Superoxide production: A procalcifying cell signalling event in osteoblastic differentiation of vascular smooth muscle cells exposed to calcification media

Recent studies showed that hydrogen peroxide (H₂O₂) enhanced bone markers expression in vascular smooth muscle cells (VSMCs) implicated in osteoblastic differentiation. This study aimed at investigating the role of NAD(P)H oxidase in vascular calcification processes. A7r5 rat VSMCs were incubated with β-glycerophosphate (10 mm) or uremic serum to induce a diffuse mineralization. H₂O₂ production by VSMCs was determinated by chemiluminescence. NAD(P)H oxidase sub-unit (p22^phox), Cbfa-1, ERK phosphorylation and bone alkaline phosphatase (ALP) expressions were measured by Western blotting. VSMCs exhibited higher production of H₂O₂ and early expression of p22^phox with β-glycerophosphate or uremic serum within 24 h of treatment. β-glycerophosphate-induced oxidative stress was associated with Cbfa-1 expression followed by ALP expression and activity, meanwhile the VSMCs expressing ALP diffusely calcified their extracellular matrix. Interestingly, diphenyleneiodonium partly prevented the osteoblastic differentiation. Results from this model strongly suggest a major implication of vascular NAD(P)H oxidase in vascular calcification supported by VSMCs osteoblastic differentiation.