Endothelial progenitor cell susceptibility to DNA damaging and DDR-modulating compounds determines endothelial differentiation accuracy.

Abstract

The clinical use of the anticancer drug doxorubicin (Dox) is limited by irreversible cardiotoxicity. The pathophysiological relevance of different cardiac cell types, including endothelial progenitor cells (EPC), in this process is unclear. Since progenitor cells are particularly relevant for tissue regeneration, we hypothesize that residual damage resulting from Dox-based therapeutic regimen may influence their endothelial differentiation accuracy. Therefore, we comparatively investigated the response of murine embryonic stem cells (mESC), endothelial progenitor cells (EC d4) and terminally differentiated endothelial-like cells (EC d6) following exposure to Dox and selected pharmacological inhibitors of DNA repair/DNA damage response (DDR) (RAD51i B02; HDACi entinostat (EST)). We show that EC d4 exhibit enhanced Dox sensitivity as compared to mESC and EC d6. EdU incorporation and replication fork progression analyses revealed pronounced agent-specific differences between mESC, EC d4 and EC d6. Furthermore, DNA damage formation varied in a drug-dependent manner, with mESC showing enhanced residual levels of DNA single-strand breaks (SSB) as compared to EC d4 and EC d6 while EC d6 revealed highest levels of DNA double-strand breaks (DSB). Dox treatment of EC d4 did not prevent their further differentiation into EC d6. However, it caused several functional impairments in the surviving EC d6 progeny, including defects in mitochondrial homeostasis, endothelial barrier function related to cell-cell adhesion factors (ZO1, VE-cadherin), cytokine response and low-density lipoprotein (LDL) uptake. This is accompanied by increased senescence. Summarizing, we demonstrate both overlapping and agent-specific responses of mESC, EC d4 and EC d6 to Dox and DNA repair/DDR inhibitors. Notably, drug treatment of EPC (EC d4) causes multiple dysfunctions in differentiated EC d6. Hence, pharmacological measures aiming to specifically protect EPC from Dox-induced damage are suggested to foster the maintenance of healthy endothelial functionality during regeneration, thereby lowering the risk of detrimental late cardiotoxicity resulting from Dox-based anticancer regimen.