Combined HIF-1α blockade and CHIR99021 treatment reverses pulmonary fibrosis via modulation endothelial-to-mesenchymal transition.

Radiation-induced pulmonary fibrosis (RIPF), a serious complication of radiotherapy, is characterized by HIF-1α-dependent endothelial-to-mesenchymal transition (EndMT) and persistent DNA damage. We demonstrate that the dual inhibition of HIF-1α and GSK-3β with 2-methoxyestradiol (2-ME) and CHIR99021 reverses fibrotic alterations in endothelial cells and pulmonary fibroblasts, accompanied by reduced DNA damage foci. In murine RIPF models, the therapy attenuated fibrosis, suppressed fibroblast activation, and preserved vascular structure. Epigenetic profiling revealed a transcriptionally active chromatin state with decreased H3K9 trimethylation and increased H3K9/H3K27 acetylation. HIF-1α deletion in collagen-expressing cells further enhanced CHIR99021-mediated reversal by restoring endothelial identity and inhibiting mesenchymal transition, as shown by lineage tracing and single-cell RNA sequencing. In fibroblasts from patients with idiopathic pulmonary fibrosis, the therapy reduced profibrotic gene expression, proliferation, and matrix remodeling. These findings highlight dual HIF-1α/GSK-3β targeting to reverse fibrosis through coordinated EndMT suppression, DNA repair, and chromatin remodeling.