Epirubicin promotes coronary endothelial senescence and dysfunction with induction of pro-inflammatory and pro-thrombotic responses: role of the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway
L Axenbeck, A Mroueh, W Fakih, L Louis, C Fesselier, V Boide-Trujillo, A Klymchenko, C Po, M Pieper, O Morel, V Schini-KerthAbstract
Background
The clinical use of epirubicin, a major anthracycline chemotherapeutic drug, is limited by cumulative cardiotoxicity that can lead to heart failure. Growing evidence suggests that endothelial dysfunction, oxidative stress and pro-inflammatory responses contribute to the pathogenesis of anthracycline-induced heart failure. However, the underlying mechanisms still remain poorly characterized. SGLT2 inhibitors, which have recently become a cornerstone of heart failure therapy across all ejection fractions, may represent an attractive candidate to mitigate epirubicin-induced heart failure.
Aim
This study investigated mechanisms underlying epirubicin-induced coronary macro- and microvascular endothelial dysfunction using in vitro and ex vivo models and explored the role of SGLT2.
Methods
Endothelial cells (ECs) isolated and cultured from porcine coronary arteries (PCA), PCA segments, and porcine left ventricle (LV) specimens were stimulated with epirubicin (100 nM). Reactive oxygen species (ROS) levels were assessed using dihydroethidium, nitric oxide (NO) using DAF-FM diacetate, senescence by SA-β-galactosidase activity, glucose uptake by 2-NBDG, NF-κB activation by immunofluorescence staining, mRNA levels by RT-PCR, protein levels by Western blot analysis, and functional consequences of endothelial activation by studying the adhesion of labelled human monocytes and platelets.
Results
Exposure of ECs to epirubicin caused a concentration-dependent formation of ROS at 1 h that was sustained up to 24 h (up to 2-fold increase vs untreated control). Both early and late pro-oxidant responses were significantly prevented by losartan (AT1R antagonist) and VAS-2870 (NADPH oxidases inhibitor), whereas empagliflozin (SGLT2 inhibitor) prevented only the sustained ROS response. ECs treated with epirubicin for 24 h showed blunted basal and bradykinin-stimulated NO formation, increased levels of SA-β-galactosidase activity (91% stained cells vs 7.8% for untreated control), increased glucose uptake (3.5-fold) and increased nuclear translocation of NF-κB (1.7-fold). In addition, they displayed stronger adhesion of human monocytes (3.3-fold) and platelets (3.5-fold). The epirubicin treatment upregulated the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and markers of AT1R/NADPH oxidases/SGLT2, senescence, endothelial dysfunction and activation. All of these responses were markedly reduced by losartan, VAS-2870 and empagliflozin.
Conclusion
Epirubicin promotes endothelial oxidative stress by activating the local angiotensin-NADPH oxidases pathway promoting SGLT2 expression. Once the protein is expressed, SGLT2 will further fuel ROS production, leading to endothelial senescence and dysfunction, as well as pro-inflammatory and pro-thrombotic responses. Inhibiting SGLT2 appears as a promising strategy to mitigate endothelial cardiotoxicity in chemotherapy.