Serum from Patients with Severe Alcoholic Liver Cirrhosis Inhibits Proliferation and Migration of Human Coronary Artery Smooth Muscle Cells

Figures

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  Figure 1. Experimental protocol of the cell culture experiments: proliferation, migration, apoptosis and gene expression in smooth muscle cells after exposure to sera from of patients with alcoholic cirrhosis or healthy controls. All steps including cell plating, starving, growth stimulation and measurements are presented, including details such as seeding-density, type of applied well-plates, serum concentrations used and time course.

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  Figure 2. Effects of patient sera on human coronary artery smooth muscle cell proliferation. Effects of 1% and 5% sera from healthy controls and patients with alcoholic liver cirrhosis (each n = 10) on proliferation of human coronary artery smooth muscle cells compared to control patients (0.1% serum). (p-values from analysis of variance (pconcentration and pgroup) and post hoc analysis: *** p ≤ 0.001 vs. healthy; † p ≤ 0.05 vs. 1% serum). Data are means ± standard error of the mean.

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  Figure 3. Effects of patient sera on apoptosis of human coronary artery smooth muscle cells. After 48 h of starvation cells were exposed for 48 h under basal (1%) and stimulated (5%) conditions; the rate of apoptosis of human coronary artery smooth muscle did not differ between groups (n = 10 per group, p-values from two-way analysis of variance: *** p ≤ 0.001 1% vs. 5% serum). Data are means ± standard error of the mean.

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  Figure 4. Effects of patient sera on migration of human coronary artery smooth muscle cells. (a) Cell migration and (b) relative cell count (percent of control) after 48 h are shown. Migration was calculated as explained in Materials and Methods. Cirrhosis markedly reduced cell migration by more than 60% (n = 10 per group, p < 0.05) (c) Representative photographs of cell migration are shown 48 h after scratch induction in 10-fold magnification. The remaining scratch gap is highlighted in the photographs by light grey shading. Scale bars correspond to 0.5 mm length. Data are means ± standard error of the mean.

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  Figure 5. Effects of patient sera on steady-state gene expression levels in human coronary vascular smooth muscle cells. Relative ribonucleic acid expression after 48 h of exposure to 1% serum from either healthy controls or patients with alcoholic liver cirrhosis, compared to that of the negative control (after starving). Retinol binding protein 1 (RBP1), osteopontin (SPP1) and Krüppel-like factor 4 (KLF4) were selected as markers associated with a synthetic phenotype (a–d). In contrast, myosin heavy chain 11 (MYH11), transgelin (SM22α), myocardin (MYOCD) and myocyte enhancer factor 2C (MEF2C) are known to be associated with a contractile phenotype (e–h). (one-sample Wilcoxon test; * p ≤ 0.05, ** p ≤ 0.01 vs. negative control; †† p ≤ 0.01, ††† p ≤ 0.001 vs. healthy controls). Data are means ± standard error of the means.