A Biphasic Effect of Alcohol on Endothelial Plasticity Through Regulation of Endothelial-to-Mesenchymal Transition

Figures

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  Fig 1.. Biphasic effect of ethanol on cytokine and hypoxia-induced EndMT in cultured endothelial cells. (a-c) SM22α, eNOS, αSMA, and Fibronectin (FN) mRNA levels determined by qRT-PCR in human coronary artery endothelial cells (HCAEC) following treatment with TGFβ1 (10 ng/ml, 7d), hypoxia (5% O2, 4d) or TGFβ1 plus hypoxia (4d), in the absence or presence of ethanol (EtOH) at concentrations indicated (0-100 mM range). (d) SM22α, eNOS, and Cdh5 mRNA levels in HCAEC treated with TGFβ1 (10 mg/ml, 2d) +/− EtOH 25 mM or 100 mM. (e) Representative western blots showing CD31, Cdh5, SM22α, and SNAIL protein expression in HCAEC treated with TGFβ1, IL-1β, or Hypoxia +/− EtOH (25 mM or 100 mM). β-actin or GAPDH were used as loading controls. Data are mean±SEM, n=3. *p<0.05 vs control (no treatment), #p<0.05 vs TGFβ1 or hypoxia.

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  Fig 2.. Differential effect of moderate and high dose ethanol on EndMT in HCAEC and HUVEC determined by immunocytochemistry. (a) CD31 expression in HCAEC treated with TGFβ1 (10ng/ml), Hypoxia (5% O2), or TGFβ+ Hypoxia in the absence or presence of moderate dose ethanol (EtOH 25 mM). (b) αSMA expression in HCAEC treated with TGFβ +/− either moderate dose ethanol (25 mM EtOH) or high dose ethanol (100 mM EtOH). (c) SM22α and (d) αSMA expression in HUVEC treated with IL1β+TGFβ2 in the absence or presence of either 25 mM EtOH or 100 mM EtOH as indicated. Representative immunofluorescence images, from at least 3 experiments, shown.

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  Fig 3.. EtOH at moderate, but not high levels, inhibits TGFβ1- and hypoxia-induced EndMT in a Notch-dependent manner. (a) αSMA mRNA levels in HCAEC treated with TGFβ1 (10 ng/ml) or exposed to hypoxic conditions (5% O2), in the absence or presence of moderate dose EtOH (25 mM, green bars) or high dose EtOH (100 mM, red bars) +/− the γ-secretase inhibitor DAPT (20 μΜ). (b) αSMA and fibronectin (FN) mRNA levels in HCAEC treated with TGFβ1 +/− the Notch ligand DLL4 (3 μg/ml) in the absence or presence of DAPT (20 μΜ). Data are mean±SEM, n=3. *p<0.05 vs control (no treatment), #p<0.05 vs TGFβ1 or hypoxia.

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  Fig 4.. EtOH at moderate, but not high levels, reverses cytokine-induced inhibition of HCAEC Cdh5 expression in a Notch-dependent manner. HCAEC were treated with inflammatory cytokines (IL1β + TGFβ1) in the absence or presence of moderate dose ethanol (25 mM EtOH) or high dose ethanol (100 mM EtOH), with or without the gamma secretase inhibitor DAPT (20 μΜ), before Cdh5 expression was determined by immunocytochemistry, (a) Representative immunofluorescence images from 2 separate experiments, and (b) cumulative quantitative data of Cdh5 fluorescent staining. Data are mean±SEM, n=6. *p<0.05 vs control (no treatment, grp 1), #p<0.05 vs IL1β + TGFβ1 (grp 2).

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  Fig 5.. EtOH dose-dependently inhibits cytokine-induced endothelial migration. HUVEC were treated with or without IL1β+TGFβ1 in the absence or presence of moderate (25 mM) and high level (100 mM) EtOH. Migration was determined by scratch wound assay. Representative phase contrast images showing scratch wounds in the different experimental groups at time zero, 3, 6, 9 and 12 h, together with bar graph of cumulative data from 3 separate experiments. Data are mean±SEM, n=3. *p<0.05 vs control (IL1β+TGFβ1 alone).

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  Fig 6.. Ligation-induced carotid remodeling. Vessels were harvested 14 days post sham-operation or ligation. (a) Representative Van Gieson stained carotid cross sections from Control and moderate Ethanol (EtOH) sham-operated Cdh5xAi9 male and female mice. (b) Representative Van Gieson stained cross sections from Ligated controls, Ligated + daily moderate EtOH (2 drink equivalent/d, BAC 15 mM), or Ligated + Binge EtOH (7 drink equivalent 2d/wk, BAC 50 mM) cohorts; males upper row, females lower row.

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  Fig 7.. Morphological analysis of carotids from sham and ligated Cdh5xAi9 transgenic mice. (a-c) left hand side; carotid adventitial (adv), medial, neointimal (NI) and lumen volumes in sham-operated controls (white bars) and sham-operated daily moderate EtOH group (green bars). (a-c) right hand side; carotid compartment volumes in sham-operated (white bars) and ligated conytrols (grey bars). Ligation injury stimulated neointimal hyperplasia and decreased lumen volume, to a greater extent in males than females. Data are mean±SEM, n=5 each for males (a) and females (b), (c) n=10, males and females pooled. *p<0.05, ** P<0.001, **** p<0.0001 vs sham-operated control.

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  Fig 8.. Differential effect of ‘daily moderate’ and ‘episodic binge’ EtOH exposures on ligation-induced carotid remodeling. Daily moderate EtOH exposure (left hand side graphs, green bars) significantly inhibited ligation-induced neointima (NI) formation and increased lumen volume, compared to controls (white bars). In contrast, 2-d binge EtOH exposure (right hand side graphs, red bars) increased adventitial volume and neointima hyperplasia. Data are mean±SEM, n=5 each for males (a) and females (b), (c) n=10, males and females pooled. *p<0.05, ** P<0.001, **** p<0.0001 vs ligated control.

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  Fig 9.. EndMT in remodeled carotids following ligation injury is reduced by moderate EtOH, but not by binge EtOH exposure. (a) (b) Representative images of immunofluorescently stained carotid cross sections from sham-operated and ligated controls, moderate EtOH, and Binge EtOH experimental groups (males). Blue = Dapi nuclear stain, red = Tm-Cdh5+, and white=αSMA+. (c) Cells co-expressing Cdh5 and αSMA (i.e., myo-endothelial cells indicative of EndMT) were quantified using QuPath bioimage analysis software in carotid cross sections post-ligation from controls (grey bars), moderate EtOH (green bars), and binge EtOH (red bars) experimental groups. Bar graphs show cumulative data expressed as % Myoendothelial cells /full cross section, % Myoendothelial cells /neointima, or Number of myoendothelial cells/full cross section. Data are mean±SEM, n=25-30 sections from 5-6 mice). *p<0.05, ** P<0.001, **** p<0.0001.