Abstract
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 extra-stimulation with macrophage-cytokine-containing media derived from paral lel cultures of allogeneic monocyte/macrophages in turn stimulated with LPS. In dystrophic calcification-like 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 phthalocyanin-positive lipidic layers (PPLs) at the surface of cells and cell-derived matrix-vesicle-like 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 devel oped enabling reliable simulations of the effects exerted on AVICs by putatively pro- or anti-calcific agents.
