mechanisms involved in this process, we suggest a reciprocal regulation between reactive oxygen species, in this case, superoxide anion, probably generated from the NADPH oxidase complex, and that by a self-perpetuating cascade, COX-2 and its vasocontractable derivatives, as thromboxane A2 and prostaglandin F2α also act "feeding" this pathway. Therefore, we suggest that the contractile profile presented by PVAT in acute sepsis (6h) is mainly due to the participation of superoxide anions, in addition to derivatives of COX-2 and that both “feed” the cycle favoring the observed contraction.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico A disfunção endotelial é uma condição crônica, que se inicia com o estresse oxidativo em resposta ao componente lisofosfatidilcolina (LPC), presente na LDL oxidada (LDLox), sendo este um evento chave para a geração de uma resposta inflamatória podendo culminar no desenvolvimento da aterosclerose e suas complicações. É reconhecido o papel fundamental das óxido nítrico sintase endotelial (eNOS) e neuronal (nNOS) na manutenção da homeostase vascular. E que seu desacoplamento, está intimamente ligado à evolução clínica da disfunção endotelial. Na busca por novas substancias que possam atuar na prevenção ou tratamento das comorbidades cardiovasculares, os estudos envolvendo componentes dietéticos tem se destacado. O butirato é produzido pelo organismo pela fermentação bacteriana de fibras no cólon e constitui a principal fonte energética para os colonócitos. Vários estudos nos últimos anos tem mostrado que o butirato possui efeitos anti-inflamatórios, anti-oxidantes, anti-aterogenicos, dentre outros efeitos. Uma vez que a disfunção endotelial é o evento inicial chave para o desenvolvimento de comorbidades cardiovasculares graves, e que o butirato possui propriedades que o tornam um excelente candidato para o estudo do seu efeito sobre a disfunção endotelial. O objetivo desse trabalho foi avaliar os efeitos do butirato de sódio sobre a disfunção endotelial causada pela LPC. Para isso foram utilizados camundongos machos C57BL6/J, com 10-12 semanas de idade, os animais foram eutanasiados e sua aorta torácica foi dividida em anéis e montados em um banho de órgãos para a avaliação da reatividade vascular. Os resultados mostraram que o butirato, em sua menor concentração de teste, foi capaz de melhorar a disfunção endotelial causada por LPC e que esse efeito se deu pela ação de nNOS presente no endotélio. Foram realizados, também, estudos in vitro de citometria de fluxo e western blot com células endoteliais (EA.Hy926) pré tratadas com LPC e depois expostas a concentrações de butirato, e em seguida, estimuladas com ACh. O tratamento com butirato levou ao aumento na produção de NO, e na diminuição de peróxido de hidrogênio (H2O2) e anion superóxido (O-2). O presente trabalho mostrou pela primeira vez que o tratamento com butirato de sódio foi capaz de reverter a disfunção endotelial causada por LPC por aumento de NO via normalização da atividade de nNOS, diminuição de ROS e pela regularização da atividade de ERK1/2. Os resultados mostram que o tratamento com butirato de sódio impede a resposta disfuncional mediada por LPC. Endothelial dysfunction is a chronic condition, which begins with oxidative stress in response to the lysophosphatidylcholine (LPC) component, present in oxidized LDL (LDLox), which is a key event for the generation of an inflammatory response that may culminate in the development of atherosclerosis and its complications. The fundamental role of endothelial (eNOS) and neuronal (nNOS) nitric oxide synthases in maintaining vascular homeostasis is recognized and that its decoupling is closely linked to the clinical evolution of endothelial dysfunction. In the search for new substances that can act in the prevention or treatment of cardiovascular comorbidities, studies involving dietary components have stood out. Butyrate is produced by the body through bacterial fermentation of fibers in the colon and is the main energy source for colonocytes. Several studies in recent years have shown that butyrate has anti-inflammatory, anti-oxidant, anti-atherogenic effects, among other effects. Since endothelial dysfunction is the key initial event for the development of severe cardiovascular comorbidities, and butyrate has properties that make it an excellent candidate for studying its effect on endothelial dysfunction. The aim of this work was to evaluate the effects of sodium butyrate on endothelial dysfunction caused by PCL. For this purpose, male C57BL6 / J mice, aged 10-12 weeks, were euthanized and their thoracic aorta was divided into rings and mounted in an organ bath to assess vascular reactivity. The results showed that butyrate, at its lowest test concentration, was able to improve endothelial dysfunction caused by LPC and that this effect was due to the action of nNOS present in the endothelium. Flow cytometry and western blot studies were also carried out with endothelial cells (EA.Hy926) pre-treated with LPC and then exposed to butyrate concentrations, and then stimulated with ACh. The treatment with butyrate led to an increase in NO production, and a decrease in hydrogen peroxide (H2O2) and superoxide anion (O2-). The present study showed for the first time that treatment with sodium butyrate was able to reverse the endothelial dysfunction caused by LPC by increasing NO via normalization of nNOS activity, decreasing ROS and by regularizing the ERK1 / 2 activity. The results show that treatment with sodium butyrate prevents the dysfunctional response mediated by LPC.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Segundo a Organização Mundial de Saúde ocorre aproximadamente 5,4 milhões de picadas por serpentes a cada ano, com 2,7 milhões de envenenamentos que levam a 100.000 mortes. No Brasil, uma das serpentes de maior importância médica é a Crotalus durissus terrificus, que ocasiona óbitos em 1,1% dos casos de envenenamento. A peçonha dessa serpente apresenta altas concentrações de fosfolipase A2, sendo essa o componente básico (CB) da Crotoxina (CTX). Essa toxina é responsável pela neurotoxicidade e miotoxicidade no acidente crotálico. Recentemente o Varespladip (LY315920), um inibidor de fosfolipase A2 sintético, tem sido estudado para neutralizar toxinas de serpentes. Portanto, para avaliar o potencial inibidor do Varespladib contra a Crotoxina, foram realizados experimentos miográficos e morfológicos, utilizando preparações nervo frênico-músculo diafragma de camundongo in vitro. As preparações foram expostas à CTX, a CB, ao Varespladib ou ao produto da pré-incubação de CTX-Varespladib ou CB-Varespladib. Os resultados do presente estudo demonstraram que o Varespladib é capaz de neutralizar os efeitos paralisantes e miotóxico da CTX e os efeitos paralisantes da CB em preparações neuromusculares de camundongos. According to the World Health Organization, approximately 5.4 million snake bites occur each year, with 2.7 million poisonings that lead to 100,000 deaths. In Brazil, one of the snakes of greatest medical importance is Crotalus durissus terrificus, which causes deaths in 1.1% of poisoning cases. The crotalic venom has high concentrations of phospholipase A2, one of which is the basic component (CB) of Crotoxin (CTX). This toxin is responsible for neurotoxicity and myotoxicity in crotalic accidents. Recently, Varespladip (LY315920), a synthetic phospholipase A2 inhibitor, has been studied to neutralize snake toxins. Therefore, to evaluate the inhibitor potential of Varespladib against Crotoxin, myographic and morphological experiments were performed, using mouse phrenic nerve-diaphragm muscle preparations in vitro. The preparations were exposed to CTX, CB, Varespladib or the pre-incubated product of CTX-Varespladib and CB-Varespladib. The results of the present study demonstrated that Varespladib is able to neutralize the paralyzing and myotoxic effects of CTX and the paralyzing effects of CB in mice neuromuscular preparations.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Evidências crescentes demonstram o aumento das taxas de doenças cardiovasculares entre pessoas vivendo com HIV/AIDS. Tanto a infecção pelo HIV-1 quanto a terapia antirretroviral estão implicadas no desenvolvimento de dislipidemias, aumento do estresse oxidativo e disfunção endotelial, sendo esses importantes eventos fisiopatológicos para o desenvolvimento da aterosclerose. O dolutegravir (DTG) é um inibidor da enzima integrase do HIV-1 que tem sido utilizado como a primeira opção de terapia antirretroviral desde 2017. No entanto, não há dados consistentes na literatura demonstrando as ações cardiovasculares e metabólicas dos inibidores da integrase, particularmente do DTG. O objetivo deste trabalho foi avaliar o impacto do tratamento subcrônico com o DTG no sistema cardiovascular, perfil lipídico, função hepática e estresse oxidativo em Camundongos C57BL/6J. Camundongos machos foram tratados com DTG (10mg/kg) ou veículo durante 28 dias, via gavagem. Curvas concentração- resposta com acetilcolina e fenilefrina foram realizadas em anéis de aorta, utilizando sistema de banho de órgãos. O tratamento com DTG gerou redução na resposta relaxante induzida pela acetilcolina. Este efeito foi associado a uma menor produção de NO nas aortas, corroborada pela redução na expressão de eNOS e nNOS também observada neste sítio. Embora não tenha sido encontrada diferença na produção de superóxido e peróxido de hidrogênio, houve aumento na formação de nitrotirosina, sugerindo maior produção de peroxinitrito nas aortas do grupo DTG. Não foram observadas alterações na glicemia e perfil lipídico sérico. No entanto, houve aumento da transaminase TGP sérica e peroxidação lipídica hepática, além de diminuição da atividade da SOD no fígado. Tais efeitos foram associados ao desenvolvimento de esteatose hepática no grupo DTG. A histopatologia cardíaca, avaliação da pressão arterial e eletrocardiográfica não demonstraram diferença entre os grupos. Os resultados deste trabalho permitem concluir que o tratamento subcrônico com DTG induziu disfunção endotelial, mediada pela diminuição da biodisponibilidade de NO, em decorrência da menor expressão das NOS. Embora não tenha sido observado alterações nos parâmetros cardíacos avaliados, o DTG induziu esteatose e maior estresse oxidativo hepático. Increasing evidence demonstrates the increased rates of cardiovascular disease among people living with HIV / AIDS. Both HIV-1 infection and antiretroviral therapy are implicated in the development of dyslipidemia, increased oxidative stress and endothelial dysfunction, these being important pathophysiological events for the development of atherosclerosis. Dolutegravir (DTG) is an inhibitor of the HIV-1 integrase enzyme that has been used as the first antiretroviral therapy option since 2017. However, there is no consistent data in the literature demonstrating the cardiovascular and metabolic actions of integrase inhibitors, particularly of the DTG. The objective of this work was to evaluate the impact of subchronic treatment with DTG on the cardiovascular system, lipid profile, liver function and oxidative stress in C57Bl / 6J mice. Male mice were treated with DTG (10mg / kg) or vehicle for 28 days, via gavage. Concentration-response curves with acetylcholine and phenylephrine were performed on aortic rings, using an organ bath system. Treatment with DTG generated a reduction in the relaxing response induced by acetylcholine. This effect was associated with a lower production of NO in the aortas, corroborated by the reduction in the expression of eNOS and nNOS also observed in this site. Although no difference was found in the production of superoxide and hydrogen peroxide, there was an increase in the formation of nitrotyrosine, suggesting greater production of peroxynitrite in the aortas of the DTG group. There were no changes in blood glucose and serum lipid profile. However, there was an increase in serum TGP transaminase and hepatic lipid peroxidation, in addition to a decrease in SOD activity in the liver. Such effects were associated with the development of fatty liver in the DTG group. Cardiac histopathology, blood pressure and electrocardiographic assessment showed no difference between groups. The results of this work allow us to conclude that the subchronic treatment with DTG induced endothelial dysfunction, mediated by the decrease in the bioavailability of NO, due to the lower expression of NOS. Although no changes were observed in the cardiac parameters evaluated, the DTG induced steatosis and increased hepatic oxidative stress.

Estudos com modelos celulares, animais e humanos demonstram de maneira consistente que as atuais terapias cirúrgicas e anti-proliferativas retardam, mas não evitam, a hiperplasia das camadas média e íntima arteriais, independentemente de um quadro de dislipidemia associado. Por outro lado, apesar do inquestionável sucesso da atual terapia hipocolesterolemiante, vários estudos sugerem que o sistema endocanabinóide pode representar um importante alvo de atuação terapêutica em doenças fibro-proliferativas vasculares. Desta forma, testamos a hipótese de que a ativação dos receptores canabinóides tipo 2 (CB2) poderia modular a enzima óxido nítrico sintase neuronal (nNOS) e prevenir o dano causado em modelo de oclusão de carótidas em camundongos C57BL6J. A artéria carótida foi ocluída sob tratamento local com agonista CB2 – JWH015. A carótida foi avaliada por técnicas histológicas e também por microscopia de fluorescência (imunofluorescência e sondas fluorescentes sensíveis ao NO e radicais superóxido). Células endoteliais humanas também foram expostas a tratamento com mesmo agonista. Neste modelo foram avaliadas a expressão proteica (eNOS, nNOS, marcadores de proliferação e receptores CB2), a produção de NO e superóxido e migração celular. Os resultados encontrados demonstram que o agonista CB2 previne a formação neoíntima no modelo estudado, com redução na densidade de células endoteliais, colágeno, metaloproteinase de matriz e radicais superóxido. Nas células endoteliais, o tratamento com JWH015 aumenta a expressão da nNOS e a produção de NO. A inibição da nNOS leva à diminuição de superóxido, indicando que esta isoforma possa estar desacoplada. Sob shear stress oscilatório (OSS), observamos que tanto o estímulo com JWH015 quanto com a inibição da nNOS levam a uma diminuição na produção de NO, que parece ser provocada pela ativação da via PI3K/AKT. Sobre a cicatrização endotelial (ensaio de fenda), encontramos que o tratamento com agonista do receptor CB2 reduziu o tempo para fechamento da fenda em repouso. Por outro lado, a inibição da enzima nNOS retardou este processo. Concluímos ainda que, para que o receptor CB2 seja utilizado como um alvo farmacológico para prevenção da formação neoíntima, mais ensaios precisam ser desenvolvidos em modelos biológicos. Studies with cell, animal, and human models consistently demonstrate that current surgical and antiproliferative therapies delay, but do not prevent, hyperplasia of the media and intima arterial layers, regardless of associated dyslipidemia. On the other hand, despite the unquestionable success of current hypocholesterolemic therapy, several studies suggest that the endocannabinoid system may represent an important target for therapeutic action in vascular fibroproliferative diseases. Thus, we tested the hypothesis that the activation of the cannabinoid type 2 (CB2) receptors could modulate the neuronal nitric oxide synthase (nNOS) enzyme and prevent the damage caused in a carotid occlusion model in C57BL6J mice. The carotid artery was occluded under local treatment with CB2-JWH015 agonist. The carotid was evaluated by histological techniques and also by fluorescence microscopy (immunofluorescence and NO-sensitive fluorescent probes and superoxide radicals). Human endothelial cells were also exposed to treatment with the same agonist. In this model, protein expression (eNOS, nNOS, proliferation markers and CB2 receptors), NO and superoxide production and cell migration were evaluated. The results showed that the CB2 agonist prevents neointima formation in the model studied, with reduction in the density of endothelial cells, collagen, matrix metalloproteinase and superoxide radicals. In endothelial cells, treatment with JWH015 increases nNOS expression and NO production. Inhibition of nNOS leads to a decrease in superoxide, indicating that this isoform may be uncoupled. Under oscillatory shear stress (OSS), we observed that both the JWH015 stimulus and the nNOS inhibition lead to a decrease in NO production, which appears to be triggered by the activation of the PI3K / AKT pathway. On endothelial healing (slit assay), we found that CB2 receptor agonist treatment reduced the time to closure of the slit at rest. On the other hand, inhibition of the nNOS enzyme delayed this process. We conclude that for the CB2 receptor to be used as a pharmacological target for the prevention of neointima formation, more trials need to be developed in biological models.

As concentrações circulantes e vasculares da interleucina (IL)-1? estão elevadas na hipertensão arterial (HA), e polimorfismos no receptor de interleucinas do tipo I (IL-1RI) estão associados a essa doença. A IL-1? promove ações diretas na vasculatura: disfunção endotelial em artérias mesentéricas de resistência (AMR), aumento da migração e proliferação de células musculares lisas vasculares, além da produção de espécies reativas de oxigênio (EROs). O papel exercido pelo IL-1RI no contexto da HA carece de elucidações. O presente trabalho testou a hipótese de que o IL-1RI promove disfunção endotelial e remodelamento vascular em decorrência da geração de EROs na hipertensão induzida por angiotensina II (ANGII). Este estudo avaliou se a ausência do IL-1RI seria capaz de prevenir o aumento da pressão arterial induzida por ANGII, disfunção endotelial, remodelamento vascular, geração de EROs e hipertrofia cardíaca. Nosso estudo demonstrou que a deleção do IL1-RI não foi capaz de prevenir o aumento da PA em camundongos nocautes submetidos a HA por ANGII. As concentrações circulantes de IL-1? estavam elevadas em animais hipertensos, sugerindo a contribuição desta citocina na cascata de sinalização mediada via IL1-RI. A ausência do IL-1RI foi capaz de prevenir a disfunção endotelial e diminuir o remodelamento vascular em AMR. O estresse oxidativo foi significativamente menor em animais nocautes para IL1-RI infundidos com ANGII. Em suma, os resultados encontrados indicam que o IL-1RI promove disfunção endotelial e remodelamento vascular em AMR na HA induzida por ANGII. Ademais, o aumento da geração de EROs é um mecanismo potencial nas mudanças funcionais e estruturais promovidas via IL1RI na HA induzida por infusão de ANGII Circulating and vascular wall concentrations of interleukin (IL)-1? are elevated in arterial hypertension (AH), and polymorphisms in the interleukin type I receptor (IL-1RI) are associated with this disease. IL-1? promotes direct actions in the vasculature: endothelial dysfunction in mesenteric arteries of resistance (AMR), increased migration and proliferation of vascular smooth muscle cells, and the production of reactive oxygen species (ROS). The role played by IL-1RI in the context of HA needs elucidation. O papel exercido pelo IL-1RI no contexto da HA carece de elucidações. The present study tested the hypothesis that IL-1RI promotes endothelial dysfunction and vascular remodeling due to the generation of ROS in angiotensin II (ANGII)-induced hypertension. This study determined whether the absence of IL-1RI would be able to prevent the increase of blood pressure (BP), endothelial dysfunction, vascular remodeling, ROS generation, and cardiac hypertrophy in ANGII-induced hypertensive mice. Our study demonstrated that IL1-RI deletion was not able to prevent the increase of PA in knockout mice subjected to HA by ANGII. Circulating concentrations of IL-1? were elevated in hypertensive animals, suggesting the contribution of this cytokine in the signaling cascade mediated via IL1-RI. The absence of IL-1RI was able to prevent endothelial dysfunction and decrease vascular remodeling in MRA. The oxidative stress was significantly lower in IL1-RI knockout animals infused with ANGII. In summary, the results indicate that IL-1RI promotes endothelial dysfunction and vascular remodeling in MRA in AH induced by ANGII. In addition, increased ROS generation is a potential mechanism in the functional and structural changes promoted via IL-1RI in AH by ANGII infusion

O diabetes mellitus (DM) está associado a diversas complicações micro e macrovasculares diretamente relacionadas a doenças cardiovasculares. A prolongada exposição à hiperglicemia e a resistência a insulina são considerados os principais fatores envolvidos nestas complicações, as quais são exacerbadas pela disfunção endotelial. Mediadores inflamatórios contribuem potencialmente para o desenvolvimento de disfunção endotelial pela geração de espécies reativas de oxigênio (EROs) que, por sua vez, estimulam a transcrição de fatores pró- inflamatórios. Receptores específicos, como os NLRs (NOD-like receptors, receptores do tipo NOD) contribuem para instalação de processo inflamatório pela ativação do complexo inflamassoma. Este regula a ativação da caspase-1 e o processamento proteolítico dos precursores pró-IL-1? e pró-IL-18 nas citocinas maduras. Diversos mediadores podem ativar o inflamassoma NLRP3 como, por exemplo, EROs e DNA mitocondrial. Pouco é conhecido sobre o envolvimento de receptores NLRP3 e DNA mitocondrial na disfunção endotelial associada ao diabetes. Testamos a hipótese que a deficiência genética do receptor NLRP3 confere resistência à ativação de processo inflamatório na vasculatura de animais com diabetes tipo 1 (DM1) e, ainda, que DNA mitocondrial contribui para a ativação vascular do inflamassoma NLRP3 e para disfunção endotelial. Foram utilizados camundongos C57Bl/6 e deficientes para NLRP3, os quais foram tratados com veículo ou submetidos a protocolo para indução de DM1 com estreptozotocina. Parâmetros vasculares funcionais foram determinados em artérias mesentéricas de resistência. Células de músculo liso vascular (CMLV) e endoteliais foram utilizadas para avaliação da ativação do inflamassoma NLRP3 por DNA mitocondrial. A geração de EROs foi avaliada pela fluorescência para o dihidroetídio e pela quimiluminescência para lucigenina. A ativação de caspase-1 e IL-1? foi avaliada por western blot e o influxo de cálcio, por fluorescência. DNA mitocondrial foi avaliado pela expressão gênica de componentes da mitocôndria. O diabetes reduziu a vasodilatação dependente de endotélio, o que não ocorreu em artérias de animais deficientes de NLRP3. Animais diabéticos apresentaram aumento da expressão vascular do receptor NLRP3, da ativação de caspase-1 eIL-1? e da geração de EROs e peróxido de hidrogênio no leito mesentérico, eventos que ocorreram em menor intensidade em camundongos deficientes de NLRP3. Houve redução na expressão proteica vascular de Nox4 (NADPH oxidase 4), bem como na expressão gênica da molécula de adesão celular vascular-1 (VCAM-1, vascular cell adhesion molecule-1) e molécula de adesão intercelular-1 (ICAM-1, intercellular adhesion molecule-1) em animais deficientes de NLRP3. Houve aumento da liberação de DNA mitocondrial citosólico no pâncreas de animais diabéticos. A incubação com o DNA mitocondrial extraído do pâncreas de animais diabéticos promoveu ativação do inflamassoma em CMLV provenientes de animais C57Bl/6, mas não em CMLV provenientes de animais deficientes de NLRP3. Esta ativação foi associada ao aumento de EROs e influxo de cálcio. Essa mesma ativação também foi observada em células endoteliais. DNA mitocondrial de camundongos diabéticos também reduziu a dilatação dependente do endotélio em artérias mesentéricas, o que foi associado à geração de EROs e ativação do inflamassoma NLRP3. Pacientes diabéticos apresentaram aumento do DNA mitocondrial circulante e ativação de caspase-1 e IL-1? no soro. Os resultados demonstram que o DNA mitocondrial pancreático de animais diabéticos promove ativação, em CMLV e células endoteliais, do inflamassoma NLRP3 através do aumento no influxo de cálcio e da geração de EROs, contribuindo para o processo de disfunção endotelial. A deficiência de NLRP3 protege os animais diabéticos contra os danos vasculares inflamatórios e disfunção endotelial. Diabetes mellitus (DM) is associated with several micro and macrovascular complications directly related to cardiovascular diseases. Prolonged exposure to hyperglycemia and insulin resistance are considered the main factors involved in these complications, which are exacerbated by endothelial dysfunction. Inflammatory mediators potentially contribute to the development of endothelial dysfunction by the generation of reactive oxygen species (ROS), which, in turn, stimulate the transcription of pro-inflammatory factors. Specific receptors such as NLRs (NOD-like receptors) contribute to the onset of inflammatory processes by the activation of a multiprotein complex called inflammasome. The NLRP3 inflammasome regulates the activation of caspase-1 and the proteolytic processing of pro-IL-1? and pro-IL-18 precursors into mature cytokines. Several mediators, such as ROS and mitochondrial DNA activate the NLRP3 inflammasome. Considering that it is not clear whether NLRP3 and mitochondrial DNA contribute to diabetes-associated endothelial dysfunction, we hypothesized that the genetic deficiency of the NLRP3 confers resistance to vascular inflammatory processes in animals with type 1 diabetes (T1D) and that mitochondrial DNA contributes to vascular activation of NLRP3 inflammasome and endothelial dysfunction. C57B1/6 and NLRP3 knockout mice were treated with vehicle or streptozotocin to induce T1D. Functional vascular parameters were determined in resistance mesenteric arteries. Cultured vascular smooth muscle cells (VSMC) and endothelial cells were used to determine NLRP3 inflammasome activation by mitochondrial DNA. ROS generation was evaluated by dihydroethidium fluorescence and by chemiluminescence for lucigenin. Caspase-1 and IL-1? activation was evaluated by western blot. Calcium influx was determined by fluorescence and mitochondrial DNA by mRNA expression of mitochondrial components. Diabetes reduced endothelium-dependent vasodilation in C57B1/6, but not in NLRP3 knockout mice. Diabetic mice presented increased vascular NLRP3 receptor expression, increased caspase-1 and IL-1? activation, as well as ROS and hydrogen peroxide generation, events that were mildly observed in NLRP3 knockout mice. There was a reduction in the vascular protein expression of Nox4 (NADPH oxidase 4) as wellas in the gene expression of VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) in NLRP3 knockout animals. There was an increase in cytosolic mitochondrial DNA release in pancreas from diabetic animals. Mitochondrial DNA from the pancreas of diabetic mice induced NLRP3 inflammasome activation in VSMC from C57B1/6 mice, but not in VSMC from NLRP3 knockout mice. This activation was associated with increased levels of ROS and calcium influx and was also detected in endothelial cells. Mitochondrial DNA from diabetic mice also decreased endothelium-dependent dilation in mesenteric arteries, which was associated with ROS generation and NLRP3 inflammasome activation. Diabetic patients exhibited increased serum mitochondrial DNA and caspase-1 and IL-1? activation. The results demonstrate that pancreatic mitochondrial DNA from diabetic mice activates the NLRP3 inflammasome in VSMC and endothelial cells by increasing calcium influx and ROS generation, contributing to endothelial dysfunction. NLRP3 deficiency prevents diabetes-related vascular inflammatory damage and endothelial dysfunction.