The commonly‐used anthracycline chemotherapy drug Doxorubicin impairs vascular endothelial function via stimulation of mitochondrial superoxide

Abstract

Doxorubicin (DOXO), a commonly used anthracycline chemotherapeutic agent, increases the risk of cardiovascular (CV) related morbidity and mortality. A key mechanism by which DOXO increases CV disease risk is by inducing endothelial dysfunction (impaired endothelium‐dependent dilation [EDD]) but: 1) the underlying mechanisms are unknown; 2) there are no established treatments for preventing these side effects.PurposeWe tested the hypotheses that: 1) DOXO impairs EDD via excess mitochondrial (mito) superoxide‐induced reductions in nitric oxide (NO) bioavailability; 2) these effects can be prevented with a mito‐targeted antioxidant.MethodsC57BL6 male mice (4mo of age) received one injection of either sham (PBS; n=4) or DOXO (10mg/ml; n=8). After 4 weeks, we assessed endothelial function (ex vivo carotid artery EDD to increasing doses of acetylcholine [ACh]) and potential underlying mechanisms: NO bioavailability (ACh dose response in the presence of the NO‐synthase inhibitor, L‐NAME); aortic total and mito‐specific superoxide production (electron paramagnetic resonance spectroscopy); aortic cytosolic, extracellular and mito isoforms of superoxide dismutase (SOD) (immunoblot); and the roles of excess total and mito‐specific superoxide in regulating EDD (ACh dose response in the presence of the SOD mimetic, TEMPOL, and the mito‐targeted antioxidant, MitoQ, respectively).ResultsMechanisms of ActionDOXO reduced maximal EDD by 41% (95±3 vs. 54±3 %; P=0.003). This reduction was mediated by a decrease in NO bioavailability, as EDD was not different between groups following incubation with L‐NAME. Vascular smooth muscle sensitivity to NO (dilatory response to sodium nitroprusside) was unaltered. DOXO increased aortic total superoxide production by 38% (5.3±0.7 to 7.3±0.5 amplitude units; P=0.003), without compensatory upregulation in aortic cytosolic or extracellular SOD. Reduced EDD with DOXO was mediated by excess superoxide production, as indicated by restoration of maximal EDD with TEMPOL (97±2%). DOXO increased aortic mito superoxide production by 50% (1.0±0.1 to 1.5±0.1 amplitude units; P=0.01) and induced a 60% reduction in aortic mito SOD (1.0±0.2 vs. 0.4±0.1 absorbance units; P=0.01). Reduced EDD with DOXO was mediated by increased mito superoxide production, as indicated by restoration of maximal EDD with MitoQ (91±8%).PreventionNext, we supplemented mice with MitoQ (250μM in drinking water; n=5) for 4 weeks following DOXO and showed that MitoQ prevented the reduction in EDD (97±1%, P=0.002 vs. DOXO alone) by preserving NO bioavailability via reduced mito superoxide (no added improvement with MitoQ incubation).ConclusionThese results suggest DOXO‐induced vascular endothelial dysfunction is mediated by excess mito superoxide‐induced reduction in NO bioavailability. Our results also demonstrate that MitoQ supplementation prevented DOXO‐induced endothelial dysfunction by suppressing mito superoxide and preserving NO bioavailability. Because MitoQ is available and safe for human use, there may be translational potential for reducing CV‐related morbidity and mortality in patients that have received DOXO chemotherapy.Support or Funding InformationNIH T32 DK007135‐44 (Z.S.C.)