Clones of interstitial cells from bovine aortic valve exhibit different calcifying potential when exposed to endotoxin and phosphate

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) α-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.