Characterization of an <i>in vitro</i> 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, Goldenberg's 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. J Atheroscler Thromb, 2003; 10: 48-56.

7-Ketocholesterol, a Major Oxysterol, Promotes Pi-Induced Vascular Calcification in Cultured Smooth Muscle Cells

“…In vitro vascular calcification was induced as previously described 12,16) , with slight modification. Briefly, confluent cells were incubated in DMEM containing 4.5 g/L glucose supplemented with 10% FBS in the presence of Pi (3 mM).…”

“…7-ketocholesterol (7kc), a major oxysterol in plaques, induces vascular SMC death with features of apoptosis, such as nuclear condensation and internucleosomal DNA fragmentation 9) , through caspase 3 activation 10) . An in vitro vascular calcification model has been developed as a useful model for analyzing the cellular mechanisms of calcification 11,12) . In this calcification model, the addition of inorganic phosphate or -glycerophosphate is used to induce calcium deposition.…”

Treatment with Vitamin K2 Combined with Bisphosphonates Synergistically Inhibits Calcification in Cultured Smooth Muscle Cells

“…In vitro calcification was induced as described previously [13][14][15][16] , with slight modifications. Briefly, confluent cells were incubated in DMEM containing 4.5 g/L glucose supplemented with 10% FBS in the presence of Pi (3 mM).…”

Atherosclerosis and Matrix Dystrophy

“…Pi-induced calcification was partially suppressed after the addition of tropoelastin, due to elastic fiber formation. Therefore, tropoelastin and elastic fibers may contribute to the inhibition of vascular calcification via the 67-kDa elastin binding protein on the cell surface (43,44). In addition to similar phenomena in endothelin-1, we demonstrated the expression of tropoelastin and lysyl oxidase at the gene level, as induced by endothelin-1, in cultured SMCs.…”