1Universitätsklinikum Bonn Medizinische Klinik und Poliklinik II Bonn, Deutschland; 2Universitätsklinikum Hamburg-Eppendorf Klinik für Kardiologie Hamburg, Deutschland; 3Institute for Cardiovascular Sciences Endothelial Signaling and Metabolism Bonn, Deutschland; 4Center for RNA Medicine Department of Clinical Medicine Copenhagen SV, Deutschland; 5The Christina Lee Brown Environment Institute Department of Medicine, University of Louisville Kentacky, USA; 6University Hospital Bonn Institute of Pharmacology and Toxicology Bonn, Deutschland; 7Cardiovascular Life Science section/Brigham and Women’s Hospital Harvard Medical School Boston, Deutschland
Rationale: Aortic valve stenosis (AVS) is a major contributor to cardiovascular death in the elderly population worldwide. MicroRNAs (miRNAs) are highly dysregulated in patients with AVS undergoing surgical aortic valve replacement (SAVR). However, a specific role and cell-cell crosstalk mediated by miRNAs and their effects on AVS are not well understood. Here, we explored the novel role of extracellular vesicles (EV)-associated-miR-145-5p, which was highly upregulated upon valvular calcification in AVS in mice and humans.
Methods: Human TaqMan miRNA arrays identified dysregulated miRNAs in aortic valve tissue explants from AVS patients compared to non-calcified valvular tissue explants of patients undergoing SAVR. Echocardiographic parameters were measured in association with the quantification of dysregulated miRNAs in murine AVS model. In vitro calcification were performed to explore the effects of EV-miR-145-5p on calcification and crosstalk in vulvular cells. To dissect molecular signature and their effect on signaling pathways, integrated OMICS analyses were performed. NGS, high-throughput transcription factor (TF)- and proteome arrays elucidated that a number of genes, miRNAs, TFs, and proteins are crucial for calcification and apoptosis, involved in the pathogenesis of AVS.
Results: Among several miRNAs deregulated in tissue explant of AVS patients, miR-145-5p was the highest dysregulated miRNA with gender-indepenent manner of patients (AUC, 0.780, p-value, 0.01). Functional miRNA array by utilizing patient-derived- and murine aortic-stenosis samples demonstrated that the expression of miR-145-5p is significantly upregulated and correlates positively with cardiac function, analyzed via echocardiography. In vitro experiments confirmed that miR-145-5p is encapsulated into EV and shuttled into valvular interstitial cells. Based on the integrated OMICs approaches, miR-145-5p interrelates with the markers of inflammation, calcification and apoptosis. In vitro calcification experiments demonstrated that miR-145-5p regulates the ALPL gene, a hallmark of calcification in vascular and valvular cells. EV-mediated shuttling of miR-145-5p suppressed the expression of ZEB2, a negative regulator of the ALPL gene, by binding to its 3' untranslated region to inhibit its translation and thereby diminishing the calcification of target valvular interstitial cells.
Conclusion: Elevated levels of pro-calcific, and pro-apoptotic EV-associated miR-145-5p contributes to the progression of AVS via a miR-145-5p/ZEB2/ALPL axis. We identified a new miR-145-5p/ZEB2/ALPL axis that contributes to the pathogenesis of AVS, and can be therapeutically targeted to minimize the burden of AVS.