Effects of ethanol on the properties of platelets and endothelial cells in model experiments

Abstract

AIM: To investigate effects of ethanol on activity markers of atherosclerosis in an in vitro endothelial cell model.

METHODS: After 24 h incubation with ethanol (0.0095%), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide, and were then incubated in direct contact with activated platelets. Following this incubation, the expression of CD40L and CD62P on platelets, and the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), urokinase plasminogen activator receptor (uPAR), and membrane-type 1 matrix metalloproteinase (MT1-MMP) on endothelial cells were measured by flow cytometry.

RESULTS: The increased expression of VCAM-1 and uPAR on endothelial cells by proinflammatory stimulation with activated platelets was significantly reduced through pre-incubation with ethanol (P < 0.05). Furthermore, platelets in direct contact with ethanol and with endothelial cells pre-incubated in ethanol showed a significant reduction in their CD40L expression (P < 0.05). Ethanol had no significant effect on ICAM-1 and MT1-MMP expression on endothelial cells.

CONCLUSION: Ethanol directly attenuates platelet activation and has significant endothelial cell-mediated effects on selected markers of atherosclerosis in vitro. These findings underline possible protective effects of ethanol on atherosclerosis.

Key Words: Platelets, Endothelial cells, Ethanol, Inflammation, Atherosclerosis

INTRODUCTION

Atherosclerosis is a progressive disease characterized by increased expression of proinflammatory markers and interactions between endothelial cells and platelets[1,2]. Platelet activation leads to an increased expression of inflammatory factors that stimulate endothelial cells and recruit additional platelets[3,4]. CD62P and CD40L are expressed on activated platelets and are directly involved in the interaction of platelets with leukocytes and endothelial cells[5]. Stimulation of endothelial cells and interaction with platelets induces the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which initiate the recruitment of neutrophils, monocytes and lymphocytes[6,7]. In turn, these result in the expression and release of matrix-degrading proteinases, the matrix metalloproteinases (MMPs)[8]. In a complex cascade, the urokinase plasminogen activator receptor (uPAR) binds and activates urokinase-type plasminogen activator, and directly degrades various proteins of the extracellular matrix and promotes vascular inflammation.

Augmented and chronic alcohol consumption is accountable for a variety of organ damage including cardiomyopathy[9], pancreatitis[10] and chronic alcoholic cirrhosis of the liver[11]. In contrast, moderate alcohol consumption may reduce the risk of coronary artery disease and total mortality[12,13]. Therefore, a strict limitation of alcohol consumption (< 30 g/d for men and < 15 g/d for women) is recommended and reasonable. Cardioprotective effects of alcohol may be mediated by antioxidative properties, an increase in high-density lipoprotein (HDL) cholesterol, as well as anti-thrombotic and vasodilatory effects. However, the underlying causative mechanisms remain unclear.

The aim of this study was to investigate the effects of ethanol on the expression of established markers of atherosclerosis on endothelial cells and platelets in an in vitro model of atherosclerosis.

MATERIALS AND METHODS

Co-incubation of platelets with ethanol pre-incubated endothelial cells

Human umbilical vein endothelial cells (HUVECs) were prepared as previously described[14,15] and cultured in endothelial cell basal medium (PromoCell) containing 2% fetal calf serum [1 μg/mL hydrocortisone (HC-500); 0.4% endothelial cell growth supplement (ECGS/H-2); 0.1 ng/mL epidermal growth factor (hEGF-0.05); 1 ng/mL basic fibroblast growth factor (hbFGF-0.5); 5 ng/mL amphotericin and 50 μg/mL gentamicin]. The confluenced endothelial cells were added to 12-well plates and incubated for 24 h with ethanol 0.0095% (Merck, Hohenbrunn, Germany). Platelets were prepared from blood of healthy probands as previously described[16,17]. Washed platelets were stimulated for 30 min with thrombin (0.5 U/mL) and lipopolysaccharide (LPS) (1000 ng/mL). Before coincubation experiments, thrombin activity was antagonized by hirudin (5 U/mL). Pretreated platelets (final concentration 2 × 10⁸/mL) were added to confluent endothelial monolayers with and without ethanol. After 60-min co-incubation under cell culture conditions, all platelets were removed by gentle washing, which was confirmed by light microscopy. After an additional 6 h of incubation of the endothelial cells, supernatant was aspirated, centrifuged at 2000 g and stored at -80 °C[18]. Following this incubation, the expression of activity markers on platelets, as well as that on endothelial cells were measured by flow cytometry.

Flow cytometric analysis

Flow cytometric analysis of platelets and endothelial cells was performed by gating in forward and side scatter. Platelets were gated back for determination of the expression of CD40L and CD62P. For the analysis of platelets, 100 μL of each sample were stained for 15 min at room temperature with 10 μL aliquots of mouse anti-human CD40L-FITC antibodies (BD Pharmingen, Heidelberg, Germany) and mouse anti-human CD62P-PE antibodies (Beckman-Coulter, Krefeld, Germany). Endothelial cells were gated back for determination of the surface expression of ICAM-1, VCAM-1, uPAR and membrane type 1 matrix MMP (MT1-MMP). For the analysis of endothelial cells, 100 μL of each sample were stained for 15 min at room temperature with 10 μL aliquots of anti-human CD54 PE-Cy5 (ICAM-1 from BD Pharmingen, Heidelberg, Germany), anti-human CD106-FITC (VCAM-1 from R&D Systems, Inc., Wiesbaden, Germany), anti-human CD87-FITC (uPAR from American diagnostica inc., Stamford, United Kingdom), anti-human MT1-MMP (Ab-1) Mouse mAb (114-IF2) (Merck Chemicals Ltd., Nottingham, United Kingdom). Corresponding isotypes (Beckman Coulter, Marseille, France) were used as a control. All flow cytometric analysis was performed on an EPICS XL-MCL analyzer (Beckman Coulter, Krefeld, Germany) equipped with an argon laser tuned at 488 nm. Mean fluorescence intensity was measured and all FACS data is expressed as MFI in this manuscript. System II Version 3.0 software was used for data acquisition and evaluation. Compensation of the four channel fluorescence was precisely adjusted using Cyto-CompTM reagents and Cyto-TrolTM control cells (Coulter Immunotech/Krefeld, Germany).

Statistical analysis

All calculations were performed using SAS release 9.2 (SAS institute Inc. Cary NC, United States). Numerical data were expressed as mean ± SD. A Student t test was applied as a parametric test. A two-tailed probability value < 0.05 was considered significant.

RESULTS

Effects of pre-incubation with ethanol on endothelial cell surface markers

HUVEC pre-incubation with ethanol resulted in decreased surface expression of VCAM-1 and uPAR under proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets compared to HUVEC without ethanol. Pre-incubation with ethanol significantly reduced surface expression of VCAM-1 from 1.809 ± 1.03 to 0.82 ± 0.13 (P = 0.045) after contact with resting platelets, from 2.16 ± 1.38 to 0.83 ± 0.11 (P = 0.043) after contact with LPS-stimulated platelets, from 1.85 ± 1.02 to 0.89 ± 0.20 (P = 0.048) after contact with LPS- and thrombin-stimulated platelets, and from 1.89 ± 1.05 to 0.92 ± 0.11 (P = 0.05) after stimulation with interleukin 1-β (Figure 1A). Pre-incubation with ethanol significantly reduced surface expression of uPAR from 0.66 ± 0.30 to 0.35 ± 0.07 (P = 0.05) after contact with thrombin-stimulated platelets, from 1.64 ± 0.37 to 0.28 ± 0.02 (P = 0.044) after contact with LPS-stimulated platelets and from 0.70 ± 0.35 to 0.34 ± 0.02 (P = 0.04) after contact with LPS- and thrombin-stimulated platelets (Figure 1B). Ethanol had no significant effects on ICAM-1 and MT1-MMP expression on endothelial cells.

Figure 1 Surface expression of vascular cell adhesion molecule-1 (A) and urokinase plasminogen activator receptor (B) on human umbilical vein endothelial cell with and without 24-h pre-incubation with ethanol. VCAM-1: Vascular cell adhesion molecule-1; uPAR: Urokinase plasminogen activator receptor; HUVEC: Human umbilical vein endothelial cells; PLT: Platelets; Thr.: Thrombin; LPS: Lipopolysaccharide; NS: Not significant.

Endothelial cell-mediated and direct effects of ethanol on platelets

Platelets in direct contact with ethanol-pre-incubated endothelial cells showed a significant reduction in their CD40L expression compared to platelets incubated with untreated endothelial cells. Surface expression of CD40L on thrombin-stimulated platelets after 1 h in direct contact with HUVECs pre-incubated with ethanol significantly decreased from 2.15 ± 0.19 to 1.98 ± 0.16 (P = 0.004) and, when stimulated with thrombin and LPS, significantly decreased from 2.20 ± 0.17 to 1.98 ± 0.14 (P = 0.03) (Figure 2A). Ethanol had no significant effects on CD62P on platelets after endothelial cell contact (Figure 2B). To discriminate between effects mediated by pre-treated endothelial cells and possible direct effects of ethanol on platelets, platelets were directly incubated with ethanol alone for 1 h. Here, ethanol incubation significantly decreased CD40L expression on thrombin-stimulated platelets from 2.31 ± 0.17 to 1.92 ± 0.26 (P = 0.01) (Figure 3A). Surface expression of CD62P on thrombin-stimulated platelets after 1 h incubation with ethanol was significantly reduced from 3.56 ± 2.17 to 1.83 ± 0.76 (P = 0.03) (Figure 3B).

Figure 2 Surface expression of CD40L (A) and CD62P (B) on platelets after 1-h adherence to human umbilical vein endothelial cell with and without ethanol pre-treatment. PLT: Platelets; Thr.: Thrombin; LPS: Lipopolysaccharide; NS: Not significant.

Figure 3 Direct effects of ethanol on surface expression of CD40L (A) and CD62P (B) on platelets. PLT: Platelets; Thr.: Thrombin; LPS: Lipopolysaccharide; NS: Not significant.

DISCUSSION

Several prospective clinical studies suggest an inverse correlation between moderate alcohol consumption and coronary artery disease. In a prospective study, Rimm et al[19] studied alcohol intake and prevalence of coronary artery disease in 51 591 males and reported that increasing alcohol intake was inversely correlated with coronary artery disease incidence (P < 0.001). Previous experimental studies investigated possible cardioprotective mechanisms of alcohol. Linn et al[20] and Goldberg et al[21] described in a representative sample of the United States adult population, that moderate alcohol consumption (one or two drinks per day) increased circulating levels of HDL by 12% on average. Gil-Bernabe et al[22] compared low- and high-dose treatment with ethanol in a mouse model and showed less dilatation of the aorta and less atherosclerotic plaques in mice treated with low-dose ethanol. Authors discussed the possible mechanism as an increased secretion of stromal cell-derived factor-1 (SDF-1) with subsequent enhanced mobilization of progenitor cells. Although the antiinflammatory signalling mechanisms of ethanol in detail still are unknown, they may be result from an increase in SDF-1 concentration in peripheral blood or an enhancement of the activity of endothelial nitric oxide synthase.

The present study provides further mechanistic insights and evidence for atheroprotective effects of ethanol by demonstrating significantly reduced expression of VCAM-1 and uPAR on endothelial cells after pre-incubation with ethanol and stimulation with activated platelets. VCAM-1 and uPAR are well established atherosclerotic markers and play an important role in the development of cardiovascular diseases. Furthermore this study investigated direct and endothelial cell-mediated effects of ethanol on platelets, and provides evidence that ethanol has significant attenuating effects on CD40L and CD62P expression on platelets. Since the aim of this study was to investigate effects of moderate alcohol consumption in an in vitro endothelial cell model, the experimental condition of alcohol applied in our study (alcohol concentration of 0.0095%) was equivalent to a human blood alcohol concentration of 0.1%. This dose of ethanol therefore is consistent with moderate alcohol consumption in real life. The present study was not designed to assess different ethanol concentrations in vitro. The possibility of different effects of different concentrations of ethanol on endothelial and platelet functions therefore cannot be excluded.

In conclusion, the findings of the present study underline possible protective effects of ethanol on atherosclerosis. Ethanol significantly decreased the expression proatherogenic markers on endothelial cells and has direct inhibitory effects on platelets. Further studies are warranted to confirm our results and to expand scientific knowledge about atheroprotective effects of ethanol on endothelial cells and platelets.

COMMENTS

Background

Moderate alcohol consumption may reduce the risk of coronary artery disease and total mortality. This study investigated the effects of ethanol on the expression of established markers of atherosclerosis on endothelial cells and platelets in an in vitro model of atherosclerosis.

Research frontiers

Ethanol, at a moderate concentration equivalent to a human blood alcohol concentration of 0.1%, significantly reduced the expression of vascular cell adhesion molecule-1 and urokinase plasminogen activator receptor, on endothelial cells after pre-incubation with ethanol and stimulation with activated platelets

Innovations and breakthroughs

Ethanol can significantly decrease the expression proatherogenic markers on endothelial cells and has direct inhibitory effects on platelets.

Applications

The study underlined possible protective effects of ethanol on atherosclerosis.

Peer review

This is a brief article regarding the possible mechanisms of ethanol on endothelial cells and platelets. This manuscript provides some insights in understanding the effect of ethanol on vessels.