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. 2016 Jan;64(1):60-8.
doi: 10.1016/j.jhep.2015.07.030. Epub 2015 Aug 5.

Hepatocyte Exosomes Mediate Liver Repair and Regeneration via sphingosine-1-phosphate

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Free PMC article

Hepatocyte Exosomes Mediate Liver Repair and Regeneration via sphingosine-1-phosphate

Hiroyuki Nojima et al. J Hepatol. .
Free PMC article

Abstract

Background & aims: Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury.

Methods: Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo.

Results: Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases.

Conclusions: Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease.

Keywords: Ischemia/reperfusion; Liver injury; Sphingolipids; Sphingosine kinase; Transplantation.

Conflict of interest statement

Conflict of interest

The authors who have taken part in this study declared no conflict of interest with respect to this manuscript.

Figures

Fig. 1
Fig. 1. Characterization of exosomes secreted by hepatocytes
(A) Analysis of exosomes by electron microscopy (bars 100 nm). (B) Size of exosomes isolated from primary hepatocytes (mean: 79 nm). (C) Western blot analysis of cell extracts prepared from primary hepatocytes (C) and exosomes isolated from primary hepatocytes (Ex) shows expression of Tsg101, CD81, CD63, early endosomes (EEA-1), or endoplasmic reticulum (Grp78) and α-actin. Shown are Western blot studies representative for three independent experiments.
Fig. 2
Fig. 2. Hepatocyte-derived exosomes induce hepatocyte proliferation in vitro
Exosomes isolated from primary hepatocytes (A), Kupffer cells (B), or sinusoidal endothelial cells (C), were added to primary hepatocytes and proliferation measured 24 h later by BrdU incorporation. Exosomes from primary hepatocytes dose-dependently increased hepatocyte proliferation. Kupffer cell exosomes reduced hepatocyte proliferation and exosomes from sinusoidal endothelial cells had no effect on proliferation. For all panels, data are mean ± SD with n = 8 (A, B) or n = 6–8 (C) per group. *p <0.05 compared to media control (0 exosomes), #p <0.05 compared to all other groups.
Fig. 3
Fig. 3. Hepatocyte-derived exosomes induce hepatocyte proliferation and liver regeneration in vivo
(A) Mice were injected intravenously with saline (control) or hepatocyte exosomes 24 h and 48 h after I/R. Ischemic lobes were taken for immunohistochemical and quantitative analysis of PCNA and H3-P staining 72 h after reperfusion. Original magnification was 400×. (B) Mice were injected intravenously with saline (control) or hepatocyte exosomes immediately after and 24 h after hepatectomy. Liver samples were taken for immunohistochemical and quantitative analysis of PCNA and H3-P staining 48 h after hepatectomy. Original magnification was 400×. (C) Liver regeneration was determined 48 h after hepatectomy by liver to body weight ratio. For all panels, data are the mean ± SD with n = 3–4 (A) or n = 4 (B, C) *p <0.05 compared to control (0 exosomes) mice. (This figure appears in colour on the web.)
Fig. 4
Fig. 4. Exosomes directly fuse with and are internalized by target hepatocytes
Hepatocyte-derived exosomes were fluorescently labeled with the cell membrane marker, PKH67, prior to incubation with primary hepatocytes for 24 h. After incubation, hepatocytes were washed and counterstained with DAPI to stain the nuclei. Control hepatocytes were treated with PBS vehicle. Shown is a representative result from four independent experiments. (This figure appears in colour on the web.)
Fig. 5
Fig. 5. Hepatocyte-derived exosomes deliver SK2 to target hepatocytes and induce hepatocyte proliferation via generation of S1P
(A) Western blot analysis of hepatocyte-derived exosomes shows expression of neutral ceramidase and SK2 proteins. Shown are Western blot studies representative for four independent experiments. (B) Primary hepatocytes were treated with media (control) or 100 μg/ml hepatocyte exosomes and cellular levels of S1P and ceramide were determined. Ceramide levels in hepatocytes were not changed by treatment with exosomes. (C) Exosomes induced S1P as determined by ELISA and mass spectrometry. The induction of S1P in hepatocytes by exosomes was prevented by pre-incubation with the SK inhibitor SPHK I2 (20 μM). For A–C, data are mean ± SD, n = 4; *p <0.05 compared to control group; #p <0.05 compared to exosome only group. (D) Exosome-induced proliferation of hepatocytes is abrogated by the SK inhibitor, SPHK I2. Cell proliferation was determined via BrdU corporation. Data are mean ± SD, n = 3. *p <0.05 compared to control group (0 exosomes, no SK inhibitor); #p <0.05 compared to exosomes only group. (E) Hepatocyte-derived exosomes deliver SK2 to target hepatocytes. Western blot analysis of SK2 in hepatocytes treated with media (control) or increasing amounts of hepatocyte-derived exosomes. SK2 expression was measured 24 h after treatment and normalized to α-actin and quantitative analysis performed. Data are mean ± SD with n = 4 per group. *p <0.05 compared to control (0 exosomes) hepatocytes. (F) SK activity in target hepatocytes was measured by kinase assays 24 h after treatment with media (control) or 100 μg/ml hepatocyte exosomes. Data are mean ± SD with n = 4 per group. *p <0.05 compared to control hepatocytes.
Fig. 6
Fig. 6. SK2 mediates the proliferative effects of hepatocyte exosomes
(A) Transfection of hepatocytes with siRNA targeting SK2 downregulates SK2 expression, while a control scrambled (scr) siRNA has no effect. Shown are Western blot studies representative of three independent experiments. (B) Exosomes derived from hepatocytes in which SK2 had been knocked down or SK2-knockout (KO) hepatocytes have no proliferative effect on target hepatocytes in vitro. (C) Biochemical inhibition of SK with FSBA prevents the proliferative effects of exosomes on hepatocytes. For all panels, data are the mean ± SD with n = 4; *p <0.05 compared to control group; #p <0.05 compared to scr siRNA group.
Fig. 7
Fig. 7. SK2 mediates the proliferative effects of hepatocyte exosomes in vivo
Mice were injected intravenously with saline (control) or hepatocyte-derived exosomes 24 h and 48 h after I/R. (A) Treatment with exosomes isolated from hepatocytes that had been transfected with a scrambled, control siRNA (scr siRNA) or siRNA targeting SK2 (SK2 siRNA). (B) Treatment with exosomes isolated from wild-type (WT) or SK2-knockout (SK2 KO) hepatocytes. Hepatocyte proliferation was determined by immunohistochemical and quantitative analysis of PCNA and H3-P staining 72 h after reperfusion. Original magnification was 400×. For all panels, data are the mean ± SD with n = 4 (A), 4–5 (B); *p <0.05 compared to control group; #p <0.05 compared to scr siRNA group (A). #p <0.05 compared to hepatocyte-derived exosomes group (B). (This figure appears in colour on the web.)
Fig. 8
Fig. 8. Hepatic I/R injury induces release of exosomes into the serum
(A) Serum exosome levels over a 96 h time course of reperfusion. Data are mean ± SD with n = 7 per group (sham group) and n = 7 per group (I/R groups). *p <0.05 compared to sham control. (B) Characterization of serum exosomes isolated 24 h after I/R injury by electron microscopy (bars, 100 nm). (C) Western blot analysis of control hepatocyte lysates (C) and exosomes isolated from serum 24 h after I/R injury (Ex) for expression of Tsg101, CD81, CD63, early endosomes (EEA-1), endoplasmic reticulum (Grp78), α-actin, SK1, and SK2. (D) Size of exosomes isolated from serum (mean: 74 nm). (E) Exosomes isolated from serum 24 h after I/R injury were added to primary hepatocytes and proliferation measured 24 h later by BrdU incorporation. Data are the mean ± SD with n = 8; *p <0.05 compared to control group (0 exosomes); #p <0.05 compared to all other groups.

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