Characterization of an in vitro Model of Calcification in Retinal Pigmented Epithelial Cells

Abstract

Little is known about the relationship at the molecular and cellular levels between vascular calcification and elastic fibers essential for elasticity. To gain a better understanding of the physiological function of elastin in vascular calcification, we developed a calcification model on cultured bovine retinal-pigmented-epithelial cells (RPEs) that do not express endogenous tropoelastin. The addition of inorganic phosphate (NaH2PO4; Pi) induced calcium deposition in RPEs. The Pi-induced calcification, as assessed by the o-cresolphthalein complexone method, Goldenbergs method, and von Kossa staining, was completely inhibited by treatment with clodronate (DMDP) and phosphonoformic acid (PFA) and was weakly suppressed by treatment with levamisole. Moreover, the osteopontin mRNA expression was upregulated in the Pi-induced calcification of RPEs. These reactions in RPEs were characteristically consistent with those already established in cultured bovine aortic smooth muscle cells (BASMCs). Furthermore, bacterially expressed tropoelastin inhibited calcium deposition in RPEs as well as in BASMCs. Finally, Pi-induced calcification was partially suppressed after the addition of tropoelastin due to elastic fiber formation. In conclusion, we suggest that this calcification model in RPEs is useful for analyzing the relation between elastic fibers and vascular calcification, and that tropoelastin and elastic fibers may contribute to the inhibition of vascular calcification.