Endothelial-Derived Oxidative Stress Drives Myofibroblastic Activation and Calcification of the Aortic Valve

Aims Oxidative stress is present in and contributes to calcification of the aortic valve, but the driving factors behind the initiation of valve oxidative stress are not well understood. We tested whether the valve endothelium acts as an initiator and propagator of oxidative stress in aortic valve disease. Methods and Results Calcified human aortic valves showed side-specific elevation of superoxide in the endothelium, co-localized with high VCAM1 expression, linking oxidative stress, inflammation, and valve degeneration. Treatment with inflammatory cytokine TNF alpha increased superoxide and oxidative stress and decreased eNOS and VE-cadherin acutely over 48 hours in aortic valve endothelial cells (VEC) and chronically over 21 days in ex vivo AV leaflets. Co-treatment of VEC with tetrahydrobiopterin (BH4) but not apocynin mitigated TNF alpha-driven VEC oxidative stress. Co-treatment of ex vivo AV leaflets with TNF alpha+BH4 or TNF alpha+peg-SOD rescued endothelial function and mitigated inflammatory responses. Both BH4 and peg-SOD rescued valve leaflets from the pro-osteogenic effects of TNF alpha treatment, but only peg-SOD was able to mitigate the fibrogenic effects, including increased collagen and alpha SMA expression. Conclusions Aortic valve endothelial cells are a novel source of oxidative stress in aortic valve disease. TNF alpha-driven VEC oxidative stress causes loss of endothelial protective function, chronic inflammation, and fibrogenic and osteogenic activation, mitigated differentially by BH4 and peg-SOD. These mechanisms identify new targets for tailored antioxidant therapy focused on mitigation of oxidative stress and restoration of endothelial protection.