The atheroprotective role of fucoidan involves the reduction of foam cell formation by altering cholesterol flux-associated factors in macrophages.

Atherosclerosis is characterized by the accumulation of lipid-laden cells in the arterial walls, resulting from dysregulation of cholesterol homeostasis in the macrophage, triggered by oxidized low-density lipoprotein (oxLDL). Previous studies have shown that fucoidan, a sulfated polysaccharide from brown seaweeds, has several atheroprotective activities, however, the mechanism of fucoidan protection is not fully understood. Thus, we investigated the effect of fucoidan on atherogenesis in apolipoprotein E-deficient (ApoE-/-) mice, on oxLDL uptake by macrophages, and on the expression of the flux-associated scavenger receptors by macrophages. Also, we examined the absorption and biodistribution of orally administered fucoidan. ApoE-/- mice fed on a cholesterol-rich diet supplemented with 1% fucoidan showed reduced dyslipidemia and atherosclerosis. Fucoidan was detected in blood and peripheral tissue after gavage, suggesting that it can exert direct systemic effects. In vitro, fucoidan reduced macrophage oxLDL uptake, which resulted in lower foam cell formation. This effect was associated with downregulation of the cholesterol influx-associated scavenger receptor (SR)-A expression, and upregulation of the cholesterol efflux-associated SR-B1 expression. In conclusion, fucoidan prevented oxLDL-mediated foam cell formation in macrophages by downregulating SR-A1/2 and by up-regulating SR-B1.

Amitriptyline efficacy in decreasing implant-induced foreign body reaction.

Beyond its actions on the nervous system, amitriptyline (AM) has been shown to lower inflammatory, angiogenic, and fibrogenic markers in a few pathological conditions in human and in experimental animal models. However, its effects on foreign body reaction (FBR), a complex adverse healing process, after biomedical material implantation are not known. We have evaluated the effects of AM on the angiogenic and fibrogenic components on a model of implant-induced FBR. Sponge disks were implanted subcutaneously in C57BL/6 mice, that were treated daily with oral administration of AM (5 mg/kg) for seven consecutive days in two protocols: treatment was started on the day of surgery and the implants were removed on the seventh day after implantation and treatment started 7 days after implantation and the implants removed 14 after implantation. None of the angiogenic (vessels, Vascular endothelial growth factor (VEGF), and interleukin-1ß (IL-1ß) or fibrogenic parameters (collagen, TGF-ß, and fibrous capsule) and giant cell numbers analyzed were attenuated by AM in 7-day-old implants. However, AM was able to downregulate angiogenesis and FBR in 14-day-old implants. The effects of AM described here expands its range of actions as a potential agent capable of attenuating fibroproliferative processes that may impair functionality of implantable devices.

Butyrate inhibits LPC-induced endothelial dysfunction by regulating nNOS-produced NO and ROS production.

Lipids oxidation is a key risk factor for cardiovascular diseases. Lysophosphatidylcholine (LPC), the major component of oxidized LDL, is an important triggering agent for endothelial dysfunction and atherogenesis. Sodium butyrate, a short-chain fatty acid, has demonstrated atheroprotective properties. So, we evaluate the role of butyrate in LPC-induced endothelial dysfunction. Vascular response to phenylephrine (Phe) and acetylcholine (Ach) was performed in aortic rings from male mice (C57BL/6J). The aortic rings were incubated with LPC (10 µM) and butyrate (0.01 or 0.1 Mm), with or without TRIM (an nNOS inhibitor). Endothelial cells (EA.hy296) were incubated with LPC and butyrate to evaluate nitric oxide (NO) and reactive oxygen species (ROS) production, calcium influx, and the expression of total and phosphorylated nNOS and ERK½. We found that butyrate inhibited LPC-induced endothelial dysfunction by improving nNOS activity in aortic rings. In endothelial cells, butyrate reduced ROS production and increased nNOS-related NO release, by improving nNOS activation (phosphorylation at Ser1412). Additionally, butyrate prevented the increase in cytosolic calcium and inhibited ERk½ activation by LPC. In conclusion, butyrate inhibited LPC-induced vascular dysfunction by increasing nNOS-derived NO and reducing ROS production. Butyrate restored nNOS activation, which was associated with calcium handling normalization and reduction of ERK½ activation.

Gluten worsens non-alcoholic fatty liver disease by affecting lipogenesis and fatty acid oxidation in diet-induced obese apolipoprotein E-deficient mice.

Obesity is closely associated with non-alcoholic fatty liver disease (NAFLD), characterized by hepatic fat accumulation and hepatocyte injury. Preclinical studies have shown exacerbated weight gain associated with an obesogenic gluten-containing diet. However, whether gluten affects obesity-induced hepatic lipid accumulation still remains unclear. We hypothesized that gluten intake could affect fatty liver development in high-fat diet (HFD)-induced obese mice. Thus, we aimed to investigate the impact of gluten intake on NAFLD in HFD-induced obese mice. Male apolipoprotein E-deficient (Apoe-/-) mice were fed with a HFD containing (GD) or not (GFD) vital wheat gluten (4.5%) for 10 weeks. Blood and liver were collected for further analysis. We found that gluten exacerbated weight gain, hepatic fat deposition, and hyperglycemia without affecting the serum lipid profile. Livers of the GD group showed a larger area of fibrosis, associated with the expression of collagen and MMP9, and higher expression of apoptosis-related factors, p53, p21, and caspase-3. The expression of lipogenic factors, such as PPARγ and Acc1, was more elevated and factors related to beta-oxidation, such as PPARα and Cpt1, were lower in the GD group compared to the GFD. Further, gluten intake induced a more significant expression of Cd36, suggesting higher uptake of free fatty acids. Finally, we found lower protein expression of PGC1α followed by lower activation of AMPK. Our data show that gluten-containing high-fat diet exacerbated NAFLD by affecting lipogenesis and fatty acid oxidation in obese Apoe-/- mice through a mechanism involving lower activation of AMPK.

Protease-activated receptor 2 enhances innate and inflammatory mechanisms induced by lipopolysaccharide in macrophages from C57BL/6 mice.

OBJECTIVE: This study was conducted to investigate the effects of the synthetic PAR2 agonist peptide (PAR2-AP) SLIGRL-NH2 on LPS-induced inflammatory mechanisms in peritoneal macrophages. METHODS: Peritoneal macrophages obtained from C57BL/6 mice were incubated with PAR2-AP and/or LPS, and the phagocytosis of zymosan fluorescein isothiocyanate (FITC) particles; nitric oxide (NO), reactive oxygen species (ROS), and cytokine production; and inducible NO synthase (iNOS) expression in macrophages co-cultured with PAR-2-AP/LPS were evaluated. RESULTS: Co-incubation of macrophages with PAR2AP (30 µM)/LPS (100 ng/mL) enhanced LPS-induced phagocytosis; production of NO, ROS, and the pro-inflammatory cytokines interleukin (IL)-1ß, tumour necrosis factor (TNF)-α, IL-6, and C-C motif chemokine ligand (CCL)2; and iNOS expression and impaired the release of the anti-inflammatory cytokine IL-10 after 4 h of co-stimulation. In addition, PAR2AP increased the LPS-induced translocation of the p65 subunit of the pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) and reduced the expression of inhibitor of NF-κB. CONCLUSION: This study provides evidence of a role for PAR2 in macrophage response triggered by LPS enhancing the phagocytic activity and NO, ROS, and cytokine production, resulting in the initial and adequate macrophage response required for their innate response mechanisms.

Vena cava presents endothelial dysfunction prior to thoracic aorta in heart failure: the pivotal role of nNOS uncoupling/oxidative stress.

Arterial endothelial dysfunction has been extensively studied in heart failure (HF). However, little is known about the adjustments shown by the venous system in this condition. Considering that inferior vena cava (VC) tone could influence cardiac performance and HF prognosis, the aim of the present study was to assess the VC and thoracic aorta (TA) endothelial function of HF-post-myocardial infarction (MI) rats, comparing both endothelial responses and signaling pathways developed. Vascular reactivity of TA and VC from HF post-MI and sham operated (SO) rats was assessed with a wire myograph, 4 weeks after coronary artery occlusion surgery. Nitric oxide (NO), H2O2 production and oxidative stress were evaluated in situ with fluorescent probes, while protein expression and dimer/monomer ratio was assessed by Western blot. VC from HF rats presented endothelial dysfunction, while TA exhibited higher acetylcholine (ACh)-induced vasodilation when compared with vessels from SO rats. TA exhibited increased ACh-induced NO production due to a higher coupling of endothelial and neuronal NO synthases isoforms (eNOS, nNOS), and enhanced expression of antioxidant enzymes. These adjustments, however, were absent in VC of HF post-MI rats, which exhibited uncoupled nNOS, oxidative stress and higher H2O2 bioavailability. Altogether, the present study suggests a differential regulation of endothelial function between VC and TA of HF post-MI rats, most likely due to nNOS uncoupling and compromised antioxidant defense.

Cholesterol Efflux-Independent Modification of Lipid Rafts by AIBP (Apolipoprotein A-I Binding Protein).

OBJECTIVE: AIBP (apolipoprotein A-I binding protein) is an effective and selective regulator of lipid rafts modulating many metabolic pathways originating from the rafts, including inflammation. The mechanism of action was suggested to involve stimulation by AIBP of cholesterol efflux, depleting rafts of cholesterol, which is essential for lipid raft integrity. Here we describe a different mechanism contributing to the regulation of lipid rafts by AIBP. Approach and Results: We demonstrate that modulation of rafts by AIBP may not exclusively depend on the rate of cholesterol efflux or presence of the key regulator of the efflux, ABCA1 (ATP-binding cassette transporter A-I). AIBP interacted with phosphatidylinositol 3-phosphate, which was associated with increased abundance and activation of Cdc42 and rearrangement of the actin cytoskeleton. Cytoskeleton rearrangement was accompanied with reduction of the abundance of lipid rafts, without significant changes in the lipid composition of the rafts. The interaction of AIBP with phosphatidylinositol 3-phosphate was blocked by AIBP substrate, NADPH (nicotinamide adenine dinucleotide phosphate), and both NADPH and silencing of Cdc42 interfered with the ability of AIBP to regulate lipid rafts and cholesterol efflux. CONCLUSIONS: Our findings indicate that an underlying mechanism of regulation of lipid rafts by AIBP involves PIP-dependent rearrangement of the cytoskeleton.

Evidence for the involvement of opioid and cannabinoid systems in the peripheral antinociception mediated by resveratrol.

Despite all the development of modern medicine, around 100 compounds derived from natural products were undergoing clinical trials only at the end of 2013. Among these natural substances in clinical trials, we found the resveratrol (RES), a pharmacological multi-target drug. RES analgesic properties have been demonstrated, although the bases of these mechanisms have not been fully elucidated. The aim of this study was to evaluate the involvement of opioid and cannabinoid systems in RES-induced peripheral antinociception. Paw withdrawal method was used and hyperalgesia was induced by carrageenan (200 µg/paw). All drugs were given by intraplantar injection in male Swiss mice (n = 5). RES (100 µg/paw) administered in the right hind paw induced local antinociception that was antagonized by naloxone, non-selective opioid receptor antagonist, and clocinnamox, µOR selective antagonist. Naltrindole and nor-binaltorfimine, selective antagonists for δOR and kOR, respectively, did not reverse RES-induced peripheral antinociception. CB1R antagonist AM251, but not CB2R antagonist AM630, antagonized RES-induced peripheral antinociception. Peripheral antinociception of RES intermediate-dose (50 µg/paw) was increased by: (i) bestatin, inhibitor of endogenous opioid degradation involved-enzymes; (ii) MAFP, inhibitor of anandamide amidase; (iii) JZL184, inhibitor of 2-arachidonoylglycerol degradation involved-enzyme; (iv) VDM11, endocannabinoid reuptake inhibitor. Acute and peripheral administration of RES failed to affect the amount of µOR, CB1R and CB2R. Experimental data suggest that RES induces peripheral antinociception through µOR and CB1R activation by endogenous opioid and endocannabinoid releasing.

Hydrogen peroxide and nitric oxide induce anticontractile effect of perivascular adipose tissue via renin angiotensin system activation.

The perivascular adipose tissue (PVAT) is located around the adventitia, composed primarily by adipocytes, stromal cells, leukocytes, fibroblasts and capillaries. It is well described that PVAT is an important modulator of the vascular tone being considered a biologically active tissue, releasing both vasoconstrictor and vasodilators factors. The literature shows that the anti-contractile effect induced by PVAT may be due to activation of the renin-angiotensin system (RAS). AIM: Investigate whether the renin-angiotensin system participates in the effect exerted by perivascular adipose tissue on the vascular tone. METHODS AND RESULTS: For this study we used thoracic aorta from Balb/c mice and performed vascular reactivity, nitric oxide and hydrogen peroxide quantification using selective probes and fluorescence microscopy, immunofluorescence to locate receptors and enzymes involved in this response. Our results demonstrated that perivascular adipose tissue induces an anti-contractile effect in endothelium-independent manner and involves Mas and AT2 receptors participation with subsequent PI3K/Akt pathway activation. This pathway culminated with nitric oxide and hydrogen peroxide production by neuronal nitric oxide synthase, being hydrogen peroxide most relevant for the anti-contractile effect of perivascular adipose tissue. CONCLUSION: For the first time in the literature, our results show the presence of Mas and AT2 receptors, as well as, nitric oxide synthase on perivascular adipose tissue. Furthermore, our results show the involvement of Mas and AT2 receptors and consequently nitric oxide synthase activation in the anti-contractile effect exerted by perivascular adipose tissue.

Wheat gluten intake increases the severity of experimental colitis and bacterial translocation by weakening of the proteins of the junctional complex.

Gluten is only partially digested by intestinal enzymes and can generate peptides that can alter intestinal permeability, facilitating bacterial translocation, thus affecting the immune system. Few studies addressed the role of diet with gluten in the development of colitis. Therefore, we investigate the effects of wheat gluten-containing diet on the evolution of sodium dextran sulphate (DSS)-induced colitis. Mice were fed a standard diet without (colitis group) or with 4·5 % wheat gluten (colitis + gluten) for 15 d and received DSS solution (1·5 %, w/v) instead of water during the last 7 d. Compared with the colitis group, colitis + gluten mice presented a worse clinical score, a larger extension of colonic injury area, and increased mucosal inflammation. Both intestinal permeability and bacterial translocation were increased, propitiating bacteria migration for peripheral organs. The mechanism by which diet with gluten exacerbates colitis appears to be related to changes in protein production and organisation in adhesion junctions and desmosomes. The protein α-E-catenin was especially reduced in mice fed gluten, which compromised the localisation of E-cadherin and ß-catenin proteins, weakening the structure of desmosomes. The epithelial damage caused by gluten included shortening of microvilli, a high number of digestive vacuoles, and changes in the endosome/lysosome system. In conclusion, our results show that wheat gluten-containing diet exacerbates the mucosal damage caused by colitis, reducing intestinal barrier function and increasing bacterial translocation. These effects are related to the induction of weakness and disorganisation of adhesion junctions and desmosomes as well as shortening of microvilli and modification of the endocytic vesicle route.

Resistance exercise mediates remote ischemic preconditioning by limiting cardiac eNOS uncoupling.

BACKGROUND: Currently viewed as a complementary non-pharmacological intervention for preventing cardiac disorders, long-term aerobic training produces cardioprotection through remote ischemic preconditioning (RIPC) mechanisms. However, RIPC triggered by acute exercise remains poorly understood. Although resistance exercise (RE) has been highly recommended by several public health guidelines, there is no evidence showing that RE mediates RIPC. Hence, we investigated whether RE induces cardiac RIPC through nitric oxide synthase (NOS)-dependent mechanism. METHODS AND RESULTS: Acute RE at 40% of the maximal load augmented systemic nitrite levels, associated with increased cardiac eNOS phosphorylation, without affecting nNOS activity. Using an experimental model of myocardial infarction (MI) through ischemia-reperfusion (IR), RE fully prevented the loss of cardiac contractility and the extent of MI size compared to non-exercised (NE) rats. Moreover, RE mitigated aberrant ST-segment and reduced life-threatening arrhythmias induced by IR. Importantly, inhibition of NOS abolished the RE-mediated cardioprotection. After IR, NE rats showed increased cardiac eNOS activity, associated with reduced dimer/monomer ratio. Supporting the pivotal role of eNOS coupling during MI, non-exercised rats displayed a marked generation of reactive oxygen species (ROS) and oxidative-induced carbonylation of proteins, whereas RE prevented these responses. We validated our data demonstrating a restoration of physiological ROS levels in NE + IR cardiac sections treated with BH4, a cofactor oxidatively depleted during eNOS uncoupling, while cardiac ROS generation from exercised rats remained unchanged, suggesting no physiological needs of supplemental eNOS cofactors. CONCLUSION: Together, our findings strongly indicate that RE mediates RIPC by limiting eNOS uncoupling and mitigates myocardial IR injury.

Modulation of nNOSser852 phosphorylation and translocation by PKA/PP1 pathway in endothelial cells.

Neuronal nitric oxide synthase (nNOS) is now considered an important player in vascular function. It has a protective role in atherosclerosis and hypertension. However, despite its importance, little is known about the mechanisms that regulate its activity in vascular cells. Here we explore the mechanisms by which nNOS is activated in endothelium. We evaluated aorta relaxation response and phosphorylation of nNOS during protein phosphatases 1 and 2 (PP1 and PP2) inhibition, in eNOS silenced mice. PP1 translocation and interaction between the nuclear inhibitor of PP1 (NIPP1) and PP1 was evaluated in endothelial EA.hy926 cells. We demonstrate here that acetylcholine (Ach)-induced relaxation is completely abolished by nNOS inhibition in eNOS silenced mice aorta which also decreased NO and H2O2 concentrations. ACh induced dephosphorylation of nNOSser852 in aorta after 20 min stimulation. Endothelial cells also showed a decrease in nNOSser852 phosphorylation during 20 min of ACh stimulation. PP2 inhibition had no effect on Ach-induced nNOSSer852 dephosphorylation in endothelial cells and did not modify Ach-induced vasodilation in aorta from eNOS silenced mice. Non-selective PP1/PP2 inhibition prevented nNOSSer852 dephosphorylation in endothelial cells and prevented Ach-induced vasodilation in eNOS silenced mice. ACh induced time-dependent PP1 and NIPP1 dissociation and PP1 translocation to cytoplasm. Protein kinase A (PKA) inhibition abolished PP1 translocation and further nNOSser852 dephosphorylation. In addition, 8-Br-cAMP reduced NIPP1/PP1 interaction, stimulated PP1 translocation and nNOSser852 dephosphorylation. Moreover, PKA Inhibition led to a decreased nNOS translocation to perinuclear region. Taken together, our results elucidate a mechanism whereby PP1 is activated by a cAMP/PKA-dependent pathway, leading to dephosphorylation of nNOSser852 and subsequent NO and possible H2O2 production resulting in endothelium-dependent vascular relaxation.

Mechanism of the vasodilator effect of mono-oxygenated xanthones: a structure-activity relationship study.

The present study characterized the mechanisms involved in the vasodilator effect of two mono-oxygenated xanthones, 4-hydroxyxanthone and 4-methoxyxanthone. 9-Xanthenone, the base structure of xanthones, was used for comparison. 4-Hydroxyxanthone and 9-xanthenone induced a concentration-dependent and endothelium-independent vasodilator effect in arteries precontracted with phenylephrine (0.1 µmol ·â€ŠL-1) or KCl (50 mmol ·â€ŠL-1). 4-Methoxyxanthone induced a concentration-dependent vasodilator effect in arteries precontracted with phenylephrine, which was partially endothelium-dependent, and involved production of nitric oxide. In endothelium-denuded arteries precontracted with KCl, the vasodilator effect of 4-methoxyxanthone was abolished. The vasodilator effect of 4-hydroxyxanthone (96.22 ± 2.10 %) and 4-methoxyxanthone (96.57 ± 12.40 %) was significantly higher than observed with 9-xanthenone (53.63 ± 8.31 %). The presence of an oxygenated radical in position 4 made 4-hydroxyxanthone (pIC50 = 4.45 ± 0.07) and 4-methoxyxanthone (pIC50 = 5.04 ± 0.09) more potent as a vasodilator than 9-xanthenone (pIC50 = 3.92 ± 0.16). In addition, 4-methoxyxanthone was more potent than the other two xanthones. Ca2+ transients in vascular smooth muscle cells elicited by high K+ were abolished by 4-hydroxyxanthone and 9-xanthenone. The endothelium-independent effect of 4-methoxyxanthone was abolished by inhibition of K+ channels by tetraethylammonium. The current work shows that an oxygenated group in position 4 is essential to achieve Emax and to increase the potency of xanthones as vasodilators. Substitution of an OH by OCH3 in position 4 increases the potency of the vasodilator effect and changes the underling mechanism of action from the blockade of L-type calcium channels to an increase in NO production and activation of K+ channels.

The activation of the cannabinoid receptor type 2 reduces neutrophilic protease-mediated vulnerability in atherosclerotic plaques.

AIMS: The activation of cannabinoid receptor type 2 (CB(2))-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB(2) pharmacological activation on markers of plaque vulnerability in vivo and in vitro. METHODS AND RESULTS: The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = 27) for ischaemic stroke. Intraplaque levels of 2-arachidonoylglycerol, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB(1) (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB(2) protein expression was reduced when compared with asymptomatic patients. In these portions, CB(2) levels were inversely correlated (r = -0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB(2) co-localized with neutrophils and MMP-9. Treatment with the selective CB(2) agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/2 phosphorylation in human neutrophils. CONCLUSION: Cannabinoid receptor type 2 receptor is down-regulated in unstable human carotid plaques. Since CB(2) activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in humans.

AIBP regulates TRPV1 activation in chemotherapy-induced peripheral neuropathy by controlling lipid raft dynamics and proximity to TLR4 in dorsal root ganglion neurons.

ABSTRACT: Nociceptive afferent signaling evoked by inflammation and nerve injury is mediated by the opening of ligand-gated and voltage-gated receptors or channels localized to cholesterol-rich lipid raft membrane domains. Dorsal root ganglion (DRG) nociceptors express high levels of toll-like receptor 4 (TLR4), which also localize to lipid rafts. Genetic deletion or pharmacologic blocking of TLR4 diminishes pain associated with chemotherapy-induced peripheral neuropathy (CIPN). In DRGs of mice with paclitaxel-induced CIPN, we analyzed DRG neuronal lipid rafts, expression of TLR4, activation of transient receptor potential cation channel subfamily V member 1 (TRPV1), and TLR4-TRPV1 interaction. Using proximity ligation assay, flow cytometry, and whole-mount DRG microscopy, we found that CIPN increased DRG neuronal lipid rafts and TLR4 expression. These effects were reversed by intrathecal injection of apolipoprotein A-I binding protein (AIBP), a protein that binds to TLR4 and specifically targets cholesterol depletion from TLR4-expressing cells. Chemotherapy-induced peripheral neuropathy increased TRPV1 phosphorylation, localization to neuronal lipid rafts, and proximity to TLR4. These effects were also reversed by AIBP treatment. Regulation of TRPV1-TLR4 interactions and their associated lipid rafts by AIBP covaried with the enduring reversal of mechanical allodynia otherwise observed in CIPN. In addition, AIBP reduced intracellular calcium in response to the TRPV1 agonist capsaicin, which was increased in DRG neurons from paclitaxel-treated mice and in the naïve mouse DRG neurons incubated in vitro with paclitaxel. Together, these results suggest that the assembly of nociceptive and inflammatory receptors in the environment of lipid rafts regulates nociceptive signaling in DRG neurons and that AIBP can control lipid raft-associated nociceptive processing.

Dietary gluten worsens hepatic steatosis by increasing inflammation and oxidative stress in ApoE-/- mice fed a high-fat diet.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disorder in the world. We have seen that gluten intake exacerbated obesity and atherosclerosis in apolipoprotein E knockout (ApoE-/-) mice. In this study, we investigated the effect of gluten consumption on inflammation and oxidative stress in the liver of mice with NAFLD. Male ApoE-/- mice were fed a gluten-free (GF-HFD) or gluten-containing (G-HFD) high-fat diet for 10 weeks. Blood, liver, and spleen were collected to perform the analyses. The animals of the gluten group had increased hepatic steatosis, followed by increased serum AST and ALT. Gluten intake increased hepatic infiltration of neutrophils, macrophages, and eosinophils, as well as the levels of chemotaxis-related factors CCL2, Cxcl2, and Cxcr3. The production of the TNF, IL-1ß, IFNγ, and IL-4 cytokines in the liver was also increased by gluten intake. Furthermore, gluten exacerbated the hepatic lipid peroxidation and nitrotyrosine deposition, which were associated with increased production of ROS and nitric oxide. These effects were related to increased expression of NADPH oxidase and iNOS, as well as decreased activity of superoxide dismutase and catalase enzymes. There was an increased hepatic expression of the NF-κB and AP1 transcription factors, corroborating the worsening effect of gluten on inflammation and oxidative stress. Finally, we found an increased frequency of CD4+FOXP3+ lymphocytes in the spleen and increased gene expression of Foxp3 in the livers of the G-HFD group. In conclusion, dietary gluten aggravates NAFLD, exacerbating hepatic inflammation and oxidative stress in obese ApoE-deficient mice.

Platelet-activating factor and protease-activated receptor 2 cooperate to promote neutrophil recruitment and lung inflammation through nuclear factor-kappa B transactivation.

Although it is well established that platelet-activated receptor (PAF) and protease-activated receptor 2 (PAR2) play a pivotal role in the pathophysiology of lung and airway inflammatory diseases, a role for a PAR2-PAFR cooperation in lung inflammation has not been investigated. Here, we investigated the role of PAR2 in PAF-induced lung inflammation and neutrophil recruitment in lungs of BALB/c mice. Mice were pretreated with the PAR2 antagonist ENMD1068, PAF receptor (PAFR) antagonist WEB2086, or aprotinin prior to intranasal instillation of carbamyl-PAF (C-PAF) or the PAR2 agonist peptide SLIGRL-NH2 (PAR2-AP). Leukocyte infiltration in bronchoalveolar lavage fluid (BALF), C-X-C motif ligand 1 (CXCL)1 and CXCL2 chemokines, myeloperoxidase (MPO), and N-acetyl-glycosaminidase (NAG) levels in BALF, or lung inflammation were evaluated. Intracellular calcium signaling, PAFR/PAR2 physical interaction, and the expression of PAR2 and nuclear factor-kappa B (NF-КB, p65) transcription factor were investigated in RAW 264.7 cells stimulated with C-PAF in the presence or absence of ENMD1068. C-PAF- or PAR2-AP-induced neutrophil recruitment into lungs was inhibited in mice pretreated with ENMD1068 and aprotinin or WEB2086, respectively. PAR2 blockade impaired C-PAF-induced neutrophil rolling and adhesion, lung inflammation, and production of MPO, NAG, CXCL1, and CXCL2 production in lungs of mice. PAFR activation reduced PAR2 expression and physical interaction of PAR2 and PAFR; co-activation is required for PAFR/PAR2 physical interaction. PAR2 blockade impaired C-PAF-induced calcium signal and NF-κB p65 translocation in RAW 264.7 murine macrophages. This study provides the first evidence for a cooperation between PAFR and PAR2 mediating neutrophil recruitment, lung inflammation, and macrophage activation.

Intense Caloric Restriction from Birth Prevents Cardiovascular Aging in Rats.

We previously demonstrated that a 50% caloric restriction (CR) from birth improves several cardiometabolic risk factors in young rats. In this study, we investigated in middle-aged rats the consequences of a 50% CR from birth on cardiometabolic risk factors, heart function/morphology, ventricular arrhythmia, and fibrillation incidence, and cardiac intracellular proteins involved with redox status and cell survival. From birth to the age of 18 months, rats were divided into an Ad Libitum (AL18) group, which had free access to food, and a CR18 group, which had food limited to 50% of that consumed by the AL18. Resting metabolic rate, blood pressure, and heart rate were recorded, and oral glucose and intraperitoneal insulin tolerance tests were performed. Blood was collected for biochemical analyses, and visceral fat and liver were harvested and weighed. Hearts were harvested for cardiac function, histological, redox status, and western blot analyses. The 50% CR from birth potentially reduced several cardiometabolic risk factors in 18-month-old rats. Moreover, compared with AL18, the CR18 group showed a ∼50% increase in cardiac contractility and relaxation, nearly three to five times less incidence of ventricular arrhythmia and fibrillation, ∼18% lower cardiomyocyte diameter, and ∼60% lower cardiac fibrosis. CR18 hearts also improved biomarkers of antioxidant defense and cell survival. Collectively, these results reveal several metabolic and cardiac antiaging effects of a 50% CR from birth in middle-aged rats.

Update on the role of cannabinoid receptors after ischemic stroke.

Cannabinoids are considered as key mediators in the pathophysiology of inflammatory diseases, including atherosclerosis. In particular, they have been shown to reduce the ischemic injury after acute cardiovascular events, such as acute myocardial infarction and ischemic stroke. These protective and anti-inflammatory properties on peripheral tissues and circulating inflammatory have been demonstrated to involve their binding with both selective cannabinoid type 1 (CB1 and type 2 (CB2) transmembrane receptors. On the other hands, the recent discoveries of novel different classes of cannabinoids and receptors have increased the complexity of this system in atherosclerosis. Although only preliminary data have been reported on the activities of novel cannabinoid receptors, several studies have already investigated the role of CB1 and CB2 receptors in ischemic stroke. While CB1 receptor activation has been shown to directly reduce atherosclerotic plaque inflammation, controversial data have been shown on neurotransmission and neuroprotection after stroke. Given its potent anti-inflammatory activities on circulating leukocytes, the CB2 activation has been proven to produce protective effects against acute poststroke inflammation. In this paper, we will update evidence on different cannabinoid-triggered avenues to reduce inflammation and neuronal injury in acute ischemic stroke.

Contribution of RAS, ROS and COX-1-derived prostanoids to the contractile profile of perivascular adipose tissue in cafeteria diet-induced obesity.

AIMS: Obesity can lead to the loss of the anticontractile properties of perivascular adipose tissue (PVAT). Given that cafeteria (CAF) diet reflects the variety of highly calorie and easily accessible foods in Western societies, contributing to obesity and metabolic disorders, we sought to investigate the impact of CAF diet on PVAT vasoactive profile and the involvement of renin-angiotensin system, oxidative stress, and cyclooxygenase pathway. MAIN METHODS: Male Balb/c mice received standard or CAF diet for 4 weeks. Oral glucose tolerance and insulin sensitivity tests were performed, and fasting serum glucose, cholesterol and triglyceride parameters were determined. Vascular reactivity, fluorescence and immunofluorescence analyzes were carried out in intact thoracic aorta in the presence or absence of PVAT. KEY FINDINGS: CAF diet was effective in inducing obesity and metabolic disorders, as demonstrated by increased body weight gain and adiposity index, hyperlipidemia, hyperglycemia, glucose intolerance and insulin insensitivity. Importantly, CAF diet led to a significant decrease in aortic contractility which was restored in the presence of PVAT, exhibiting therefore a contractile profile. The contractile effect of PVAT was associated with the activation of AT1 receptor, reactive oxygen species, cyclooxygenase-1, thromboxane A2 and prostaglandin E2 receptors. SIGNIFICANCE: These findings suggest that the contractile profile of PVAT involving the renin-angiotensin system activation, reactive oxygen species and cyclooxygenase-1 metabolites may be a protective compensatory adaptive response during early stage of CAF diet-induced obesity as an attempt to restore the impaired vascular contraction observed in the absence of PVAT, contributing to the maintenance of vascular tone.