A Novel Ex Vivo Model of Aortic Valve Calcification. A Preliminary Report

Abstract

Background: No pharmacological treatment exists to prevent or stop the calcification process of aortic valves causing aortic stenosis. The aim of this study was to develop a robust model of induced calcification in whole aortic valve leaflets which could be suitable for studies of the basic mechanisms and for testing potentially inhibitory drugs.Methods: Pig hearts were obtained from a commercial abattoir. The aortic valve leaflets were dissected free and randomized between experimental groups. Whole leaflets were cultured in individual wells. Two growth media were used for cultivation: standard growth medium and an antimyofibroblastic growth medium. The latter was employed to inhibit contraction of the leaflet into a ball-like structure. Calcification was induced in the growth medium by supplementation with an osteogenic medium. Leaflets were cultivated for four weeks and medium was changed every third day. To block calcification, the inhibitor SNF472 (a formulation of the hexasodium salt of myo-inositol hexaphosphate hexasodium salt) was used at concentrations between 1 and 100 µM. After cultivation for four weeks the leaflets were snap frozen in liquid nitrogen and kept at −80 °C until blind assessment of the calcium amount in leaflets by inductively coupled plasma optical emission spectroscopy. For statistical analysis, a Kruskal–Wallis test with Dunn’s post-test was applied.Results: Osteodifferentiation with calcium accumulation was in principle absent when standard medium was used. However, when the antimyofibroblastic medium was used, a strong calcium accumulation was induced (p = 0.006 compared to controls), and this was blocked in a dose-dependent manner by the calcification inhibitor SNF472 (p = 0.008), with an EC50 of 3.3 µM.Conclusion: A model of experimentally induced calcification in cultured whole leaflets from porcine aortic valves was developed. This model can be useful for studying the basic mechanisms of valve calcification and to test pharmacological approaches to inhibit calcification.

Organ Culture Model of Aortic Valve Calcification

“…The use of intact valve leaflets has the advantage that the VIC are retained in the physiological arrangement with respect to their association with the extracellular matrix and their relationship with the VEC. Intact valve leaflets from pigs and mice have previously been used to assess the effects of pro-calcific stimuli, including inorganic phosphate osteogenic media, osteogenic media supplemented with TGFβ1 and mechanical injury (11,12,(21)(22)(23)(24). These previous studies have largely relied upon osteogenic media, which contains β-glycerophosphate, dexamethasone, and ascorbic acid, to drive the calcification response.…”

Cardiomyogenic Differentiation Potential of Human Dilated Myocardium-Derived Mesenchymal Stem/Stromal Cells: The Impact of HDAC Inhibitor SAHA and Biomimetic Matrices

“…In addition, the MSC isolated from different types of adult tissues can have specific features: human fetal heart MSC was shown to have cardiac immunophenotyping and differentiation potential to CM, endothelial, or smooth muscle cells [ 42 , 43 , 44 ]. Human heart myocardium-derived MSCs can be used to investigate the human heart pathologies and/or possible ways of heart regeneration [ 45 , 46 ]. Other authors also suggested that adult human heart tissue-derived MSC have better cardio regenerating capabilities compared to the bone marrow or other types of adult MSC [ 47 ].…”

“…In this study, the dilated myocardium-derived hmMSC showed different properties compared to the healthy hmMSC: they were bigger in size, slower proliferated, showed more pronounced osteogenic differentiation than chondro and adipo, but had similar MSC-typical surface markers [ 56 , 57 ]. The strong osteogenic differentiation of both types of the hmMSC might be an age-related sign: both cell types were isolated from elder men (55–65 years old) myocardiums that often show increased calcium deposits causing calcification and stiffening of the valve cusps in vivo [ 45 ]. Moreover, the dilated hmMSC had significantly (1.5-fold) higher level of HDACs activity, which could negatively affect dilated myocardium.…”

Models and Techniques to Study Aortic Valve Calcification in Vitro, ex Vivo and in Vivo. An Overview

“…We have developed a reliable model of cultured whole leaflets from porcine valves ( Zabirnyk et al, 2020 ). Shortly, after animal sacrifice in an authorized abattoir, the hearts are transported on ice to the laboratory where the aortic valve leaflets are dissected free.…”