Sustained mesenchymal reprogramming of endothelial cells after completion of chemotherapy.

Cardiovascular disease is the prevailing cause of death among cancer survivors. Remarkable increase in cardiac disease burden in this group is likely due to the effects of cancer itself and, especially, cardiotoxic cancer treatments. Of the cardiotoxic cancer treatments, anthracyclines are notoriously known for causing vascular damage and severe cardiovascular complications. A defining feature of cardiac damage by doxorubicin (Dox), a prototypical anthracycline, is that it typically progresses after completion of chemotherapy. As the nature of delayed deterioration is not understood, we focused in this study on the events that occur upon completion of chemotherapy. We adopted the Dox treatment/washout model to examine its lasting effects on endothelial cells. Our ChIP sequencing, transcriptomic, reporter plasmid, and Smad2/3 phosphorylation experiments demonstrated enhanced activity of the canonical TGF-β and activin pathways during Dox washout. Another notable feature was sustained mesenchymal reprogramming with significant upregulation of transcripts characteristic of fibroblastic and smooth muscle lineages, both in endothelial cultures and cardiac microvascular endothelial cells in vivo. We utilized a selective ALK4/5/7 receptor kinase inhibitor, SB431542 (SB), to probe the role of the canonical TGF-β/activin pathways in endothelial-to-mesenchymal reprogramming by Dox. When present during Dox washout, SB blocked increased expression of both mesenchymal transcripts and protein markers, and prevented cytoskeletal changes and fibronectin production by the treated endothelial cells. Cytoskeletal rearrangements led to increased endothelial monolayer permeability that was abolished by SB treatment. Thus, increased production of ALK4/5 receptor ligands, TGF-β2 and activin, and heightened Smad2/3 activation response to these ligands during Dox washout leads to sustained mesenchymal reprogramming of endothelial cells and compromised endothelial barrier function.