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Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate ([PP.sub.i]), and regulated generation and homeostasis of extracellular [PP.sub.i] is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular [PP.sub.i] generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular [PP.sub.i] mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate ([P.sub.i]) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular [PP.sub.i]. Attenuated [PP.sub.i] accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades [PP.sub.i]. Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular [P.sub.i] alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated [P.sub.i] induced decreases in the ATP release pathway(s) that supports ENPP1 activity: this resulted in markedly reduced rates of [PP.sub.i] accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) [alpha]-actin, SM22[alpha], and calponin. Notably, addition of exogenous ATP (or [PP.sub.i] per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular [PP.sub.i] generation mediated by both ANK- and ATP release-dependent mechanisms serves as a critical regulator of VSMC calcification. ectonucleotide pyrophosphatase/phosphodiesterase-1; ANK; adenosine 3',5'-cyclic monophosphate; ATP release doi:10.1152/ajpcell.00419.2009.

The role of atherosclerotic calcification in plaque rupture remains controversial. In previous analyses using finite element model analysis, circumferential stress was reduced by the inclusion of a calcium deposit in a representative human anatomical configuration. However, a recent report, also using finite element analysis, suggests that microscopic calcium deposits increase plaque stress. We used mathematical models to predict the effects of rigid and liquid inclusions (modeling a calcium deposit and a lipid necrotic core, respectively) in a distensible material (artery wall) on mechanical failure under uniaxial and biaxial loading in a range of configurations. Without inclusions, stress levels were low and uniform. In the analytical model, peak stresses were elevated at the edges of a rigid inclusion. In the finite element model, peak stresses were elevated at the edges of both inclusions, with minimal sensitivity to the wall distensibility and the size and shape of the inclusion. Presence of both a rigid and a soft inclusion enlarged the region of increased wall stress compared with either alone. In some configurations, the rigid inclusion reduced peak stress at the edge of the soft inclusion but simultaneously increased peak stress at the edge of the rigid inclusion and increased the size of the region affected. These findings suggest that the presence of a calcium deposit creates local increases in failure stress, and, depending on relative position to any neighboring lipid pools, it may increase peak stress and the plaque area at risk of mechanical failure. plaque rupture; vulnerable plaque; atherosclerosis; vascular calcification

Much remains to be discovered about the etiology of heart valve disease and the molecular level mechanisms that drive it. The MAPK/ERK pathway influences calcification in many cell types and has been linked to the expression of a contractile phenotype in valvular interstitial cells (VICs). However, a direct correlation between MAPK/ERK pathway activity and VIC calcification has not been previously described. Thus the role of the MAPK pathway in the calcification of VIC cultures was investigated by measuring ERK activation in both calcifying and noncalcifying VIC environments and then, conversely, analyzing the effects of ERK pathway inhibition on VIC calcification and phenotype. Prolonged elevation of phosphorylated ERK-1/2 was found in calcifying VIC cultures, whereas directly blocking phosphorylation of ERK-1/2 resulted in a dramatic decrease in nodule number, nodule size, and total calcified area. Application of the ERK pathway inhibitor was also associated with a dramatic decrease in apoptosis, which may have contributed to the decreased nodule formation obtained via ERK inhibition. Real-time PCR analysis revealed that calcified samples exhibited significantly elevated expression of several myofibroblastic and osteoblastic markers, while ERK inhibition substantially reduced the expression of these markers, often to levels comparable to the noncalcifying control. These data suggest that the MAPK pathway plays an important role in regulating the phenotype and calcification of VICs, wherein sustained pathway activation is associated with increased VIC calcification. These findings may be used to further elucidate the mechanisms of valvular disease and identify potential treatment targets. heart valve; signaling pathways; extracellular matrix; mineralization

Comparison of RNA fingerprinting of the avian eggshell gland (ESG) without and with an egg revealed upregulation of a 382-bp cDNA fragment that showed high homology to the mammalian glypican 4 (GPC4). The gene sequence revealed a conserved glypican signature, a glycosyl phosphatidyl inositol-anchorage site, and cystein residues, most of which were conserved. GPC-4 was expressed in the ESG in a circadian fashion only during the period of eggshell calcification, when maximal mechanical strain was imposed. Removal of the egg just before to its entry into the ESG, with consequent elimination of the mechanical strain, caused reduction in the gene expression. Artificial application of the mechanical strain induced expression of the GPC-4 gene that was related to the level of the strain. GPC-4 expression was strain dependent in other parts of the oviduct. In the ESG, GPC-4 was expressed exclusively by the glandular epithelium and not by the pseudostratified epithelium facing the lumen. In summary, we cloned the avian homologue of GPC-4, established its pattern of expression in the avian ESG, and demonstrated for the first time that this gene is regulated by mechanical strain. heparan-sulfate proteoglycans; calcification

Abstract--Calcified structures of summer flounder, Paralichthys dentatus, were evaluated to identify the best age determination method. Scales, the currently preferred structure, were compared with opercular bones and to right and [...]

Previous studies have shown a reduction in corpus callosum area in advanced Alzheimer's disease, but it is unclear whether callosal atrophy is present in the transitional phase between health and [...]

Aortic stenosis characterized by calcific valvular disease is prevalent in the United states and Europe. Clinical studies have shown that the prognosis of this disease is extremely poor with a survival rate of less than 50 percent. Recent investigations have identified aortic-jet velocity and valve calcification as predictors of the outcome of the asymptomatic aortic stenosis in adults.

The combination of hypercalcaemia, hypercalciuria, and nephrocalcinosis with and without renal impairment is rare in paediatric clinical practice. However, this constellation of findings has been reported in three children with [...]

E.O. Kajander and N. Ciftcioglu's article on the role of nanobacteria in renal stone formation can used in other studies involving diseases associated with pathologic extraskeletal calcification. These nanobacteria, which are classified under the Proteobacteria family, have been found to create carbonate apatite crystals at neutral pH and at physiologic phosphate and calcium concentrations.

Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects. Key Words: skeletal structure; bone; calcein; zebrafish; BMP.