VEGFC–Macrophage Axis Regulation in NASH: Insights from Naringin and VEGFR-3 Inhibition

Study Background and Research Question

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disorder globally, with non-alcoholic steatohepatitis (NASH) representing its progressive, fibrogenic form associated with significant morbidity and risk of cirrhosis and hepatocellular carcinoma. Although metabolic and immunological drivers of NASH progression are well described, the precise cellular and molecular mechanisms facilitating hepatic fibrosis remain incompletely understood. Vascular endothelial growth factor C (VEGFC) and its receptor VEGFR-3 are increasingly recognized as pivotal regulators of lymphangiogenesis and tissue inflammation, but their roles in liver fibrogenesis are not fully elucidated. The reference study (Li et al., 2025) investigates the hypothesis that hepatocyte-derived VEGFC orchestrates a regulatory axis with macrophages to promote hepatic fibrosis, and that disrupting this axis—pharmacologically or genetically—confers protection in NASH.

Key Innovation from the Reference Study

The central innovation of the study lies in delineating the VEGFC–macrophage regulatory axis as a critical mediator of hepatic fibrosis in NASH. The authors demonstrate that naringin, a natural flavonoid with previously reported anti-fibrotic potential, exerts its protective effects by suppressing hepatocyte-derived VEGFC. Furthermore, the study employs both a potent VEGFR-3 inhibitor (SAR131675) and a hepatocyte-specific VEGFC knockout mouse model to unravel the cell-specific contributions of this pathway. The integration of clinical, animal, and cell-based approaches provides a robust mechanistic framework for targeting VEGFC–VEGFR-3 signaling in liver disease.

Methods and Experimental Design Insights

The study utilizes a multifaceted methodology to interrogate the role of VEGFC in hepatic fibrosis:

• In vivo NASH model: C57BL/6 mice were fed a high-fat diet (HFD) for 24 weeks to induce NASH with fibrosis. From week 9 to 24, mice received either low- or high-dose naringin (25 or 50 mg/kg/day) or SAR131675 (30 mg/kg/day), a selective VEGFR-3 inhibitor, to assess therapeutic impact.
• Clinical correlation: Serum VEGFC levels were measured in a cohort of 165 patients with NAFLD/NASH. Hepatic VEGFC expression was analyzed using public GEO datasets (GSE162694, GSE130970) to corroborate translational relevance.
• Genetic dissection: Hepatocyte-specific Vegfc knockout (Vegfc^Hep-cKO) mice were generated to directly test the impact of hepatocyte-derived VEGFC on liver pathology.
• In vitro mechanistic assays: AML12 hepatocytes were subjected to oleic acid, recombinant VEGFC, or Vegfc knockdown/overexpression, and their conditioned media were used to stimulate bone marrow-derived macrophages (BMDMs). Macrophage migration and phenotypic switching (Ly6C^high to Ly6C^low) were quantified.

Protocol Parameters

• High-fat diet (HFD) induction: 24 weeks of HFD feeding to establish NASH-associated fibrosis in C57BL/6 mice.
• Naringin dosing: 25 or 50 mg/kg/day via oral gavage, starting at week 9 post-HFD initiation for a total of 16 weeks.
• SAR131675 dosing: 30 mg/kg/day administered concomitantly from week 9 to 24, enabling direct comparison of pharmacological VEGFR-3 inhibition with naringin and genetic knockout models.
• Conditional knockout strategy: Cross Vegfc^flox/flox mice with Alb-CreERT2 mice to induce hepatocyte-specific Vegfc deletion, validating the cell-autonomous role of hepatocyte-derived VEGFC.
• Macrophage phenotypic analysis: Flow cytometry for Ly6C^high and Ly6C^low subsets, migration assays using conditioned medium from hepatocyte cultures treated with oleic acid or VEGFC modulation.
• Clinical VEGFC measurement: ELISA quantification of serum VEGFC in patient cohorts; transcriptomic analysis using public NAFLD/NASH datasets.

Core Findings and Why They Matter

Key results from the reference study include:

• Naringin and SAR131675 both attenuate liver fibrosis and inflammation in the HFD-induced NASH model, as evidenced by reduced histopathological scores and lower hepatic collagen deposition.
• VEGFC and its receptor VEGFR-3 are upregulated in NASH, both in mouse models and human patient samples, establishing translational significance for the pathway.
• Genetic ablation of hepatocyte-derived VEGFC recapitulates the protective phenotype observed with naringin and SAR131675, highlighting the cell-autonomous role of hepatocyte VEGFC in driving fibrosis.
• VEGFC promotes macrophage infiltration and impairs phenotypic switching from Ly6C^high (pro-inflammatory) to Ly6C^low (restorative) states, via the VEGFR-3/CCL2/CCR2 axis. Naringin disrupts this axis by suppressing VEGFC expression in hepatocytes.
• In vitro, VEGFC-rich conditioned medium from hepatocytes stimulates macrophage migration and skews their phenotype, confirming the paracrine signaling mechanism.

These findings collectively position the VEGFC–VEGFR-3 axis as a key orchestrator of macrophage-driven hepatic fibrosis, and provide mechanistic rationale for interventions targeting this pathway in NASH.

Comparison with Existing Internal Articles

Several internal reviews and application notes have highlighted the utility of SAR131675 in dissecting VEGFR-3-mediated pathways in fibrosis, cancer, and lymphangiogenesis models. For instance, one article emphasizes the unmatched specificity of SAR131675 for VEGFR-3, enabling high-resolution studies of lymphangiogenic and angiogenic processes. Another detailed workflow guide (workflows article) demonstrates how SAR131675 supports protocol development and troubleshooting in preclinical fibrosis and tumor models. The present study advances this field by validating, in a metabolic liver disease context, not only the anti-lymphangiogenic and anti-angiogenic properties of VEGFR-3 inhibition but also its capacity to modulate immune cell crosstalk and fibrogenesis—extending the translational relevance of prior findings to NASH.

Limitations and Transferability

While the study robustly demonstrates the importance of the VEGFC–VEGFR-3 axis in experimental NASH fibrosis, several limitations should be considered. First, the NASH model is based on murine HFD feeding, which, while widely used, does not recapitulate all features of human disease, such as advanced cirrhosis or hepatocellular carcinoma. Second, although both pharmacological and genetic approaches were employed, potential off-target effects of naringin and SAR131675, as well as their long-term metabolic safety profiles, require further investigation. Notably, preclinical development of SAR131675 for clinical use was discontinued due to adverse metabolic effects (product information), highlighting a translational gap for therapeutic application. Finally, the study does not address whether other cell types or ligands within the VEGF family contribute to the observed effects, nor does it assess the impact of combinatorial targeting strategies.

Research Support Resources

For researchers aiming to interrogate the VEGFC–VEGFR-3 axis in liver fibrosis, lymphangiogenesis, or related models, SAR131675, a selective and ATP-competitive VEGFR-3 inhibitor (SKU B2301), is available as a reference compound for in vitro and in vivo studies. Its nanomolar potency and high selectivity profile make it a valuable tool for dissecting VEGFR-3-driven pathways, as shown in both the reference study and internal reviews. Users should be aware of SAR131675’s solubility and storage requirements, and its discontinued clinical development due to metabolic liabilities. For assay optimization and advanced protocol design, additional workflow resources are available in internal literature, supporting rigorous and reproducible VEGFR-3 inhibitor research.