We have recently demonstrated that during lipotoxicity, hepatocytes release extracellular vesicles (EVs) enriched in miRNAs (Science Sig. Oct 2013). Our aim is to investigate if extracellular vesicles
released by hepatocytes during lipotoxicity may modulate hepatic HSC phenotype by delivering specific microRNAs. Methods. Human hepatoma cells (HepG2), and primary mouse hepatocytes were exposed to the saturated free fatty acid (FFA) palmitic acid for up to 24 hrs. EVs and EV-free supernatant were isolated from cell-free supernatants by ultracentrifuga-tion and quantitated by flow cytometry. HSC chemotaxis and chemokinesis were assessed by Boyden’s chamber and wound healing assay, respectively. HSC proliferation was assessed by BrDu-FITC staining and quantitation FK228 manufacturer of pro-fibrogenic transcripts was performed for cell activation. EVs internalization and delivery of miRNAs into HSC was addressed by immuno-fluorescence. Specific PPAR-γ-targeting miRNAs identified JQ1 research buy and quantified in EVs and HSCs by qPCR. Depletion of miRNAs from EVs was achieved by anti-miRNA and specific siRNA on maternal cells. A functional analysis of miRNA was assessed by miRNA mimics. Results. Hepatocyte-derived EVs released during
lipotoxicity are efficiently internalized by HSCs resulting in their activation, as shown by marked up-regulation of pro-fibrogenic genes, such as Collagen-I, α-SMA and TIMP-2, proliferation (EVs vs. EVs-free supernatant, p<0.04), chemo-taxis (EVs MCE公司 vs. EV-free supernatant, p<0.001) and chemokinesis (EVs vs. EVs-free supernatant, p<0.002), mainly after 16-24 hrs. These changes were associated with suppression of PPAR-γ expression in HSC. EVs internalization results in delivery of their miRNA content into HSCs. Lipotoxic hepatocyte-derived EVs miRNA content included various miRNAs that are known inhibitors of PPAR-γ expression with miR-128a being the most effective. Further loss- and gain-of-function studies identified miR-128a as a central modulator of the
effects of EVs on PPAR-γ inhibition and HSC activation. Conclusion. Our study demonstrates that EVs released by hepatocytes during lipotoxicity are critical signals that contribute to HSC activation in a process involving delivery of specific miRNAs and modulation of PPAR-γ expression. These results uncover a novel miRNA-regulated pathway committing HSC activation during lipotoxicity and have important implications for development of therapeutic strategies for patients with NAFLD. Disclosures: Akiko Eguchi – Grant/Research Support: Gilead The following people have nothing to disclose: Davide Povero, Nadia Panera, Anna Alisi, Valerio Nobili, Ariel E. Feldstein Background/Aims: Hepatic stellate cell (HSC) activation is required for fibrogenesis therefore understanding mechanisms governing HSC activation are important.