CDC42 Plays a Crucial Role in the Early Regulation of Hepatic Stellate Cell Activation

Academic Background

Liver fibrosis is a pathological process associated with various liver diseases, which may eventually progress to cirrhosis—a life-threatening condition. Currently, there are no effective treatments to halt the progression of liver fibrosis. Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis. In healthy livers, HSCs are in a quiescent state and primarily involved in retinoid storage. However, in injured livers, HSCs become activated and transform into myofibroblast-like cells, producing large amounts of extracellular matrix, such as collagen and fibronectin, ultimately leading to the development of liver fibrosis and cirrhosis. Therefore, inhibiting the activation of HSCs is considered a potential therapeutic target for liver fibrosis.

The activation of HSCs occurs in two phases: the initiation phase and the perpetuation phase. The initiation phase involves changes in the morphology, gene expression, and phenotype of HSCs, making them responsive to fibrogenic stimuli. The perpetuation phase further promotes and maintains the activation of HSCs through fibrogenic stimuli. However, most current research focuses on the perpetuation phase, while the mechanisms underlying the initiation phase remain poorly understood. Additionally, HSCs that have been activated and subsequently deactivated are more easily reactivated than those that have never been activated. Therefore, understanding the regulatory mechanisms of the initiation phase is crucial for continuously suppressing the activation of HSCs.

Source of the Paper

This research paper, titled “CDC42 is crucial for the early regulation of hepatic stellate cell activation,” was authored by Hideto Yuasa et al. and published in the journal American Journal of Physiology-Cell Physiology (January 28, 2025). The research team hails from multiple institutions, including the Department of Anatomy and Regenerative Biology at Osaka Metropolitan University Graduate School of Medicine, the Research Institute for Light-Induced Acceleration System at Osaka Metropolitan University, and the Department of Hepatology at Osaka Metropolitan University Graduate School of Medicine. The study received funding from several sources, including the Japan Society for the Promotion of Science (JSPS).

Research Workflow and Results

1. Research Workflow

a) Animal Model and Liver Injury Induction

The study first utilized a C57BL/6J mouse model, inducing liver injury through intraperitoneal injection of carbon tetrachloride (CCl4). Mice were euthanized on days 1, 4, and 7 after injection, and liver samples were collected for analysis.

b) Isolation and Culture of Primary Hepatic Stellate Cells (pHSCs)

Primary HSCs were isolated from C57BL/6J mice and cultured in both normal dishes and E-cadherin-coated dishes. Morphological changes in HSCs under different culture conditions were observed using microscopy and immunofluorescence staining.

c) CDC42 Activation and Inhibition Experiments

The study used CDC42 inhibitors (ML141 and ZCL278) and activators (Rho/Rac/CDC42 Activator I) to modulate CDC42 activity in HSCs. Changes in HSC morphology and molecular marker expression following CDC42 activity modulation were analyzed via Western blotting and immunofluorescence staining.

d) Analysis of Human Liver Tissue Samples

The study also analyzed liver tissue samples from 18 patients with liver fibrosis. Transmission electron microscopy (TEM) and three-dimensional reconstruction techniques were employed to observe the morphological characteristics of HSCs at different stages of fibrosis.

2. Key Findings

a) Morphological Changes of HSCs During Liver Injury

The study found that after CCl4 treatment, the cell processes and HSC spines of HSCs shortened, and the cells adopted an oval shape. Subsequently, HSCs differentiated into two activated forms: flattened and complex shapes. During the recovery phase, HSCs regained their quiescent stellate morphology.

b) Role of CDC42 in Maintaining the Morphology of Quiescent HSCs

In vitro experiments revealed that CDC42 activation could maintain the morphological characteristics of quiescent HSCs, while CDC42 inhibition caused HSCs to lose these features. Moreover, CDC42 activation suppressed the expression of markers associated with activated HSCs, such as α-smooth muscle actin (α-SMA).

c) Role of CDC42 in Liver Fibrosis In Vivo

In vivo experiments showed that HSCs in mice treated with the CDC42 inhibitor ML141 lost the morphological characteristics of quiescent HSCs and were unable to revert from the activated state back to the quiescent state. Additionally, HSCs around fibrotic areas in human liver tissues exhibited morphological changes indicative of early activation.

3. Conclusions and Implications

The study concluded that CDC42 is a key regulator of morphological and molecular changes during HSC activation. CDC42 activation can maintain the morphological characteristics of quiescent HSCs and inhibit their activation. This discovery provides a new target for the development of therapeutic agents against liver fibrosis, offering significant scientific value and clinical application potential.

4. Highlights of the Study

• Key Discovery: CDC42 plays a critical regulatory role in the early stages of HSC activation, maintaining the morphological characteristics of quiescent HSCs.
• Methodological Innovation: The study utilized three-dimensional reconstruction techniques and transmission electron microscopy to observe morphological changes in HSCs under different states in detail.
• Application Value: CDC42 serves as a novel target for treating liver fibrosis, providing a theoretical basis for the development of new therapeutic drugs.

Other Valuable Information

The study also found that morphological changes in HSCs around fibrotic areas are closely related to the progression of liver fibrosis. This finding offers new insights for the early diagnosis of liver fibrosis. Furthermore, the study revealed that CDC42 suppresses HSC activation by regulating adhesion between HSCs and hepatocytes. This mechanism provides a new perspective for understanding the occurrence and development of liver fibrosis.

Summary

Through detailed observation of morphological changes in HSCs, this study elucidates the critical role of CDC42 in liver fibrosis. Not only does the research provide new insights into the mechanisms of liver fibrosis, but it also identifies potential targets for the development of therapeutic drugs targeting liver fibrosis. In the future, further investigation into the regulatory mechanisms of CDC42 and its applications in liver fibrosis treatment will hold significant scientific and clinical importance.