Abstract 371: TNFα Drives Endothelial Dysfunction and Oxidative Stress via eNOS Uncoupling in Aortic Valve Disease

Abstract

Objective: Oxidative stress (OxS) is known to contribute to late stages of aortic valve disease (AVD), but its role in early-stage valve inflammation and as a driver of valve pathology has not been investigated. Here we show that inflammation drives increased OxS via eNOS uncoupling. Using an ex vivo model of AVD, we show that targeting eNOS uncoupling with tetrahydrobiopterin or superoxide dismutase can mitigate later-stage valve degeneration by blocking the downstream effects of eNOS uncoupling. Methods and Results: We first observed evidence of eNOS uncoupling in the endothelium of calcified human aortic valve leaflets, which showed increased superoxide co-localized with cells positive for CD31 and VCAM-1, suggesting a connection between endothelial inflammatory activation and increased oxidative stress. We then showed that inflammatory cytokine TNF-α caused increased superoxide, free radicals, and hydrogen peroxide in aortic valve endothelial cells, as soon as 15 minutes after treatment. TNF-α did not cause increased oxidative stress in endothelial cells from the aorta, pointing to a valve-specific mechanism. TNF-α-driven oxidative stress decreased valve endothelial cell nitric oxide secretion, lowered eNOS and VE-cadherin protein levels, and increased expression of inflammatory adhesion molecules (VCAM1, ICAM1). Addition of eNOS co-factor tetrahydrobiopterin (BH4) mitigated the increases in OxS caused by TNF-α, demonstrating that eNOS uncoupling was the primary pathophysiological mechanism at play. Ex vivo, we used porcine aortic valve leaflets to screen the ability of BH4, catalase, and superoxide dismutase to protect against the downstream effects of eNOS uncoupling. We found that all three antioxidants mitigated increases in valve OxS, improved endothelial function, and protected against extracellular matrix remodeling. TNF-α+SOD also protected against increases in myofibroblastic protein α-smooth muscle actin and chronic elevation of VCAM1. Conclusions: These results present endothelial inflammatory oxidative stress as a new mechanism that connects early valve pathology with later stages of degeneration. Targeting these mechanisms via tailored antioxidant therapy could provide new avenues for treatment of CAVD.