on October 2, 2008
Published online before print October 2, 2008, doi: 10.1161/ATVBAHA.108.174342
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Submitted on July 18, 2008
Accepted on September 21, 2008
Clones of Interstitial Cells From Bovine Aortic Valve Exhibit Different Calcifying Potential When Exposed to Endotoxin and Phosphate
Marcello Rattazzi *;
From the Dipartimento di Medicina Clinica e Sperimentale (M.R., E.F., E.B., G.Z., I.B., M.P., G.P.F., C.A., P.P.), Dipartimento di Scienze Cardiologiche (L.I., G.T., G.G.), Toraciche e Vascolari, Dipartimento di Scienze Biomediche Sperimentali (S.S.), Università degli Studi di Padova, Treviso, Italy.
* To whom correspondence should be addressed. E-mail: marcello.rattazzi{at}unipd.it.
Objective—Our purpose was to study in vitro whether phenotypically-distinct interstitial cell clones from bovine aortic valve (BVIC) possess different calcifying potential in response to endotoxin (lipopolysaccharide [LPS]) and phosphate (Pi).
Methods and Results—Among various clones of BVIC obtained by limited dilution technique we selected 4 clones displaying different growth patterns and immunophenotypes. Uncloned and cloned cells were treated with combinations of LPS (100 ng/mL) and Pi (2.4 mmol/L). Uncloned BVIC showed increased alkaline phosphatase activity (ALP) after treatment with LPS, which resulted in calcification after addition of Pi. Among BVIC clones, only Clone 1 (fibroblast-like phenotype) showed a relevant increase in ALP after LPS treatment in parallel with prevention of smooth muscle (SM) 
-actin accumulation. No effect was observed in clonal cells harboring a more stable SM cell-like profile (Clone 4). None of the isolated clones calcified but mineralization was induced in the presence of LPS plus Pi when Clone 1 was cocultured with Clone 4 or after seeding on type I collagen sponges.
Conclusion—Endotoxin and phosphate can act as valve calcification promoters by targeting specific fibroblast-like interstitial valve cells that possess a unique procalcific potential.
Key words: aortic valve • calcification • clones • endotoxin • phosphate