Extracellular vesicles from steatotic hepatocytes influence stellate cells in liver fibrosis

Background:

Liver fibrosis, caused by fatty degeneration, is a major cause of liver failure, and is also associated with higher levels of hepatocellular carcinoma. Although the pathophysiologic process leading to liver fibrosis is not completely clarified, stellate cells appear to play an important role. In this study, we have investigated a possible interplay between hepatocytes under normal and steatotic conditions. Our hypothesis was that extracellular vesicles (EV) isolated from hepatocytes could influence the behavior and phenotype of stellate cells.

Methods:

By high speed centrifugation, EV were isolated from the conditioned media of hepatocellular carcinoma cell line HepG2, under baseline conditions (C-EV) or after induction of steatosis by a mixture of linoleic and oleic acid (FA-EV). The EV were incubated with the stellate TWNT4 cell line and expression of matrix remodeling markers was determined by qPCR and western blotting. Migration of TWNT4 cells was investigated using Boyden chambers. Determination of the cell content was done by mass spectrometry.

Results:

The migration of TWNT4 cells towards sera obtained from patients with clinically diagnosed NASH was increased compared to sera from matched controls. Induction of steatosis in HEPG2 cells resulted in increased EV release compared to basal conditions. Chemotactic migration of TWNT4 cells was increased towards both C-EV and FA-EV. TWNT4 cells were incubated with the EV obtained from resting and steatotic HEPG2 cells. Interestingly, when TWNT4 cells were incubated with FA-EV, chemotaxis towards CCL5 was reduced. Measurement of matrix remodeling markers after treatment revealed that the expression of the collagen type 1a1 gene (COL1A1) did not change after EV-treatment, yet RNA levels of matrix metalloprotease 2 (MMP2) were reduced. Likewise, the expression of the markers MMP14, tissue inhibitor of MMP (TIMP1) and platelet-derived growth factor (PDGF) was decreased, albeit only after treatment with FA-EV. Differentiation of stellate cells to myofibroblasts is known to drive fibrosis. Incubation of TWNT4 cells with FA-EV led to a decreased expression of the myofibroblast marker a-smooth muscle cell actin. As well, the co-incubation of TWNT4 cells with FA-EV did not lead to an increase in apoptosis.

Conclusion:

Based on our current observations, we conclud that EV from resting or steatotic HepG2 cells can influence the production of matrix remodeling markers and migration of TWNT4 cells.