Journal: The American Journal of Pathology

Article Title: Fibrogenic Signaling Is Suppressed in Hepatic Stellate Cells through Targeting of Connective Tissue Growth Factor (CCN2) by Cellular or Exosomal MicroRNA-199a-5p

doi: 10.1016/j.ajpath.2016.07.011

Figure Lengend Snippet: Exosomal miR-199a-5p dampens fibrogenic signaling in activated hepatic stellate cells (HSCs). A: Nanoparticle tracking analysis of exosomes isolated from the conditioned medium of passage 6 (P6) mouse HSCs that were cultured for 48 hours in serum-free medium. The insets show a typical video still obtained during data collection (left inset) and a Western blot for CD81 of the purified exosome preparation (right inset). B: Transmission electron microscopy of exosomes from quiescent [day 2 (D2)] or activated (P6) HSCs collected from the cells after 48-hour culture in, respectively, medium that contained 1% fetal bovine serum (FBS) (exosome-depleted) or serum-free medium (left column). The outer margins of the exosomes are shown by the white dotted line. Production of exosomes during HSC activation in vitro; cells were maintained for 48 hours in 1% FBS (exosome-depleted) before harvesting the exosomes (right column). C: RT-PCR analysis of miR-199a-5p levels in exosomes purified from D2 HSCs [cultured for 48 hours in medium that contained 1% FBS (exosome depleted)] or P6 HSCs (cultured for 48 hours in serum-free medium), with data normalized to let-7a expression as previously validated.23D: Using the co-culture system previously described,10 recipient P6 mouse HSCs (maintained in medium that contained 10% FBS) were transfected with Fire-Ctx plasmids containing either wild-type or mutant connective tissue growth factor (CCN2) 3′ untranslated region (3′-UTR) (Figure 1) and co-cultured for 24 hours with D1 mouse HSCs, some of which had been pretreated with 10 μmol/L GW4869. The activity of firefly luciferase in lysates of recipient cells, normalized to that of Renilla luciferase, is shown 24 hours after the central divider had been removed from the microwells, as described.10E: Fibrogenic gene expression and protein production in P6 HSCs, with or without transfection of the cells with a miR-199a-5p antagomir for 24 hours in medium that contained 10% FBS, followed by 24-hour serum-starvation and then treatment under serum-free conditions with exosomes (8 μg/mL) purified from D2 HSCs [cultured for 48 hours in medium that contained 1% FBS (exosome depleted)] or P6 HSCs (cultured for 48 hours in serum-free medium). CCN2 isoforms are discussed in Figure 3E. F: PKH26 Exo is hepatic uptake of PKH26-stained exosomes [collected from D2 HSCs that were maintained for 2 days in medium that contained exosome-depleted serum (1%)] 4 hours after i.v. injection in CCl4-treated mice (175 μL in 1325 μL of corn oil/kg, 3 times per week for 2 weeks) (top image). Control (Ctrl) is liver from a CCl4-treated mouse that was not injected with exosomes. Imaging of DAPI-stained isolated liver cells (blue) revealing uptake in vivo of PKH26-stained exosomes (red) by activated HSCs but not hepatocytes (middle and bottom image). Data are typical of 3 independent experiments performed in triplicate. ∗∗P < 0.01, ∗∗∗P < 0.001 versus D3; †††P < 0.001 versus D2; ‡P < 0.05, ‡‡P 0.01, and ‡‡‡P < 0.001 versus Ctrl. Scale bars = 100 nm (B). Col. 1(α1), collagen 1(α1); Hep., hepatocytes; Mut, mutant; RLU, relative luminescence units; WT, wild-type.

Article Snippet: Purified exosomes were evaluated for size and frequency by NanoSight nanoparticle tracking analysis (Malvern Instruments, Westborough, MA).

Techniques: Isolation, Cell Culture, Western Blot, Purification, Transmission Assay, Electron Microscopy, Activation Assay, In Vitro, Reverse Transcription Polymerase Chain Reaction, Expressing, Co-Culture Assay, Transfection, Mutagenesis, Activity Assay, Luciferase, Staining, Injection, Imaging, In Vivo