Aortic valve disease augments vesicular microRNA-145-5p to regulate the calcification of valvular interstitial cells via cellular crosstalk

Abstract

AbstractRationaleAortic 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, miRNA-dependent mechanisms regulating inflammation and calcification or miRNA-mediated cell-cell crossstalk during the pathogenesis of AVS are still poorly understood. Here, we explored the role of extracellular vesicles (EV)-associatedmiR-145-5p, which we showed to be highly upregulated upon valvular calcification in AVS in mice and humans.MethodsHuman 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 a murine AVS model.In vitrocalcification experiments were performed to explore the effects ofEV-miR-145-5pon calcification and crosstalk in valvular cells. To dissect molecular miRNA signatures and their effect on signaling pathways, integrated OMICS analyses were performed. RNA sequencing (RNA-seq), high-throughput transcription factor (TF) and proteome arrays showed that a number of genes, miRNAs, TFs, and proteins are crucial for calcification and apoptosis, which are involved in the pathogenesis of AVS.ResultsAmong several miRNAs dysregulated in valve explants of AVS patients,miR-145-5pwas the most highly gender-independently dysregulated miRNA (AUC, 0.780, p-value, 0.01). MiRNA arrays utilizing patient-derived- and murine aortic-stenosis samples demonstrated that the expression ofmiR-145-5pis significantly upregulated and correlates positively with cardiac function based on echocardiography.In vitroexperiments confirmed thatmiR-145-5pis encapsulated into EVs and shuttled into valvular interstitial cells. Based on the integrated OMICs results,miR-145-5pinterrelates with markers of inflammation, calcification, and apoptosis.In vitrocalcification experiments demonstrated thatmiR-145-5pregulates theALPLgene, a hallmark of calcification in vascular and valvular cells. EV-mediated shuttling ofmiR-145-5psuppressed the expression ofZEB2, a negative regulator of theALPLgene, by binding to its 3’ untranslated region to inhibit its translation, thereby diminishing the calcification of target valvular interstitial cells.ConclusionElevated levels of pro-calcific and pro-apoptotic EV-associatedmiR-145-5pcontribute to the progression of AVS via theZEB2-ALPLaxis, which could potentially be therapeutically targeted to minimize the burden of AVS.Clinical SignificanceWhat is known?Aortic valve stenosis (AVS) is the most prevalent structural heart valve disease requiring surgical or interventional valve replacement. Currently, no medical treatment option is available to slow, halt, or reverse the progression of the disease.AVS induces pressure overload on the left ventricle (LV), resulting in concentric hypertrophy and LV dysfunction.AVS is not an exclusively degenerative disease that leads to fibrosis and calcification of the valve cusps but rather a chronic inflammatory disease, in which mechanical strain and shear stress lead to endothelial dysfunction and immune cell infiltration, which induces chronic inflammation, apoptosis and differentiation of valvular interstitial cells into osteoblast-like cells.Increasing osteoblastic differentiation and the formation of macrocalcifications are hallmarks of the later stages of AVS.What is the new information we provide?During aortic valve stenosis, expression pattern of vesicle-associated regulatory miRNAs is altered.Patient-derived aortic valve tissue demonstrated an increased expression ofmiR-145-5pin humans, as well as in aortic valve explants from an experimental murine AVS model.MiR145-5pcontributes to calcification of the aortic valve through ZEB2, a transcriptional repressor of ALPL, in valvular interstitial cells.Extracellular vesicular shuttling ofmiR-145-5pcontributes to valvular cell-cell crosstalk and plays a role in the pathogenesis of AVS.