Adiponectin-enriched liver-derived ECM attenuates fibrogenic features of activated hepatic stellate cells in a multicellular 3D model

Abstract

Adiponectin (ADPN), an adipocytokine expressed in multiple tissues including the liver by quiescent hepatic stellate cells (HSCs) has recognized antifibrotic properties. However, most studies rely on simplified 2D systems that fail to capture the liver microenvironment. This study investigated the antifibrotic effects of ADPN in a multicellular 3D liver microtissue (MT) model. A 2D HSCs culture was first used to confirm activation of HSCs by 2 ng/mL TGF-β and determine the optimal ADPN dose. Five groups were included: non-activated HSCs, activated HSCs, and activated HSCs containing ADPN at 2.5, 5, or 10 µg/mL embedded in human fibrotic liver ECM. Cell viability, migration, and fibrotic marker expression were assessed. Subsequently, 3D model was generated using Huh-7, HUVEC, and LX-2 cells combined with fibrotic ECM-derived microparticles (MPs) with or without ADPN. Cell proliferation, migration, gene/protein expression, and hepatic functional markers were evaluated. In 2D cultures, ADPN reduced viability and migration of activated HSCs and down-regulated α-SMA protein and ACTA-2 , COL1A1 , and FN1 gene expression in a dose-dependent manner. Based on these results, 5 µg/mL ADPN was selected for 3D experiments. ADPN-enriched MTs showed reduced proliferation and migration, reflected by decreased Ki-67 expression, reduced expansion and limited sprouting. This was accompanied by lower α-SMA, collagen I, and vimentin expression; decreased MKI67 , ACTA-2 , COL1A1 , FN1 , and VIM expression; increased PPARG ; and modulation of MMP-1 and MMP-2 . ADPN also enhanced hepatocyte-associated functional markers, including ALB, CYP3A4, and CYP7A1 expression, increased urea production, and reduced AFP levels. ADPN modulates fibrosis by inhibiting HSC activation, proliferation and migration, and ECM remodeling, while improving hepatic function.