Exosomes mediate intercellular transfer of pro-fibrogenic connective tissue growth factor (CCN2) between hepatic stellate cells, the principal fibrotic cells in the liver

Abstract

Background: Fibrogenic pathways in the liver are principally regulated by hepatic stellate cells (HSC), which produce and respond to fibrotic mediators such as connective tissue growth factor (CCN2). The aim of this study was to determine whether CCN2 is shuttled between HSC in membranous nanovesicles, or "exosomes."

Methods: Exosomes were incubated with HSC after isolation from conditioned medium of control or CCN2-green fluorescent protein (GFP)-transfected primary mouse HSC or human LX-2 HSC. Some exosomes were stained fluorescently with PKH26. HSC co-culture experiments were performed in the presence of GW4869 exosome inhibitor. CCN2 or CCN2-GFP were evaluated by quantitative real-time polymerase chain reaction or Western blot.

Results: HSC-derived exosomes contained CCN2 or CCN2 mRNA, each of which increased in concentration during HSC activation or after transfection of HSC with CCN2-GFP. Exosomes, stained with either PKH26 or purified from CCN2-GFP-transfected cells, were taken up by activated or quiescent HSC resulting in CCN2-GFP delivery, as shown by their direct addition to recipient cells or by the GW4869-dependency of donor HSC.

Conclusion: CCN2 is packaged into secreted, nano-sized exosomes that mediate its intercellular transfer between HSC. Exosomal CCN2 may amplify or fine tune fibrogenic signaling and, in conjunction with other exosome constituents, may have utility as a noninvasive biomarker to assess hepatic fibrosis.

Figure 1. CCN2 mRNA and protein are present in mouse HSC exosomes

(A) Quantitative RT-PCR of CCN2 mRNA in exosomes isolated from primary mouse HSC on Day 3-14 of culture. Data are from three independent experiments and expressed as mean ± s.e.m. *P<0.05 as determined by Student’s t-test using SIGMA PLOT 11.0 software (SPSS Inc., Chicago, IL). (B) Western blot for CCN2 in exosomes isolated from 24-hour conditioned medium from individual T-75 flasks containing primary mouse HSC on days 3-9 of culture (5 g total exosomal protein/lane). (C) CCN2 Western blot of exosomes isolated from P5 mouse HSC. Data are representative of three independent determinations.

Figure 2. Uptake of exosomal CCN2-GFP by mouse HSC or human LX-2 cells

(A) Uptake of PKH26-labeled HSC-derived exosomes by activated HSC after 24 hours. PKH26 is in red and DAPI nuclear stain is in blue. Data are representative of five independent experiments. (B) Quantitative RT-PCR of GFP mRNA in donor HSC transfected with a CCN2-GFP fusion vector (left), exosomes isolated from CCN2-GFP-transected HSC (center) or recipient HSC after incubation with the exosomes for 24 hours (right). (C) Western blotting with anti-GFP to detect CCN2-GFP in lysates of CCN2-GFP-transfected donor HSC (left), CCN2-GFP in their secreted exosomes (center), or CCN2-GFP or CD9-GFP in lysates of recipient cells incubated with CCN2-GFP- or CD9-GFP-containing exosomes for 2 or 6 hours in the presence of absence of cylcoheximide (right). (D) Uptake by LX-2 cells of PKH26-labeled LX-2 cell-derived exosomes. Data are representative of 4 independent experiments. (E) qRT-PCR for GFP mRNA in LX-2 cells incubated for 2-24 hours with exosomes isolated from CCN2-GFP-transected LX-2 cells. (F) Western blotting with anti-GFP to detect CCN2-GFP in lysates of recipient LX-2 cells incubated for 2-24 hours with CCN2-GFP-containing LX-2 cell-derived exosomes.

Figure 3. Exosomal delivery of CCN2-GFP to Day 2 mouse HSC

PKH26 fluorescence and CCN2, GFP or α-SMA immunofluorescence in Day 2 HSC receiving no exosomes, exosomes from control activated HSC, or exosomes from CCN2-GFP-transfected activated HSC.

Figure 4. Exosomal communication between neighboring human LX-2 cells

(A) Direct GFP fluorescence in donor LX-2 cells over-expressing CCN2-GFP or in co-cultured recipient LX-2 cells incubated in the presence or absence of 10 M GW4869 for 48 hours. (B) Quantification of total GFP-positive recipient cells in (A) assessed in triplicate wells from 3 independent experiments **p< 0.001.