Reproducible In Vitro Tissue Culture Model to Study Basic Mechanisms of Calcific Aortic Valve Disease: Comparative Analysis to Valvular Interstitials Cells.

Andreas Weber,Payam Akhyari,Friederike Schöttler,Melissa Pfaff,Artur Lichtenberg,Vera Schmidt

doi:10.3390/biomedicines9050474

Andreas Weber, Payam Akhyari + Show 4 more

Open Access

https://doi.org/10.3390/biomedicines9050474

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Abstract

The hallmarks of calcific aortic valve disease (CAVD), an active and regulated process involving the creation of calcium nodules, lipoprotein accumulation, and chronic inflammation, are the significant changes that occur in the composition, organization, and mechanical properties of the extracellular matrix (ECM) of the aortic valve (AV). Most research regarding CAVD is based on experiments using two-dimensional (2D) cell culture or artificially created three-dimensional (3D) environments of valvular interstitial cells (VICs). Because the valvular ECM has a powerful influence in regulating pathological events, we developed an in vitro AV tissue culture model, which is more closely able to mimic natural conditions to study cellular responses underlying CAVD. AV leaflets, isolated from the hearts of 6–8-month-old sheep, were fixed with needles on silicon rubber rings to achieve passive tension and treated in vitro under pro-degenerative and pro-calcifying conditions. The degeneration of AV leaflets progressed over time, commencing with the first visible calcified domains after 14 d and winding up with the distinct formation of calcium nodules, heightened stiffness, and clear disruption of the ECM after 56 d. Both the expression of pro-degenerative genes and the myofibroblastic differentiation of VICs were altered in AV leaflets compared to that in VIC cultures. In this study, we have established an easily applicable, reproducible, and cost-effective in vitro AV tissue culture model to study pathological mechanisms underlying CAVD. The valvular ECM and realistic VIC–VEC interactions mimic natural conditions more closely than VIC cultures or 3D environments. The application of various culture conditions enables the examination of different pathological mechanisms underlying CAVD and could lead to a better understanding of the molecular mechanisms that lead to VIC degeneration and AS. Our model provides a valuable tool to study the complex pathobiology of CAVD and can be used to identify potential therapeutic targets for slowing disease progression.

Highlights

Calcific aortic valve disease (CAVD), a major cause of aortic stenosis (AS), is the most frequent type of valvular disorder worldwide [1,2]
The cellular components of the aortic valve include a monolayer of valvular endothelial cells (VECs) on the outer surface of the leaflets and valvular interstitial cells (VICs), which populate each of the three layers [6,9]
Degeneration of AV Leaflets Progresses over Time

Summary

Introduction

Calcific aortic valve disease (CAVD), a major cause of aortic stenosis (AS), is the most frequent type of valvular disorder worldwide [1,2]. Due to a lack of medical treatment, aortic valve replacement, performed surgically (SAVR) or transcatheterally (TAVI) remains the gold standard of the treatment of symptomatic aortic valve stenosis [4,5]. Amongst the hallmarks of CAVD are the significant changes that occur in the composition, organization, Biomedicines 2021, 9, 474. Biomedicines 2021, 9, 474 and mechanical properties of the highly organized extracellular matrix (ECM) of the aortic valve (AV) [11,12]. The cellular components of the aortic valve include a monolayer of valvular endothelial cells (VECs) on the outer surface of the leaflets and valvular interstitial cells (VICs), which populate each of the three layers [6,9]. VICs can differentiate into myofibroblast-like (aVIC) or osteoblast-like (obVIC)

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