Impact of Oxidative Stress and Protein S-Glutathionylation in Aortic Valve Sclerosis Patients with Overt Atherosclerosis

Figures

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  Figure 1. Imbalance of systemic glutathione homeostasis and endothelial dysfunction. (A) Box plot representing the ratio between the reduced (GSH) and the oxidized (GSSG) forms of glutathione in patients with normal aortic valve leaflet (No-AVSc; n = 29) and aortic valve sclerosis (AVSc; n = 29). (B) Box plot representing ADMA levels in patients with No-AVSc (n = 29) and AVSc (n = 29).

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  Figure 2. Aortic valve protein S-glutathionylation. (A,B) Dot-blot evaluation of total glutathione (GSH) expression in aortic valve leaflets (controls n = 3; AVSc n = 6) and relative quantification showed in the bar graph (dot-blot analyses using ImageJ; plugin: Dot-blot Analyzer v1.0). (C,D) Representative images showing histological analysis of human aortic valve in Fibrosa and Spongiosa layers. No-AVSc: patients with a normal aortic valve leaflet. AVSc: patients with aortic valve sclerosis. Black arrows indicate positive cells for GSH staining.

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  Figure 3. In vitro model of protein S-glutathionylation. (A) Bar graph representing the ratio between the reduced (GSH) and the oxidized (GSSG) form of glutathione (GSH/GSSG ratio) in human umbilical vein endothelial cells (HUVEC) after 4 h of treatment (2-AAPA 50 and 100 μM) and 24 h of recovery (n = 3). (B) Western blot representative images of HUVEC treated with 2-AAPA (50 and 100 μM) for 4 h and 24 h of recovery (n = 4); GSH is shown in green and β-actin is shown in red; Bar graph representing Western blot quantification by ImageJ. (* p < 0.05 vs. Untreated; ** p < 0.001 vs. Untreated; ## p < 0.001 vs. Dimethyl Sulfoxide (DMSO)). (C) Western blot evaluation of histone 2AX phosphorylation (γH2AX) in HUVEC after 2-AAPA (50 and 100 μM) treatment for 4 h and 24 h of recovery (n = 4); Bar graph representing Western blot quantification by ImageJ. (** p < 0.001 vs. Untreated; # p < 0.05 vs. DMSO; ## p < 0.01 vs. DMSO). Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) has been used for normalization.

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  Figure 4. Protein S-glutathionylation and endothelial-to-mesenchymal transition. Quantitative Polymerase Chain Reaction (qPCR) of the gene encoding endothelial markers (A) CD31, (B) VE-cadherin and mesenchymal marker (C) ACTA2 after 4 h of 2-AAPA (50 μM) treatment and 48 h of recovery (n = 3). qPCR data are expressed as log2 fold change (logFC) in comparison to untreated cells.