Direct Extracellular Matrix Modulation Attenuates Intestinal Fibrosis via a Fibronectin‐Targeted Approach

ABSTRACT Intestinal fibrosis can progress independently of inflammation, driven by a self‐perpetuating cycle of extracellular matrix (ECM)‐myofibroblast interactions. However, due to the lack of reliable therapeutic targets within the ECM, the current strategy predominantly focuses on intracellular aspects of myofibroblasts, neglecting the regulation of the ECM itself. In this study, we first performed matrisomic analysis on human (ileal/colonic) and animal (decellularized/native) intestines, identifying fibronectin as the only ECM component consistently elevated in fibrotic versus normal gut tissues across all conditions. Subsequently, immunofluorescence co‐staining identified fibronectin as the principal structural scaffold of the fibrotic intestinal ECM. Furthermore, fibroblast‐specific Fn1 ablation ameliorates intestinal fibrosis and transforms refractory fibrotic thickening into reversible inflammatory thickening in both innate and adaptive immune‐driven models. Mechanistically, domain‐specific inhibitors (pUR4, polymerization inhibition; R1R2, collagen binding inhibition; ATN161, integrin engagement inhibition), combined with ECM‐mimetic platforms, demonstrated that fibronectin blockade directly inhibited its matrix assembly and impaired subsequent collagen fibrillogenesis—the major deposited component in fibrosis. Additionally, fibronectin‐depleted ECM diminished α5β1 integrin‐mediated mechanotransduction, thereby suppressing fibroblast activation and disrupting the self‐perpetuating cycle of intestinal fibrosis. Thus, fibronectin inhibition directly impedes ECM accumulation and ameliorates intestinal fibrosis, offering a new dimension for therapeutic intervention and an immediately druggable target in fibrotic diseases.