Ethanol Enhances Endothelial Rigidity by Targeting VE-Cadherin—Implications for Acute Aortic Dissection

Figures

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  Figure 1. Cell migration. HAoECs were plated on 12-well-plates and exposed to nicotine (Nic) at 100 nM, angiotensin II (Ang II) at 100 nM, or ethanol (EtOH) at 171.2 mM (1% v/v). (A) The cell-covered area was analyzed using live-cell imaging over 24 h. EtOH caused reduced cell migration capacity vs. control. (B) Representative imaging of cell cultures after 12 and 24 h, showing the cell-covered area between the two cell fractions. * p < 0.05 vs. control applying one-way ANOVA with Bonferroni correction. (n = 6).

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  Figure 2. Cell permeability and metabolic viability. (A/B) Transwell assay for endothelial permeability. Human aortic endothelial cells (HAoECs) were seeded on assay inserts to form a monolayer within 3 days. Subsequently, cells were (A) starved for 6 h or (B) not starved before being exposed to the experimental conditions for 24 h. The permeability of the monolayer was tested using fluorescein isothiocyanate–dextran (FITC-dextran) by analyzing the fluorescence molecules that passed through the monolayer after 20 min. Starvation and EtOH in non-starved conditions caused an enhanced permeability. (C/D) MTT assay for metabolic viability. HAOECs were seeded in 96-well plates and (C) starved for 6 h or (D) not starved before being exposed to the experimental conditions for 24 h. Absorbance at 450 nm was measured following addition of WST-1. Starvation caused a decline in metabolic viability, whereas none of the risk factors for developing an acute aortic dissection (AAD) altered the metabolic viability in non-starved conditions. * p < 0.05 individual group vs. control or non-starved control (=control u.s., A,C) applying ANOVA with Bonferroni correction. (n = 7–12).

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  Figure 3. Mechanical cell adherence. (A) Schematic illustration of the hanging drop assay. Human aortic endothelial cells (HAoECs) were cultivated on a culture plate and then exposed to the experimental conditions for 24 h. The cells were trypsinized in cell culture medium under ongoing exposure and drops containing 8000 cells were pipetted onto the lid of a cell culture plate and incubated for 4 h. Three representative images were taken from each drop w/wo trituration to study cell adherence under increased mechanical stress. (B,C) The bar graphs show the percentage for given groups of cells in clusters. For each treatment, ±2000 cells were evaluated; data are presented as the average of the three image sections from each of six independent experiments without (B) and with (C) titration, as shown in (D). * p < 0.05 vs. control, # p < 0.05 vs. without titration applying one-way ANOVA with Bonferroni correction. (n = 6).

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  Figure 4. Transcriptional and protein expression levels of complexing junctional proteins. (A–E) Transcriptional levels of the complexing junctional proteins of tight and adherens junctions (TJs and AJs, respectively) following exposure to the risk factors for developing an aortic dissection (AD). The transcriptional levels were analyzed by applying the ΔΔCT method. Data are presented as fold-change vs. GAPDH. (F) Protein expression level of VE-cadherin following exposure to ethanol (EtOH) using Western blotting normalized to ß-Actin. (G) Immunohistochemistry for VE-cadherin in hAoECs normalized for fluorescence per cell, indicating marked up-regulation of VE-cadherin vs. control. (H) Representative images of VE-cadherin staining for quantification in G. Scale bar is 100 µm, magnification 400×. * p < 0.05 vs. control by applying ANOVA with Bonferroni correction. n = 7–10 (A–E), n = 3 (F), and n = 4 (G).

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  Figure 5. Schematic illustration of a potential pathomechanism for the development of endothelial tears in acute aortic dissection (AAD). (a) Under physiological conditions, adherens junctions (AJs) provide stability to the endothelial cell layer by connecting the cytoskeletons of adjacent cells. (b) Exposure of the endothelial cell layer to the risk factors for the development of AAD increases the expression of VE-cadherin, leading to an increase in the rigidity of the layer. (c) This results in the decreased deformability of the endothelial cell layer during expansion of the aorta as part of the cardiac cycle. In particular, at elevated systolic blood pressures, the endothelial cell layer “breaks” and an endothelial entry tear may be more likely to develop. Figure created with BioRender.com.