title: Ultrastructural and spectrophotometric study on the effects of putative triggers on aortic valve interstitial cells in in vitro models simulating metastatic calcification.
abstract: Metastatic calcification of cardiac valves is a common complication in patients affected by chronic renal failure. In this study, primary bovine aortic valve interstitial cells (AVICs) were subjected to pro-calcific treatments consisting in cell stimulation with (i) elevated inorganic phosphate (Pi = 3 mM), to simulate hyperphosphatemic conditions; (ii) bacterial endotoxin lipopolysaccharide (LPS), simulating direct effects by microbial agents; and (iii) conditioned media (CM) derived from cultures of either LPS-stimulated heterogenic macrophages (commercial murine RAW264.7 cells) or LPS-stimulated fresh allogenic monocytes/macrophages (bCM), simulating consequent inflammatory responses, alone or combined. Compared to control cultures, spectrophotometric assays revealed shared treatment-dependent higher values of both calcium amounts and alkaline phosphatase activity for cultures involving the presence of elevated Pi. Ultrastructurally, shared peculiar pro-calcific degeneration patterns were exhibited by AVICs from these latter cultures irrespectively of the additional treatments. Disappearance of all cytomembranes and concurrent formation of material showing positivity to Cuprolinic Blue and co-localizing with silver precipitation were followed by the outcropping of such a material, which transformed in layers outlining the dead cells. Subsequent budding of these layers resulted in the formation of bubbling bodies and concentrically laminated calcospherulae mirroring those in actual soft tissue calcification. In conclusion, the in vitro models employed appear to be reliable tools for simulating metastatic calcification and indicate that hyperphosphatemic-like conditions could trigger valve calcification per se, with LPS and allogenic macrophage-derived secretory products acting as possible calcific enhancers via inflammatory responses.
authors.affiliation: Department of Experimental Clinical Medicine, University of Udine, Piazzale Kolbe 3, Udine, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Bonetti
authors.forename: Alberto
authors.initials: A
authors.lastname: Della Mora
authors.forename: Magali
authors.initials: M
authors.lastname: Contin
authors.forename: Franco
authors.initials: F
authors.lastname: Tubaro
authors.forename: Maurizio
authors.initials: M
authors.lastname: Marchini
authors.forename: Fulvia
authors.initials: F
authors.lastname: Ortolani
mesh_terms: D000818:Animals; D001021:Aortic Valve / Q000473:pathology* / Q000648:ultrastructure; D001024:Aortic Valve Stenosis / Q000503:physiopathology*; D002114:Calcinosis / Q000503:physiopathology*; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008955:Models, Cardiovascular; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D017077:Culture Media, Conditioned; D008070:Lipopolysaccharides; D010710:Phosphates; D002118:Calcium