Inhibition of inflammatory mediators by polyphenolic plant extracts in human intestinal Caco-2 cells.

The mitogen-activated protein kinases (MAPK) and nuclear factor kappaB (NF-kappaB) are involved in transduction cascades that play a key role in inflammatory response. We tested the ability of preselected natural polyphenolic extracts (grape seed, cocoa, sugar cane, oak, mangosteen and pomegranate) to modulate intestinal inflammation using human intestinal Caco-2 cells treated for 4h with these extracts and then stimulated by cytokines for 24 or 48h. The effect of polyphenolic extracts, at 50mumol of gallic acid equivalent/l, was investigated on inflammation-related cellular events: (i) NF-kappaB activity (cells transfected with a NF-kappaB-luciferase construct), (ii) activation of Erk1/2 and JNK (western blotting), (iii) secretion of interleukin 8 (IL-8) (ELISA), (iv) secretion of prostaglandin (PG) E(2) (ELISA), (v) production of NO (Griess method). Results show that: (i) sugar cane, oak and pomegranate extracts inhibited NF-kappaB activity (from 1.6 to 1.9-fold) (P<0.001); (ii) pomegranate slightly inhibited Erk1/2 activation (1.3-fold) (P=0.008); (iii) oak and pomegranate decreased NO synthesis by 1.5-fold (P<0.001) and that of IL-8 by 10.3 and 6.7-fold respectively; (iv) pomegranate and cocoa decreased PGE(2) synthesis by 4.6 (P<0.0001) and 2.2-fold (P=0.001), respectively. We suggest that pomegranate extract could be particularly promising in dietary prevention of intestinal inflammation

Neuraminidase-1: A Sialidase Involved in the Development of Cancers and Metabolic Diseases

Sialidases or neuraminidases (NEU) are glycosidases which cleave terminal sialic acid residues from glycoproteins, glycolipids and oligosaccharides. Four types of mammalian sialidases, which are encoded by different genes, have been described with distinct substrate specificity and subcellular localization: NEU-1, NEU-2, NEU-3 and NEU-4. Among them, NEU-1 regulates many membrane receptors through desialylation which results in either the activation or inhibition of these receptors. At the plasma membrane, NEU-1 also associates with the elastin-binding protein and the carboxypeptidase protective protein/cathepsin A to form the elastin receptor complex. The activation of NEU-1 is required for elastogenesis and signal transduction through this receptor, and this is responsible for the biological effects that are mediated by the elastin-derived peptides (EDP) on obesity, insulin resistance and non-alcoholic fatty liver diseases. Furthermore, NEU-1 expression is upregulated in hepatocellular cancer at the mRNA and protein levels in patients, and this sialidase regulates the hepatocellular cancer cells&rsquo; proliferation and migration. The implication of NEU-1 in other cancer types has also been shown notably in the development of pancreatic carcinoma and breast cancer. Altogether, these data indicate that NEU-1 plays a key role not only in metabolic disorders, but also in the development of several cancers which make NEU-1 a pharmacological target of high potential in these physiopathological contexts

Early Alterations of Intra-Mural Elastic Lamellae Revealed by Synchrotron X-ray Micro-CT Exploration of Diabetic Aortas

International audienceDiabetes is a major concern of our society as it affects one person out of 11 around the world. Elastic fiber alterations due to diabetes increase the stiffness of large arteries, but the structural effects of these alterations are poorly known. To address this issue, we used synchrotron X-ray microcomputed tomography with in-line phase contrast to image in three dimensions C57Bl6J (control) and db/db (diabetic) mice with a resolution of 650 nm/voxel and a field size of 1.3 mm3. Having previously shown in younger WT and db/db mouse cohorts that elastic lamellae contain an internal supporting lattice, here we show that in older db/db mice the elastic lamellae lose this scaffold. We coupled this label-free method with automated image analysis to demonstrate that the elastic lamellae from the arterial wall are structurally altered and become 11% smoother (286,665 measurements). This alteration suggests a link between the loss of the 3D lattice-like network and the waviness of the elastic lamellae. Therefore, waviness measurement appears to be a measurable elasticity indicator and the 3D lattice-like network appears to be at the origin of the existence of this waviness. Both could be suitable indicators of the overall elasticity of the aorta

X‐ray microtomography reveals a lattice‐like network within aortic elastic lamellae

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Revealing the elasticity of an individual aortic fiber during ageing at nanoscale by in situ atomic force microscopy †

International audienceArterial stiffness is a complex process affecting the aortic tree that significantly contributes to cardiovascular diseases (systolic hypertension, coronary artery disease, heart failure or stroke). This process involves a large extracellular matrix remodeling mainly associated with elastin content decrease and collagen content increase. Additionally, various chemical modifications that accumulate with ageing have been shown to affect long-lived assemblies, such as elastic fibers, that could affect their elasticity. To precisely characterize the fiber changes and the evolution of its elasticity with ageing, high resolution and multimodal techniques are needed for precise insight into the behavior of a single fiber and its surrounding medium. In this study, the latest developments in atomic force microscopy and the related nanomechanical modes are used to investigate the evolution and in a near-physiological environment, the morphology and elasticity of aorta cross sections obtained from mice of different ages with an unprecedented resolution. In correlation with more classical approaches such as pulse wave velocity and fluorescence imaging, we demonstrate that the relative Young's moduli of elastic fibers, as well as those of the surrounding areas, significantly increase with ageing. This nanoscale characterization presents a new view on the stiffness process, showing that, besides the elastin and collagen content changes, elasticity is impaired at the molecular level, allowing a deeper understanding of the ageing process. Such nanomechanical AFM measurements of mouse tissue could easily be applied to studies of diseases in which elastic fibers suffer pathologies such as atherosclerosis and diabetes, where the precise quantification of fiber elasticity could better follow the fiber remodeling and predict plaque rupture. † Electronic supplementary information (ESI) available. Se

Characterization of the <i>in vitro</i> model of aging.

<p>Young, intermediate and aged fibroblasts are represented by passage 3 (white), 5 (gray) and 8 (black bars), respectively. Data shown are representative of three independent experiments. (A) Acid β-galactosidase detection during <i>in vitro</i> aging. Acid β-galactosidase positive senescent cells appear in blue. The histogram reports the senescent/total cells ratio in the three conditions. This ratio was set to 100% for p3. (B) Fibroblasts proliferation is decreased during <i>in vitro</i> aging. Cells were incubated with the Uptiblue reagent and proliferation was evaluated by measuring the absorbance at 560nm. The results are expressed as a percentage of p3 condition value. (C) Evaluation of ROS production during <i>in vitro</i> aging. ROS production was detected by fluorescence using the Image-iT LIVE Green Reactive Oxygen Species Detection Kit. (λ _excitation 495nm / λ _emission 529nm). The results are expressed as a percentage of p3 condition value. (D) Evalution of MMP-1 secretion during <i>in vitro</i> aging. MMP-1 secretion was determined by ELISA. The results are expressed as a percentage of p3 condition value. *, <i>p<</i>0.05, ***, <i>p</i><0.01.</p

Transmembrane Peptides as a New Strategy to Inhibit Neuraminidase-1 Activation

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Analysis of protein expression and plasma membrane localization of Neu-1 and PPCA.

<p>Young, intermediate and aged fibroblasts were represented by passage 3 (white), 5 (grey) and 8 (black bars). (A) Analysis of Neu-1 and PPCA protein levels during aging. Cells were stimulated or not for 24 hours with 50 μg/mL of EDP before protein extraction. Neu-1 and PPCA were then analyzed by Western blotting using anti-Neu-1 and anti-PPCA antibodies. Densitometric analysis was performed using the Quantity One software. The results were expressed as the ratio Neu-1/β-actin and PPCA/β-actin and normalized to the control expressed in percent. Dotted lines (100%) represent the values of the unstimulated controls. (B) Analysis of Neu-1 and PPCA localization during aging. Neu-1 and PPCA cell localizations were analyzed by confocal microscopy. Bar, 10 μm.</p

Impact of <i>in vitro</i> aging on EDP biological effects.

<p>Young and aged fibroblasts were represented by passage 3 (white) and 8 (black bars), respectively. Dotted lines (100%) represent the values of the unstimulated controls. (A) Impact of <i>in vitro</i> aging on EDP-mediated fibroblast proliferation. Cells were stimulated or not with 50 μg/mL of EDP or 200 μg/mL of scramble or VGVAPG peptides during 48h. Proliferation was analyzed by counting using Trypan Blue exclusion. (B) Impact of <i>in vitro</i> aging on EDP-induced MMP-1 secretion on fibroblasts. MMP-1 secretion was evaluated by ELISA. Cells were stimulated with 50 μg/mL of EDP or 200 μg/mL of scramble or VGVAPG peptides during 24h. (C) Impact of <i>in vitro</i> aging on EDP-induced ROS production. Cells were stimulated for 30 minutes with 50 μg/mL of EDP or 200 μg/mL of scramble or VGVAPG peptides. ROS were detected by the Image-iT LIVE Green Reactive Oxygen Species Detection Kit as described. (D) Impact of <i>in vitro</i> aging on EDP-induced ERK1/2 activation. ERK1/2 phosphorylation was analyzed by Western blotting after 30 min of stimulation with 50 μg/mL of EDP. Cells extracts were analyzed using anti-phospho-ERK1/2 (T202/Y204) and anti-ERK1/2 antibodies. The blots are presented in the left panel. The corresponding densitometric analysis is provided on the right panel. *, <i>p<</i>0.05, ** <i>p<</i>0.02, *** <i>p</i><0.01.</p

Impact of aging on EDP-induced sialidase activity.

<p>Young, intermediate and aged fibroblasts were represented by passage 3 (white), 5 (gray) and 8 (black bars). (A) Basal sialidase activity increases with age. Sialidase activity assay was performed on crude membrane using the chromogenic substrate <i>para-</i>nitrophenyl-NANA for 4 hours at 37°C. (B) EDP-mediated sialidase activity is altered with age. Cells were stimulated or not with 50 μg/mL of EDP for 30 minutes then crude membrane were prepared and used to measure sialidase activity. Histogram represent the normalized results relative to the control expressed in percent. Each data is representative of three independent experiments. ***, <i>p</i><0.01. Dotted lines represent the values of the unstimulated controls.</p