Abstract 459: Protein Glutathionylation Promotes Calcification via Oxidative DNA Damage in Human Valve Endothelial Cells

Abstract

Background: Calcific aortic valve disease (CAVD) is one of the most common form of heart valve disease and affects 3% of the population. The initial phase, called aortic valve sclerosis (AVSc), is linked to inflammation and oxidative stress with direct consequences on endothelial cells. Thus, we aimed to evaluate the effects of protein glutathionylation caused by oxidative damage in endothelial cells. Methods: We analysed blood samples from 58 subjects, 29 with AVSc and 29 without (no-AVSc). The levels of oxidized (GSSG) and reduced (GSH) forms of glutathione were measured by a liquid chromatography-tandem mass spectrometry method and their ratio (GSH/GSSG) represent a recognized an oxidative stress status index. 2-AAPA, a glutathione reductase inhibitor, was used to increase GSSG levels. Human umbilical vein endothelial cells (HUVEC) were employed to create a model of protein glutathionylation. Valve endothelial cells (VEC) were isolated from pig and human aortic valves and were used to test and to validate this model. Results: Subjects with AVSc had a circulating GSH/GSSG ratio lower than no-AVSc (17.0±1.7 vs 24.4±2.6, respectively; p=0.02), indicating an increased systemic oxidative stress status in AVSc. Protein glutathionylation was increased more than 9-fold in human specimens from AVSc compared to no-AVSc subjects (p=0.03). In HUVECs, 2-AAPA significantly reduced the GSH/GSSG ratio (p=0.003) and induced an increment in protein glutathionylation of 1.91±0.17 fold compared to untreated cells (p=0.006). In pig VECs, 2-AAPA caused a 2-fold increment in reactive oxygen species production and an activation of the histone H2AX. In human VECs, protein glutathionylation caused an upregulation of smooth muscle actin (+0.7±0.16; p=0.004) and a downregulation of vascular endothelial cadherin (-0.4±0.02; p<0.0001) and, after 7 days, treated VECs were able to calcify at a greater extent (+2.83±0.4) than untreated cells (p<0.0001). Conclusion: These preliminary results show for the first time that protein glutathionylation could play a direct role in CAVD development via oxidative DNA damage. Restoring the proper balance of GSH and GSSG may be beneficial to reduce the endothelial damage occurring in patients affected by CAVD.