NTN1 cell-autonomously promotes fibrotic response in HSCs.

Liver fibrosis is a hallmark of progressive liver diseases including metabolic dysfunction-associated steatohepatitis (MASH). Activated hepatic stellate cells (HSCs) are responsible for the excessive synthesis of extracellular matrix(ECM) proteins and collagen deposition in the liver, leading to progressive loss of hepatic functions. However, the nature of paracrine and autocrine signals driving HSC activation and fibrotic response within the liver microenvironment remains incompletely delineated. Here we uncover Netrin-1 (NTN1) as a crucial HSC-derived autocrine factor that promotes HSC activation and liver fibrosis. Hepatic NTN1 mRNA and protein levels were elevated during fibrosis associated with MASH and CCl4-induced liver injury. Hepatic NTN1 overexpression exacerbated diet-induced MASH pathologies and CCl4-induced liver fibrosis. Conversely, HSC-specific conditional ablation of NTN1 alleviated the progression of liver fibrosis. Mechanistic studies using cultured HSCs demonstrated that NTN1 promoted HSC activation in a cell-autonomous manner via a UNC5B and Ca2+-dependent signaling pathway. These findings illustrate the causative role of NTN1-mediated autocrine signaling in driving HSC activation and fibrotic response during liver disease, suggesting potential therapeutic targets for anti-fibrosis therapy. Overall design: To critically assess the role of Netrin-1 in mediating HSC activation and liver fibrosis, we generated mice with HSC-specific ablation of Ntn1 using Lrat-Cre, an established genetic tool for conditional gene inactivation in HSCs, and subjected them to Gan diet feeding for 5 months. We also generated a recombinant adenovirus-associated virus (AAV) vector under the control of thyroxine binding globulin (TBG) promoter that directs liver-specific overexpression of Netrin-1. We transduced mice with control AAV-TBG-Vector or AAV-TBG-Ntn1 and subjected them to Gan diet feeding for 5 months.