Endothelial-mesenchymal transition represents a key process during atherosclerotic plaque calcification

Study objective The endothelial-mesenchymal transition (End-MT) has been recently proposed as a key process during plaque evolution and calcification. In our hypothesis, microenvironment cues stimulate the endothelial cell conversion into MSC-like cells that lately differentiate toward the osteogenic lineage, contributing to the calcium accumulation within the intima. The main objective of the present study was to investigate the expression of SLUG, a key transcription factor regulating the End-MT, in human atherosclerotic lesions of abdominal aortic aneurysms (AAA) and carotid plaques (CP). Then we addressed the End-MT in endothelial cells, investigating its possible association with the calcification process in presence of inflammatory soluble mediators and in co-culture models with vascular resident cells. Methods SLUG was analysed in FFPE tissues of AAA and CP patients. End-MT was explored in Human Umbilical Vein Endothelial Cells (HUVEC) in presence of the inflammatory cytokine TNF-α and the members of the TGF- β superfamily (the canonical TGF-β1 and TGF-β3) for 7 days. Mesenchymal Stem Cells (MSCs) were ex-vivo isolated from AAA and CP biopsies, characterized and used for transwell co-culture assays with HUVEC. After 7-days, HUVEC were tested for osteogenic differentiation by calcium stain with Alizarin Red and by mRNA levels of SLUG, MMP-9, CD31 and RUNX-2. Considering that SLUG is a potential target of miR-30 family, we also detected miR-30a/d variations in HUVEC after each culture conditions. Results SLUG analysis by IHC revealed intense stain in AAA and CP tissues, mostly within inflammatory infiltrates and in endothelial cells. TNF-α and TGF-β3 effectively promoted End-MT, as supported by the spindle-shape morphology and by the increase of SLUG and MMP-9 mRNA. TNF-α also stimulated RUNX-2 expression in HUVEC. In addition, HUVEC demonstrated osteogenic property as shown by mineralization assay and RUNX-2 expression, especially after culture with AAA- and CP-MSC. Interestingly, the HUVEC osteogenic phenotype was accompanied by SLUG up-regulation and slight decrease of CD31. Finally, End-MT and osteogenic differentiation decreased miR-30a and miR-30d expression in HUVEC. Conclusions Our results support the occurrence of the End-MT process in human atherosclerotic disease and highlight that the endothelial cells actively govern the plaque formation as well as the late calcification, by switching into the mesenchymal cell intermediate. Future investigations will address the potential therapeutic strategies targeting the main End-MT mediators in order to inhibit the calcification of the atherosclerotic plaque.