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.

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.

Dual effect of amitriptyline in the control of vascular tone: Direct blockade of calcium channel in smooth muscle cells and reduction of TLR4-dependent NO production in endothelial cells.

BACKGROUND AND PURPOSE: Amitriptyline (AM) is a classical and typical tricyclic antidepressant drug. Despite its well-known effects on the nervous system, it has been described to work as a TLR4 antagonist and several clinical works suggested some unexpected cardiovascular effects. The role of amitriptyline on vascular tone is not clear, thus we hypothesized that amitriptyline has a double effect on vascular tone by both endothelial TLR4-dependent nitric oxide down-regulation and calcium channel blockade in smooth muscle cells. EXPERIMENTAL APPROACH: Changes in isometric tension were recorded on a wire myograph. NO production was evaluated by fluorescence microscopy and flow cytometry in the mouse aorta and EAhy926 cells using DAF fluorescence intensity. Calcium influx was evaluated in A7r5 cells by flow cytometry. Western blot was used to analyze eNOS and nNOS phosphorylation. KEY RESULTS: AM reduced PE-induced contraction by calcium influx diminution in smooth muscle cells (F/F0 = 225.6 ± 15.9 and 118.6 ± 17.6 to CT and AM, respectively). AM impaired Ach-dependent vasodilation (Emax = 95.8 ± 1.4; 78.1 ± 1.8; 60.4 ± 2.9 and -7.4 ± 1.0 for CT, 0.01, 0,1 and 1 µmol/L AM, respectively) through reduction of calcium influx and NO availability and TLR4 antagonism in a concentration-dependent manner. AM or TLR4 gene deletion significantly reduced NO production (Fluorescence = 9503 ± 871.7, 2561 ± 282, 4771 ± 728 and 1029 ± 103 to CT, AM, TLR4-/- and AM + TLR4-/-, respectively) by an increase in nNOSser852 and reduction in eNOSser1177 phosphorylation in endothelial cells. CONCLUSIONS AND IMPLICATIONS: Our data show that amitriptyline impaired vascular function through two different mechanisms: blockade of TLR4 in endothelial cells and consequent decrease in NO production and calcium influx reduction in smooth muscle and endothelial cells. We also suggest, for the first time, nNOS activity reduction by AM in non-neuronal cells.

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.

High-Carbohydrate Diet Enhanced the Anticontractile Effect of Perivascular Adipose Tissue Through Activation of Renin-Angiotensin System.

The perivascular adipose tissue (PVAT) is an active endocrine organ responsible for release several substances that influence on vascular tone. Increasing evidence suggest that hyperactivation of the local renin-angiotensin system (RAS) in the PVAT plays a pivotal role in the pathogenesis of cardiometabolic diseases. However, the local RAS contribution to the PVAT control of vascular tone during obesity is still not clear. Since the consumption of a high-carbohydrate diet (HC diet) contributes to obesity inducing a rapid and sustained increase in adiposity, so that the functional activity of PVAT could be modulated, we aimed to evaluate the effect of HC diet on the PVAT control of vascular tone and verify the involvement of RAS in this effect. For that, male Balb/c mice were fed standard or HC diet for 4 weeks. Vascular reactivity, histology, fluorescence, and immunofluorescence analysis were performed in intact thoracic aorta in the presence or absence of PVAT. The results showed that HC diet caused an increase in visceral adiposity and also in the PVAT area. Phenylephrine-induced vasoconstriction was significantly reduced in the HC group only in the presence of PVAT. The anticontractile effect of PVAT induced by HC diet was lost when aortic rings were previously incubated with angiotensin-converting enzyme inhibitor, Mas, and AT2 receptors antagonists, PI3K, nNOS, and iNOS inhibitors, hydrogen peroxide (H2O2) decomposing enzyme or non-selective potassium channels blocker. Immunofluorescence assays showed that both Mas and AT2 receptors as well as nNOS and iNOS isoforms were markedly expressed in the PVAT of the HC group. Furthermore, the PVAT from HC group also exhibited higher nitric oxide (NO) and hydrogen peroxide bioavailability. Taken together, these findings suggest that the anticontractile effect of PVAT induced by HC diet involves the signaling cascade triggered by the renin-angiotensin system through the activation of Mas and AT2 receptors, PI3K, nNOS, and iNOS, leading to increased production of nitric oxide and hydrogen peroxide, and subsequently opening of potassium channels. The contribution of PVAT during HC diet-induced obesity could be a compensatory adaptive characteristic in order to preserve the vascular function.