Effects of Halofuginone on Fibrosis, Inflammation, and Angiogenesis in a Primary Human Chronic Subdural Hematoma External Membrane Fibroblast Model

Introduction: This study aimed to investigate the antifibrotic, anti-inflammatory, redox-modulating, and antiangiogenic effects of halofuginone in an in vitro model of the chronic subdural hematoma (CSDH) external membrane using primary human fibroblasts and brain microvascular endothelial cells. Methods: Primary fibroblast cultures were generated from residual external membrane and dura mater tissues obtained during routine CSDH surgery (n = 11). To reproduce the inflammatory–fibrotic microenvironment of CSDH, cells were stimulated with TGF-β1 and IL-17A and treated with halofuginone (0.01–0.5 µM). Fibrotic and inflammatory responses were evaluated by qPCR analysis of COL1A1, COL3A1, ACTA2, CTGF, TGFB1, and IL17A. Redox status was assessed by intracellular reactive oxygen species, oxidative stress index, and total antioxidant capacity. Angiogenic activity was quantified by VEGF ELISA. Endothelial permeability was assessed using a FITC–dextran assay, and fibroblast contractility was evaluated using a three-dimensional collagen gel contraction assay. Figures highlight the 0.1 and 0.5 µM concentrations, where effects were most pronounced. Results: Halofuginone suppressed profibrotic gene expression in a dose-dependent manner, with maximal effects at 0.1–0.5 µM, leading to marked reductions in COL1A1, COL3A1, ACTA2, and CTGF. TGFB1 and IL17A transcript levels were decreased by approximately 40–70%. Oxidative stress was attenuated, as indicated by reduced intracellular reactive oxygen species and a progressive decrease in oxidative stress index. In brain microvascular endothelial cells, halofuginone reduced VEGF secretion and endothelial permeability. Additionally, fibroblast-mediated collagen gel contraction was inhibited by approximately 35–62%. Conclusion: Halofuginone concurrently modulates fibrotic, inflammatory, oxidative, and angiogenic pathways involved in CSDH external membrane pathology. These findings support the pharmacological targetability of the external membrane and identify halofuginone as a promising candidate for focused preclinical and adjunct therapeutic strategies.