TMAO (trimethylamine N-oxide) and its precursors are ingested from meat and other diets. Increased serum concentrations of TMAO have been associated with higher risk for atherosclerosis, myocardial infarction and heart failure. However, the underlying molecular mechanisms are incompletely understood. Inflammation and subsequent endothelial-to-mesenchymal transition (EndMT), a process where endothelial cells (ECs) lose their function and phenotype and gain a mesenchymal character – contribute to the development of atherosclerotic plaques. However, a potential uptake of TMAO into ECs has not been reported yet. Renal cells are capable to uptake TMAO via an organic cation transporter, namely Solute Carrier Family 22 Member 2 (SLC22A2) but this transporter is not expressed by endothelial cells. In this study we aimed to study the potential uptake of TMAO into ECs and the impact of TMAO on EndMT.

Using tandem mass spectrometry we found that TMAO uptake into primary human endothelial cells is increased by time, resulting in a TMAO uptake of 0.5 nmol per total mg protein within the first 30 minutes and of 5.8 nmol per total mg protein within 24 hours. The uptake of TMAO resulted in a significant increase of inflammation such as upregulation of the immune cell adhesion molecule E-Selectin by 77.7-fold. A prolonged treatment with TMAO for 3 days resulted in an increase of EndMT indicated by a 6.8-fold increase of the mesenchymal marker Versican and a decrease of 92% of the endothelial marker LYVE1 (both p<0.05). For identification of a potential TMAO transporter in human endothelial cells, we determined the expression of all members of Solute Carrier Family 22 (SLC22) in ECs. ECs express four potential transporters, namely SLC22A4, SLC22A5, SLC22A17 and SLC22A23, whereas the 18 other family members show only very low or absent expression levels in ECs. SLC22A5 was reported to be a transporter for carnitine – a precursor of TMAO. We decided therefore to evaluate the role of this potential TMAO carrier in the cellular uptake of TMAO. Overexpression of SLC22A5 in ECs resulted in a 36% higher intracellular TMAO concentration after 30 minutes of TMAO treatment. SLC22A5 knockdown in ECs prevented TMAO induced inflammation (-77,8% induction of E-Selectin) and EndMT (-98,3% induction of Versican). 

In summary, our data demonstrate the uptake of TMAO into ECs. This leads to inflammation and EndMT. Furthermore, the experiments reveal that the organic cation transporter SLC22A5 regulates the uptake of TMAO into ECs and thereby modulates TMAO-mediated vascular inflammation and EndMT.