Extracellular Vesicles From Steatotic Hepatocytes Provoke Pro-Fibrotic Responses in Stellate Cells.
Background and aims
High caloric dietary intake is associated with hepatic steatosis and chronic hepatocyte damage leading ultimately to liver fibrosis and cirrhosis with organ failure. Although the pathophysiologic process orchestrating liver fibrosis is not completely clarified, pivotal steps are the activation and transdifferentiation of hepatic stellate cells. In this study, we aim to assess the direct interplay between hepatocytes and hepatic stellate cells under normal and steatotic conditions and hypothesize that extracellular vesicles (EV) isolated from hepatocytes can directly manipulate the phenotype of stellate cells.
Methods
By high speed centrifugation, EV were isolated from conditioned media of the hepatocellular carcinoma cell line HepG2, under baseline conditions (C-EV) or after induction of steatosis by linoleic and oleic acid for 24 hours (FA-EV). Migration of the stellate cell line TWNT4 towards respective EV as well as sera of NASH patients was investigated using Boyden chambers. TWNT4 phenotype alterations after incubation with EV was determined by qPCR, western blotting and immunofluorescence staining.
Results
HepG2 cells released more EV after treatment with fatty acids. Chemotactic migration of TWNT4 cells was increased specifically towards FA-EV. Prolonged incubation of TWNT4 cells with FA-EV induce expression of proliferation markers and a myofibroblast-like phenotype. Whereas the expression of the collagen type 1 1 gene did not change after FA-EV-treatment, expression of the myofibroblast markers e.g. -smooth muscle cell actin and TIMP1 were significantly increased.
Conclusion
We concluded that EV from steatotic HepG2 cells can influence the behavior and phenotype of TWNT4 cells as well as the expression of remodeling markers and guides directed migration. These findings imply EV as operational, intercellular communicators in the pathophysiology of steatosis associated liver fibrosis.
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Posted 22 Sep, 2020
On 14 Oct, 2020
Received 13 Oct, 2020
Received 13 Oct, 2020
Invitations sent on 28 Sep, 2020
On 28 Sep, 2020
On 28 Sep, 2020
On 22 Sep, 2020
On 21 Sep, 2020
On 18 Sep, 2020
On 16 Sep, 2020
Extracellular Vesicles From Steatotic Hepatocytes Provoke Pro-Fibrotic Responses in Stellate Cells.
Posted 22 Sep, 2020
On 14 Oct, 2020
Received 13 Oct, 2020
Received 13 Oct, 2020
Invitations sent on 28 Sep, 2020
On 28 Sep, 2020
On 28 Sep, 2020
On 22 Sep, 2020
On 21 Sep, 2020
On 18 Sep, 2020
On 16 Sep, 2020
Background and aims
High caloric dietary intake is associated with hepatic steatosis and chronic hepatocyte damage leading ultimately to liver fibrosis and cirrhosis with organ failure. Although the pathophysiologic process orchestrating liver fibrosis is not completely clarified, pivotal steps are the activation and transdifferentiation of hepatic stellate cells. In this study, we aim to assess the direct interplay between hepatocytes and hepatic stellate cells under normal and steatotic conditions and hypothesize that extracellular vesicles (EV) isolated from hepatocytes can directly manipulate the phenotype of stellate cells.
Methods
By high speed centrifugation, EV were isolated from conditioned media of the hepatocellular carcinoma cell line HepG2, under baseline conditions (C-EV) or after induction of steatosis by linoleic and oleic acid for 24 hours (FA-EV). Migration of the stellate cell line TWNT4 towards respective EV as well as sera of NASH patients was investigated using Boyden chambers. TWNT4 phenotype alterations after incubation with EV was determined by qPCR, western blotting and immunofluorescence staining.
Results
HepG2 cells released more EV after treatment with fatty acids. Chemotactic migration of TWNT4 cells was increased specifically towards FA-EV. Prolonged incubation of TWNT4 cells with FA-EV induce expression of proliferation markers and a myofibroblast-like phenotype. Whereas the expression of the collagen type 1 1 gene did not change after FA-EV-treatment, expression of the myofibroblast markers e.g. -smooth muscle cell actin and TIMP1 were significantly increased.
Conclusion
We concluded that EV from steatotic HepG2 cells can influence the behavior and phenotype of TWNT4 cells as well as the expression of remodeling markers and guides directed migration. These findings imply EV as operational, intercellular communicators in the pathophysiology of steatosis associated liver fibrosis.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6