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.

1. Introduction

Anthracycline derivatives such as doxorubicin (Dox) are widely used anticancer drugs [1], which act as type II topoisomerase (Topo II) poisons, thereby leading to the formation of highly cytotoxic DNA double-strand breaks (DSBs) [2]. DSBs are potent triggers of the DNA damage response (DDR), which regulates cell cycle progression, DNA repair and apoptosis, thereby defining the balance between survival and death-related mechanisms [3]. In addition, generation of reactive oxygen species (ROS), DNA intercalation, inhibition of DNA helicases and chromatin damage contribute to Dox-induced cytotoxicity [4, 5]. It is believed that at clinically relevant low Dox concentrations the inhibition of topoisomerase II (TopoII) isoforms and subsequent formation of DSB is particularly relevant for its cytotoxicity. By contrast, using higher Dox concentrations, oxidative stress and DNA intercalation likely become more relevant as toxic mode of action [6].

Cumulative and irreversible cardiotoxicity ultimately leading to cardiomyopathy and congestive heart failure is the clinically most relevant adverse effect of anthracyclines [1]. In view of the low antioxidative capacity of cardiomyocytes, mitochondria-related iron-dependent and iron-independent ROS formation may be of pathophysiological relevance for Dox-induced cardiac damage, with p53-regulated mechanisms of senescence and cell death being involved [7–11]. Hence, chemoprevention of anthracycline-mediated oxidative stress has been considered to achieve cardio-protection [10–12]. Yet, antioxidants failed to demonstrate substantial cardioprotective potency following anthracycline-based anticancer therapy [4, 13, 14]. By contrast, the EDTA analogue dexrazoxane, which is a strong catalytic inhibitor of topoisomerase II, is able to prevent anthracycline-induced cardiac damage [15, 16]. Based on data obtained from the use of dexrazoxane derivatives that are lacking ion-chelating activity but still harboring Topo II inhibitory potency, it is hypothesized that Topo II inhibition is the primary mechanism of cardioprotection.