The Role of CTGF in Liver Fibrosis Induced in 3D Human Liver Spheroids

Abstract

Connective tissue growth factor (CTGF) is involved in the regulation of extracellular matrix (ECM) production. Elevated levels of CTGF can be found in plasma from patients with liver fibrosis and in experimental animal models of liver fibrosis, but the exact role of CTGF in, e.g., diet-induced human liver fibrosis is not entirely known. To address this question, we utilized a 3D human liver co-culture spheroid model composed of hepatocytes and non-parenchymal cells, in which fibrosis is induced by TGF-β1, CTGF or free fatty acids (FFA). Treatment of the spheroids with TGF-β1 or FFA increased COL1A1 deposition as well as the expression of TGF-β1 and CTGF. Recombinant CTGF, as well as angiotensin II, caused increased expression and/or production of CTGF, TGF-β1, COL1A1, LOX, and IL-6. In addition, silencing of CTGF reduced both TGF-β1- and FFA-induced COL1A1 deposition. Furthermore, we found that IL-6 induced CTGF, COL1A1 and TGF-β1 production, suggesting that IL-6 is a mediator in the pathway of CTGF-induced fibrosis. Taken together, our data indicate a specific role for CTGF and CTGF downstream signaling pathways for the development of liver inflammation and fibrosis in the human 3D liver spheroid model.

Mechanisms of 5-HT receptor antagonists in the regulation of fibrosis in a 3D human liver spheroid model

“…ATP content was measured on the day of harvest in at least 4 spheroids per condition as previously described 21 . The CellTiter Glo Luminescent Cell Viability Assay Kit (Promega, Madison, WI) was used according to the manufacturer’s instructions.…”

“…Total RNA isolation was performed with QIAzol lysis reagent (Qiagen, Hilden, Germany) using 48 spheroids essentially as previously described 21 . After addition of the lysis chloroform, the mixture was thoroughly mixed and centrifuged.…”

“…The immunohistochemistry was performed essentially as previously described 21 . Spheroids were fixed for 2 h at room temperature in 4% paraformaldehyde.…”

“…In previous studies, we have demonstrated that this human 3D spheroid model when treated with FFA becomes steatotic and develops a fibrotic phenotype with an increased COL1A1 deposition, TGF-β1, CTGF, and CYP2E1 production. We showed that the model can be used to study pathogenesis of NASH and liver fibrosis and for drug screening to treat NASH 20 , 21 . In this study, we used the model to investigate the role of 5-HT, 5-HT signalling pathways, and the 5-HT 2A receptor in the development of FFA-induced liver fibrosis.…”

Liver fibrosis: pathological features, clinical treatment and application of therapeutic nanoagents

“…CTGF is also an important tissue fibrosis-promoting protein, mainly produced by aHSCs, which has been shown to play an important role in cell proliferation, differentiation, migration, adhesion and ECM synthesis. 54 TGF-b1 releases regulatory proteins, such as CTGF. In turn, CTGF regulates the production of TGF-b1 and further activates HSCs in rat models, creating a positive feedback loop.…”