Enzymes of the purinergic signaling system exhibit diverse effects on the degeneration of valvular interstitial cells in a 3‐D microenvironment

Abstract

Calcific aortic valve disease is an active disease process with lipoprotein deposition, chronic inflammation, and progressive leaflet degeneration. Expression of ectonucleotidases, a group of membrane-bound enzymes that regulate the metabolism of ATP and its metabolites, may coregulate the degeneration process of valvular interstitial cells (VICs). The aim of this study was to investigate the role of the enzymes of the purinergic system in the degeneration process of VICs. Ovine VICs were cultivated in vitro under different prodegenerative conditions and treated with inhibitors of ectonucleoside triphosphate diphosphohydrolase 1 (CD39)/ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and 5'-nucleotidase (CD73), as well as with adenosine and adenosine receptor agonists. Experiments were performed both in 2-dimensional (2-D) and 3-dimensional (3-D) cell-culture models. Our main findings were that VICs continuously release ATP. Inhibition of ATP hydrolyzing enzymes (CD39 and ENPP1) resulted in profound prodegenerative effects with a vigorous up-regulation of CD39, ENPP1, and CD73, as well as TGF-β1 and osteopontin at the gene level. In our 3-D model, the effect was more pronounced than in 2-D monolayers. Increasing adenosine levels, as well as stimulating the adenosine receptors A and A, exhibited strong prodegenerative effects, whereas conversely, lowering adenosine levels by inhibition of CD73 resulted in protective effects against degeneration. Dysregulation of any one of these enzymes plays an important role in the degeneration process of VICs. Stimulation of ATP and adenosine has prodegenerative effects, whereas lowering the adenosine levels exerts a protective effect.-Weber, A., Barth, M., Selig, J. I., Raschke, S., Dakaras, K., Hof, A., Hesse, J., Schrader, J., Lichtenberg, A., Akhyari, P. Enzymes of the purinergic signaling system exhibit diverse effects on the degeneration of valvular interstitial cells in a 3-D microenvironment.

Nucleotide ecto-enzyme metabolic pattern and spatial distribution in calcific aortic valve disease; its relation to pathological changes and clinical presentation

“…[10] Adenosine was also attributed to have pro-stenotic properties by the stimulation of ovine VIC degeneration by A2aR and A2b receptor (A2bR) activation. [66] On the contrary, treatment with A2bR agonist significantly reduced the calcification volume of progenitor cell-derived teratomas from patients with CD73 deficiency. [65] Also in our recent work, we have shown that CD73−/− mice spontaneously developed aortic valve dysfunction increasing peak aortic flow and valvular Fig.…”

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

“…Further, there is a lot of literature, to which current results and outcome measures can be compared [ 26 , 41 , 42 ]. Artificially created 3D environments of VICs are more physiologically relevant and predictive than 2D cultures and exhibit a higher degree of structural complexity and homeostasis but are time consuming, labor intensive, and expensive [ 27 , 33 , 41 , 42 ]. Furthermore, 3D cultures created from specific tissues (e.g., basement membrane extracts) can contain undesirable components such as viruses or growth factors.…”

“…Furthermore, 3D cultures created from specific tissues (e.g., basement membrane extracts) can contain undesirable components such as viruses or growth factors. It also must be mentioned that the microscopical analysis of 3D cultures is accompanied by technical challenges, while 2D cultures can be analyzed by almost any kind of imaging [ 27 , 33 , 41 , 42 ].…”

“…Valvular interstitial cells (VICs) were isolated as described previously [ 33 ]. Briefly, excised leaflets were washed in cooled PBS, cut into small pieces, put into gelatine-coated (0.5%) cell culture flasks and cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose with GlutaMAX supplement (Invitrogen, Carlsbad, CA, USA) including 10% fetal calf serum (FCS; Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL P/S, 1% non-essential amino acids (Sigma-Aldrich, St. Louis, MO, USA), and 1 μg/mL amphotericin B at 37 °C and 5% CO 2 to allow the VICs to emigrate.…”

Identification of CD34+/PGDFRα+ Valve Interstitial Cells (VICs) in Human Aortic Valves: Association of Their Abundance, Morphology and Spatial Organization with Early Calcific Remodeling

“…The mammalian heart built of cardiomyocytes also contains a large population of non-myocyte cells (e.g., interstitial cells, endothelial cells) [12,13]. Recently, VICs have been discussed in the literature as important players in the degeneration of the heart valves, participating in the pathogenesis of CAVD [11,[14][15][16]. These double-faced cells seem to be crucial in homeostasis regulation but, on the other hand, are responsible for aortic valve fibrosis and osteogenic degeneration, which is associated with activation of developmental transcriptional regulatory pathways [17][18][19][20].…”