Despite many efforts, cardiovascular diseases are one of the most common causes of morbidity and mortality worldwide. Atherosclerosis is often to blame for their development, and the importance of the immune system in its forming and progression is being increasingly emphasized. With vascular endothelial cell activation, adhesion molecules are expressed on their surface, and pro-inflammatory cytokines and chemokines are released into the surrounding area. These attract vascular monocytes, which begin to adhere to the endothelium. Endothelial cell activation, monocyte adhesion and the resulting inflammation are supposed to be stimulated by thrombin and titanium dioxide nanoparticles. Due to thrombin’s ability to trigger increased expression of adhesion molecules on endothelial cells, it is supposed to be an important factor in atherogenesis. TiO2 nanoparticles are used in many industries, including biomedicine. Their widespread use means a greater possibility of human exposure. Since nanoparticles accumulated in the body can trigger inflammation and tissue damage, assessing their toxicity to different organ systems is crucial. By developing new in vitro models, we can significantly contribute to a more complete toxicological assessment of substances and predict their impact on human health.
With this master's thesis, we defined the ability of thrombin and TiO2 nanotubes to activate the vascular endothelial cell line 2H-11 using an in vitro cell model. This was first verified by determining the extent of adhesion of WEHI-274.1 vascular monocytes to the endothelium using confocal fluorescence microscopy. Then, we determined the expression of the adhesion proteins ICAM-1 and VCAM-1 on the endothelium exposed to thrombin and TiO2 nanotubes, by labelling them with primary fluorescent antibodies and ascertained the percentage of the surface of the imaged endothelium covered by the antibody signal.
The results showed that the number of adhered monocytes increased linearly with time, regardless of the condition to which the endothelium was exposed. By statistical testing we were unable to confirm the differences in the number of adhered monocytes between thrombin or TiO2 nanotubes and the negative control, for which we used unexposed endothelium. Compared to the negative control, the data also indicated a less extensive expression of adhesion proteins in endothelium exposed to thrombin and a more extensive expression of adhesion proteins in endothelium exposed to TiO2 nanotubes, but at the given number of measurements, differences in the expression of ICAM-1 and VCAM-1 were not yet statistically significant.
The lack of differences between control and exposed cells was mainly attributed to the strong activation of the endothelial cells 2H-11 by themselves. It turned out that they are more similar to tumour than healthy endothelium. Perhaps in the future, by perfecting our in vitro model, an effective method for determining the safety of substances and their impact on the environment and human health will be developed.
|
