Background
Calcifying aortic valve stenosis (AS) is the most common valvular heart disease in humans. Our current pathophysiological concept of AS development suggests that local chronic inflammation induces fibrosis and calcification of the valve cusps. Underlying remodeling processes are driven by endothelial to mesenchymal transition (EndMT) of aortic valvular endothelial cells (VECs). During EndMT, valvular endothelial cells lose their endothelial characteristics and acquire a mesenchymal phenotype. Various signaling pathways, led by distinct signaling molecules, for instance TGFβ, BMPs, and inflammatory cytokines, regulate this transition. Targeting these pathways with anti-inflammatory agents or inhibitors of EndMT could offer novel therapeutic strategies for AS. For in vitro experiments various protocols have been used to study EndMT, however, the optimal induction protocol for this process remains a topic of active debate. The aim of this study is to systematically test various substances including pro-inflammatory cytokines to establish a comprehensive protocol for EndMT induction in vitro.
Methods and results
In order to induce EndMT in VECs the following substances were tested: (1) TGFβ1, (2) the combination of TGFβ1 + IL1β, (3) TNFα, (4) BMP-2, (5) BMP-4, (6) BMP-6, and (7) BMP-9. As marker genes to quantify effectiveness of EndMT, expression of predominantly endothelial (PECAM, vWF and NOS3) and mesenchymal (VIMENTIN, ACTA2 and VCAM1) genes was examined via qPCR. The combination of TGFβ1 + IL1β (2) and TNFα (3) were shown to be the most effective strategies, leading to a significant suppression of vWF and NOS3 and a significant overexpression of VCAM1. In contrast, the BMPs showed only minor changes in gene expression in comparison to the negative control. Consequently, subsequent experiments focused on TGFβ1 + IL1β (2) and TNFα (3). A dosing experiment for TGFβ1 showed that a concentration of 2 ng/mL led to the same EndMT induction as 10 ng/mL. In a proteomic analysis the most reliable induction of EndMT was shown with concomitant TGFβ1 + IL1β stimulation leading to a robust suppression of endothelial (NOS3 and vWF) and upregulation of mesenchymal (VCAM1, TAGLN) proteins. Furthermore, it was shown that it is beneficial to use medium free of hydrocortisone and VEGF for EndMT induction, as these components stabilize the phenotype of endothelial cells, which is desired for under regular culture.
Conclusion
The most pronounced and consistent EndMT induction was achieved with the concomitant stimulation of TGFβ and IL1β. A concentration of 2 ng/mL TGFβ1 is sufficient. Furthermore, the medium should not contain hydrocortisone and VEGF.
