Differential responses by aortic valve interstitial cells using in vitro models mimicking metastatic and dystrophic calcification: critical role of inorganic phosphate and additional effects of other pro-calcific agents including LDLs

Calcific aortic valve stenosis (CAVS) is the most severe valvulopathy affecting western world population. In the present thesis, in vitro models stimulating metastatic or dystrophic calcification in cultured bovine aortic valve interstitial cells (AVICs) were studied. Multivaried stimulations of primary cultures of AVICs were performed using inorganic phosphate (Pi) at critical concentrations, bacterial endotoxin lipopolysaccharide (LPS), and conditioned medium (CM) from cultures of allogeneic LPS-stimulated macrophages. Spectrophotometric estimations revealed calcification primarily to depend on elevated Pi levels (≥ 2.0 mM), which were comparable with the most elevated normophosphatemic levels and hyperphosphatemic ones. Pi-dependent effects resulted to be enhanced by adding LPS and CM together but not alone. Ultrastructurally, the calcific process consisted in a distinct AVIC degeneration as found for actual CAVS, in which the crucial event was a progressive plasmamembrane and organelle membrane colliquation that culminated in the generation of an acid-phospholipid-rich material outlining dying cells and their remnants and acting as major hydroxyapatite (HA) nucleator. AVIC remnants included rounded paracrystalline calcospherulae mirroring those described for CAVS. Consistently, Raman micro-spectroscopy applied to calcifying cultured AVICs revealed peripheral localization of HA and acidic phospholipids. Formation of calcific nodules also in the presence of apoptosis inhibitors and negative immunoreactivity to cleaved caspase 3 and annexin V led to exclude apoptosis to occur for calcifying AVICs. Conversely, surviving AVICs were found to undergo initial autophagocytosis, as revealed by immunopositivity to marker MAP1-LC3A, and subsequent derangement as revealed by ultrastructural detection of unusual hypertrophy of endoplasmic reticulum correlating with incubation time course and Pi concentration. In CAVS-affected valves atherosclerosis-like features were detected, with (i) Raman analysis showing colocalization between HA, phospholipids, cholesterol and carotenoids, and (ii) parallel electron microscopy revealing the presence of typical intra- and extracellular cholesterol crystals. Thus, the effects exerted by native low density lipoproteins in both native (nLDL) and aggregated (agLDL) form were explored on cultured AVICs using thin layer chromatography (TLC), spectrophotometry, histochemistry and electron microscopy. The chromatographically detected amounts of internalized esterified cholesterol differently correlated with the spectrophotometrically calcium estimations. Concerning pro-calcific effects, nLDLs and agLDLs alone induced mild calcification, whereas their combination with pro-calcific Pi+LPS+CM mixture unexpectedly provoked negligible calcification for nLDLs but prominent calcification for agLDLs. In conclusion, LDLs are further potential candidates for conditioning AVIC-dependent calcification in addition to Pi and other pro-calcific factors.