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068

BONE TYPE ALKALINE PHOSPHATASE IS EXPRESSED BY CALCIFIED HOMOGRAFTS

P Mathieu, A Pépin, J Perron, F Dagenais, R Baillot

Sainte-Foy, Quebec

OBJECTIVES: Calcification of valve allografts (VA) is associated with early graft degeneration and failure. Mechanisms leading to calcification of VA leaflets are still poorly understood. Osseous calcification is closely associated with alkaline phosphatase (ALP) activity. Therefore, we tested the hypothesis that bone type ALP activity might be implicated in the calcification process of VA leaflets.

METHODS: Human valve were obtained from calcified explanted VA (n=8), and normal aortic valve (control) from transplant explanted hearts (n=12).

RESULTS: Von Kossa staining showed that explanted VA were mostly calcified at the junction of leaflets with either the aortic wall or the septal muscle, whereas free margin of leaflets were free of calcification. Explanted VA expressed the bone glycoprotein, osteonectin, indicating an osteoblastic activity. Calcified VA were found to express an enzymatic activity for ALP, and immnohistochemistry studies showed that bone type specific ALP was expressed within calcified areas. To further elucidate the role of bone type ALP activity in the calcification process of VA, leaflets interstitial cells (ICs) were isolated and cultured from all calcified VA. ICs did not calcify sponteanously, however when the growth medium was supplemented with b-glycerophosphate (2 mM) (calcifying medium) it induced the formation of calcified nodules. Calcified nodules expressed osteonectin and bone type ALP. Activity for ALP increased significantly over 14 days of culture with the calcifying medium and correlated with the calcium content (r =0,5; p=0,01). Levamisol (an ALP inhibitor) inhibited the b-glycerophosphate-induced calcification process as well as the expression of osteoblastic differentiation markers.

CONCLUSIONS: Therefore, calcification of VA is closely associated with bone type specific ALP activity, and furthermore, in vitro data suggest that ALP activity is an essential step leading to calcification of valve ICs.

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