Mechanisms mediating vascular calcification remain incompletely understood. Nanometer scale objects hypothesized to be a type of bacteria (nanobacteria) are associated with calcified geological specimens, human kidney stones, and psammona bodies in ovarian cancer. Experiments were designed to evaluate human vascular tissue for the presence of similar nanometer-scale objects. Calcified human aneurysms ( n = 8), carotid plaques ( n = 2), femoral arterial plaques ( n = 2), and cardiac valves ( n = 2) and noncalcified aneurysms from patients with bicuspid aortic valve disease ( n = 2) were collected as surgical waste from the Heart Hospital of Austin, Austin, Texas, and Mayo Clinic, Rochester, Minnesota. Whole mounts or adjacent sections from each specimen were examined by electron microscopy, stained for calcium phosphate, or stained with a commercially available antibody (8D10). Filtered (0.2 μm) homogenates of aneurysms were cultured and costained with 8D10 antibody followed by PicoGreen to detect DNA or incubated with [3H]uridine. Staining for calcium phosphate was heterogeneously distributed within all calcified tissues. Immunological staining with 8D10 was also heterogeneously distributed in areas with and without calcium phosphate. Analysis of areas with positive immunostaining identified spheres ranging in size from 30 to 100 nm with a spectral pattern of calcium and phosphorus (high-energy dispersive spectroscopy). Nanosized particles cultured from calcified but not from noncalcified aneurysms were recognized by a DNA-specific dye and incorporated radiolabeled uridine, and, after decalcification, they appeared via electron microscopy to contain cell walls. Therefore, nanometer-scale particles similar to those described as nanobacteria isolated from geological specimens and human kidney stones can be visualized in and cultured from calcified human cardiovascular tissue.

Rzewuska-Lech, Ewa, Muthuvel Jayachandran, Lorraine A. Fitzpatrick, and Virginia M. Miller. Differential effects of 17␤-estradiol and raloxifene on VSMC phenotype and expression of osteoblast-associated proteins. Am J Physiol Endocrinol Metab 289: E105-E112, 2005. First published February 15, 2005 doi:10.1152/ajpendo.00366.2004.-Several studies demonstrate an association between osteoporosis and arterial calcific disease, both of which being common in elderly women. Estradiol and raloxifene, a selective estrogen receptor modulator, prevent bone loss in postmenopausal women. Little is known regarding how these agents affect arterial calcification. The aim of this study was to determine whether or not 17␤-estradiol and raloxifene reduced vascular smooth muscle cell (VSMC) differentiation and expression of bone-associated proteins during phosphate-induced calcification in vitro. Aortic VSMC were cultured from adult, gonadally intact, and ovariectomized (OVX) female pigs. Calcifying medium was added, and cells were treated with solvent (control), 17␤-estradiol (E2), or raloxifene. Extent of calcification and phenotypic expression of bone-associated proteins [matrix gla protein (MGP), osteoprotegerin (OPG), and bone sialoprotein (BSP)] were examined at 3-day intervals over 2 wk. Calcium content increased in all groups but was greater in VSMC derived from intact compared with OVX animals. E 2 reduced calcification and preserved a contractile phenotype. Expression of OPG significantly decreased with time; this decrease was significantly greater in VSMC derived from OVX compared with gonadally intact pigs. E 2 and raloxifene preserved expression of OPG only in VSMC from intact pigs. Expression of MGP increased significantly with time and was not affected by E 2 or raloxifene treatments. E2 treatment significantly inhibited synthesis of BSP in cells from both groups. In conclusion, E2 slows differentiation of VSMC induced by excess phosphate. Effectiveness of raloxifene to preserve expression of bone cell-associated proteins depends on the hormonal status of the tissue donor. vascular smooth muscle cells; estrogen; raloxifene; selective receptor modulator; smooth muscle phenotype

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