## 5 Discussion  
The main tasks in our study were the following. (i) To analyze the metabolism of rivaroxaban in HUVECs in vitro. To accomplish this task, an analysis of rivaroxaban and its metabolites in cell culture media was performed using both UPLC-ESI-MS and UPLC-QTOF-MS methods. (ii) To determine the impact of rivaroxaban and the hsa-miR-24-3p mimic on the expression of _CYP4F2_ and its potential transcription factors, _HNF4a_, _PXR_ and _CAR_, and CYP4F2 protein levels in endothelial cell cultures. The commercial human umbilical vein endothelial cell culture, HUVEC, was chosen as a model system, as it is convenient for studying various aspects of endothelial function, \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Samples Transfected with miRNA Mimics**} & \multicolumn{3}{c}{**Concentration of CYP4F2**} \\ \cline{2-4}  & **Median, ng/mL** & **Minimum–Maximum Value, ng/mL** & \(p\) **Value** \\ \hline Negative control * & 20 & 9–42 & 0.029 \\ \hline hsa-miR-24-3p & 3 & 3–6 & \\ \hline \hline \end{tabular}  
* Samples transfected with miRNA, which does not contain target genes.  
\end{table} 
Table 6: Changes in the concentration of CYP4F2 protein after transfection with hsa-miR-24-3p mimic in HUVECs compared to negative control. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Treated Samples**} & \multicolumn{3}{c}{**Changes in _CYP4F2_** **Act**} \\ \cline{2-4}  & **Median** & **Minimum–Maximum Value** & \\ \hline Control sample & 15 & 15–16 & \\ \hline
1 uM dexamethasone & 16 & 16–17 & \\ \hline \hline \end{tabular}
\end{table} 
Table 7: Relative changes in the expression (\(\Delta\)Ct) of _CYP4F2_ after exposure to 1 uM dexamethasone in HUVECs compared to control cells. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Samples Transfected with miRNA Mimics**} & \multicolumn{3}{c}{**Changes in _CYP4F2_** **Act**} \\ \cline{2-4}  & **Median** & **Minimum–Maximum Value** & \\ \hline Negative control * & 15.5 & 15–16 & \\ \hline hsa-miR-24-3p & 16.2 & 16–17 & \\ \hline \hline \end{tabular}  
* Samples transfected with random sequence miRNA mimic molecule, which does not have target genes in human cell lines.  
\end{table} 
Table 5: Relative changes in the expression (\(\Delta\)Ct) of _CYP4F2_ after transfection with hsa-miR-24-3p in HUVECs compared to negative control. including angiogenesis, oxidative stress, hypoxia and inflammatory mechanisms under normal and pathological conditions associated with CVD pathogenesis. Rivaroxaban was detected in all HUVECs studied and treated with different concentrations: 0.1, 0.2, 0.5, 1, 2, 5 and 10 uM. The selection of rivaroxaban concentrations was based on the studies of [PERSON] et al. and [PERSON] et al. for the detection of rivaroxaban's peak concentration in plasma 2-4 h after 20 mg tablet administration. The concentrations presented in ng/mL were converted to molar concentrations (uM) [12,34]. According to the study of [PERSON] et al., 2013, the retention time of rivaroxaban during the performance of reversed-phase liquid chromatography was 2.37 min [35]. A similar retention time (2.34 min) was determined in our study. Based on the \(m\)/z of metabolites presented in other studies, the following metabolites were identified in the cell media of HUVECs from our study: M-1, M-2, M-5, M-8, M-10, M-11 and M-18 [36]. Rivaroxaban and its metabolites are usually analyzed in liver cells [35,36]. It is worth noting that rivaroxaban is also metabolized in HUVECs, as the results of this study revealed. The HUVEC culture medium samples treated with different concentrations of rivaroxaban were analyzed using the LC-QTOF-MS system. The analysis revealed a peak at 5.4-5.5 min, which decreased with an increase in the concentration of rivaroxaban. No data were found about the newly identified substance associated with rivaroxaban. The molecular formula of the new compound is C\({}_{22}\)H\({}_{43}\)NO.