Effects of concurrent training and N-acetylcysteine supplementation on cardiac remodeling and oxidative stress in middle-aged spontaneously hypertensive rats.

BACKGROUND: This study evaluated the effects of concurrent isolated training (T) or training combined with the antioxidant N-acetylcysteine (NAC) on cardiac remodeling and oxidative stress in spontaneously hypertensive rats (SHR). METHODS: Six-month-old male SHR were divided into sedentary (S, n = 12), concurrent training (T, n = 13), sedentary supplemented with NAC (SNAC, n = 13), and concurrent training with NAC supplementation (TNAC, n = 14) groups. T and TNAC rats were trained three times a week on a treadmill and ladder; NAC supplemented groups received 120 mg/kg/day NAC in rat chow for eight weeks. Myocardial antioxidant enzyme activity and lipid hydroperoxide concentration were assessed by spectrophotometry. Gene expression of NADPH oxidase subunits Nox2, Nox4, p22 phox, and p47 phox was evaluated by real time RT-PCR. Statistical analysis was performed using ANOVA and Bonferroni or Kruskal-Wallis and Dunn. RESULTS: Echocardiogram showed concentric remodeling in TNAC, characterized by increased relative wall thickness (S 0.40 ± 0.04; T 0.39 ± 0.03; SNAC 0.40 ± 0.04; TNAC 0.43 ± 0.04 *; * p < 0.05 vs T and SNAC) and diastolic posterior wall thickness (S 1.50 ± 0.12; T 1.52 ± 0.10; SNAC 1.56 ± 0.12; TNAC 1.62 ± 0.14 * mm; * p < 0.05 vs T), with improved contractile function (posterior wall shortening velocity: S 39.4 ± 5.01; T 36.4 ± 2.96; SNAC 39.7 ± 3.44; TNAC 41.6 ± 3.57 * mm/s; * p < 0.05 vs T). Myocardial lipid hydroperoxide concentration was lower in NAC treated groups (S 210 ± 48; T 182 ± 43; SNAC 159 ± 33 *; TNAC 110 ± 23 *# nmol/g tissue; * p < 0.05 vs S, # p < 0.05 vs T and SNAC). Nox 2 and p22 phox expression was higher and p47 phox lower in T than S [S 1.37 (0.66-1.66); T 0.78 (0.61-1.04) *; SNAC 1.07 (1.01-1.38); TNAC 1.06 (1.01-1.15) arbitrary units; * p < 0.05 vs S]. NADPH oxidase subunits did not differ between TNAC, SNAC, and S groups. CONCLUSION: N-acetylcysteine supplementation alone reduces oxidative stress in untreated spontaneously hypertensive rats. The combination of N-acetylcysteine and concurrent exercise further decreases oxidative stress. However, the lower oxidative stress does not translate into improved cardiac remodeling and function in untreated spontaneously hypertensive rats.

Expression of Hv1 proton channels in myeloid-derived suppressor cells (MDSC) and its potential role in T cell regulation.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population with high immunosuppressive activity that proliferates in infections, inflammation, and tumor microenvironments. In tumors, MDSC exert immunosuppression mainly by producing reactive oxygen species (ROS), a process triggered by the NADPH oxidase 2 (NOX2) activity. NOX2 is functionally coupled with the Hv1 proton channel in certain immune cells to support sustained free-radical production. However, a functional expression of the Hv1 channel in MDSC has not yet been reported. Here, we demonstrate that mouse MDSC express functional Hv1 proton channel by immunofluorescence microscopy, flow cytometry, and Western blot, besides performing a biophysical characterization of its macroscopic currents via patch-clamp technique. Our results show that the immunosuppression by MDSC is conditional to their ability to decrease the proton concentration elevated by the NOX2 activity, rendering Hv1 a potential drug target for cancer treatment.

Topiramate treatment in Wistar rats during childhood induces sex-specific vascular dysfunction in adulthood.

The present study determined whether treatment during childhood with topiramate (TPM), a new generation antiepileptic drug, results in altered aortic reactivity in adult male and female rats. We also sought to understand the role of endothelium-derived contractile factors in TPM-induced vascular dysfunction. Male and female Wistar rats were treated with TPM (41 mg/kg/day) or water (TPM vehicle) by gavage during childhood (postnatal day, 16-28). In adulthood, thoracic aorta reactivity to phenylephrine (phenyl), as well as aortic thickness and expression of cyclooxygenases (COX-1 and COX-2), NOX2, and p47phox were evaluated. The aortic response to phenyl was increased in male and female rats from the TPM group when compared with the control group. In TPM male rats, the hyperreactivity to phenyl was abrogated by the inhibition of NADPH oxidase and COX-2, while in female rats, responses were restored only by inhibition of COX-2. In addition, TPM male rats presented aortic hypertrophy and increased expression of NOX-2 and p47phox, while TPM female rats showed increased COX-2 aortic expression. Taken together, for the first-time, the present study provides evidence that treatment with TPM during childhood causes vascular dysfunction in adulthood, and that the mechanism underlying the vascular effects of TPM is sex-specific.

High-Fat and Combined High-Fat and Sucrose Diets Promote Cardiac Oxidative Stress Independent of Nox2 Redox Regulation and Obesity in Rats.

BACKGROUND/AIMS: Oxidative stress is associated with cardiometabolic alterations, and the involvement of excess glucose and fatty acids has been demonstrated in this process. Thus, the aim of this study was to investigate the effects of different hypercaloric diets on cardiac oxidative stress. METHODS: Wistar rats were randomized into four groups: control (C), high-sucrose (HS), high-fat (HF), and high-fat with sucrose (HFS). Nutritional assessment, food profiles, histological analysis, comorbidities, and cardiovascular characteristics were determined. Cardiac oxidative stress was analyzed by malondialdehyde (MDA) and carbonylated proteins, and the cardiac protein expression levels of type 1 angiotensin receptor (AT-1), nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2), superoxide dismutase (SOD 1 e 2), glutathione peroxidase (GPX), and catalase (CAT) were determined by western blot. RESULTS: The HF group showed an increase in adiposity; however, it did not present adipocyte hypertrophy and comorbidities. Cardiac MDA and carbonylated protein levels were higher in the HF and HFS compared with the C group. The levels of oxidant and antioxidant proteins showed no difference between the groups. CONCLUSION: HF and HFS dietary interventions promoted cardiac oxidative stress, in the presence and absence of obesity, respectively. However, this process was neither mediated by the pro-oxidants AT1 and Nox2, nor by the quantitative reduction of antioxidant enzymes.

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.

(-)-Epicatechin protects thoracic aortic perivascular adipose tissue from whitening in high-fat fed mice.

High adipose tissue (AT) accumulation in the body increases the risk for many metabolic and chronic diseases. This work investigated the capacity of the flavonoid (-)-epicatechin to prevent undesirable modifications of AT in mice fed a high-fat diet. Studies were focused on thoracic aorta perivascular AT (taPVAT), which is involved in the control of blood vessel tone, among other functions. Male C57BL/6J mice were fed for 15 weeks a high-fat diet with or without added (-)-epicatechin (20 mg per kg body weight per d). In high-fat diet fed mice, (-)-epicatechin supplementation: (i) prevented the expansion of taPVAT, (ii) attenuated the whitening of taPVAT (according to the adipocyte morphology, diameter, and uncoupling-protein 1 (UCP-1) levels) and (iii) blunted the increase in plasma glucose and cholesterol. The observed taPVAT modifications were not associated with alterations in the aorta wall thickness, aorta tumor necrosis factor-alpha (TNF-α) and NADPH-oxidase 2 (NOX2) expression, and endothelial nitric oxide synthase (eNOS) phosphorylation levels. In summary, our results indicate (-)-epicatechin as a relevant bioactive protecting from the slow and silent development of metabolic and chronic diseases as they are associated with excessive fat intake.

Reactive oxygen species and nitric oxide imbalances lead to in vivo and in vitro arrhythmogenic phenotype in acute phase of experimental Chagas disease.

Chagas Disease (CD) is one of the leading causes of heart failure and sudden death in Latin America. Treatments with antioxidants have provided promising alternatives to ameliorate CD. However, the specific roles of major reactive oxygen species (ROS) sources, including NADPH-oxidase 2 (NOX2), mitochondrial-derived ROS and nitric oxide (NO) in the progression or resolution of CD are yet to be elucidated. We used C57BL/6 (WT) and a gp91PHOX knockout mice (PHOX-/-), lacking functional NOX2, to investigate the effects of ablation of NOX2-derived ROS production on the outcome of acute chagasic cardiomyopathy. Infected PHOX-/- cardiomyocytes displayed an overall pro-arrhythmic phenotype, notably with higher arrhythmia incidence on ECG that was followed by higher number of early afterdepolarizations (EAD) and 2.5-fold increase in action potential (AP) duration alternans, compared to AP from infected WT mice. Furthermore, infected PHOX-/- cardiomyocytes display increased diastolic [Ca2+], aberrant Ca2+ transient and reduced Ca2+ transient amplitude. Cardiomyocyte contraction is reduced in infected WT and PHOX-/- mice, to a similar extent. Nevertheless, only infected PHOX-/- isolated cardiomyocytes displayed significant increase in non-triggered extra contractions (appearing in ~75% of cells). Electro-mechanical remodeling of infected PHOX-/-cardiomyocytes is associated with increase in NO and mitochondria-derived ROS production. Notably, EADs, AP duration alternans and in vivo arrhythmias were reverted by pre-incubation with nitric oxide synthase inhibitor L-NAME. Overall our data show for the first time that lack of NOX2-derived ROS promoted a pro-arrhythmic phenotype in the heart, in which the crosstalk between ROS and NO could play an important role in regulating cardiomyocyte electro-mechanical function during acute CD. Future studies designed to evaluate the potential role of NOX2-derived ROS in the chronic phase of CD could open new and more specific therapeutic strategies to treat CD and prevent deaths due to heart complications.

Effects of progesterone treatment on endothelium-dependent coronary relaxation in ovariectomized rats.

AIM: Although progesterone (P4) has a beneficial effect on the cardiovascular system, P4 actions on the coronary bed have not yet been fully elucidated. This study evaluated the effect of progesterone treatment on endothelium-dependent coronary vascular reactivity in Wistar rats. MAIN METHODS: Eight-week-old adult rats were divided into Sham, Ovariectomized (OVX), Ovariectomized and progesterone treated (OVX P4). The OVX P4 group received daily doses of progesterone (2 mg/kg/day). Vascular reactivity was assessed by a modified Langendorff technique. The intensity of eNOS, Akt, and gp91phox protein expression was quantified by Western blotting. Superoxide anion (O2●-) and hydrogen peroxide (H2O2) production was measured by dihydroethidium and 2',7'-dichlorofluorescein, respectively. KEY FINDINGS: Treatment with P4 was able to prevent the reduction in baseline coronary perfusion pressure induced by ovariectomy. We observed that endothelium-dependent coronary vasodilation was reduced in the OVX group and potentiated in the OVX P4 group. Following the inhibition of the nitric oxide (NO) pathway, the bradykinin-induced relaxing response was potentiated in the OVX P4 group. With regard to the combined inhibition of NO and prostanoids pathways, the OVX P4 group showed a greater relaxing response, similar to what was found upon individual inhibition of NO. After the combined inhibition of NO, prostanoids and epoxyeicosatrienoic acids' pathways, the vasodilatory response induced by BK was abolished in all groups. SIGNIFICANCE: Treatment with P4 prevented oxidative stress induced by ovariectomy. These results suggest that progesterone has a beneficial action on the coronary vascular bed.

Maternal endotoxemia induces renal collagen deposition in adult offspring: Role of NADPH oxidase/TGF-ß1/MMP-2 signaling pathway.

Maternal endotoxemia has been shown to increase renal collagen deposition in the offspring. Renal fibrosis is a hallmark of progressive chronic kidney disease. It was investigated whether maternal reactive oxygen species (ROS) leads to renal fibrosis or exacerbates unilateral ureteral obstruction (UUO)-induced renal fibrosis in the offspring of dams treated with lipopolysaccharide (LPS). Furthermore, it was studied the role of matrix metalloproteinases (MMPs) in these changes. Adults Wistar rats were obtained from dams submitted to LPS administration through the third part of gestation. To evaluate the role of maternal ROS, part of the dams received α-tocopherol simultaneously with LPS. Part of the offspring in each group was submitted to UUO at adulthood when sub-groups were treated with NADPH oxidase inhibitor, apocynin. Maternal LPS administration increased proteinuria, systolic arterial pressure and renal collagen deposition in adult offspring. LPS offspring rats also presented higher MMP-2 activity in parallel to a decreased renal cortical TIMP-2 content. These changes were correlated to increased amounts of TGF-ß1 and NOX2. Maternal α-tocopherol treatment prevented collagen deposition and reduced arterial pressure in adult offspring. α-Tocopherol also inhibited maternal endotoxemia-induced changes in TGF-ß1/NOX2/MMP-2 signaling. UUO led to increased collagen deposition in the contralateral kidneys of LPS offspring, which was correlated to increased NADPH oxidase activity and prevented by NADPH oxidase inhibition. In summary, maternal endotoxemia led to alterations in the TGF-ß1/NOX2/MMP-2 signaling pathway in renal tissue concomitant with collagen deposition, therefore contributing to hypertension in adult offspring.

Chronic Mercury Exposure in Prehypertensive SHRs Accelerates Hypertension Development and Activates Vasoprotective Mechanisms by Increasing NO and H2O2 Production.

Mercury is a heavy metal associated with cardiovascular diseases. Studies have reported increased vascular reactivity without changes in systolic blood pressure (SBP) after chronic mercury chloride (HgCl2) exposure, an inorganic form of the metal, in normotensive rats. However, we do not know whether individuals in the prehypertensive phase, such as young spontaneously hypertensive rats (SHRs), are susceptible to increased arterial blood pressure. We investigated whether chronic HgCl2 exposure in young SHRs accelerates hypertension development by studying the vascular function of mesenteric resistance arteries (MRAs) and SBP in young SHRs during the prehypertensive phase. Four-week-old male SHRs were divided into two groups: the SHR control group (vehicle) and the SHR HgCl2 group (4 weeks of exposure). The results showed that HgCl2 treatment accelerated the development of hypertension; reduced vascular reactivity to phenylephrine in MRAs; increased nitric oxide (NO) generation; promoted vascular dysfunction by increasing the production of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2); increased Gp91Phox protein levels and in situ levels of superoxide anion (O2·-); and reduced vasoconstrictor prostanoid production compared to vehicle treatment. Although HgCl2 accelerated the development of hypertension, the HgCl2-exposed animals also exhibited a vasoprotective mechanism to counterbalance the rapid increase in SBP by decreasing vascular reactivity through H2O2 and NO overproduction. Our results suggest that HgCl2 exposure potentiates this vasoprotective mechanism against the early establishment of hypertension. Therefore, we are concluding that chronic exposure to HgCl2 in prehypertensive animals could enhance the risk for cardiovascular diseases.

N-Methyl-d-Aspartate Receptor Modulation by Nicotinamide Adenine Dinucleotide Phosphate Oxidase Type 2 Drives Synaptic Plasticity and Spatial Memory Impairments in Rats Exposed Pre- and Postnatally to Ethanol.

Aims: Pre- and/or early postnatal ethanol exposure (prenatal alcohol exposure [PAE]) impairs synaptic plasticity as well as memory formation, but the mechanisms underlying these effects remain unclear. Both long-term potentiation (LTP) and spatial memory formation in the hippocampus involve the nicotinamide adenine dinucleotide phosphate oxidase type 2 (NOX2) enzyme. Previous studies have reported that N-methyl-d-aspartate receptor (NMDAR) activation increases NOX2-mediated superoxide generation, resulting in inhibition of NMDAR function, but whether NOX2 impacts NMDAR function in PAE animals leading to impaired LTP and memory formation remains unknown. We aim to evaluate whether the NOX2-NMDAR complex is involved in the long-lasting deleterious effects of PAE on hippocampal LTP and memory formation. Results: Here we provide novel evidence that PAE animals display impaired NMDAR-dependent LTP in the cornus ammonis field 1 (CA1) and NMDAR-mediated LTP in the dentate gyrus (DG). Moreover, PAE rats displayed increased NMDAR-mediated transmission in both hippocampal areas. Interestingly, NOX2 pharmacological inhibition restored NMDAR-mediated transmission and LTP in the CA1, but not in the DG. PAE also induced overexpression of NOX2 and CaMKII isoforms, but did not modify the content or the redox state of the N-methyl-d-aspartate receptor subunit-1 (NR1) subunit of NMDAR in both areas of the hippocampus. In addition, adolescent PAE rats orally fed the antioxidant and free radical scavenger apocynin exhibited significantly improved spatial memory acquisition. Innovation and Conclusion: By showing in PAE animals NOX2 overexpression and increased NMDAR-mediated transmission, which might lead to impaired synaptic plasticity and memory formation in a region-specific manner, we provide an important advance to our current understanding of the cellular mechanisms underlying PAE-dependent defective hippocampal function.

Arachidonic Acid and Nitroarachidonic: Effects on NADPH Oxidase Activity.

Arachidonic acid (AA) is a polyunsaturated fatty acid that participates in the inflammatory response mainly through bioactive-lipids formation in macrophages and also in the phagocytic NADPH oxidase 2 (NOX2) activation. NOX2 is the enzyme responsible for a huge superoxide formation in macrophages, essential to eliminate pathogens inside the phagosome. The oxidase is an enzymatic complex comprised of a membrane-bound flavocytochrome b 558 (gp91phox/p22phox), three cytosolic subunits (p47phox, p40phox and p67phox) and a Rac-GTPase. The enzyme becomes active when macrophages are exposed to appropriate stimuli that trigger the phosphorylation of cytosolic subunits and its migration to plasmatic membrane to form the active complex. It is proposed that AA stimulates NOX2 activity through AA interaction with different components of the NADPH oxidase complex. In inflammatory conditions, there is an increase in reactive oxygen and nitrogen species that results in the production of nitrated derivatives of AA, such as nitroarachidonic acid (NO2-AA). NO2-AA is capable to inhibit NOX2 activity by interfering with p47phox migration to the membrane without affecting phosphorylation of cytosolic proteins. Also, NO2-AA is capable to interact with protein disulfide isomerase (PDI), which is involved on NOX2 active complex formation. It has been demonstrated that NO2-AA forms a covalent adduct with PDI that could prevent the interaction with NOX2 and it would explain the inhibitory effects of the fatty acid upon NOX2. Together, current data indicate that AA is an important activator of NOX2 formed in the early events of the inflammatory response, leading to a massive production of oxidants that may, in turn, promote NO2-AA formation and shutting down the oxidative burst. Hence, AA and its derivatives could have antagonistic roles on NOX2 activity regulation.

Role of NADPH oxidase-2 in the progression of the inflammatory response secondary to striatum excitotoxic damage.

BACKGROUND: During excitotoxic damage, neuronal death results from the increase in intracellular calcium, the induction of oxidative stress, and a subsequent inflammatory response. NADPH oxidases (NOX) are relevant sources of reactive oxygen species (ROS) during excitotoxic damage. NADPH oxidase-2 (NOX-2) has been particularly related to neuronal damage and death, as well as to the resolution of the subsequent inflammatory response. As ROS are crucial components of the regulation of inflammatory response, in this work, we evaluated the role of NOX-2 in the progression of inflammation resulting from glutamate-induced excitotoxic damage of the striatum in an in vivo model. METHODS: The striata of wild-type C57BL/6 J and NOX-2 KO mice (gp91Cybbtm1Din/J) were stereotactically injected with monosodium glutamate either alone or in combination with IL-4 or IL-10. The damage was evaluated in histological sections stained with cresyl violet and Fluoro-Jade B. The enzymatic activity of caspase-3 and NOX were also measured. Additionally, the cytokine profile was identified by ELISA and motor activity was verified by the tests of the cylinder, the adhesive tape removal, and the inverted grid. RESULTS: Our results show a neuroprotective effect in mice with a genetic inhibition of NOX-2, which is partially due to a differential response to excitotoxic damage, characterized by the production of anti-inflammatory cytokines. In NOX-2 KO animals, the excitotoxic condition increased the production of interleukin-4, which could contribute to the production of interleukin-10 that decreased neuronal apoptotic death and the magnitude of striatal injury. Treatment with interleukin-4 and interleukin-10 protected from excitotoxic damage in wild-type animals. CONCLUSIONS: The release of proinflammatory cytokines during the excitotoxic event promotes an additional apoptotic death of neurons that survived the initial damage. During the subsequent inflammatory response to excitotoxic damage, ROS generated by NOX-2 play a decisive role in the extension of the lesion and consequently in the severity of the functional compromise, probably by regulating the anti-inflammatory cytokines production.

Exercise training improves vascular reactivity in ovariectomized rats subjected to myocardial infarction.

The aim of this study was to evaluate the effects of exercise training (ET) on the aortic vascular reactivity of ovariectomized and infarcted rats. The animals were divided into 5 groups: Control, Ovariectomized + SHAM sedentary (OVX+SHAMSED), OVX+SHAM and ET (OVX+SHAMET), OVX + Myocardial Infarction sedentary (OVX+MISED), and OVX + MI and ET (OVX+MIET). ET protocol (60 minutes/day, 5x/week) in a motorized treadmill began 15 days after MI and lasted 8 weeks. The endothelium-dependent and endothelium-independent vascular reactivity were evaluated as well as the role of the reactive oxygen species (ROS). Superoxide and nitric oxide (NO) production were analyzed in situ using DHE and DAF-2 fluorescence, respectively. The expression of gp91phox and of the antioxidant enzymes were evaluated by western blotting in the thoracic aorta samples. MI promoted a significant increase in the contractile response and impaired endothelium-mediated relaxation. However, ET prevented the impairment in the vascular reactivity in MI animals. In addition, the protein expression of gp91phox and superoxide production increased and the NO production decreased in the OVX+MISED group but not in the OVX+MIET group. Therefore, ET improves vascular reactivity in MI ovariectomized rats by preventing the increase in the expression of gp91phox and the decrease in the antioxidant enzymes, resulting in a normal ROS and NO production. Thus, ET can be an effective therapeutic strategy for improving the MI-induced vascular alterations in estrogen deficiency condition.

OxHDL controls LOX-1 expression and plasma membrane localization through a mechanism dependent on NOX/ROS/NF-κB pathway on endothelial cells.

Systemic inflammatory diseases enhance circulating oxidative stress levels, which results in the oxidation of circulating high-density lipoprotein (oxHDL). Endothelial cell function can be negatively impacted by oxHDL, but the underlying mechanisms for this remain unclear. Some reports indicate that the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is also a receptor for oxHDL. However, it is unknown if oxHDL induces increased LOX-1 expression at the plasma membrane, as an event that supports endothelial dysfunction. Therefore, the aims of this study were to determine if oxHDL induces plasma-membrane level changes in LOX-1 and, if so, to describe the underlying mechanisms in endothelial cells. Our results demonstrate that the incubation of arterial or vein endothelial cells with oxHDL (and not HDL) induces the increase of LOX-1 expression at the plasma membrane; effect prevented by LOX-1 inhibition. Importantly, same results were observed in endothelial cells from oxHDL-treated rats. Furthermore, the observed oxHDL-induced LOX-1 expression is abolished by the down-regulation of NOX-2 expression with siRNA (and no others NOX isoforms), by the pharmacological inhibition of NAD(P)H oxidase (with DPI or apocynin) or by the inhibition of NF-κB transcription factor. Coherently, LOX-1 expression is augmented by the incubation of endothelial cells with H2O2 or GSSG even in absence of oxHDL, indicating that the NOX-2/ROS/ NF-κB axis is involved. Interestingly, oxHDL incubation also increases TNF-α expression, cytokine that induces LOX-1 expression. Thus, our results suggest a positive feedback mechanism for LOX-1 receptor during inflammatory condition where an oxidative burst will generate oxHDL from native HDL, activating LOX-1 receptor which in turn will increase the expression of NOX-2, TNF-α and LOX-1 receptor at the plasma membrane. In conclusion, oxHDL-induced translocation of LOX-1 to the plasma membrane could constitute an induction mechanism of endothelial dysfunction in systemic inflammatory diseases.

Dietary (-)-epicatechin affects NF-κB activation and NADPH oxidases in the kidney cortex of high-fructose-fed rats.

Inflammation involves the activation of redox-sensitive transcription factors, e.g., nuclear factor κB (NF-κB). Administration of (-)-epicatechin to high-fructose-fed rats prevented NF-κB activation and up-regulation of the NADPH oxidase 4 (NOX4) in the kidney cortex. These results add mechanistic insights into the action of (-)-epicatechin diminishing inflammatory responses.

HIF-1α-regulated MIF activation and Nox2-dependent ROS generation promote Leishmania amazonensis killing by macrophages under hypoxia.

Increasing attention is given to the finding that macrophages under hypoxia are capable of controlling infection by the intracellular protozoan parasite Leishmania amazonensis. The hypoxia-inducible factor (HIF)-1α has been shown to play an essential role in this enhanced innate immune response. Our study aimed to explore the HIF-1α-dependent mechanisms leading to reduced survival of the parasites residing in macrophages under low oxygen conditions. Hypoxia triggered (P < 0.01) NADPH oxidase 2 (Nox2) expression and reactive oxygen species (ROS) production in J774 macrophages upon 24-h infection with L. amazonensis. Furthermore, increased (P < 0.01) expression levels of HIF-1α and macrophage migration inhibitory factor (MIF) were detected in the infected cells grown at 3% oxygen tension. We found that either HIF-1α silencing, Nox2 inhibition or MIF antagonism caused a significant (P < 0.05) reversal of the improved leishmanicidal activity displayed by the hypoxic phagocytes. Taken together, our current results suggest that, under conditions of limited availability of oxygen, activation of the HIF-1α/MIF axis via Nox2/ROS induction promotes killing of L. amazonensis amastigotes by macrophages. Such protective mechanism might operate in L. amazonensis-infected tissues where low oxygen levels prevail.

Sex differences in subcutaneous adipose tissue redox homeostasis and inflammation markers in control and high-fat diet fed rats.

The development of obesity-related metabolic disorders is more evident in male in comparison with female subjects, but the mechanisms are unknown. Several studies have shown that oxidative stress is involved in the pathophysiology of obesity, but the majority of these studies were performed with male animals. The aim of this study was to evaluate the sex-related differences in subcutaneous adipose tissue redox homeostasis and inflammation of rats chronically fed a high-fat diet. NADPH oxidase (NOX), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase activities were evaluated in the subcutaneous adipose tissue (SC) of adult male and female rats fed either a standard chow (SCD) or a high-fat diet (HFD) for 11 weeks. NOX2 and NOX4 messenger RNA (mRNA) levels, total reduced thiols, interleukin (IL)-1ß, tumor necrosis factor α (TNF-α), and IL-6 were also determined. Higher antioxidant enzyme activities and total reduced thiol levels were detected in SC of control male compared with female rats. Chronic HFD administration increased NOX activity and NOX2 and NOX4 mRNA levels and decreased SOD and GPx activities only in male animals. IL-1ß, TNF-α, and IL-6 levels, as well as Adgre1, CD11b, and CD68 mRNA levels, were also higher in SC of males after HFD feeding. In SC of females, catalase activity was higher after HFD feeding. Taken together, our results show that redox homeostasis and inflammation of SC is sexually dimorphic. Furthermore, males show higher oxidative stress in SC after 11 weeks of HFD feeding owing to both increased reactive oxygen species (ROS) production through NOX2 and NOX4 and decreased ROS detoxification.

Chronic iron overload induces vascular dysfunction in resistance pulmonary arteries associated with right ventricular remodeling in rats.

Although iron excess is toxic to the vasculature and even that pulmonary hypertension has been reported in this scenario, the role of iron overload per se remains to be clarified. This study aimed to test the effects of chronic iron-overload in rats on the morphophysiology of resistance pulmonary arteries (RPA) and right ventricle (RV) remodeling. Rats were injected with saline or iron-dextran (10, 100 and 200 mg/kg/day i.p.) for 28 days. Our results indicated increased circulating iron with significant lung deposits. Moreover, rats treated with the highest dose exhibited RV dysfunction and hypertrophy; inward remodeling and increased vasoconstriction of the RPA. Vascular hyperreactivity was accompanied by reduced nitric oxide (NO), and was reversed by incubation with Dimethylsulfoxide, Catalase and Tempol. The NADPH oxidase subunit gp91phox was increased due to iron-overload, and incubation with angiotensin II type-1 receptor (AT1) antagonist losartan not only reduced oxidative stress but also restored vascular function. Thus, we concluded that AT1 pathway plays a role in pulmonary vascular dysfunction by increasing oxidative stress and reducing NO bioavailability, thereby contributing to vascular remodeling and pulmonary hypertension of iron-overload. This finding should instigate future studies on the beneficial impacts of in vivo blockade of AT1 receptor under iron overload.

Metaplasticity in the Visual Cortex: Crosstalk Between Visual Experience and Reactive Oxygen Species.

Metaplasticity is the regulation of synaptic plasticity based on the history of previous synaptic activation. This concept was formulated after observing that synaptic changes in the visual cortex are not fixed, but dynamic and dependent on the history of visual information flux. In visual cortical neurons, sustained synaptic stimulation activate the enzymatic complex NOX2, resulting in the generation of reactive oxygen species (ROS). NOX2 is the main molecular structure responsible for translating neural activity into redox modulation of intracellular signaling pathways involved in plastic changes. Here, we studied the interaction between NOX2 and visual experience as metaplastic factors regulating synaptic plasticity at the supergranular layers of the mouse visual cortex. We found that genetic inhibition of NOX2 reverses the polarizing effects of dark rearing from LTP to LTD. In addition, we demonstrate that this process relies on changes in the NMDA receptor functioning. Altogether, this work indicates a role of ROS in the activity-dependent regulation of cortical synaptic plasticity.SIGNIFICANCE STATEMENT Synaptic plasticity in the visual cortex is modulated by the history of sensory experience and this modulation has been defined as metaplasticity. Dark rearing facilitates synaptic potentiation as a mechanism optimizing the range of synaptic modification. This process requires the production of reactive oxygen species mediated by the enzymatic complex NOX2. If the activity of NOX2 is inhibited, then visual deprivation results in synaptic depression. These findings increase our knowledge about metaplasticity and help in our understanding of how neural activity modulates cellular mechanisms of synaptic change.