A simple and non-radioactive technique to study the effect of monophosphoesters on matrix vesicle-mediated calcification

Abstract

A simple and non-radioactive technique based on O-cresolpthalein complexone assay was developed to study in vitro non-radioactive calcium ( 40 Ca) deposition by isolated matrix vesicles. Using this technique, the effect of various phosphoester substrates including ATP, AMP and β-GP on in vitro MV-calcification was studied. O-cresolpthalein complexone assay with non-radioactive calcium demonstrated that AMP or β-GP were more effective in promoting calcium deposition by isolated MVs than ATP. The application of this nonradioactive technique, which is highly sensitive and simple, would offer a useful alternative approach to the routinely used radiometric biomineralization assay which employs radioactive 45 Ca.

End stage renal disease‐induced hypercalcemia may promote aortic valve calcification via Annexin VI enrichment of valve interstitial cell derived‐matrix vesicles

“…Transmission electron microscopy showed accumulation of vesicular bodies, ranging from 150 to 250 nm in diameter in the ECM of CAVD tissue. Many vesicles displayed spindle‐like projections of hydroxyapatite, as previously reported in MVs derived from chondrocytes (Figure g,h; [Garimella, Sipe, & Anderson, ]). Vesicles displaying this distinctive morphology were not observed in the control tissue, despite the presence of “empty” vesicles of comparable sizes (Figure e,f).…”

Phosphodiesterase Activity of Alkaline Phosphatase in ATP-initiated Ca2+ and Phosphate Deposition in Isolated Chicken Matrix Vesicles

“…Although PP i inhibits hydroxyapatite seeding (62), the uptake of P i and calcium by MVs is not significantly affected by the presence of ATP or PP i at low concentrations (51,61,64,65). In this report, we used ADPR to detect the enzymes that are able to release PP i from ATP.…”

Membrane Vesicles Nucleate Mineralo-organic Nanoparticles and Induce Carbonate Apatite Precipitation in Human Body Fluids

“…Fluorescence Spectroscopy-MVs were quantified as before (49,50) by measuring the concentration of total proteins using a commercial Bradford protein assay (Bio-Rad). To detect lipid membranes, we mixed a fraction of MVs corresponding to 0.5 g of MV proteins with 0.1 mM 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindotricarbocyanine iodide (DiR; Molecular Probes) in a final volume of 1 ml.…”

“…Calcium Deposition Assay-The ability of MVs to calcify was assessed using a non-radioactive calcium phosphate deposition assay (49). Briefly, phospholipid-rich MV fractions corresponding to 30 g of total MV proteins were added into DMEM and incubated in cell culture conditions.…”