Background:

Calcific aortic valve disease (CAVD) is the most common heart valve diseases requiring surgical or interventional treatment, worldwide, with a prevalence of >3% in humans over 75 years. Thought to be a purely degenerative disease, it is now clear that inflammation and osteoblastic differentiation of valvular interstitial cells (VICs) are hallmarks of the disease. Micro RNAs (miRNAs) have been shown to play a crucial role during osteoblastic differentiation of cardiovascular (CV) cells. Moreover, miRNAs can be transported between different cell types of the CV system via extracellular vesicles (EVs). Although the role of intercellular communication via EVs has been studied in many types of CV diseases, it remains unknown if EV bound miRNA transfer plays a role during the progression of CAVD.

Purpose:

We aimed to identify dysregulated miRNAs in the tissue of patients with CAVD, investigate their mechanistic function and elucidate whether these miRNAs could be transferred between valvular cells via EVs.

Methods and results:

We analyzed miRNA expression in explanted aortic valve cusps from patients with CAVD (undergoing aortic valve replacement surgery for aortic valve stenosis, AVS) and compared to controls (non-calcified cusps, from patients undergoing surgery for aortic regurgitation) with the help of miRNA arrays. We found a significantly different miRNA expression profile in patients with CAVD (Fig 1A). We further analyzed aortic valve tissue from mice after wire-injury induced AVS. A total of 17 miRNAs overlapped in human and murine AVS (Fig 1C). Three miRNAs from the miR-145/143 cluster were upregulated in stenotic tissue with miR-145-5p being the most significant (Fig 1A). Isolated large EVs from this tissue also showed an upregulation in patients with CAVD (Fig 1D). We isolated valvular endothelial and interstitial cells to determine the origin of the increased expression. VICs showed a markedly higher expression level of miR-145-5p (Fig 1E). In vitro calcification of VICs led to an increase of miR-145-5p expression (Fig 1F). Further analysis revealed an upregulation of alkaline phosphatase (ALPL), which is essential for valvular calcification (Fig 1F). Target prediction showed a binding site for miR-145-5p in the mRNA of ZEB2/SIP1, a known transcriptional repressor of ALPL. Expression levels of ZEB2 were significantly lower in calcified VICs. Transfection of VICs with miR-145-5p mimic led to reduced expression of ZEB2 and increased expression of ALPL (Fig 1G). MiR-145-5p therefore promotes calcification via inhibition of the ZEB2, which leads to an increased expression of ALPL. In transfer experiments we are able to show that indeed, EVs generated from VICs are taken up by target VICs. Transfer of miR-145-5p was evaluated by transfection with fluorescently labelled miR-145-5p and copy number experiments, proving uptake of miR-145-5p into target cells (Fig 1H).

Conclusion:

We identified a new signalling pathway in stenotic aortic valves, contributing to valvular calcification. MiR-145-5p induces calcification in VICs via a newly identified miR-145-5p/ZEB2/ALPL axis. Furthermore, miRNA transfer between valvular cells contribute to osteoblastic differentiation and disease progression. Further mechanistical and in vivo studies in our established wire-injury mouse model of aortic valve stenosis will reveal if this pathway is targetable with miR-145-5p inhibitors to influence disease progression.