Long noncoding RNA H19 is elevated in aortic valve stenosis and governs angiogenic response and valvular endothelial cell phenotypes through endothelial to mesenchymal transition
K Maus, Y Zhou, S Uchida, S Zimmer, A Pfeifer, G Nickenig, Dr HosenAbstract
Background
Endothelial to mesenchymal transition (EndMT) is a transdifferentiation process of cells that is associated with the development of aortic valve stenosis (AVS) in the elderly. It has been shown that long noncoding RNAs are dysregulated in this process and may play a crucial role in EndMT-mediated AVS. Here, we aim to elucidate the role of long noncoding RNA H19 in the pathogenesis of AVS with a particular focus on the EndMT process.
Methods and results
RNA sequencing of aortic valve tissue explanted from AVS patients with coronary artery disease (CAD) revealed that H19 was significantly dysregulated. EndMT was induced in vitro in vascular and valvular endothelial cells (VECs) via TNFα and TGFß+IL1β stimulation for 5 d, and expression of endothelial marker genes vWF and eNOS and mesenchymal marker genes αSMA, SM22, Vimentin, and SNAI2 was confirmed by RT-qPCR. Loss of function of H19 followed by EndMT induction demonstrated distinct changes in gene expression profile and protein levels, which were detected by qRT-PCR, immunoblotting, and immunofluorescence. Further experiments indicated that loss of H19 may promote EndMT. Functional assays in vascular and valvular endothelial cells suggested that H19 is a critical regulator of angiogenic sprouting and migration. To unveil the specific molecular signature in angiogenesis a RT2 profiler array was performed to identify the regulation of angiogenesis with 84 angiogenesis-related genes. Our assay revealed JAG1 as a potential dysregulated candidate with a 4.43-fold regulation. Further gene expression analyses via RT-qPCR and protein expression analyses via immunofluorescence and immunoblotting confirmed the H19-dependent expression of JAG1 in the EndMT process. In addition, gene expression analysis in primary VECs isolated from the aortic valves of AVS patients revealed the differential expression of several potential H19 downstream regulators of EndMT, namely JAG1, NOTCH1, and STAT3. The analysis hints at potential pathways being involved in the H19-mediated EndMT promoting AVS.
Conclusion
Our study suggests that H19 regulates the EndMT process, promoting AVS by influencing EndMT marker expression and VEC cellular function via regulation of the potential downstream target JAG1. Further elucidation of the downstream pathway and investigation in a murine model of AVS should confirm H19-mediated regulation of EndMT in the development of AVS.GraphicalAbstract