The aim of this study was to determine whether exosomes contribute to HSC activation during liver fibrosis. Methods/Results:

Exosomes were isolated from human serum and conditional media of TSEC (immortalized mouse liver endothelial cells) by differential ultracentrifugation, and characterized based on size (90-110 nm) and marker (TSG101, LAMP1, CD63 and CD81) characteristics. Incubation of LX2 HSC cell line with human serum derived exosomes was associated with increased AKT phosphorylation (8.3-fold, n=6, p<0.05), increased mRNA levels of smooth muscle actin (1.7fold, n=3, p<0.05), fibronectin (1.8-fold, n=3, p<0.05) and collagen (1.7-fold, n=3, p<0.05), and increased cell migration (2.5-fold, n=3, p<0.05) in Transwell assays. Exosome DNA Damage inhibitor activation of LX2 cells required exosome endocytosis since inhibition of endocytosis with transfection of the dominant negative dynamin GTPase construct Dyn2K44A or by the pharmacological inhibitor, dynasore significantly attenuated exosome-in-duced AKT phosphorylation (72% and 90%, respectively, n=3, p<0.05). Exosome biotinylation studies showed that internalized exosomes target initially to early endosomes and subsequently to lysosomes based on double immunofluorescence FK506 cell line staining using early endosome marker, EEA and lysosome marker, LAMP1 (Pearson coefficients of colocalization= 0.32 and 0.36, respectively, n=5). Western blot analysis

of exo-somes for enrichment of molecules implicated in HSC activation revealed presence of sphingosine kinase 1 (SK1), an enzyme that produces sphingosine-1 phosphate (S1P). Indeed, exo-somes derived from conditioned media of TSEC overexpressing SK1 further increased LX2 cell S1P levels (2-fold, n=6, p<0.05)

and LX-2 migration (2-fold, n=3, p<0.05) suggesting that S1P generated by exosomes may selleck chemicals promote HSC activation. Finally, S1P levels were increased in serum of mice with CCl4 induced liver fibrosis (1.4-fold, n=17, p<0.05) and SK1 mRNA levels were upregulated in human liver cirrhosis patient samples (2.5-fold, n=3, p<0.05). Conclusion: These findings advance our understanding of exosome-mediated HSC activation and identify potential molecular targets for attenuating this process. Disclosures: The following people have nothing to disclose: Ruisi Wang, Sheng Cao, Usman Yaqoob, Thiago de Assuncao, Vijay Shah BACKGROUND: Liver fibrosis is characterized by extensive accumulation of extracellular matrix, mostly Collagen Type I. Bone marrow(BM)-derived fibrocytes, designated as CD45 and Col1a1 expressing cells (CD45+Col1a1+ cells), are recruited to fibrotic liver in response to chronic liver injury. However, the role of fibrocytes in liver fibrosis remains unclear. AIM: The contribution of BM-derived fibrocytes to experimental liver fibrosis was studied in fibrocyte deleted BM chimeric mice, in which fibrocyte death was induced throughout liver injury by overexpression of DTA in CD45+Col1a1+ cells.