Ethanol-stimulated differentiated functions of human or mouse hepatic stellate cells are mediated by connective tissue growth factor

Abstract

Background and Aims-Connective tissue growth factor (CTGF) expression is intimately associated with hepatic fibrotic pathophysiology. In this study, CTGF production and action was investigated in ethanol-treated mouse primary hepatic stellate cells (HSC) or human LX-2 cells.

Fibrogenic Signaling Is Suppressed in Hepatic Stellate Cells through Targeting of Connective Tissue Growth Factor (CCN2) by Cellular or Exosomal MicroRNA-199a-5p

“…Primary HSCs were isolated, essentially as we have previously described, 33 by buoyant-density centrifugation from normal male Swiss Webster mice (6 to 8 weeks), and spent medium from the cultured cells was replaced with fresh Dulbecco's modified Eagle's medium, F12, and 10% fetal bovine serum (FBS) medium on D1 and every other day as needed. HSCs were split 1:3 every 5 days and used at passages 0 to 6 (P0 to P6).…”

“…Our previous studies found that, when isolated from normal (nonfibrotic) animals, cells studied within 24 hours of brief culture do not exhibit characteristics typical of activated cells (eg, a-SMA, CCN2) and thereafter gradually transition to a highly activated phenotype during the ensuing 7 to 20 days of culture. 10,22,23,33,34 In this study, CCN2 and miR-199a-5p expression were measured in primary HSCs until D20 of culture. In some experiments, triplicate wells of cells in serum-free medium were incubated for up to 48 hours in the presence of 0 to 3 ng/mL of transforming growth factor (TGF)-b1.…”

“…Human LX-2 cells (a kind gift from Dr. Scott Friedman, Icahn School of Medicine at Mount Sinai, New York, NY) were maintained in culture as described. 33 …”

“…33 LX-2 cells were co-transfected with miR-199a-5p mimic and either the parental lentivector or the vector containing the wild-type CCN2 3 0 -UTR (see above). Cells were also transfected with pRL-CMV vector to control for transfection efficiency.…”

Transforming Growth Factor-β (TGF-β)-mediated Connective Tissue Growth Factor (CTGF) Expression in Hepatic Stellate Cells Requires Stat3 Signaling Activation

“…Expression of CTGF in HSCs is up-regulated by numerous profibrotic factors, including TGF-␤ (4,18,19), endothelin-1 (20), PDGF-BB, acetaldehyde (21), ethanol (22), and the Th2 cytokine IL-13 (23,24), with TGF-␤ being the most important trigger as elevated TGF-␤ levels are observed in almost all fibrotic liver diseases, irrespective of etiology. Although in vitro experiments have shown that TGF-␤ plays only a minor role in CTGF production in quiescent HSCs (20,23,24), activated HSCs as well as established HSC cell lines secrete high levels of CTGF in response to TGF-␤ treatment (22,23).…”