Download on calcium deposition (a) and medium

Calcification of Vascular Smooth Muscle Cell Cultures
by Takeo Wada, Marc D. McKee, Susie Steitz, and Cecilia M. Giachelli
Circulation Research
Volume 84(2):166-178
February 5, 1999
Copyright © American Heart Association, Inc. All rights reserved.
Time course of calcium deposition in the cell layer (a) and medium phosphorus (b) and calcium
(c) concentrations in cultured BASMCs. Cells were cultured in growth medium (Grow M; ○) or
calcification medium (Calc.
Takeo Wada et al. Circ Res. 1999;84:166-178
Copyright © American Heart Association, Inc. All rights reserved.
Effects of β-glycerophosphate (β-GP) on calcium deposition in the cell layer (a) and medium
phosphorus (b) and calcium (c) concentrations in BASMC cultures.
Takeo Wada et al. Circ Res. 1999;84:166-178
Copyright © American Heart Association, Inc. All rights reserved.
Morphology (a through c) and alkaline phosphatase activity (d) of BASMCs. a, By light
microscopy, opaque areas were observed about 10 days after switching to calcification medium
(×40 magnification). b, Von Kossa staining (black) shows diffusive and granular calcification
predominantly in the extracellular matrix regions of the cultures.
Takeo Wada et al. Circ Res. 1999;84:166-178
Copyright © American Heart Association, Inc. All rights reserved.
Electron microscopic analysis showing ultrastructural features of BASMCs after culturing for 10
days in calcification medium containing 10 mmol/L β-glycerophosphate. a, Cells (smooth muscle
cells [SMCs]) are generally fusiform in morphology and show a diverse array of intracellular
organelles that include Golgi elements (G), rough endoplasmic reticulum (rER), mitochondria
(m), glycogen (gly), and extended nuclei (Nu) often with prominent nucleoli. b, At other sites in
the cultures where BASMCs appear actively engaged in the synthesis and secretion of
extracellular matrix, the Golgi apparatus is extensively developed (Golgi, dashed line) with
multiple layering of saccules whose associated spherical and cylindrical distensions are typical
of cells producing the fibrillar collagens. m indicates mitochondria. c, BASMCs depositing
collagen fibrils (Coll) are generally better spread, show a prominent Golgi complex with
abundant cisternae of rough endoplasmic reticulum (rER), and have a well-developed
cytoskeleton with ramifying bundles of microfilaments (mf) coursing throughout the cytoplasm
and inserting into the cell membrane.
Takeo Wada et al. Circ Res. 1999;84:166-178
Copyright © American Heart Association, Inc. All rights reserved.
Electron micrographs selected to show various calcification (Cal) events associated with the
collagenous (Coll) extracellular matrix produced by BASMCs (SMCs) after 10 days in
calcification medium. a, Electron-dense calcified deposits frequently appear as discrete,
micrometer-sized masses within the collagen-rich extracellular matrix surrounding the cells.
Takeo Wada et al. Circ Res. 1999;84:166-178
Copyright © American Heart Association, Inc. All rights reserved.
Effects of levamisole on calcium deposition (a) and medium phosphorus (b) and calcium (c)
concentrations in BASMCs. Calcium and phosphorus levels were measured at day 10 by the ocresolphthalein complexone method and the phosphomolybdate complex method, respectively.
Takeo Wada et al. Circ Res. 1999;84:166-178
Copyright © American Heart Association, Inc. All rights reserved.
Effects of osteopontin (a) and vitronectin and fibronectin (b) on calcification of BASMC cultures.
Takeo Wada et al. Circ Res. 1999;84:166-178
Copyright © American Heart Association, Inc. All rights reserved.
Effects of added osteopontin on alkaline phosphatase activity of BASMCs (a) and phosphorus
concentration in the medium (b) and the direct effects of levamisole and osteopontin (OPN) on
alkaline phosphatase (ALP) activity (c). a and b, Cells were cultured in growth medium (Grow M.)
or calcification medium (Calc.
Takeo Wada et al. Circ Res. 1999;84:166-178
Copyright © American Heart Association, Inc. All rights reserved.
Effects of osteopontin (OPN) on calcium deposition (a) and medium phosphorus (b) and calcium
(c) concentrations in BASMC cultures at various initial calcium concentrations. a, Calcium level
in cell layer extracts was measured at day 10 by the o-cresolphthalein complexone method and
normalized to cellular protein content. b and c, Calcium and phosphorus levels in the medium
were measured at day 0 (pre) and day 10 (vehicle and OPN) by the o-cresolphthalein complexone
method and phosphomolybdate complex method, respectively, and are presented as
mean±SEM; n=3.
Takeo Wada et al. Circ Res. 1999;84:166-178
Copyright © American Heart Association, Inc. All rights reserved.
Localization of endogenous and exogenous osteopontin in mineralizing BASMC cultures. a,
After immunocytochemical labeling for endogenous osteopontin, gold particles are diffusely
located over calcified areas (CAL) of the extracellular matrix.
Takeo Wada et al. Circ Res. 1999;84:166-178
Copyright © American Heart Association, Inc. All rights reserved.