Melatonin prevents overproduction of reactive oxygen species and vascular dysfunction induced by cyclophosphamide.

AIMS: Overproduction of reactive oxygen species (ROS) is a pathologic hallmark of cyclophosphamide toxicity. For this reason, antioxidant compounds emerge as promising tools for preventing tissue damage induced by cyclophosphamide. We hypothesized that melatonin would display cytoprotective action in the vasculature by preventing cyclophosphamide-induced oxidative stress. MATERIALS AND METHODS: Male C57BL/6 mice (22-25 g) were injected with a single dose of cyclophosphamide (300 mg/kg; i.p.). Mice were pretreated or not with melatonin (10 mg/kg/day, i.p.), given during 4 days before cyclophosphamide injection. Functional (vascular reactivity) and oxidative/inflammatory patterns were evaluated at 24 h in resistance arteries. The antioxidant action of melatonin was assessed in vitro in cultured vascular smooth muscle cells (VSMCs) of mesenteric arteries. KEY FINDINGS: Cyclophosphamide induced ROS generation in both mesenteric arterial bed (MAB) and cultured VSMCs, and this was normalized by melatonin. Cyclophosphamide-induced ROS generation and lipoperoxidation in the bladder and kidney was also prevented by melatonin. Increased levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were detected in the MAB of cyclophosphamide-treated mice, all of which were prevented by melatonin. Functional assays using second-order mesenteric arteries of cyclophosphamide-treated mice revealed a decrease in vascular contractility. Melatonin prevented vascular hypocontractility in the cyclophosphamide group. Melatonin partially prevented the decrease in myeloperoxidase (MPO) and N-acetyl-beta-D-glucosaminidase (NAG) activities in the MAB of the cyclophosphamide group. SIGNIFICANCE: Melatonin may constitute a novel and promising therapeutic approach for management of the toxic effects induced by cyclophosphamide in the vasculature.

Reactive Oxygen Species Are Central Mediators of Vascular Dysfunction and Hypertension Induced by Ethanol Consumption.

Consumption of high amounts of ethanol is a risk factor for development of cardiovascular diseases such as arterial hypertension. The hypertensive state induced by ethanol is a complex multi-factorial event, and oxidative stress is a pathophysiological hallmark of vascular dysfunction associated with ethanol consumption. Increasing levels of reactive oxygen species (ROS) in the vasculature trigger important processes underlying vascular injury, including accumulation of intracellular Ca2+ ions, reduced bioavailability of nitric oxide (NO), activation of mitogen-activated protein kinases (MAPKs), endothelial dysfunction, and loss of the anticontractile effect of perivascular adipose tissue (PVAT). The enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a central role in vascular ROS generation in response to ethanol. Activation of the renin-angiotensin-aldosterone system (RAAS) is an upstream mechanism which contributes to NADPH oxidase stimulation, overproduction of ROS, and vascular dysfunction. This review discusses the mechanisms of vascular dysfunction induced by ethanol, detailing the contribution of ROS to these processes. Data examining the association between neuroendocrine changes and vascular oxidative stress induced by ethanol are also reviewed and discussed. These issues are of paramount interest to public health as ethanol contributes to blood pressure elevation in the general population, and it is linked to cardiovascular conditions and diseases.

AT1 receptors modulate ethanol-induced loss of anticontractile effect of perivascular adipose tissue.

Ethanol consumption activates renin-angiotensin-aldosterone system (RAAS), which plays a major role in the pro-contractile and hypertensive effects linked to ethanol. We hypothesized that ethanol consumption induces loss of the anticontractile effect of perivascular adipose tissue (PVAT)through RAAS-mediated mechanisms. We examined the contribution of angiotensin II type 1 receptors (AT1R) to ethanol-induced PVAT dysfunction. With this purpose, male Wistar Hannover rats were treated with ethanol 20 % (in volume ratio) and/or losartan (antagonist of AT1R; 10 mg/kg/day, gavage) for 9 weeks. Losartan prevented the increase in blood pressure and the loss of the anticontractile effect of PVAT induced by ethanol consumption. PVAT dysfunction occurred after 3 and 9 weeks of treatment with ethanol in an endothelium-dependent manner. Blockade of AT1R prevented ethanol-induced reduction of adiponectin levels in PVAT from ethanol-treated rats. Functional assays revealed that ethanol impaired the anticontractile effect of PVAT-derived angiotensin (1-7) and endothelial nitric oxide (NO). In conclusion, AT1R are implicated in ethanol-induced loss of the anticontractile effect of PVAT. In PVAT, AT1R activation decreases the production of adiponectin, a PVAT-derived factor that promotes vasorelaxation in an endothelium-dependent manner. In the endothelium, AT1R favors the production of superoxide (O2•-) leading to a reduction in NO bioavailability. These responses impair the vasodilator action induced by PVAT-derived angiotensin (1-7), which occurs via Mas receptors located in endothelial cells. Ethanol-induced PVAT dysfunction favors vascular hypercontractility, a response that could contribute to the hypertensive state associated with ethanol consumption.

Aspergillus fumigatus Extracellular Vesicles Display Increased Galleria mellonella Survival but Partial Pro-Inflammatory Response by Macrophages.

Fungal extracellular vesicles (EVs) mediate intra- and interspecies communication and are critical in host-fungus interaction, modulating inflammation and immune responses. In this study, we evaluated the in vitro pro- and anti-inflammatory properties of Aspergillus fumigatus EVs over innate leukocytes. A. fumigatus EVs induced a partial proinflammatory response by macrophages, characterized by increased tumor necrosis factor-alpha production, and increased gene expression of induced nitric oxide synthase and adhesion molecules. EVs induce neither NETosis in human neutrophils nor cytokine secretion by peripheral mononuclear cells. However, prior inoculation of A. fumigatus EVs in Galleria mellonella larvae resulted in increased survival after the fungal challenge. Taken together, these findings show that A. fumigatus EVs play a role in protection against fungal infection, although they induce a partial pro-inflammatory response.

Extracellular Vesicles from Candida haemulonii var. vulnera Modulate Macrophage Oxidative Burst.

Members of the Candida haemulonii species complex are multidrug-resistant emergent yeast pathogens able to cause superficial and invasive infections in risk populations. Fungal extracellular vesicles (EVs) play a critical role in the pathogenicity and virulence of several species and may perform essential functions during infections, such as carrying virulence factors that behave in two-way communications with the host, affecting survival and fungal resistance. Our study aimed to describe EV production from Candida haemulonii var. vulnera and evaluate whether murine macrophage RAW 264.7 cells respond to their stimuli by generating an oxidative response after 24 h. For this purpose, reactive oxygen species detection assays demonstrated that high concentrations of yeast and EVs (1010 particles/mL) of Candida haemulonii did not change macrophage viability. However, the macrophages recognized these EVs and triggered an oxidative response through the classical NOX-2 pathway, increasing O2•- and H2O2 levels. However, this stress did not cause lipid peroxidation in the RAW 264.7 cells and neither lead to the activation of the COX-2-PGE2 pathway. Thus, our data suggest that low concentrations of C. haemulonii EVs are not recognized by the classical pathway of the oxidative burst generated by macrophages, which might be an advantage allowing the transport of virulence factors via EVs, not identified by the host immune system that could work as fine tube regulators during infections caused by C. haemulonii. In contrast, C. haemulonii var. vulnera and high EV concentrations activated microbicidal actions in macrophages. Therefore, we propose that EVs could participate in the virulence of the species and that these particles could be a source of antigens to be exploited as new therapeutic targets.

Mineralocorticoid receptors contribute to ethanol-induced vascular hypercontractility through reactive oxygen species generation and up-regulation of cyclooxygenase 2.

The effects on blood pressure produced byethanol consumption include both vasoconstriction and activation of the renin-angiotensin-aldosterone system (RAAS), although the detailed relationship between these processes is yet to be accomplished. Here, we sought to investigate the contribution of mineralocorticoid receptors (MR) to ethanol-induced hypertension and vascular hypercontractility. We analyzed blood pressure and vascular function of male Wistar Hannover rats treated with ethanol for five weeks. The contribution of the MR pathway to the cardiovascular effects of ethanol was evaluated with potassium canrenoate, a MR antagonist (MRA). Blockade of MR prevented ethanol-induced hypertension and hypercontractility of endothelium-intact and -denuded aortic rings. Ethanol up-regulated cyclooxygenase (COX)2 and augmented vascular levels of both reactive oxygen species (ROS) and thromboxane (TX)B2, a stable metabolite of TXA2. These responses were abrogated by MR blockade. Hyperreactivity to phenylephrine induced by ethanol consumption was reversed by tiron [a scavenger of superoxide (O2∙-)], SC236 (a selective COX2 inhibitor) or SQ29548 (an antagonist of TP receptors). Treatment with the antioxidant apocynin prevented the vascular hypercontractility, as well as the increases in COX2 expression and TXA2 production induced by ethanol consumption. Our study has identified novel mechanisms through which ethanol consumption promotes its deleterious effects in the cardiovascular system. We provided evidence for a role of MR in the vascular hypercontractility and hypertension associated with ethanol consumption. The MR pathway triggers vascular hypercontractility through ROS generation, up-regulation of COX2 and overproduction of TXA2, which will ultimately induce vascular contraction.

Ethanol consumption favors pro-contractile phenotype of perivascular adipose tissue: A role for interleukin-6.

Perivascular adipose tissue (PVAT) exerts anticontractile effect, but under non-physiological conditions it may contribute to vascular dysfunction by releasing pro-inflammatory cytokines. Since PVAT is an important source of interleukin (IL)-6, we evaluated whether this cytokine would contribute to ethanol-induced vascular dysfunction. With this purpose, male C57BL/6 wild-type (WT) or IL-6-deficient mice (IL-6-/-) were treated with ethanol for 12 weeks. Increased blood pressure was evidenced after 4 and 6 weeks of treatment with ethanol in WT and IL-6-/- mice, respectively. In WT mice, ethanol increased plasma and PVAT levels of IL-6. Ethanol favoured pro-contractile phenotype of PVAT in mesenteric arteries from WT, but not IL-6-deficient mice. Functional studies showed that tiron [(a scavenger of superoxide (O2-)] reversed the pro-contractile effect of PVAT in mesenteric arteries from ethanol-treated mice. Ethanol increased the levels of O2- in PVAT from WT mice. Ethanol-induced increase in O2- generation was higher in arteries with PVAT from WT mice when compared to IL-6-deficient mice. Treatment with ethanol augmented myeloperoxidase activity in the mesenteric arterial bed (MAB; with or without PVAT) from WT, but not IL-6-deficient mice. In conclusion, IL-6 contributes to the pro-contractile effect of PVAT by a mechanism that involves increase in ROS generation. Additionally, IL-6 mediates intravascular recruitment of neutrophils in response to ethanol and plays a role in the early stages of ethanol-induced hypertension. Collectively, our findings provide novel evidence for a role of IL-6 in the vascular dysfunction induced by ethanol.

Comparative study between apocynin and protocatechuic acid regarding antioxidant capacity and vascular effects.

Reactive oxygen species (ROS) derived from NOX enzymes activity play an important role in the development of cardiovascular diseases. Compounds able to decrease oxidative stress damage are potential candidates as drugs and/or supplements for hypertension treatment. Here, we aimed to compare in vitro ROS scavenging potency, effective NOX inhibition and effects on vascular reactivity of apocynin to another phenolic compound, protocatechuic acid, in vascular cells from spontaneously hypertensive rat (SHR), where redox signaling is altered and contributes to the development and/or maintenance of hypertension. We evaluated the in vitro antioxidant capacity and free radical scavenging capacity of both phenolic compounds. Moreover, we investigated the effect of both compounds on lipid peroxidation, lucigenin chemiluminescence, nitric oxide (NO•) levels and ROS concentration in vascular cells of SHR or human umbilical vein endothelial cell (HUVEC). Apocynin and protocatechuic acid presented antioxidant capacity and ability as free radical scavengers, decreased thiobarbituric acid reactive substances (TBARS) in aortic cells from SHR, and increased NO• concentration in isolated HUVEC. Both compounds were able to reduce lucigenin chemiluminescence and increased the potency of acetylcholine in aorta of SHR. However, in SHR aortas, only apocynin diminished the contraction induced by phenylephrine. In conclusion, these results strongly reinforce the potential application of substances such as apocynin and protocatechuic acid that combine abilities as scavenging and/or prevention of ROS generation, establishment of NO bioactivity and modulation of vascular reactivity. Due to its phytochemical origin and low toxicity, its potential therapeutic use in vascular diseases should be considered.

Inhibition of inducible nitric oxide synthase protects against the deleterious effects of sub-lethal sepsis and ethanol in the cardiorenal system.

We tested the hypothesis that ethanol would aggravate the deleterious effects of sub-lethal cecal ligation and puncture (SL-CLP) sepsis in the cardiorenal system and that inhibition of inducible nitric oxide synthase (iNOS) would prevent such response. Male C57BL/6 mice were treated with ethanol for 12 weeks. One hour before SL-CLP surgery, mice were treated with N6-(1-iminoethyl)-lysine (L-NIL, 5 mg/kg, i.p.), a selective inhibitor of iNOS. A second dose of L-NIL was administered 24 h after SL-CLP surgery. Mice were killed 48 h post surgery and the blood, the renal cortex, and the left ventricle (LV) were collected for biochemical analysis. L-NIL attenuated the increase in serum creatinine levels induced by ethanol, but not by SL-CLP. Ethanol, but not SL-CLP, increased creatine kinase (CK)-MB activity and L-NIL did not prevent this response. In the renal cortex, L-NIL prevented the redox imbalance induced by ethanol and SL-CLP. Inhibition of iNOS also decreased lipoperoxidation induced by ethanol and SL-CLP in the LV. L-NIL prevented the increase of pro-inflammatory cytokines and reactive oxygen species induced by ethanol and (or) SL-CLP in the cardiorenal system, suggesting that iNOS modulated some of the molecular mechanisms that underlie the deleterious effects of both conditions in the cardiorenal system.

Imipramine attenuates anxiety- and depressive-like effects of acute and prolonged ethanol-abstinence in male rats by modulating SERT and GR expression in the dorsal hippocampus.

AIMS: Considering that serotoninergic agents attenuate symptoms of anxiety and are used to treat depression, we investigated whether subchronic treatment with imipramine, a serotonin/noradrenaline reuptake inhibitor, would prevent the anxiogenic-like behaviour induced by acute and/or chronic ethanol withdrawal. We also investigated whether those changes were related to the disfunctioning of hypothalamic-pituitary-adrenal (HPA) axis and serotonergic neurotransmission. MAIN METHODS: 264 Male Wistar rats were treated with ethanol 6% (vol./vol.) for 21 days. Acute ethanol withdrawal was induced by abrupt discontinuation of treatment and sustained for 48 h. Protracted abstinence was sustained for an additional period of 21 days. Behavioural tests included the Elevated Plus Maze (EPM) or Light/Dark Box (LDB) after acute abstinence, and the Forced Swim Test (FST) after protracted abstinence. Imipramine (15 mg/kg, i.p.) was administered 24, 19 and 1 h before EPM or LDB tests. KEY FINDINGS: Acute abstinence decreased exploration of the open arms of the EPM, without changing exploration of LDB. Additionally, chronic abstinent rats displayed more time immobile in the FST, when compared to control animals. These effects were attenuated by imipramine treatment, without changing basal response. Imipramine prevented protracted abstinence -induced decrease in glucocorticoid receptor (GR) and serotonin transporter (SERT) expression in the dorsal hippocampus. SIGNIFICANCE: Our findings indicate that chronic ethanol withdrawal affects the hippocampal serotonergic system by decreasing serotonin transporter expression. It also disturbs the HPA axis functioning through an imbalance on GR and mineralocorticoid (MR) expression.

Inducible nitric oxide synthase (iNOS) mediates ethanol-induced redox imbalance and upregulation of inflammatory cytokines in the kidney.

Overexpression of the inducible isoform of the enzyme nitric oxide synthase (iNOS) has been associated to pathological processes in the kidney. Ethanol consumption induces the renal expression of iNOS; however, the contribution of this enzyme to the deleterious effects of ethanol in the kidney remains elusive. We examined whether iNOS plays a role in the renal dysfunction and oxidative stress induced by ethanol consumption. With this purpose, male C57BL/6 wild-type (WT) or iNOS-deficient (iNOS-/-) mice were treated with ethanol (20% v/v) for 10 weeks. Treatment with ethanol increased the expression of Nox4 as well as the concentration of thiobarbituric acid reactive substances and the levels of tumor necrosis factor α in the renal cortex of WT but not iNOS-/- mice. Augmented serum levels of creatinine and increased systolic blood pressure were found in WT and iNOS-/- mice treated with ethanol. WT mice treated with ethanol showed increased production of reactive oxygen species and myeloperoxidase activity, but these responses were attenuated in iNOS-/- mice. We concluded that iNOS played a role in ethanol-induced oxidative stress and pro-inflammatory cytokine production in the kidney. These are mechanisms that may contribute to the renal toxicity induced by ethanol.

Melatonin reverses the loss of the anticontractile effect of perivascular adipose tissue in obese rats.

Perivascular adipose tissue (PVAT) undergoes functional changes in obesity. Increased oxidative stress is a central mechanism whereby obesity induces loss of the anticontractile effect of PVAT. Melatonin is an antioxidant that displays vasoprotective action in cardiovascular disease. Here, we sought to investigate whether melatonin would restore the anticontractile effect of periaortic PVAT in obesity. Male Wistar Hannover rats were treated for 10 weeks with a high-calorie diet. Melatonin (5 mg/kg/d, p.o., gavage) was administered for 2 weeks. Functional findings showed that obesity-induced loss of the anticontractile effect of PVAT and treatment with melatonin reversed this response. Tiron [a scavenger of superoxide anion (O2 - )] restored the anticontractile effect of PVAT in aortas from obese rats, suggesting a role for reactive oxygen species (ROS) in such response. Decreased superoxide dismutase (SOD) activity and augmented levels of ROS were detected in periaortic PVAT from obese rats. These responses were accompanied by decreased levels of nitric oxide (NO) in PVAT. Treatment with melatonin restored SOD activity, decreased ROS levels, and increased NO bioavailability in PVAT from obese rats. Here, we first reported the beneficial effects of melatonin in periaortic PVAT in obesity. Melatonin reversed the adverse effects of obesity in PVAT that included overproduction of ROS, reduced SOD activity, and decreased bioavailability of NO. Therefore, PVAT may constitute an important target for the treatment of obesity-induced vascular dysfunction and melatonin emerges as a potential tool in the management of the vascular complications induced by obesity.

Nebivolol Prevents Up-Regulation of Nox2/NADPH Oxidase and Lipoperoxidation in the Early Stages of Ethanol-Induced Cardiac Toxicity.

Changes in redox state are described in the early stages of ethanol-induced cardiac toxicity. Here, we evaluated whether nebivolol would abrogate ethanol-induced redox imbalance in the heart. Male Wistar rats were treated with a solution of ethanol (20% v/v) for 3 weeks. Treatment with nebivolol (10 mg/kg/day; p.o. gavage) prevented the increase of both superoxide (O2•-) and thiobarbituric acid reactive substances (TBARS) in the left ventricle of rats chronically treated with ethanol. Neither ethanol nor nebivolol affected the expression of Nox4, p47phox, or Rac-1. Nebivolol prevented ethanol-induced increase of Nox2 expression in the left ventricle. Superoxide dismutase (SOD) activity as well as the concentration of reduced glutathione (GSH) was not altered by ethanol or nebivolol. Augmented catalase activity was detected in the left ventricle of both ethanol- and nebivolol-treated rats. Treatment with nebivolol, but not ethanol increased eNOS expression in the left ventricle. No changes in the activity of matrix metalloproteinase (MMP)2 or in the expressions of MMP2, MMP9, and tissue inhibitor metalloproteinase (TIMP)1 were detected after treatment with ethanol or nebivolol. However, ethanol increased the expression of TIMP2, and this response was prevented by nebivolol. Our results provided novel insights into the mechanisms underlying the early stages of the cardiac injury induced by ethanol consumption. We demonstrated that Nox2/NADPH oxidase-derived ROS play a role in ethanol-induced lipoperoxidation and that this response was prevented by nebivolol. In addition, we provided evidence that MMPs are not activated in the early stages of ethanol-induced cardiac toxicity.

Ethanol and cyclophosphamide induce similar nephrotoxic effects: possible role for Nox4 and superoxide.

We tested the hypothesis that ethanol consumption would aggravate the renal damage induced by cyclophosphamide (CYP). Male C57BL/6 J mice from control (n = 8) and CYP (n = 12) groups had free access to filtered water and standard rodent chow for 12 weeks. Then, 24 h before euthanasia mice received an intraperitoneal injection of saline or CYP (300 mg/kg). Mice from ethanol (n = 8) and CYP + ethanol (n = 12) groups had free access to increasing doses of ethanol for 12 weeks. Twenty-four hours before euthanasia, mice from ethanol and CYP + ethanol groups received an intraperitoneal injection of saline or CYP, respectively. Ethanol, CYP, or the association of both drugs augmented serum levels of creatinine and increased the levels of superoxide ([Formula: see text]) generation and thiobarbituric acid reactive substances in the renal cortex. Upregulation of Nox4 and increased activity of superoxide dismutase were detected in the renal cortex of mice treated with ethanol, CYP, or the combination of these drugs; however, these molecular alterations induced by CYP were not potentiated by ethanol consumption. Our findings revealed that chronic ethanol consumption had no potentiating effect on the nephrotoxic effects displayed by CYP. It is possible that the combination of these drugs showed no synergistic effect because they share the same molecular mechanisms of renal toxicity.

Interleukin-10 limits the initial steps of the cardiorenal damage induced by ethanol consumption.

AIMS: Reactive oxygen species (ROS) and pro-inflammatory cytokines play a critical role in organ damage induced by ethanol consumption. Interleukin (IL)-10 maintain tissue homeostasis through restriction of excessive inflammatory responses and inhibition of ROS generation. These responses limit unnecessary tissue damage in the cardiorenal system. We hypothesized that IL-10 would limit the deleterious effects induced by ethanol consumption in the cardiorenal system. MATERIALS AND METHODS: Male C57BL/6J wild-type (WT) or IL10-deficient mice (IL-10-/-) were treated with ethanol (20% v/v) for 6 weeks. KEY FINDINGS: IL-10 deficiency was associated with an increased mortality rate. Ethanol consumption decreased plasma levels of IL-10 in WT mice. Increased levels of IL-6 were detected in the aorta from IL-10-deficient mice, but not WT mice. No alterations in the levels of urea, creatinine, sodium, potassium or creatine kinase (CK)-MB were found after treatment with ethanol. Augmented concentration of thiobarbituric acid reactive substances (TBARS) was found in the left ventricle (LV) of IL-10-deficient mice, but not WT mice. Increased levels of superoxide anion (O2-) were found in the renal cortex of both WT and IL-10-deficient mice. Renal cortex from WT mice chronically treated with ethanol showed decreased levels of H2O2. No changes in the expression of Nox1, Nox4 or catalase were found in the renal cortex from ethanol-treated mice. SIGNIFICANCE: IL-10 limited the production of ROS and the synthesis of pro-inflammatory cytokines induced by ethanol in the cardiorenal system. These findings provided novel evidence that IL-10 counteracted the initial mechanisms whereby ethanol induces its cardiorenal damages.

Acute restraint stress increases blood pressure and oxidative stress in the cardiorenal system of rats: a role for AT1 receptors.

We evaluate whether acute restraint stress may affect the oxidative state of the cardiorenal system and the possible contribution of angiotensin II/AT1 receptors in such response. Male Wistar rats were restrained for 60 min within wire mesh chambers. Some rats were treated with losartan (selective AT1 receptor antagonist, 10 mg/kg, p.o., gavage) 30 min before being stressed. Biochemical analyses were conducted after the 60-min period of restraint. Treatment with losartan prevented the increase in mean arterial pressure (MAP), but not heart rate (HR) induced by acute stress. Phenylephrine-induced contraction of endothelium-intact aortas was not affected by acute stress. Losartan prevented the increase in both superoxide anion (O2•-) and hydrogen peroxide (H2O2) levels induced by acute stress in the aorta and renal cortex. Similarly, the augmented activity of superoxide dismutase (SOD) induced by acute stress in the aorta and renal cortex was prevented by losartan. Enhanced levels of O2•- and thiobarbituric acid reactive species (TBARS) were detected in the left ventricle (LV) of stressed rats, but losartan did not prevent these responses. Similarly, losartan did not inhibited stress-induced decrease in the concentration of nitrate/nitrite (NOx) and H2O2 in the left ventricle. Stress increased ROS generation and affected the enzymatic antioxidant system in the cardiorenal system. In addition to its well-known cardiovascular changes during acute stress, angiotensin II also induces ROS generation in the cardiorenal system in a tissue-specific manner. The increase in oxidative stress mediated by angiotensin II/AT1 receptors could be one mechanism by which acute stress predisposes to cardiorenal dysfunctions.

Are Reactive Oxygen Species Important Mediators of Vascular Dysfunction?

Reactive Oxygen Species (ROS) are reactive derivatives of oxygen metabolism. The ROS generation can be mediated by distinctive enzymatic systems including NADPH oxidases. The components of this enzyme are expressed in endothelial and vascular smooth muscle cells, adventitial fibroblasts, and infiltrating monocytes/macrophages. Oxidative stress is a molecular dysregulation in ROS generation/elimination, which plays a key role in the development of vascular dysfunction in distinctive conditions including hypertension. It is characterized by vascular inflammation, a loss of NO bioavailability and endothelial dysfunction. Considering that oxidative stress is a key mediator of vascular dysfunction, antioxidant therapy with classic antioxidants seemed to be a promising alternative for the treatment of vascular diseases. In this sense, some commonly used drugs for the treatment of cardiovascular diseases such as Angiotensin Converting Enzyme (ACE) inhibitors or angiotensin receptor AT1 antagonists showed antioxidant effects that might have contributed, at least in part, to the beneficial effects of these drugs on the treatment of cardiovascular diseases. The effectiveness of these drugs shows that ROS are in fact important mediators of vascular dysfunction and that angiotensin II plays a critical role in such response.

Treatment with nitrite prevents reactive oxygen species generation in the corpora cavernosa and restores intracavernosal pressure in hypertensive rats.

Hypertension is a risk factor for erectile dysfunction (ED) and both conditions are associated with oxidative stress. Given that nitrite is described to display antioxidant effects, we hypothesized that treatment with nitrite would exert antioxidant effects attenuating both reactive oxygen species (ROS) generation in the corpora cavernosa (CC) and ED induced by hypertension. Two kidney, one clip (2K1C) hypertension was induced in male Wistar rats. Treatment with sodium nitrite (15 mg/kg/day, p.o., gavage) was initiated two weeks after surgery to induce hypertension and maintained for four weeks. Nitrite abrogated both the decrease in intracavernosal pressure and endothelial dysfunction of the CC induced by hypertension. Treatment with nitrite decreased hypertension-induced ROS generation in the CC assessed in situ using the fluorescent dye dihidroethidium (DHE) and with the lucigenin assay. Western immunoblotting analysis revealed that nitrite prevented the increase in Nox1 expression in the CC from 2K1C rats. Decreased concentrations of hydrogen peroxide (H2O2) were found in the CC from hypertensive rats and treatment with nitrite prevented this response. Treatment with nitrite increased the fluorescence of DAF-2DA in the CC from sham-operated rats and restored nitric oxide (NO) levels in the CC from 2K1C rats. In summary, we found novel evidence that nitrite reversed the decrease in intracavernosal pressure induced by 2K1C hypertension. This response was partially attributed to the antioxidant effect of nitrite that blunted ROS generation and endothelial dysfunction in the CC. In addition, nitrite-derived NO may have promoted direct protective actions against hypertension-induced CC dysfunction.

Ethanol Withdrawal Alters the Oxidative State of the Heart Through AT1-Dependent Mechanisms.

AIMS: We investigated the cardiac effects of ethanol withdrawal and the possible role of AT1 receptors in such response. METHODS: Male Wistar rats were treated with increasing doses of ethanol (3 to 9%, vol./vol.) for 21 days. The cardiac effects of ethanol withdrawal were investigated 48 h after abrupt discontinuation of ethanol. Some animals were orally treated with losartan (10 mg/kg/day), a selective AT1 receptor antagonist. RESULTS: Ethanol withdrawal did not affect serum levels of creatine kinase (CK)-MB. Losartan prevented ethanol withdrawal-induced increase in superoxide anion (O2•-) production in the left ventricle (LV). However, ethanol withdrawal did no alter the levels of thiobarbituric acid reactive substances (TBARS) or the expression of Nox1, Nox2 or Nox4 were found in the LV. Ethanol withdrawal reduced the concentration of hydrogen peroxide (H2O2) in the LV and this response was prevented by losartan. Ethanol withdrawal increased catalase activity in the LV and losartan attenuated this response. No changes on superoxide dismutase (SOD) activity or expression were detected in the LV during ethanol withdrawal. The expression of AT1, AT2 or angiotensin converting enzyme (ACE) was not affected by ethanol withdrawal. Similarly, no changes on the expression of ERK1/2, SAPK/JNK, COX-1 or COX-2 were found in the LV during ethanol withdrawal. CONCLUSIONS: Ethanol withdrawal altered the cardiac oxidative state through AT1-dependent mechanisms. Our findings showed a role for angiotensin II/AT1 receptors in the initial steps of the cardiac effects induced by ethanol withdrawal.

Perivascular adipose tissue contributes to lethal sepsis-induced vasoplegia in rats.

It is well established that sepsis induces vascular hyporesponsiveness to vasoconstrictors. Perivascular adipose tissue (PVAT) displays anti-contractile action in various blood vessels. We hypothesized that sepsis would increase the anti-contractile effect of PVAT aggravating sepsis-induced vasoplegia. Male Wistar Hannover rats were subjected to lethal sepsis by cecal ligation and puncture (CLP) method. Aorta or PVAT were collected for functional or biochemical assays 6 h after CLP surgery. Functional experiments showed that sepsis increased the anti-contractile action of PVAT in both endothelium-intact and endothelium-denuded aortas. Carboxy-PTIO, L-NAME and ODQ reversed the hypocontractility mediated by PVAT in aortas from septic rats. Inhibition of nNOS and iNOS with 7-nitroindazole and 1400 W attenuated PVAT-mediated hypocontractility during sepsis. Similar results were found in the presence of indomethacin and Ro1138452, a selective prostacyclin IP receptor antagonist. However, neither tiron nor catalase affected phenylephrine-induced contraction in aortas from septic rats. Increased levels of superoxide anion (O2•-) and 6-keto-prostaglandin F1α (stable product of prostacyclin) were detected in PVAT from septic rats. In situ quantification of reactive oxygen species and nitric oxide (NO) using fluorescent dyes revealed increased levels of both in PVAT from septic rats. The novelty of our study is that PVAT contributes to sepsis-induced vasoplegia by releasing NO and prostacyclin. These findings suggested that signaling pathways in PVAT may be considered as potential novel pharmacological therapeutic targets during sepsis-induced vasoplegia.