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 membraneous nanovesicles, or “exosomes”. Methods Exosomes were incubated with HSC after isolation from conditioned medium of control or CCN2-GFP-transfected primary mouse HSC or human LX-2 HSC. Some exosomes were flourescently stained with PKH26. HSC co-culture experiments were performed in the presence of GW4869 exosome inhibitor. CCN2 or CCN2-GFP were evaluated by qRT-PCR 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. Conclusions CCN2 is packaged into secreted nano-sized exosomes which mediate its intercellular transfer between HSC. Exosomal CCN2 may amplify or fine-tune fibrogenic signaling and may, in conjunction with other exosome constituents, have utility as a noninvasive biomarker to assess hepatic fibrosis.

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

“…11,19,60,61 Whereas most previous studies have focused on these mechanisms as they relate to exosomal pathways in cancer and immune cells, including HCCs, 15,17,18 emerging evidence from this and other laboratories has revealed that fibrogenic cells are also regulated by exosomal networks. 10,22,23,31,34 In addition to revealing that miR-199a-5p targets the CCN2 3 0 -UTR in the same cells as which it is produced, the studies reported here indicate that miR199a-5p is released from HSCs in exosomes and that exosomal miR-199a-5p concentrations reflect those of their producer cells, being expressed at high levels in exosomes from quiescent HSCs and at low levels in exosomes from activated HSCs. We found that delivery of endogenous exosomal miR-199a-5p from quiescent HSCs to activated HSCs resulted in down-regulation of CCN2 3 0 -UTR activity and that exosomes from quiescent HSCs are preferentially targeted to the activated HSC population in vivo in CCl 4 -injured liver and suppress fibrogenic gene expression in activated HSCs in vitro in a miR-199a-5pedependent manner.…”

“…CCN2 expression becomes suppressed in recipient activated HSCs because of the action of exosomal miR-199a-5p and/or miR-214, which directly bind to the CCN2 3 0 -UTR, and exosomal Twist1, which transcriptionally drives production of endogenous miR-199a-5p and/or miR-214 from DNM3os. Exosomes also play a role in delivery of profibrogenic molecules to quiescent or partially activated HSCs from injured hepatocytes 31 or fully activated HSCs 22 (not illustrated).…”

“…Our previous studies found that, when isolated from normal (nonfibrotic) animals, cells studied within 24 hours of brief culture do not exhibit characteristics typical of activated cells (eg, a-SMA, CCN2) and thereafter gradually transition to a highly activated phenotype during the ensuing 7 to 20 days of culture. 10,22,23,33,34 In this study, CCN2 and miR-199a-5p expression were measured in primary HSCs until D20 of culture. In some experiments, triplicate wells of cells in serum-free medium were incubated for up to 48 hours in the presence of 0 to 3 ng/mL of transforming growth factor (TGF)-b1.…”

Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells

“…In the liver, some exosomes that include miRNAs take part in the pathogenesis of liver fibrosis by modulating the epigenetic regulation of HSC (21). Exosome-mediated intercellular transfer of connective tissue growth factor (CCN2) by HSC is also implicated in fine-tuning of liver fibrosis (22). Conversely, exosomes derived from human umbilical cord mesenchymal cells attenuate liver fibrosis in mice by suppressing the Smad signaling pathway (23).…”

Exosome: An Emerging Participant in the Development of Liver Disease

“…LSEC-derived exosomes containing sphingosine kinase 1 (SK1) can adhere to and then enter HSCs after binding to HSCs through the fibronectin/α5β1-integrin complex; afterwards, the exosomes result in an enhanced migration ability of target HSCs via the SK1/S1P pathway, thus participating in liver fibrosis (17). Moreover, simultaneous, with the above-described process, the activated HSCs can secrete exosomes that carry a high level of a connective tissue growth factor (CCN2), which in turn augments the fibrosis signal and activates more HSCs to accelerate the liver fibrosis process through interaction with other components (23). In contrast, quiescent HSCs can release exosomes that deliver Twist1 (a 21-kDa bHLH transcription factor) (24) and miR-214 (25) to activated HSCs.…”