Introduction: Basic calcium phosphate (BCP) crystals (octacalcium phosphate (OCP), carbapatite (CA) and hydroxyapatite (HA)) are associated with severe forms of osteoarthritis (OA). In advanced OA, cartilage shows chondrocyte apoptosis, overexpression of annexin V (A5), and BCP crystal deposition within matrix vesicles. We assessed in vitro whether BCP crystals and overexpression of A5 increased chondrocyte apoptosis.
Methods: Apoptosis was induced by BCP crystals, TNFα (20ng/ml) and Fas-Ligand, (20ng/ml, FasL) in normal articular chondrocytes (control) and in A5 overexpressed chondrocytes, performed by adenovirus infection. Apoptosis was assessed by caspase 3 (Cas3) activity, and DNA fragmentation.
Results: All BCP crystals, TNFα and FasL induced chondrocyte apoptosis as demonstrated by decreased cell viability and increased Cas3 activity and DNA fragmentation. TUNEL positive staining chondrocytes were increased by OCP (12,4 ± 5,2 %), CA (9,6 ± 2,6%) and HA (9,2 ± 3,0 %) crystals and TNFα (9,6 ± 2,4 %) stimulation compared to control (3,1 ± 1,9%). BCP crystals increased Cas3 activity in a dose-dependent fashion. BCP crystal-induced chondrocyte apoptosis was independent from TNFα and interleukin-1β pathways but required cell-crystal contact and intralysosomal crystal dissolution. Indeed, pre-incubation with ammonium chloride, a lysosomial inhibitor of BCP crystal dissolution, significantly decreased BCP crystal-induced Cas3 activity. Finally, overexpression of A5 enhanced BCP crystal- and TNFα-induced chondrocyte apoptosis.
Conclusion: Overexpression of A5 and the presence of BCP crystals observed in advanced OA contributed to chondrocyte apoptosis. Our results suggest a new pathophysiologic mechanism for calcium-containing crystal arthropathies.
- annexin V
- basic calcium phosphate crystals
- nitric oxide