Question: 

The aim of the project is to analyse ethanol effects on leukocyte and macrophage differentiation, cardiovascular commitment and the expression of pro-inflammatory cytokines in embryoid bodies derived from mouse embryonic stem (ES) cells. Ethanol is a very common stimulant and also prevalent as a solvent in many pharmaceuticals. The effects of high amounts of alcohol are well described (addiction, liver disease, poly-neuropathy etc). It is important to study also the effects of low doses of ethanol intake, because especially during very early stages of pregnancy gestation frequently has not yet been diagnosed.

Methods: 

ES-cell derived embryoid bodies (EBs) were used as an in vitro model for early stages of embryogenesis. The expression of vascular CD31 and macrophage CD68 was analysed by immunohistochemistry. mRNA expression of TLR4 (toll like receptor 4), TNFalpha (tumor necrosis factor alpha) and VEGF (vascular endothelial growth factor) was assessed by real time RT PCR. Reactive oxygen species (ROS) were determined by the fluorescence dye DCF (2',7'- dichlorodihydrofluorescein-diactate).

Results: 

The growth of EBs was inhibited starting at an ethanol concentration of 1,7 mM. A significant increase of dead cells in the supernatant was detected after 10 days at the same ethanol concentration. The differentiation of cardiomyocytes was impaired as well at day 10 and the beating frequency was decreased. The differentiation of endothelial cells was significantly impaired at 0,34 mM and higher ethanol concentrations. The mRNA expression of VEGF was decreased which correlates with the reduction of the CD31 positive cell area at day 4 + 6 of differentiation. The number of CD68 positive cells was first slightly increased at low ethanol concentrations of 0,17mM and reduced at higher concentrations of 17,1mM. The inflammation markers TLR4 and TNFa were upregulated at low concentrationes of 0,17mM ethanol. Ethanol stimulated the generation of ROS. Hence the inhibition of endothelial cell differentiation by ethanol may be due to oxidative stress.

Conclusion: 

Ethanol stimulates ROS generation and the inflammatory response already at low concentrations (0,17mM). At high (0,34–1,7 mM) concentrations endothelial and cardiomyocyte differentiation is suppressed. CD68 positive macrophages were inhibited at higher ethanol concentrations as well.