Calcification of Vascular Smooth Muscle Cell Cultures

Abstract

—Calcification of vascular tissue is a common complication in aging, atherosclerosis, diabetes, renal failure, aortic stenosis, and prosthetic valve replacement. Osteopontin is a noncollagenous adhesive protein routinely found at sites of dystrophic calcification and synthesized at high levels by macrophages in calcified aortic valves and atherosclerotic plaques. In the present study, we have characterized the calcification of bovine aortic smooth muscle cell (BASMC) cultures in vitro and have studied the effects of exogenous osteopontin on mineral deposition. Induction of calcification in BASMC cultures was alkaline phosphatase–dependent and was characterized by a multilayer cell morphology. Mineral deposition occurred in the basal matrix of multilayered areas as indicated by von Kossa staining, and transmission electron microscopy and electron diffraction identified the mineral as apatite. Ultrastructural analysis of the cultures showed the presence of extracellular matrix vesicles, calcifying collagen fibrils, and nodular-type calcifications similar to those found in calcified heart valves and atherosclerotic plaques. Purified osteopontin (0.05 to 5 μg/mL) dose dependently inhibited calcification of BASMC cultures, whereas vitronectin and fibronectin had no effect. In contrast to the inhibitory mechanism of levamisole on mineral deposition, osteopontin did not inhibit alkaline phosphatase activity or reduce phosphorus levels in the culture medium. Addition of calcium to the cultures overcame the inhibitory effect of osteopontin on BASMC culture calcification and resulted in decreased levels of calcium in the culture medium and increased levels in the cell layer. Moreover, using high-resolution, colloidal-gold immunocytochemistry, osteopontin was found intimately associated with growing apatite crystals. These data indicate that the effect of osteopontin, although calcium-dependent, was not mediated by simple calcium chelation but most likely by direct interaction of osteopontin with crystal surfaces. These studies suggest that BASMCs can be used to model vascular calcification in vitro and that soluble osteopontin released near sites of vascular calcification may represent an adaptive mechanism aimed at preventing vascular calcification.