Targeting Nestin+ hepatic stellate cells ameliorates liver fibrosis by facilitating TβRI degradation

Abstract

Background & aims: Liver fibrosis is a wound healing response that arises from various aetiologies. The intermediate filament protein Nestin has been reported to participate in maintaining tissue homeostasis during wound healing responses. However, little is known about the role Nestin plays in liver fibrosis. This study investigated the function and precise regulatory network of Nestin during liver fibrosis.

Methods: Nestin expression was assessed via immunostaining and quantitative real-time PCR (qPCR) in fibrotic/cirrhotic samples. The induction of Nestin expression by transforming growth factor beta (TGFβ)-Smad2/3 signalling was investigated through luciferase reporter assays. The functional role of Nestin in hepatic stellate cells (HSCs) was investigated by examining the pathway activity of profibrogenic TGFβ-Smad2/3 signalling and degradation of TGFβ receptor I (TβRI) after interfering with Nestin. The in vivo effects of knocking down Nestin were examined with an adeno-associated virus vector (serotype 6, AAV6) carrying short-hairpin RNA targeting Nestin in fibrotic mouse models.

Results: Nestin was mainly expressed in activated HSCs and increased with the progression of liver fibrosis. The profibrogenic pathway TGFβ-Smad2/3 induced Nestin expression directly. Knocking down Nestin promoted caveolin 1-mediated TβRI degradation, resulting in TGFβ-Smad2/3 pathway impairment and reduced fibrosis marker expression in HSCs. In AAV6-treated murine fibrotic models, knocking down Nestin resulted in decreased levels of inflammatory infiltration, hepatocellular damage, and a reduced degree of fibrosis.

Conclusion: The expression of Nestin in HSCs was induced by TGFβ and positively correlated with the degree of liver fibrosis. Knockdown of Nestin decreased activation of the TGFβ pathway and alleviated liver fibrosis both in vitro and in vivo. Our data demonstrate a novel role of Nestin in controlling HSC activation in liver fibrosis.

Lay summary: Liver fibrosis has various aetiologies but represents a common process in chronic liver diseases that is associated with high morbidity and mortality. Herein, we demonstrate that the intermediate filament protein Nestin plays an essential profibrogenic role in liver fibrosis by forming a positive feedback loop with the TGFβ-Smad2/3 pathway, providing a potential therapeutic target for the treatment of liver fibrosis.

Keywords: AAV6; Cav-1; Liver fibrosis; Nestin; TβRI degradation.