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Fibrosis Disease: From Mechanisms and Therapeutic Targets to Animal Models

Scientist on March 3, 2025
in Discovery & Innovation, Research Services

This blog post was written by WuXi AppTec Discovery Services, a service provider that delivers preclinical discoveries through world class capabilities, directed by teams with a reputation for collaboration and scientific excellence. Their services are available on the Scientist.com marketplace.

Fig.1. Tissular diversity of fibrosis1

Fibrosis is a pathological process characterized by the excessive deposition of extracellular matrix (ECM) within an organ, leading to significant damage to its structure and function, which can potentially impact multiple organs such as the lungs, liver, heart, kidneys and skin (Fig.1). Notable examples of fibrotic diseases include idiopathic pulmonary fibrosis (IPF), liver cirrhosis, cystic fibrosis (CF) and myelofibrosis1,2 , ultimately causing organ failure and death if left untreated.

Mechanism

Fig.2. The etiology of fibrosis in different tissues or organs2

Fibrosis results from the abnormal proliferation of connective tissue due to factors such as infections, alcohol abuse, environmental exposures and genetic mutations. This leads to the disruption of normal tissue architecture and an uncontrolled wound-healing response. Fibrosis is a pathological process that begins with upregulating inflammatory mediators and recruiting neutrophils, eosinophils and macrophages to the damaged site to clear debris and necrotic areas. Subsequently, fibrogenic cytokines in the microenvironment, such as fibroblast growth factors (FGFs) and platelet-derived growth factors (PDGFs), transform fibroblasts and other mesenchymal cells into myofibroblasts, which secrete ECM components. Normally, activated myofibroblasts are cleared from the wound site through apoptosis once the injury is repaired; however, in fibrosis, these myofibroblasts remain persistently active, causing ECM excessive deposition, which stiffens the tissue, reduces oxygen availability and causes further cellular damage. This is exacerbated by dysfunctional endothelial cells and disrupted intercellular interactions, such as abnormal angiogenesis with the specific characteristics of fibrosis varying depending on the organ involved and its unique tissue microenvironment (Fig. 2). Despite extensive studies, the mechanisms driving organ-specific fibrosis have not been fully elucidated, posing challenges for developing antifibrotic drugs.2

Therapeutic Targets

The development and progression of fibrosis involve the abnormal activation of specific cells and signaling pathways, making them potential targets for therapeutic intervention in fibrotic diseases.

Abnormal Cell Types
Fibrosis is the result of complex interactions between different cell types. In many fibrotic conditions, myofibroblasts play a central role in producing ECM through secretion and contraction. Additionally, even cells like basal and apoptotic secretory epithelial cells can contribute to the activation of fibroblasts, further promoting the fibrotic process. Endothelial cell damage leading to metabolic disarray can cause abnormal angiogenesis. Immune cells like T lymphocytes, macrophages and neutrophils play a significant role in the development of fibrosis by activating fibroblasts through the release of various cytokines and growth factors, contributing to the progression of diseases such as nonalcoholic steatohepatitis (NASH), IPF and chronic kidney disease (CKD), where abnormal immune cell behavior is a key factor in the fibrotic process.1

Abnormal Signaling Pathways
The abnormality of the cells mentioned above is associated with overactivation of multiple signaling pathways in the microenvironment. For example, the transforming growth factor-beta (TGF-β) signaling pathway regulates fibrosis through Smad-dependent and independent pathways. The platelet-derived growth factor/receptor (PDGF/PDGFR) signaling pathway promotes cell division by activating downstream signals, while fibroblast growth factor/receptor (FGF/FGFR) signaling impacts multiple pathways including RAS-ERK, PI3K-AKT and JAK/STAT. These abnormally activated pathways contribute to tissue fibrogenesis by regulating the activation, metabolism, inflammation and cross-linking of ECM by myofibroblasts. Other pathways, such as the vascular endothelial growth factor/receptor (VEGF/VEGFR), connective tissue growth factor (CTGF) and WNT/β-catenin also play roles (Figure 3).2

Fig.3. Interactions between growth factors-associated signaling pathways and a summary of related target drugs2

Therapeutic Targets
By targeting these abnormal cells and signaling pathways, scientists have developed various antifibrotic drugs, such as pirfenidone, which targets the TGF-β signaling pathway and inhibits immune cells like macrophages and neutrophils; FGF21 analogs (with therapeutic potential in NASH models); ruxolitinib, targeting the JAK/STAT signaling pathway (used for treating myelofibrosis); and nintedanib, a multi-target tyrosine kinase inhibitor targeting macrophages and lymphocytes. These drugs and targets provide multiple strategies for treating fibrotic diseases. As research advances on these cells and pathways, more potential targets will be applied in the development of anti-fibrotic drugs.3-5

Fibrotic Animal Models

Animal models are crucial for improving our understanding of pathogenesis, enabling researchers to identify therapeutic targets and novel drugs. Simulating fibrotic diseases in animal models offers valuable insights into translating basic discoveries into clinical applications.6-7

WuXi Biology Integrated Platform for Fibrosis Drug Development
Over the past three years, WuXi Biology has supported the submission of more than 40 IND applications for fibrosis-related drugs to the FDA/NMPA. With a repository of over 500 types of animal models, our platform spans a broad spectrum of therapeutic areas, including immunology/inflammation; fibrosis; diseases of the lung, liver and kidney; metabolic disorders; central nervous system disorders; and conditions affecting the skin, eyes and ears. This extensive range of models facilitates fibrosis drug development for clients worldwide (Fig.4).

Fig.4. WuXi Biology Integrated Platform to Support Fibrosis Drug Development
Fig. 5. A. Mouse Models of Bleomycin-Induced Pulmonary Fibrosis; B. Pulmonary Function and Body Weight Measurements of Test Mice

Case Study 1: Mouse Models of Bleomycin-Induced Pulmonary Fibrosis

A bleomycin-induced mouse model is widely used to study pulmonary fibrosis, because bleomycin causes direct damage to alveolar epithelial cells in the lungs and triggers an inflammatory response. This response eventually leads to the development of pulmonary fibrosis, mirroring the pathological features seen in human IPF, making it a relevant model for this condition.6

Fig.6. Total cells, soluble collagen, TIMP-1, lung hydroxyproline content in BALF from bleomycin-treated mice

The WuXi Biology team selected 8-10 week old C57BL/6 mice, injected bleomycin through the trachea and randomly administered nintedanib on day 7. At the conclusion of the experiment, lung tissues and bronchoalveolar lavage fluid (BALF) were harvested to assess pathological features of pulmonary fibrosis (Figure 5A). Employing whole-body plethysmography (WBP) — a non-invasive technique to assess lung function and body weight changes (Figure 5B) — significant mitigation of fibrosis was noted in the nintedanib-treated mice, as evidenced by decreased numbers of fibrotic inflammatory cells, lower soluble collagen levels, a marked drop in tissue inhibitor of metalloproteinases-1 (TIMP-1) levels and improved hydroxyproline levels (a marker of collagen content) in the lung tissue (Figure 6).

Further histological examination of the mouse lung tissues was performed to assess inflammation and fibrosis severity. Masson’s trichrome staining revealed normal lung architecture in the control (sham) group, whereas the bleomycin-induced (model) group exhibited loss of alveolar structures and increased collagen deposition. In the nintedanib (60mpk QD)-treated group, collagen deposition was substantially decreased, and alveolar integrity was preserved. Histological analysis using hematoxylin and eosin (H&E) staining revealed a reduction in inflammatory cell infiltration in the nintedanib-treated group compared to the model group. Additionally, myofibroblast marker α-SMA staining, which was prominently expressed in the model group, showed a significant reduction in the treatment group (Figure 7). These results confirm that the team effectively induced a mouse model of pulmonary fibrosis with bleomycin, and that nintedanib treatment efficaciously mitigated the severity of pulmonary fibrosis.

Fig.7. Analysis of modified Ashcroft score, areas of lung fibrosis and inflammation, and α-SMA positive staining in lung tissues of mouse models
Fig. 8. A. Mouse models with CCl4-induced liver fibrosis; B. Weight changes in mice following CCl4 administration**

Case Study 2: Mouse Models of CCl4-Induced Liver Fibrosis

Carbon tetrachloride (CCl4) is a potent hepatotoxin that induces hepatocellular damage and inflammation, which are precursors to liver fibrosis. This chemical is extensively used as a classical model to elucidate the underlying pathological mechanisms of liver fibrosis and to assess the efficacy of antifibrotic drugs.7

Fig.9. Serum levels of ALT and AST, and hepatic expression of Col1a1, Col3a1, and α-SMA in CCL4-treated mice

The WuXi Biology team implemented a CCl4-induced liver fibrosis model by administering CCl4 intraperitoneally to C57BL/6 mice aged 8-10 weeks, twice weekly. Two weeks post-induction, the mice were stratified based on liver function parameters into various groups and a four-week treatment regimen was commenced with Tropifexor, a selective farnesoid X receptor (FXR) agonist (Figure 8A). Throughout this period, liver function and body weight were meticulously monitored (Figure 8B).

Subsequent liver function tests revealed significant increases in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the CCl4-treated group. These levels were substantially reduced following treatment. Gene and protein expression studies revealed marked increases in type I (Col1a1) and type III (Col3a1) collagen, as well as α-SMA expression in the model group. Post-treatment, these markers significantly decreased, indicating a reduction in fibrosis (Figure 9).

Further histopathological evaluation of liver tissues in the model group showed elevated α-SMA expression, a marker of myofibroblast activation. This expression was notably reduced in the treatment group. Additionally, Picrosirius Red (PSR) staining demonstrated extensive liver fibrosis and abnormal collagen distribution in the model group, with significant alleviation observed after treatment with Tropifexor (Figure 10).

Fig.10. Analysis of α-SMA immunostaining and Picrosirius Red (PSR) staining to assess fibrosis and collagen distribution in liver tissues

Conclusion

Fibrosis, resulting from a multifactorial and complex process, presents substantial unmet clinical needs. Understanding the pathogenesis, identifying therapeutic targets and developing animal models are crucial for creating effective therapeutic strategies for bringing new hopes to patients with fibrotic diseases. WuXi Biology’s integrated platform to support fibrosis drug development offers comprehensive services from drug screening, in vivo/ex vivo analysis, mechanism research to clinical biomarkers. By providing end-to-end support across various disease areas, WuXi Biology empowers global partners to advance their fibrosis drug development efforts effectively.

References
  1. Bhattacharya, M, Ramachandran, P. Nat Immunol 24, 1423 – 1433 (2023).
  2. Zhao, M., Wang, L. et al. Sig Transduct Target Ther 7, 206 (2022).
  3. Marlies Wijsenbeek, Vincent Cottin. The New England Journal of Medicine 383:958-968(2024).
  4. Arif, M., Basu, A. et al. Advanced science (Weinheim, Baden-Wurttemberg, Germany), 10(16), e2207454.
  5. Qian, T., Fujiwara, et al. Gastroenterology, 162(4), 1210 – 1225.
  6. Song, S., Fu, Z. et al. The European respiratory journal, 59(5).
  7. Zhang, K., Zhang, M. X., et al. Military Medical Research, 10(1), 56.
animal models cardiomyopathy colitis crohn’s drug development fibrosis ibd in vivo ipf nafld nash