PU.1 Facilitates Endothelial-to-Mesenchymal Transition in Cardiac Endothelial Cells

The endothelial-to-mesenchymal transition (EndMT) plays a critical role in cardiac fibrosis pathogenesis. However, the molecular mechanisms driving EndMT remain poorly understood. This study investigates the regulatory function of the transcription factor PU.1 in EndMT using primary cardiac endothelial cells.

Immunofluorescence was performed to assess characteristic protein markers in cultured cells. PU.1 knockdown was achieved through siRNA transfection. Key gene expression was quantified at mRNA and protein levels. EndMT progression was evaluated via migration and tube formation assays. Additionally, immunoprecipitation was utilized to examine PU.1 interaction with phosphorylated Smad3 (p-Smad3).

TGF-β1-induced EndMT is coupled with a significant upregulation of PU.1 expression. PU.1 silencing attenuated EndMT, evidenced by elevated CD31/VE-cadherin and reduced α-SMA/N-cadherin/FSP-1 levels under TGF-β1 stimulation. PU.1 knockdown functionally impaired cell migration while promoting vascular lumenogenesis. Conversely, forced PU.1 expression was sufficient to drive EndMT in cardiac endothelial cells. Mechanistically, our data suggest that PU.1 enhances Smad3 phosphorylation, potentially through direct binding to and stabilization of the p-Smad3 protein.

PU.1 drives EndMT in cardiac endothelial cells by enhancing Smad3 phosphorylation and stability. These results elucidate novel molecular pathways in EndMT and identify PU.1 as a potential therapeutic target for attenuating cardiac fibrosis.