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Research article
Extracellular Vesicles From Steatotic Hepatocytes Provoke Pro-Fibrotic Responses in Stellate Cells.
Rory Koenen
Universiteit Maastricht Cardiovascular Research Institute Maastricht
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9955-9730
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
stellate cell, liver fibrosis, extracellular vesicles, non-alcoholic fatty liver disease
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This preprint is under consideration at Molecular Medicine. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Extracellular Vesicles From Steatotic Hepatocytes Provoke Pro-Fibrotic Responses in Stellate Cells.
Rory Koenen
Universiteit Maastricht Cardiovascular Research Institute Maastricht
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9955-9730
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Revision
Version 1
Posted 22 Sep, 2020
No community comments so far
Editorial decision: Major revision
On 14 Oct, 2020
Review #1 received
Received 13 Oct, 2020
Review #2 received
Received 13 Oct, 2020
Reviewers invited
Invitations sent on 28 Sep, 2020
Reviewer #1 agreed
On 28 Sep, 2020
Reviewer #2 agreed
On 28 Sep, 2020
Editor assigned
On 22 Sep, 2020
Editor invited
On 21 Sep, 2020
Submission checks complete
On 18 Sep, 2020
First submitted
On 16 Sep, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Medical Genetics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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