Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration

Abstract

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl 4 -induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FNintegrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.

Pathogenesis of Nonalcoholic Steatohepatitis

“…Exosomes are small membrane vesicles that signal stress to distant cells. Lipotoxic hepatocytes 51 , particularly those committed to apoptosis 52 , increase release of exosomes that signal other hepatocytes 53 , Kupffer cells 52 , hepatic stellate cells, 54 and sinusoidal endothelial cells 50 . Through this process, dying hepatocytes induce an integrated regenerative response that assures their replacement 53 .…”

Hepatic sinusoids in liver injury, inflammation, and fibrosis: new pathophysiological insights

“…While HSC communicate with SEC via exosomes, the same seems to be true vice versa. Wang et al [77] published a study showing that SEC-derived exosomes containing SK1 regulate HSC signaling and migration through Fibronectin-integrin-dependent exosome adherence and dynamin-2-dependent exosome internalization. In more detail, SK1 has been shown to be an SEC-derived exosome protein activated by FGF-2, which has been known to be important in the release of exosomes in liver fibrosis.…”

“…Exosomes overexpressing SK1 contained more S1P, an HSC chemotactic factor, and led to HSC migration via AKT activation in the target cell. Adhesion and endocytosis have been shown to be mediated through fibronectin (on exosomes) -integrin (on HSC) interaction and dynamin-2, respectively [77]. Thereby, Wang et al not only highlighted the potential role of exosomes in HSC-SEC crosstalk and liver fibrogenesis, they also added important knowledge about how these vesicles may interact with their target cells.…”

Exosome: An Emerging Participant in the Development of Liver Disease

“…They can be secreted and ingested by a wide variety of cells (8,14). After the exosomes arrive at their destination, the information is transmitted from the exosomes to the recipient cells in 1 of 3 ways: 1) through a ligand-receptor interaction on the cell membrane surface (15); 2) through the fusion of the exosome membrane with the recipient cell membrane (16); and 3) through an internalization of exosomes via cellular endocytosis (17) (Figure 1). Exosomes are released by the donor cells and then captured by the recipient cells.…”

“…Pathogenesis of the liver involves various types of liver cells; of these, HSCs are the key link in the occurrence and development of hepatic fibrosis. 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).…”