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.

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.

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.

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.

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.

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.

Chronic ethanol consumption increases reactive oxygen species generation and the synthesis of pro-inflammatory proteins in the heart through TNFR1-dependent mechanisms.

We evaluated the role of tumor necrosis factor (TNF)-α receptor 1 (TNFR1) on ethanol-induced cardiac dysfunction. Male C57BL/6J wild-type (WT) or TNFR1-deficient mice (TNFR1-/-) were treated with ethanol (20% v/v) for 10 weeks. Increased protein expression of TNFR1 and NFκB p65 was detected in the left ventricle (LV) of WT mice chronically treated with ethanol. Echocardiographic analysis showed that ethanol consumption increased left ventricular posterior wall end-diastolic diameter and left ventricular posterior wall end-systolic diameter in WT, but not TNFR1-/- mice. Increased levels of TNF-α, interleukin (IL)-6, superoxide anion (O2-), thiobarbituric acid reactive substances (TBARS) as well as increased nitrotyrosine immunostaining were detected in the LV from WT, but not TNFR1-/- mice. Conversely, treatment with ethanol decreased nitrate/nitrite (NOx) concentration in the LV. Histopathological analysis showed that ethanol did not induce inflammatory infiltrates, necrosis or edema in the LV. No differences in the ventricular expression of iNOS, Nox2 or COX-2 as well as in the activity of superoxide dismutase (SOD), myeloperoxidase (MPO) and N-acetyl-beta-D-glucosaminidase (NAG) were found after treatment with ethanol. Our study provided novel evidence that ethanol consumption augmented the production of reactive oxygen species (ROS) and the synthesis of pro-inflammatory proteins in the LV through TNFR1-dependent mechanisms. These findings provided novel mechanistic insights about the contribution of TNFR1 in the initial steps of the cardiac damage induced by ethanol.

Chronic ethanol consumption induces micturition dysfunction and alters the oxidative state of the urinary bladder.

Oxidative stress is pointed out as a major mechanism by which ethanol induces functional and structural changes in distinctive tissues. We evaluated whether ethanol consumption would increase oxidative stress and cause micturition dysfunction. Male C57BL/6J mice were treated with 20% ethanol (v/v) for 10 weeks. Our findings showed that chronic ethanol consumption reduced micturition spots and urinary volume in conscious mice, whereas in anaesthetized animals cystometric analysis revealed reduced basal pressure and increased capacity, threshold pressure, and maximum voiding. Treatment with ethanol reduced the contraction induced by carbachol in isolated bladders. Chronic ethanol consumption increased the levels of oxidant molecules and thiobarbituric acid reactive species in the mouse bladder. Upregulation of Nox2 was detected in the bladder of ethanol-treated mice. Increased activity of both superoxide dismutase and catalase were detected in the mouse bladder after treatment with ethanol. Conversely, decreased levels of reduced glutathione were detected in the bladder of ethanol-treated mice. The present study first demonstrated that chronic ethanol consumption induced micturition dysfunction and that this response was accompanied by increased levels of oxidant molecules in the mousebladder. These findings suggest that ethanol consumption is a risk factor for vesical dysfunction.

Cannabidiol prevents haloperidol-induced vacuos chewing movements and inflammatory changes in mice via PPARγ receptors.

The chronic use of drugs that reduce the dopaminergic neurotransmission can cause a hyperkinetic movement disorder called tardive dyskinesia (TD). The pathophysiology of this disorder is not entirely understood but could involve oxidative and neuroinflammatory mechanisms. Cannabidiol (CBD), the major non-psychotomimetic compound present in Cannabis sativa plant, could be a possible therapeutic alternative for TD. This phytocannabinoid shows antioxidant, anti-inflammatory and antipsychotic properties and decreases the acute motor effects of classical antipsychotics. The present study investigated if CBD would attenuate orofacial dyskinesia, oxidative stress and inflammatory changes induced by chronic administration of haloperidol in mice. Furthermore, we verified in vivo and in vitro (in primary microglial culture) whether these effects would be mediated by PPARγ receptors. The results showed that the male Swiss mice treated daily for 21 days with haloperidol develop orofacial dyskinesia. Daily CBD administration before each haloperidol injection prevented this effect. Mice treated with haloperidol showed an increase in microglial activation and inflammatory mediators in the striatum. These changes were also reduced by CBD. On the other hand, the levels of the anti-inflammatory cytokine IL-10 increased in the striatum of animals that received CBD and haloperidol. Regarding oxidative stress, haloperidol induced lipid peroxidation and reduced catalase activity. This latter effect was attenuated by CBD. The combination of CBD and haloperidol also increased PGC-1α mRNA expression, a co-activator of PPARγ receptors. Pretreatment with the PPARγ antagonist, GW9662, blocked the behavioural effect of CBD in our TD model. CBD also prevented LPS-stimulated microglial activation, an effect that was also antagonized by GW9662. In conclusion, our results suggest that CBD could prevent haloperidol-induced orofacial dyskinesia by activating PPARγ receptors and attenuating neuroinflammatory changes in the striatum.

Dysregulated mitogen-activated protein kinase and matrix metalloproteinase in ethanol-induced cavernosal dysfunction.

We evaluated the effects of ethanol consumption on the mitogen-activated protein kinases (MAPK) and metalloproteinases (MMP) pathways in the rat cavernosal smooth muscle (CSM). Male Wistar rats were treated with ethanol (20% v/v) for 6 weeks. Quantitative real-time polymerase chain reaction experiments showed that ethanol consumption did not alter mRNA levels of p38MAPK, SAPK/JNK, ERK1/2, MMP-2, or MMP-9 in the rat CSM. Western immunoblotting experiments revealed decreased protein expression of p38MAPK and phosphorylation of SAPK/JNK in the CSM from ethanol-treated rats. Additionally, ethanol consumption decreased the expression of MMP-2. Functional assays showed that SP600125, an inhibitor of SAPK/JNK, prevented the increase in endothelin (ET)-1-induced contraction in the CSM from ethanol-treated rats. Treatment with ethanol decreased MMP-2 activity, but did not change net MMP activity in the rat CSM. Ethanol consumption increased the circulating levels of MMP-2, MMP-9, and TIMP-2 as well as the MMP-9/TIMP-1 ratio. The major finding of our study is that ethanol consumption down-regulates both MAPK and MMP pathways in the rat CSM, whereas it increases the circulating levels of MMP-9. Additionally, we found that SAPK/JNK plays a role in ethanol-induced increase on ET-1 contraction in the isolated rat CSM.

Melatonin: Antioxidant and modulatory properties in age-related changes during Trypanosoma cruzi infection.

The purpose of this study was to investigate the effects of melatonin on selected biomarkers of innate and humoral immune response as well as the antioxidant/oxidant status (superoxide dismutase-SOD and reduced glutathione levels (GSH) to understand whether age-related changes would influence the development of acute Trypanosoma cruzi (T. cruzi) infection. Young- (5 weeks) and middle-aged (18 months) Wistar rats were orally treated with melatonin (gavage) (05 mg/kg/day), 9 days after infection. A significant increase in both SOD activity and GSH levels was found in plasma from all middle-aged melatonin-treated animals. Melatonin triggered enhanced expression of major histocompatibility class II (MHC-II) antigens on antigen-presenting cell (APC) and peritoneal macrophages in all treated animals. High levels of CD4+ CD28-negative T cells (*P<.05) were detected in middle-aged control animals. Melatonin induced a significant reduction (***P<.001) in CD28-negative in CD4+ and CD8+ T cells in middle-aged control animals. Contrarily, the same group displayed upregulated CD4+ CD28+ T and CD8+ CD28+ T cells. Melatonin also triggered an upregulation of CD80 and CD86 expression in all young-treated groups. Significant percentages of B and spleen dendritic cells in middle-aged infected and treated animals were observed. Our data reveal new features of melatonin action in inhibiting membrane lipid peroxidation, through the reduction in 8-isoprostane, upregulating the antioxidant defenses and triggering an effective balance in the antioxidant/oxidant status during acute infection. The ability of melatonin to counteract the immune alterations induced by aging added further support to its use as a potential therapeutic target not only for T. cruzi infection but also for other immunocompromised states.

Acute restraint stress induces endothelial dysfunction: role of vasoconstrictor prostanoids and oxidative stress.

We hypothesized that acute stress would induce endothelial dysfunction. Male Wistar rats were restrained for 2 h within wire mesh. Functional and biochemical analyses were conducted 24 h after the 2-h period of restraint. Stressed rats showed decreased exploration on the open arms of an elevated-plus maze (EPM) and increased plasma corticosterone concentration. Acute restraint stress did not alter systolic blood pressure, whereas it increased the in vitro contractile response to phenylephrine and serotonin in endothelium-intact rat aortas. NG-nitro-l-arginine methyl ester (l-NAME; nitric oxide synthase, NOS, inhibitor) did not alter the contraction induced by phenylephrine in aortic rings from stressed rats. Tiron, indomethacin and SQ29548 reversed the increase in the contractile response to phenylephrine induced by restraint stress. Increased systemic and vascular oxidative stress was evident in stressed rats. Restraint stress decreased plasma and vascular nitrate/nitrite (NOx) concentration and increased aortic expression of inducible (i) NOS, but not endothelial (e) NOS. Reduced expression of cyclooxygenase (COX)-1, but not COX-2, was observed in aortas from stressed rats. Restraint stress increased thromboxane (TX)B(2) (stable TXA(2) metabolite) concentration but did not affect prostaglandin (PG)F2α concentration in the aorta. Restraint reduced superoxide dismutase (SOD) activity, whereas concentrations of hydrogen peroxide (H(2)O(2)) and reduced glutathione (GSH) were not affected. The major new finding of our study is that restraint stress increases vascular contraction by an endothelium-dependent mechanism that involves increased oxidative stress and the generation of COX-derived vasoconstrictor prostanoids. Such stress-induced endothelial dysfunction could predispose to the development of cardiovascular diseases.

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.

Biotransformation of ent-pimaradienoic acid by cell cultures of Aspergillus niger.

Microbial transformation stands out among the many possible semi-synthetic strategies employed to increase the variety of chemical structures that can be applied in the search for novel bioactive compounds. In this paper we obtained ent-pimaradienoic acid (1, PA, ent-pimara-8(14),15-dien-19-oic acid) derivatives by fungal biotransformation using Aspergillus niger strains. To assess the ability of such compounds to inhibit vascular smooth muscle contraction, we also investigated their spasmolytic effect, along with another five PA derivatives previously obtained in our laboratory, on aortic rings isolated from male Wistar rats. The microbial transformation experiments were conducted at 30°C using submerged shaken liquid culture (120 rpm) for 10 days. One known compound, 7α-hydroxy ent-pimara-8(14),15-dien-19-oic acid (2), and three new derivatives, 1ß-hydroxy ent-pimara-6,8(14),15-trien-19-oic acid (3), 1α,6ß,14ß-trihydroxy ent-pimara-7,15-dien-19-oic acid (4), and 1α,6ß,7α,11α-tetrahydroxy ent-pimara-8(14),15-dien-19-oic acid (5), were isolated and identified on the basis of spectroscopic analyses and computational studies. The compounds obtained through biotransformation (2-5) did not display a significant antispasmodic activity (values ranging from 0% to 16.8% of inhibition); however the previously obtained diterpene, methyl 7α-hydroxy ent-pimara-8(14),15-dien-19-oate (8), showed to be very effective (82.5% of inhibition). In addition, our biological results highlight the importance to study the antispasmodic potential of a large number of novel diterpenes, to conduct further structure-activity relationship investigations.

Ethanol consumption increases endothelin-1 expression and reactivity in the rat cavernosal smooth muscle.

AIMS: We investigated the effects of chronic ethanol consumption on the cavernosal smooth muscle (CSM) reactivity to endothelin-1 (ET-1) and the expression of ET system components in this tissue. METHODS: Male Wistar rats were treated with heavy dose of ethanol (20% v/v) for 6 weeks. Reactivity experiments were performed in the isolated rat CSM. Plasma and CSM nitrate generation and also superoxide anion generation in rat CSM were measured by chemiluminescence. Protein and mRNA levels of pre-pro-ET-1, endothelin-converting enzyme-1 (ECE-1), ETA and ETB receptors, eNOS, nNOS and iNOS were assessed by western immunoblotting and quantitative real-time polymerase chain reaction, respectively. RESULTS: Chronic ethanol consumption increased plasma ET-1 levels and the contractile response induced by this peptide in the isolated CSM. The relaxation induced by acetylcholine, but not IRL1620, a selective ETB receptor agonist, was reduced in CSM from ethanol-treated rats. BQ123, a selective ETA receptor antagonist, produced a rightward displacement of the ET-1 concentration-response curves in CSM from control, but not ethanol-treated rats. Reduced levels of nitrate were found in the plasma and CSM from ethanol-treated rats. Ethanol consumption increased superoxide anion generation in the rat CSM. The mRNA levels of pre-pro-ET-1, ECE-1, ETA and ETB receptors, eNOS, nNOS and iNOS were not altered by ethanol consumption. Protein levels of ET-1, ETA receptor and iNOS were higher in the CSM from rats chronically treated with ethanol. CONCLUSION: The major findings of the present study are that heavy ethanol consumption increases plasma ET-1 levels and the contraction induced by the peptide in the CSM. Increased CSM reactivity to ET-1 and altered protein levels of ET-1 and ETA receptors could play a role in the pathogenesis of erectile dysfunction associated with chronic ethanol consumption.

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.

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.

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.