Calcification of human valve interstitial cells is dependent on alkaline phosphatase activity.
OBJECTIVE
The calcification of heart valves is associated with valve degeneration and failure, but the mechanisms involved are poorly understood. The presence of lamellar bone has been demonstrated in calcified aortic valves. Since osseous calcification is closely associated with alkaline phosphatase (ALP) activity, it was hypothesized that ALP activity might be implicated in the calcification of isolated leaflet interstitial cells (ICs).


METHODS
Human valve leaflet ICs were isolated from transplant-explanted hearts at the time of transplantation (n = 12).


RESULTS
Isolated leaflet ICs expressed the fibroblast-specific antigen (100% of cells) and smooth muscle (SM) alpha-actin (70-80% of cells), but osteoblastic markers were not expressed. Cultured ICs did not calcify spontaneously, however when the growth medium was supplemented with beta-glycerophosphate (an organic phosphate) it induced the formation of calcified nodules that expressed osteonectin and ALP, but not SM alpha-actin. Beta-glycerophosphate-induced calcification of ICs showed a time-dependent effect on the calcium content of treated cells over a 14-day period. ALP activity was considerably increased in beta-glycerophosphate-treated ICs, and this correlated with the calcium content (r = 0.5: p = 0.01). Levamisol (an ALP inhibitor) inhibited the beta-glycerophosphate-induced calcification process, as well as the expression of osteoblastic differentiation markers.


CONCLUSIONS
Isolated and cultured leaflet ICs did not calcify spontaneously, though organic phosphate induced the formation of calcified nodules that expressed osteoblastic markers. The calcification of isolated ICs was seen to be dependent on ALP activity.
Mathieu
Patrick
P
Department of Cardiac Surgery, Laval Hospital, Sainte-Foy, Québec, Canada. Patrick.Mathieu@chg.ulaval.ca
Voisine
Pierre
P
Pépin
Andrée
A
Shetty
Rahoul
R
Savard
Nadia
N
Dagenais
François
F
D000199:Actins / Q000032:analysis; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000502:physiology*; D001021:Aortic Valve / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D002114:Calcinosis / Q000139:chemically induced / Q000201:enzymology*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003239:Connective Tissue Cells / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005994:Glycerophosphates / Q000494:pharmacology; D006349:Heart Valve Diseases / Q000201:enzymology*; D006801:Humans; D007978:Levamisole / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects; D015676:Osteonectin / Q000032:analysis; D013997:Time Factors
D000199:Actins; D004791:Enzyme Inhibitors; D005994:Glycerophosphates; D015676:Osteonectin; D007978:Levamisole; D000469:Alkaline Phosphatase; C031463:beta-glycerophosphoric acid