Title : In vitro model of co-cultivation of endothelial and mast cells to study atherogenesis mechanisms
Abstract:
Atherosclerosis is accompanied with inflammation, affecting the vascular endothelial transport. The mechanisms of mast cells (MSc) secretome influence on transendothelial transport is unclear. The aim was to study the effect of MCs-derived biological factors on the transendothelial transport of LDLs. EA.hy926 cells (upper chamber) were co-cultured with activated MCs (HMC-1, lower chamber). Test substances (LDLs, CRP, TNFα, signaling pathway inhibitors etc.) were added to the upper chamber and incubated for 24h. The transport rate was calculated as the % of LDLs transferred to the lower chamber. Samples were analyzed biochemically and immunologically. On average, 25 repeats per point was performed in each experiment. Two-way analysis of variance and Tukey's post hoc test were used for statistics. After 24h, up to 15% of the proteins passed through the endothelial cell monolayer. Transport without the endothelial monolayer was used as control: It was more rapid and reached maximum after 2h (25-40% of the protein amount). LDLs transport across the endothelium involves clathrin- and caveolin-dependent transcytosis, as shown by confocal microscopy, flow cytometry and inhibitors. Pro-inflammatory concentrations of CRP and TNFα enhanced transendothelial transport. Although MCs-derived TNFα activating the transport of LDLs, the MCs suppressed this process. The key role of histamine in mediating MCs suppressive effect on transendothelial transport was shown using H1-receptor inhibitors. We showed that MCs can modulate the physiological state of blood vessels endothelium via histamine. In vitro model is suitable for further study of the influence of MCs and pro-atherogenic factors on the endothelium barrier. We consider MCs and their mediators as pro-atherogenic factors enhancing the rate of LDLs influx into the arterial intima. By modulating the activity of MCs, their mediators, or receptors it is possible to influence atherogenic processes. This research was funded by the Russian Science Foundation (RSCF), grant number 25-74-31017.
