Increased Large Elastic Artery Stiffening with The Anthracycline Chemotherapy Drug Doxorubicin: Potential Role of Excess Mitochondrial Superoxide

Abstract

The highly effective, yet proteotoxic, chemotherapeutic agent Doxorubicin (DOXO) increases the risk of cardiovascular diseases (CVDs). A key antecedent to CVDs with DOXO is aortic stiffening (assessed in vivo via aortic pulse wave velocity [aPWV]), but the underlying mechanisms driving this increase in stiffness are currently unknown. Aortic stiffening can occur due to: 1) adverse changes in extracellular matrix (ECM) proteins; and 2) the formation of proteotoxic advanced glycation end products (AGEs) that form crosslinks between ECM proteins—both of these can be driven by inflammation and excess mitochondrial (mito) superoxide production.Purpose1) To investigate underlying molecular mechanisms of DOXO‐induced aortic stiffening. 2) To determine if increased arterial stiffness is partially attenuated by decreasing excess mito superoxide using the mito‐targeted antioxidant, MitoQ.MethodsC57BL6 young adult (4mo of age) male mice received one injection of either sham (PBS; n=4) or DOXO (10mg/ml in PBS; n=8). aPWV was assessed prior to and 4 weeks following sham/DOXO administration. To assess aortic stiffness independent of humoral factors and vasomotor tone, ex vivo aortic intrinsic mechanical stiffness (elastic modulus via wire myography) was measured following sacrifice. ECM proteins (elastin and collagen) and AGEs were measured via immunoblot. Lastly, aortic abundance of pro‐inflammatory proteins was assessed using a multiplex ELISA.ResultsDOXO increased aPWV from 330.2 ± 19.89 cm/sec (baseline) to 446.1 ± 32.62 at 4w (P < 0.05), whereas no difference was observed over time in the sham group. Aortic intrinsic mechanical stiffness was ~125% higher after DOXO vs. sham (6140 ± 507 vs. 2556 ± 417 kPa; P < 0.01). Elastin was 52% lower after DOXO vs. sham (0.61 ± 0.09 vs. 1.28 ± 0.24 A.U.; P < 0.05), whereas no difference was observed in collagen. Doxorubicin treatment did not affect AGE levels. Pro‐inflammatory Interferon‐γ (0.60 ± 0.09 vs. 0.34 ± 0.04 pg/ml; P < 0.05), Interleukin‐1β (0.16 ± 0.01 vs. 0.12 ± 0.01 pg/ml; P < 0.05), Interleukin‐6 (0.14 ± 0.01 vs. 0.11 ± 0.01 pg/ml; P < 0.05) and Tumor Necrosis Factor‐α (0.13 ± 0.05 vs. 0.04 ± 0.01 pg/ml; P < 0.05) were ~30–225% greater after DOXO vs. sham. Next, to determine that DOXO‐induced aortic stiffening is mediated by excess production of mito superoxide, we supplemented mice with MitoQ (250 μM in drinking water; n=5) for 4w following DOXO. The DOXO‐induced increase in aPWV was attenuated with MitoQ (374.4 ± 15.78 cm/sec [baseline] vs. 420.9 ± 17.52 [4w]; P = 0.18). Furthermore, MitoQ mitigated the greater aortic intrinsic mechanical stiffness observed in the DOXO only group (4239 ± 761 vs. 6140 ± 507 kPa; P < 0.05).ConclusionThese data suggest DOXO induces aortic stiffening, in part, by increasing aortic intrinsic wall stiffness, reducing elastin and increasing inflammation. Importantly, aortic stiffening was partially prevented by supplementation with the mito‐targeted antioxidant, MitoQ, suggesting an important role for the involvement of excessive mito superoxide in DOXO‐induced aortic stiffening.Support or Funding InformationSG‐089, Undergraduate Research Opportunities Program (D.A.H.)NIH T32 DK007135 (Z.S.C.)