Interaction between liver sinusoidal endothelial cells and hepatic stellate cells under high fatty acids and high fructose conditions
Embargo Date
2027-11-18
OA Version
Citation
Abstract
BACKGROUND: Fibrosis is a pathological process characterized by excessive accumulation of extracellular matrix (ECM) proteins, such as collagen, in the tissues affected. Liver fibrosis is a progression marker of metabolic-associated fatty liver disease (MAFLD). MAFLD can be induced in mice by feeding them an obesogenic diet, which includes high fat and fructose. HSCs play important roles in retinoid and lipid storage and metabolism, cytokine and growth factor secretion, and potential antigen presentation under quiescent conditions. However, once activated, HSCs differentiate into myofibroblasts and become the main contributor to liver fibrosis due to excessive collagen expression, thereby promoting scar tissue formation.
Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells with multiple functions, including chylomicron clearance, immune cell recruitment, endocytosis, and maintenance of hepatic stellate cells (HSCs) quiescence. However, under chronic liver injury, LSECs convert to a pro-inflammatory, pro-thrombotic, pro-angiogenic, and pro-fibrotic phenotype. Dysfunctional LSECs exhibit elevated levels of various inflammation biomarkers such as interleukins, C-C motif chemokines (CCLs), and C-X-C motif chemokines (CXCLs), as well as signaling molecules like platelet-derived growth factors (PDGFs) and growth arrest-specific six protein (GAS6). These molecules subsequently disrupt liver metabolism and induce activation of HSCs.
The interaction between LSECs and activated HSCs under high-fat and high-fructose conditions exacerbates liver inflammation and fibrosis. Understanding the intricate molecular mechanisms involved in the crosstalk between LSECs and HSCs during NAFLD-associated liver fibrosis is crucial for developing targeted therapeutic strategies. This study aims to investigate how LSECs and HSCs interact under high-fat and high-fructose conditions, shedding light on pathways that contribute to liver fibrosis progression in metabolic disorders. METHOD: For the co-culture study, human liver sinusoidal endothelial cells (hLSECs) were initially treated with fructose (20 mM) or a palmitate/oleate (P/O) (100 µM) for 48 hours in a cell culture medium. Subsequently, the cells were starved and exposed to fructose, P/O, and neutrophil elastase (NE, 10 nM) for an additional day. Post-treatment, hLSECs were harvested for mRNA expression analysis, and the conditioned medium was collected. A wound healing assay was conducted to assess hepatic stellate cells (hHSCs) activation and migration. Confluent plates of hHSCs were scratched with a p1000 pipette tip, followed by overnight treatment with the collected conditioned medium. Images were captured using a Keyence-9000 microscope.
To evaluate the direct effects of fructose and free fatty acids on hHSCs, the cultured hHSCs were first pretreated with fructose (20 mM) or P/O (100 µM) for 72 hours in a cell culture medium. After starvation, the cells were treated with NE (30 nM) for 24 hours. Subsequently, the cells were harvested for further experiments, including a wound healing assay to evaluate hHSCs activation and migration and immunofluorescence (IF) staining to assess vimentin expression. Images were acquired using a Keyence-9000 microscope and analyzed by Image J. RESULT: Our data indicate that, under combined treatment with fructose and palmitate/oleate (P/O), LSECs exhibit significantly increased levels of adhesion molecules (VCAM1, ICAM1, selectin-E), inflammatory cytokines (IL-1b, IL-6, IL-8), STING signaling-related biomarkers (STING, TNFa, IFN-b1), chemokines (CCL2, CCL3, CXCL1, CXCL2, CXCL5, CXCL16), and HSC activating molecules (Gas6, PDGFb, PDGFd).
Furthermore, fructose, fatty acid, and NE treatments, whether individually or combined, significantly induce HSC migration and vimentin expression. NE induces these effects via the PAR2/ROCK signaling pathway. Inhibition of the AXL receptor also demonstrates a protective effect by attenuating fructose, fatty acid, and NE-induced cell migration and vimentin expression.
CONCLUSION: Our data demonstrate that under high-fat and high-fructose exposure conditions, the interaction between LSECs and HSCs involves two main components. Firstly, fructose and fatty acids stimulate the expression and secretion of HSC activators from LSECs, thereby activating HSCs and promoting their migration. Secondly, within this pro-inflammatory environment, LSECs enhance the recruitment of immune cells whose secretions, including NE, in conjunction with fructose and fatty acids, further activate HSCs and induce migration.
Description
2024