Enhanced TGF-beta signaling and mesenchymal transcriptional activity in endothelial cells after completion of chemotherapy

Our previous in vitro and in vivo studies have demonstrated the role of the TGF-beta/Smad3 pathway in endothelial damage by chemotherapeutic agent doxorubicin (Dox) but the downstream mechanisms remain unknown. We hypothesized that both TGF-beta pathway and activation of mesenchymal transcriptional program persist in endothelial cells after removal of Dox from culture media. Human umbilical vein endothelial cells (HUVEC) were treated with sub-apoptotic concentrations of Dox and a selective inhibitor of the TGF-beta canonical pathway SB431542 (SB) followed by removal of Dox from the media (the washout period). Activity of the canonical TGF-beta pathway, expression of mesenchymal transcripts/proteins, and endothelial function assays were evaluated during both Dox treatment and washout. We detected enhanced activity of the canonical TGF-beta pathway upon Dox removal from media using RNA sequencing, CAGA 12 reporter plasmid experiments, and phosphorylation of Smad3, a transcription factor in the pathway. SB abolished the activity of the pathway when given concurrently with Dox or during the Dox washout period only. Further studies revealed increased expression of mesenchymal transcripts of both smooth muscle ( Tagln, Cnn1, Tpm1 ) and fibroblastic ( Cdh2, Fn1, Col1a2, Acta2 ) lineages in endothelial cells upon Dox washout. Mesenchymal proteins encoded by these transcripts were also upregulated. Again, SB prevented the upregulated expression of mesenchymal transcripts/proteins when given either along with Dox or during washout only. We further examined the role of the TGF-beta pathway in endothelial dysfunction observed after removal of Dox from the culture media. Specifically, pretreatment with Dox caused cytoskeletal changes that were characterized, in particular, by expanded filamentous actin network. Additionally, endothelial monolayer permeability was increased during the Dox washout period, and SB prevented both cytoskeletal and endothelial permeability defects in Dox-pretreated cells. Likewise, endothelial cell migration was affected by Dox pretreatment in the TGF-beta pathway dependent manner. To conclude, enhanced activity of the canonical TGF-beta pathway persists after completion of Dox therapy and contributes to sustained mesenchymal reprogramming and functional defects in endothelial cells. This work was supported by grant R15HL133873 from NIH National Heart, Lung, and Blood Institute. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.