Background:

Calcific aortic valve disease (CAVD) is the most common heart diseases, requiring interventional or surgical valve implantation/replacement if symptomatic. At this time, no pharmacological treatment for halting or reversing CAVD is available. The prevalence of this disease is over 2% in patients older than 60 years of age and the 2-year mortality rate of symptomatic, severe aortic valve stenosis (AVS) is greater than 50%. In recent years, our understanding of disease mechanisms and pathology in this area has changed greatly. It has become clear that CAVD is not purely a degenerative process, but that tightly regulated mechanisms underlie disease initiation and progression. Under physiological conditions, the ventricular and the aortic side of the valve are exposed to dramatically different blood flow patterns. The flow on the ventricular side is characterized by being high in magnitude, pulsatile and laminar, whereas the flow pattern on the aortic side is low in magnitude and oscillatory. Calcification of the valve is frequently only seen on the fibularis side of the valve cusps, suggesting a possible involvement of flow patterns in disease initiation and progression.

Purpose:

We aim to elucidate the effect of different flow patterns on ncRNA and gene expression in valvular endothelial cells (VECs) and investigate the effects on endothelial dysfunction, endothelial to mesenchymal transition (EndMT) and immune cell adhesion. 

Methods and results:

In initial experiments we compared VECs exposed to laminar flow with no-flow controls with the help of an Ibidi pump system. We can show that flow-dependent changes in miRNA and gene expression indeed occur. We exposed human VECs to high laminar flow (20 dyn/cm²) and performed miRNA arrays as well as gene expression analysis via TaqMan qPCR. Known shear sensitive genes (e.g. KLF2, KLF4) are upregulated, adhesion molecules (e.g. VCAM1, ICAM1) and genes promoting EndMT (e.g. transforming growth factor beta, TGF-b) are downregulated (Fig 1 A). In a miRNA array, we observed a large number of miRNAs that where differentially up- or downregulated in a flow dependent manner (Fig 1B). We have also established an EndMT model mimicking calcifying conditions, present in the diseased aortic valve (Fig 1C). For this, we subject human VECs to osteogenic medium (OM) with added TGF-b1 or BMP-2. Treating human VECs with this medium results in an upregulation of specific markers for EndMT (e.g. SNAI2) and decrease in endothelial markers (e.g. PECAM1, CDH5). We are currently in the course of exposing VECs to different laminar, pulsatile laminar or oscillatory flow rates, mimicking flow conditions on the ventricular and aortic side of the aortic valve cusps. RNA isolated from these cells will be analyzed by whole transcriptomic sequencing to identify differential expression of genes and noncoding RNAs (long ncRNAs, circular RNAs etc.). Flow-induced or repressed ncRNA candidates will be further analyzed in our EndMT model to unravel their mechanistic role during disease pathogenesis. 

Conclusion:

Initial endothelial dysfunction is one of the initiating factors of CAVD. Different flow conditions on the aortic and ventricular valve cusps seem to have protective or detrimental effects on the endothelium. Systematic analysis of the effect of different flow conditions on valvular endothelial cells will identify involved genes/proteins that might be targetable to ameliorate disease initiation and progression.