NOX-derived ROS generation drives endothelial-to-mesenchymal transition in human pulmonary endothelial cells exposed to sera from patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive lung disease marked by extracellular matrix deposition, oxidative stress, and profound microvascular remodeling. Endothelial dysfunction, particularly via endothelial-to-mesenchymal transition (EndMT), has been implicated in fibrotic progression but remains insufficiently characterized. In this study, human pulmonary microvascular endothelial cells (HPMECs) were exposed to 5% serum from patients with IPF or healthy donors to model disease-associated vascular alterations. IPF serum stimulated a robust increase in reactive oxygen species (ROS) production and proliferation, concomitant with downregulation of endothelial markers (von Willebrand factor, CD31) and upregulation of mesenchymal markers (α-smooth muscle actin, collagen I), consistent with EndMT induction. Notably, pharmacological inhibition of NADPH oxidase (NOX) with diphenyleneiodonium markedly attenuated ROS generation, phenotypic switching, and junctional disruption observed under IPF serum exposure. Similarly, inhibition of protein kinase C (PKC) by chelerythrine suppressed ROS production and proliferative responses, implicating PKC-dependent pathways in ROS-mediated endothelial injury. Immunofluorescence analyses confirmed structural reorganization, revealing loss of endothelial junctional integrity and accumulation of mesenchymal proteins, both reversed by NOX inhibition. Together, these findings establish IPF serum-derived factors as potent drivers of endothelial oxidative stress and EndMT via NOX- and PKC-dependent mechanisms. Targeting these redox-sensitive pathways may represent a promising therapeutic strategy to mitigate vascular dysfunction, tissue remodeling, and disease progression in IPF.