Abstract Body: Purpose: Endothelial-to-mesenchymal transition (EndMT) is a critical process in the pathogenesis of vascular fibrosis and remodeling cardiovascular diseases. Emerging evidence suggests that endothelial metabolic reprogramming plays a key role in this process. The glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) regulates glycolytic flux and may link metabolism to epigenetic regulation. To investigate this mechanism, we employed a subretinal fibrosis model as a representative of microvascular fibrosis.
Methods: Endothelial lineage tracing using Cdh5-EYFP mice identified EndMT-derived myofibroblasts expressing PFKFB3. Microvascular fibrosis was induced by laser injury in heterozygous Pfkfb3^+/- mice and controls. Fibrotic lesions were evaluated by immunostaining for collagen I and α-SMA. In vitro, endothelial cells were treated with TGF-β2 to induce EndMT, followed by PFKFB3 knockdown. EndMT markers were quantified by qPCR and Western blot, and histone acetylation/lactylation levels were analyzed to assess metabolic-epigenetic coupling.
Results: Heterozygous Pfkfb3 deficiency significantly reduced EndMT and fibrosis in the microvascular injury model. In vitro, loss of PFKFB3 function attenuated TGF-β2-induced EndMT, accompanied by decreased histone acetylation and lactylation. These findings suggest that PFKFB3-driven glycolysis promotes epigenetic reprogramming that facilitates endothelial transition and fibrotic activation.
Conclusions: PFKFB3 acts as a metabolic regulator of EndMT and microvascular fibrosis by regulating histone modifications. Subretinal fibrosis serves as a unique and quantifiable model of microvascular fibrosis, offering mechanistic insights applicable to systemic vascular and cardiac fibrosis. Targeting endothelial PFKFB3 may represent a promising antifibrotic strategy across cardiovascular diseases.