Exploration of microRNAs from blood extracellular vesicles as biomarkers of exposure to polycyclic aromatic hydrocarbons.

Exposure to polycyclic aromatic hydrocarbons (PAHs), ubiquitously environmental contaminant, leads to the development of major toxic effects on human health, such as carcinogenic and immunosuppressive alterations reported for the most studied PAH, i.e., benzo(a)pyrene (B(a)P). In order to assess the risk associated with this exposure, it is necessary to have predictive biomarkers. Thus, extracellular vesicles (EVs) and their microRNA (miRNA) contents, have recently been proposed as potentially interesting biomarkers in Toxicology. Our study here explores the use of vesicles secreted and found in blood fluids, and their miRNAs, as biomarkers of exposure to B(a)P alone and within a realistic occupational mixture. We isolated EVs from primary human cultured blood mononuclear cells (PBMCs) and rat plasma after PAH exposure and reported an increased EV production by B(a)P, used either alone or in the mixture, in vitro and in vivo. We then investigated the association of this EV release with the blood concentration of the 7,8,9,10-hydroxy (tetrol)-B(a)P reactive metabolite, in rats. By performing RNA-sequencing (RNA-seq) of miRNAs in PBMC-derived EVs, we analyzed miRNA profiles and demonstrated the regulation of the expression of miR-342-3p upon B(a)P exposure. We then validated B(a)P-induced changes of miR-342-3p expression in vivo in rat plasma-derived EVs. Overall, our study highlights the feasibility of using EVs and their miRNA contents, as biomarkers of PAH exposure and discusses their potential in environmental Toxicology.

Small RNA-sequencing reveals the involvement of microRNA-132 in benzo[a]pyrene-induced toxicity in primary human blood cells.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental contaminants, triggering deleterious effects such as carcinogenicity and immunosuppression, and peripheral blood mononuclear cells (PBMCs) are among the main cell types targeted by these pollutants. In the present study, we sought to identify the expression profiles and function of miRNAs, gene regulators involved in major cellular processes recently linked to environmental pollutants, in PBMC-exposed to the prototypical PAH, benzo[a]pyrene (B[a]P). Using small RNA deep sequencing, we identified several B[a]P-responsive miRNAs. Bioinformatics analyses showed that their predicted targets could modulate biological processes relevant to cell death and survival. Further studies of the most highly induced miRNA, miR-132, showed that its up-regulation by B[a]P was time- and dose-dependent and required aryl hydrocarbon receptor (AhR) activation. By evaluating the role of miR-132 in B[a]P-induced cell death, we propose a mechanism linking B[a]P-induced miR-132 expression and cytochromes P-450 (CYPs) 1A1 and 1B1 mRNA levels, which could contribute to the apoptotic response of PBMCs. Altogether, this study increases our understanding of the roles of miRNAs induced by B[a]P and provides the basis for further investigations into the mechanisms of gene expression regulation by PAHs.

Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

Recent evidence supporting that adipose tissue (AT)-derived extracellular vesicles (EVs) carry an important part of the AT secretome led us to characterize the EV-adipokine profile. In addition to evidencing a high AT-derived EV secretion ability that is further increased by obesity, we identify enrichment of oligomeric forms of adiponectin in small EVs (sEVs). This adipokine is mainly distributed at the EV external surface as a result of nonspecific adsorption of soluble adiponectin. EVs also constitute stable conveyors of adiponectin in the blood circulation. Adiponectin-enriched sEVs display in vitro insulin-sensitizing effects by binding to regular adiponectin receptors. Adoptive transfer of adiponectin-enriched sEVs in high-fat-diet-fed mice prevents animals from gaining weight and ameliorated insulin resistance and tissue inflammation, with major effects observed in the AT and liver. Our results therefore provide information regarding adiponectin-related metabolic responses by highlighting EVs as delivery platforms of metabolically active forms of adiponectin molecules.

Sonic Hedgehog receptor Patched deficiency in astrocytes enhances glucose metabolism in mice.

OBJECTIVE: Astrocytes are glial cells proposed as the main Sonic hedgehog (Shh)-responsive cells in the adult brain. Their roles in mediating Shh functions are still poorly understood. In the hypothalamus, astrocytes support neuronal circuits implicated in the regulation of energy metabolism. In this study, we investigated the impact of genetic activation of Shh signaling on hypothalamic astrocytes and characterized its effects on energy metabolism. METHODS: We analyzed the distribution of gene transcripts of the Shh pathway (Ptc, Gli1, Gli2, and Gli3) in astrocytes using single molecule fluorescence in situ hybridization combined with immunohistofluorescence of Shh peptides by Western blotting in the adult mouse hypothalamus. Based on the metabolic phenotype, we characterized Glast-CreERT2-YFP-Ptc-/- (YFP-Ptc-/-) mice and their controls over time and under a high-fat diet (HFD) to investigate the potential effects of conditional astrocytic deletion of the Shh receptor Patched (Ptc) on metabolic efficiency, insulin sensitivity, and systemic glucose metabolism. Molecular and biochemical assays were used to analyze the alteration of key pathways modulating energy metabolism, insulin sensitivity, glucose uptake, and inflammation. Primary astrocyte cultures were used to evaluate a potential role of Shh signaling in astrocytic glucose uptake. RESULTS: Shh peptides were the highest in the hypothalamic extracts of adult mice and a large population of hypothalamic astrocytes expressed Ptc and Gli1-3 mRNAs. Characterization of Shh signaling after conditional Ptc deletion in the YFP-Ptc-/- mice revealed heterogeneity in hypothalamic astrocyte populations. Interestingly, activation of Shh signaling in Glast+ astrocytes enhanced insulin responsiveness as evidenced by glucose and insulin tolerance tests. This effect was maintained over time and associated with lower blood insulin levels and also observed under a HFD. The YFP-Ptc-/- mice exhibited a lean phenotype with the absence of body weight gain and a marked reduction of white and brown adipose tissues accompanied by increased whole-body fatty acid oxidation. In contrast, food intake, locomotor activity, and body temperature were not altered. At the cellular level, Ptc deletion did not affect glucose uptake in primary astrocyte cultures. In the hypothalamus, activation of the astrocytic Shh pathway was associated with the upregulation of transcripts coding for the insulin receptor and liver kinase B1 (LKB1) after 4 weeks and the glucose transporter GLUT-4 after 32 weeks. CONCLUSIONS: Here, we define hypothalamic Shh action on astrocytes as a novel master regulator of energy metabolism. In the hypothalamus, astrocytic Shh signaling could be critically involved in preventing both aging- and obesity-related metabolic disorders.

Phenotyping of circulating extracellular vesicles (EVs) in obesity identifies large EVs as functional conveyors of Macrophage Migration Inhibitory Factor.

OBJECTIVE: Obesity-associated metabolic dysfunctions are linked to dysregulated production of adipokines. Accumulating evidence suggests a role for fat-derived extracellular vesicles (EVs) in obesity-metabolic disturbances. Since EVs convey numerous proteins we aimed to evaluate their contribution in adipokine secretion. METHODS: Plasma collected from metabolic syndrome patients were used to isolate EV subtypes, namely microvesicles (MVs) and exosomes (EXOs). Numerous soluble factor concentrations were measured successively on total, MV- and EXO-depleted plasma by multiplexed immunoassays. RESULTS: Circulating MVs and EXOs were significantly increased with BMI, supporting a role of EVs as metabolic relays in obesity. Obesity was associated with dysregulated soluble factor production. Sequential depletion of plasma MVs and EXOs did not modify plasma levels of these molecules, with the exception of Macrophage Migration Inhibitory Factor (MIF). Half of plasma MIF circulated within MVs, and this MV secretory pathway was conserved over different MIF-producing cells. Although MV-associated MIF triggered rapid ERK1/2 activation in macrophages, these functional MV-MIF effects specifically relied on MIF tautomerase activity. CONCLUSION: Our results emphasize the importance of reconsidering MIF-metabolic actions with regard to its MV-associated form and opening new EV-based strategies for therapeutic MIF approaches.

Extracellular vesicles and cardiovascular disease therapy.

Cardiovascular disease (CVD) constitutes one of the leading causes of mortality worldwide, therefore representing a major public health concern. Despite recent advances in the treatment of patients with acute myocardial infarction (AMI), such as bypass surgery or percutaneous coronary intervention, pathological cardiac remodeling often predisposes survivors to fatal heart failure. In this context, the proven efficacy of stem cell-regenerative therapies constitutes a promising therapeutic perspective with is nevertheless slow down by safety and ethical concerns. Recent studies have underscored the capacity of stem cell-derived extracellular vesicles (EV) to recapitulate the regenerative properties of their parental cells therefore offering a therapeutic alternative to cell therapy in cardiovascular regenerative medicine. In this article, we review the functional relevance of using stem cell-derived EV as therapeutically agents and detail the identified molecular pathways that they used to exert their effects. We also discuss the advantages of using such an acellular regenerative therapy, in regard with parental stem cells, and address the limitations, which would need to be resolved, before their clinical translation.