PAI-1 Overexpression in Valvular Interstitial Cells Contributes to Hypofibrinolysis in Aortic Stenosis

Figures

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  Figure 1. Valvular lipids accumulation together with plasminogen activator inhibitor 1 (PAI-1) and nuclear factor-κB (NF-κB) expression. Representative microphotographs of lipids accumulation in (A) control valve and (B) stenotic valve, original magnification 4×. Regions of interest (ROI) are marked in yellow. Valvular expression of (C) PAI-1 in control leaflets, (D) PAI-1 in stenotic leaflets. (E) NF-κB in control leaflets, and (F) colocalization (yellow) of PAI-1 (green) and NF-κB (red). IgG isotype control for healthy (G) and stenotic (H) valves. Red arrowhead indicates aortic side of the leaflet; yellow arrowheads indicate the immunopositive areas. Scale bar 200 μm, original magnification 4×.

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  Figure 2. Valvular expression of fibrinolytic proteins and inhibitors. Representative microphotographs showing valvular expression of plasminogen, α2-antiplasmin, tissue plasminogen activator (tPA), and D-dimer in control (left panel) and stenotic aortic leaflets (right panel). Red arrowhead indicates aortic side of the leaflet; yellow arrowheads indicate the immunopositive areas. Scale bar 200 μm, original magnification 4×.

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  Figure 3. Valvular PAI-1 correlated with lipids accumulation and AS severity. The scatterplots showing associations between (A) valvular PAI-1 expression and lipids accumulation, (B) valvular PAI-1 expression and mean transvalvular pressure gradient (PGmean). Associations between continuous variables were calculated using Pearson’s or Spearman’s correlation coefficients. N = 50, p-values of <0.05 were considered statistically significant.

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  Figure 4. Expression of fibrinolytic proteins in valve interstitial cells (VICs) according to different culture conditions. Representative microphotographs of VICs cultured in different conditions tested for (A) PAI-1 expression and IgG isotype control, (B) plasminogen, α2-antiplasmin or tPA expression, and IgG isotype control. Cell nuclei are stained blue (DAPI), protein expression is green. Scale bar 20 µm, original magnification 40×. The experiment was repeated three times using VICs isolated from different valves.

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  Figure 5. Effect of BAY 11-7082 and TM5275 on PAI-1 inhibition in VICs cultures. (A) Representative microphotographs of PAI-1 and tPA immunostaining in VICs stimulated with TNF−α or LDL in combination with NF−κB inhibitor (BAY 11-7082) or PAI-1 inhibitor (TM5275). Cell nuclei are stained blue (DAPI), PAI−1 is green. Scale bar 20 µm, original magnification 40×. The dot plots show (B) PAI−1antigen levels and (C) clot lysis time (CLT) in VICs supernatants from different culture conditions. Data presented as mean ± SD. ** p < 0.01 and *** p < 0.001 compared to VICs cultured in the calcification medium supplemented with LDL. Statistical analysis was performed by ANOVA and post-hoc tests. (D) Box plot shows relative gene expression of SERPINE1 in VICs cultured in different conditions. Real-time PCR data are presented as mRNA expression fold change. All experiments were repeated three times using cells, supernatants or lysates from different VICs cultures; all samples were tested in triplets.