Endothelial-to-mesenchymal transition derived fibronectin mediates pulmonary fibrosis in mice following chronic exposure to ozone-oxidized 6PPD.

The release of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylene-diamine (6PPD) and its environmental transformation products poses a significant respiratory health risk. While 6PPD-quinone has been widely studied, the toxicological profiles of other oxidation derivatives remain poorly understood. In the present study, we simulated the atmospheric aging of 6PPD via ozone oxidation (O3-6PPD) and identified N-(1,3-dimethylbutyl)-N'-phenyl-p-quinone-diimine (6QDI) as a major transformation product. Compared to the parent compound 6PPD, O3-6PPD exhibited a reduced hydrodynamic diameter and enhanced cytotoxicity. In male C57BL/6 J mice, sub-chronic inhalation of O3-6PPD at human-relevant exposure levels induced dose-dependent respiratory dysfunction and aberrant lung remodeling, characterized by inflammatory infiltration, collagen deposition, and disrupted microvascular architecture. Integrated transcriptomic analysis revealed coordinated alterations in fibrotic and angiogenic signaling pathways, with fibronectin (Fn1) acting as a central regulator. Mechanistically, O3-6PPD activated TGFβ-dependent endothelial-to-mesenchymal transition (EndMT), resulting in excessive Fn1 deposition. This accumulation drove epithelial dysfunction via cross-talk and accelerated fibrogenesis. These adverse effects were effectively attenuated by blocking TGFβR1 or inhibiting Fn1 binding. Collectively, this study identifies a novel vascular-driven mechanism underlying O3-6PPD-induced pulmonary fibrosis, providing new insights into the health risks of tire-wear-related transformation products and potential vascular-targeted therapeutic strategies.