Microvasculature remodeling in the mouse lower gut during inflammaging

Inflammaging is defined as low-grade, chronic, systemic inflammation in aging, in the absence of overt infection. Age-associated deterioration of gastrointestinal function could be ascribed to the inflammaging, although evidence is yet to emerge. Here we show that microvessels in aging mouse intestine were progressively deprived of supportive structures, microvessel-associated pericytes and adherens junction protein vascular endothelial (VE)-cadherin, and became leaky. This alteration was ascribed to up-regulation of angiopoetin-2 in microvascular endothelial cells. Up-regulation of the angiopoietin-2 was by TNF-α, originated from M2-like residential CD206 + macrophages, proportion of which increases as animal ages. It was concluded that antigenic burdens encountered in intestine throughout life create the condition of chronic stage of inflammation, which accumulates M2-like macrophages expressing TNF-α. The TNF-α induces vascular leakage to facilitate recruitment of immune cells into intestine under the chronic inflammatory setting. © Author(s) 2017.1

Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury

<div><p>Drug- and alcohol-induced liver injury are a leading cause of liver failure and transplantation. Emerging evidence suggests that extracellular vesicles (EVs) are a source of biomarkers because they contain unique proteins reflecting the identity and tissue-specific origin of the EV proteins. This study aimed to determine whether potentially hepatotoxic agents, such as acetaminophen (APAP) and binge alcohol, can increase the amounts of circulating EVs and evaluate liver-specific EV proteins as potential biomarkers for liver injury. The circulating EVs, isolated from plasma of APAP-exposed, ethanol-fed mice, or alcoholic hepatitis patients versus normal control counterparts, were characterized by proteomics and biochemical methods. Liver specific EV proteins were analyzed by immunoblots and ELISA. The amounts of total and liver-specific proteins in circulating EVs from APAP-treated mice significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs from APAP-exposed mice revealed that the amounts of liver-specific and/or hepatotoxic proteins were increased compared to those of controls. Additionally, the increased protein amounts in EVs following APAP exposure returned to basal levels when mice were treated with <i>N</i>-acetylcysteine or glutathione. Similar results of increased amounts and liver-specific proteins in circulating EVs were also observed in mice exposed to hepatotoxic doses of thioacetamide or d-galactosamine but not by non-hepatotoxic penicillin or myotoxic bupivacaine. Additionally, binge ethanol exposure significantly elevated liver-specific proteins in circulating EVs from mice and alcoholics with alcoholic hepatitis, compared to control counterparts. These results indicate that circulating EVs in drug- and alcohol-mediated hepatic injury contain liver-specific proteins that could serve as specific biomarkers for hepatotoxicity.</p></div

Proteomic analysis of circulating EVs isolated from APAP-exposed mice and confirmation of various proteins by immunoblot analysis.

<p>(A) Proteomic analysis of EV proteins isolated from Control or APAP-exposed mice using 2D LC-MS/MS. Differentially expressed proteins were identified by bioinformatic analysis, as described. (B) Classification based on the subcellular location of the identified proteins. Comparison of proteomic data from the EVs in this study with those of rat primary hepatocytes, as reported previously by our group[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172463#pone.0172463.ref030" target="_blank">30</a>]. (C) The 380 proteins in EVs from control plasma and 431 proteins in EVs from APAP plasma were classified by tissuespecific origin and categorized according to the DAVID program. Some proteins are classified to more than two organs. (D) Comparison of differentially expressed proteins in circulating EVs from APAP-treated groups with the previously reported results[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172463#pone.0172463.ref039" target="_blank">39</a>] from hepatocytes treated with APAP, based on biological process analysis. (E) Confirmation of liver-specific proteins, inflammation-related proteins, and exosomal marker proteins in circulating EVs by immunoblot analysis, as indicated. (F) Protein levels of ASS1 and CES1 were measured in whole liver lysates from the indicated groups.</p

Analysis of liver- or muscle-specific proteins in circulating EVs from mice with liver or muscle injury.

<p>Wild-type male BALB/c mice (6 weeks old) were treated with a single ip injection of APAP (300 mg/kg) or intramuscular injection with 0% or 0.5% BPVC into the right and left tibialis anterior muscles and were sacrificed at 24 h post treatment (n = 10/group). (A) Representative H&E staining of formalin-fixed muscle sections for all indicated groups. (B) Plasma ALT and AST levels. (C) Analysis of the total protein amounts in circulating EVs isolated from the indicated groups. (D) Immunoblot analyses of liver-specific or muscle-specific proteins in circulating EVs from each group, as indicated. (E) The amounts of ALB or STN1 in circulating EVs from each group were respectively measured by ELISA specific for mouse proteins. *<i>P</i> < 0.05.</p

1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation

Abstract Cancer patients treated with chemotherapy often experience a rapid decline of blood neutrophils, a dose-limiting side effect called chemotherapy-induced neutropenia. This complication brings about dose reductions or cessation of chemotherapy during treatment of cancer patients because a rapid decline of neutrophil counts increases susceptibility to infection. Here, we found that 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation via the inhibition of neutrophil-attracting chemokine production in macrophages using in vivo and in vitro approaches. A single intraperitoneal administration of gemcitabine induced the migration of circulating neutrophils into the peritoneal cavity in normal mice, and PLAG effectively decreased neutrophil migration by inhibiting the expression of adhesion molecules, L-selectin and LFA-1. Inhibition of CXCR2 by its antagonist, reparixin, abrogated gemcitabine-induced neutrophil migration, indicating that chemokines produced by gemcitabine mainly support neutrophil activation. In vitro experiments demonstrated that PLAG inhibited NADPH oxidase 2 (NOX2)-mediated reactive oxygen species production induced by gemcitabine, which is the upstream of MIP-2 and/or CXCL8. Importantly, PLAG down-regulated gemcitabine-induced membrane translocation of the cytosolic NOX subunit, Rac1, and phosphorylation of p47phox. The activation of upstream signaling molecules of p47phox phosphorylation, phospholipase C β3 and protein kinase C, were effectively regulated by PLAG. We also demonstrated that 1-palmitoyl-2-linoleic-3-hydroxyl-rac-glycerol (PLH), the natural form of diacylglycerol, has no effects on gemcitabine-induced CXCL8 production and dHL-60 migration, suggesting that an acetyl group at the third position of the glycerol backbone may have a key role in the regulation of neutrophil activation. Altogether, this study suggests the potential of PLAG as a therapeutic strategy to modulate chemotherapy-induced neutrophil activation for cancer patients undergoing chemotherapeutic treatment

The amounts of total and liver-specific proteins in circulating EVs were increased in binge ethanol-exposed mice and alcoholics.

<p>(A) Wild-type male BALB/c mice (6 weeks old) were treated twice with saline (CON) or 6 g ethanol/kg via oral gavage at 12-h interval and were sacrificed 1 h after the second dose (n = 10/group). Representative H&E staining of formalin-fixed liver sections. (B) Protein amounts of the circulating EVs isolated from control or alcohol-exposed mice, as indicated. *<i>P</i> < 0.05. (C) Detection of the liver-specific proteins in circulating EVs from the indicated mouse plasma by immunoblot analysis. (D) Analysis of the protein amounts in circulating EVs isolated from the sera of healthy controls (n = 9), who never drank alcohol due to religious reasons, and alcoholics (n = 13) with hepatitis. (E) The amounts of liver-specific protein ALB in circulating EVs from the indicated groups were measured by ELISA for human ALB protein. *<i>P</i> < 0.05. (F) Detection of the liver-specific proteins in circulating EVs from the different groups by immunoblot analyses, as indicated. *<i>P</i> < 0.05.</p