Pro-calcific responses by aortic valve interstitial cells in a novel in vitro model simulating dystrophic calcification

Etiopathogenetic mechanisms in calcific aortic valve stenosis are still poorly understood despite this being the third major cause of heart disease in western world. In prior in vitro cultures simulating metastatic calcification, procalcific effects on aortic valve interstitial cells (AVICs) resulted by adding bacterial endotoxin lipopolysaccharide (LPS) at high inorganic phosphate (Pi) levels. Here we accomplished improved in vitro models simulating either metastatic (Pi = 2.6 mM) or dystrophic calcification (Pi = 1.3 mM), in which LPSstimulated bovine AVICs underwent extrastimulation with macrophagecytokinecontaining media derived from parallel cultures of allogeneic monocyte/macrophages in turn stimulated with LPS. In dystrophic calcificationlike cultures, lower calcium amount was spectrometrically assessed with parallel reduced alkaline phosphatase activity with respect to metastatic calcificationlike cultures, with an about threefold slower progression of mineralization. Hydroxyapatite crystal precipitation was ultrastructurally found to correlate with AVIC degeneration processes culminating with the formation of phthalocyaninpositive lipidic layers (PPLs) at the surface of cells and cellderived matrixvesiclelike bodies, acting as calcium nucleators according to a pattern mirroring those we had previously found in in vivo conditions. In conclusion, an in vitro model has been developed enabling reliable simulations of the effects exerted on AVICs by putatively proor anticalcific agents.