Matrix vesicles: structure, composition, formation and function in calcification.

Matrix vesicles (MVs) induce calcification during endochondral bone formation. Experimental methods for structural, compositional, and functional analysis of MVs are reviewed. MV proteins, enzymes, receptors, transporters, regulators, lipids and electrolytes are detailed.

Differential uptake and selective permeability of fusarochromanone (FC101), a novel membrane permeable anticancer naturally fluorescent compound in tumor and normal cells.

The differential accumulation of fluorescent molecules in tumorigenic versus normal cells is a well-reported phenomenon and is the basis for photodiagnostic therapy. Through the use of confocal microscopy, the kinetic uptake and accumulation of fusarochromanone (FC101) was determined in two lines of living tumorigenic cells of mesenchymal-epithelial origin and normal fibroblast cells. Like other fluorescent cationic molecules, FC101 showed increased accumulation in tumorigenic cells; however, unlike other molecules, it appeared to be accumulated in a time-dependent manner.

Differential effects of zinc and magnesium ions on mineralization activity of phosphatidylserine calcium phosphate complexes.

Mg(2+) and Zn(2+) are present in the mineral of matrix vesicles (MVs) and biological apatites, and are known to influence the onset and progression of mineral formation by amorphous calcium phosphate (ACP) and hydroxyapatite (HAP). However, neither has been studied systematically for its effect on mineral formation by phosphatidylserine-Ca(2+)-Pi complexes (PS-CPLX), an important constituent of the MV nucleation core. Presented here are studies on the effects of increasing levels of Mg(2+) and Zn(2+) on the process of mineral formation, either when present in synthetic cartilage lymph (SCL), or when incorporated during the formation of PS-CPLX.

Mineralization of annexin-5-containing lipid-calcium-phosphate complexes: modulation by varying lipid composition and incubation with cartilage collagens.

Matrix vesicles (MVs) in the growth plate bind to cartilage collagens and initiate mineralization of the extracellular matrix. Native MVs have been shown to contain a nucleational core responsible for mineral formation that is comprised of Mg(2+)-containing amorphous calcium phosphate and lipid-calcium-phosphate complexes (CPLXs) and the lipid-dependent Ca(2+)-binding proteins, especially annexin-5 (Anx-5), which greatly enhances mineral formation. Incorporation of non-Ca(2+)-binding MV lipids impedes mineral formation by phosphatidylserine (PS)-CPLX.

Analysis and molecular modeling of the formation, structure, and activity of the phosphatidylserine-calcium-phosphate complex associated with biomineralization.

The nucleational core of matrix vesicles contains a complex (CPLX) of phosphatidylserine (PS), Ca(2+), and inorganic phosphate (P(i)) that is important to both normal and pathological calcification. Factors required for PS-CPLX formation and nucleational activity were studied using in vitro model systems and molecular dynamic simulations. Ca(2+) levels required for and rates of PS-CPLX formation were monitored by light scattering at 340 nm, assessing changes in amount and particle size.

Inhibitory effects of fusarochromanone on melanoma growth.

Fusarochromanone is a toxic metabolite produced by Fusarium equiseti, a fungus present in decaying cereal plants in northern latitudes; it has been detected in various food grains. Fusarochromanone has been shown to have both stimulatory and inhibitory effects on various mammalian cells, depending on the concentration used. Whether these cytotoxic effects can be used in the clinical treatment of tumors remains to be established.

In vitro modeling of matrix vesicle nucleation: synergistic stimulation of mineral formation by annexin A5 and phosphatidylserine.

Annexins A5, A2, and A6 (Anx-A5, -A2, and -A6) are quantitatively major proteins of the matrix vesicle nucleational core that is responsible for mineral formation. Anx-A5 significantly activated the induction and propagation of mineral formation when incorporated into synthetic nucleation complexes made of amorphous calcium phosphate (ACP) and Anx-A5 or of phosphatidylserine (PS) plus ACP (PS-CPLX) and Anx-A5. Incorporation of Anx-A5 markedly shortened the induction time, greatly increasing the rate and overall amount of mineral formed when incubated in synthetic cartilage lymph.

Kinetic analysis of mineral formation during in vitro modeling of matrix vesicle mineralization: effect of annexin A5, phosphatidylserine, and type II collagen.

Matrix vesicles (MVs) are involved in de novo mineral formation by nearly all vertebrate tissues. The driving force for MV mineralization is a nucleational core composed of three principal constituents: (i) amorphous calcium phosphate (ACP), complexed in part with phosphatidylserine (PS) to form (ii) calcium-phosphate-lipid complexes (CPLX), and (iii) annexin A5 (AnxA5), the principal lipid-dependent Ca(2+)-binding protein in MVs. We describe methods for reconstituting the nucleational core using a biomimetic approach and for analyzing the kinetics of its induction of mineral formation.

Chondrocytes isolated from tibial dyschondroplasia lesions and articular cartilage revert to a growth plate-like phenotype when cultured in vitro.

We report here a comparative study of the development and behavior of chondrocytes isolated from normal growth plate tissue, tibial dyschondroplasic lesions, and from articular cartilage. The objective of these studies was to determine whether the properties exhibited by chondrocytes in dysplasic lesions or in articular cartilage were due to their cellular phenotype, their environment, or both. We had previously analyzed the electrolytes and amino acid levels in the extracellular fluid of avian growth plate chondrocytes.

Separation and quantification of chicken and bovine growth plate cartilage matrix vesicle lipids by high-performance liquid chromatography using evaporative light scattering detection.

Matrix vesicles (MV) are lipid bilayer-enclosed nanoscale structures that initiate extracellular mineral formation in most vertebrate species. Little attention has been given to differences between species in membrane lipid composition or to how new mineral is formed in MV. To explore more precisely the lipids of MV isolated from avian and bovine species, we developed a new high-performance liquid chromatography (HPLC) method used in combination with evaporative light scattering detection (ELSD) to quantify their lipid composition.

Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens.

The mechanism of matrix vesicle (MV) mineralization was studied using MVs isolated from normal growth plate tissue, as well as several putative intermediates in the MV mineralization pathway--amorphous calcium phosphate (ACP), calcium phosphate phosphatidylserine complex (CPLX) and hydroxyapatite (HAP). Radionuclide uptake and increase in turbidity were used to monitor mineral formation during incubation in synthetic cartilage lymph (SCL). Inhibitors of phosphate (Pi) metabolism, as well as replacing Na(+) with various cations, were used to study MV Pi transport, which had been thought to be Na(+)-dependent.

Modeling of matrix vesicle biomineralization using large unilamellar vesicles.

Stable, large unilamellar vesicles (LUV) have been constructed that model matrix vesicles (MV) in inducing de novo mineral formation when incubated in synthetic cartilage lymph (SCL). Using a dialysis method for incorporation of predetermined pure lipid, electrolyte and protein constituents, the detergent n-octyl beta-D-glucopyranoside enabled formation of stable, impermeable LUV with a diameter ( approximately 300 nm), lipid composition (phosphatidylcholine-phosphatidylserine-cholesterol, 7:2:2, molar ratio) and enclosed inorganic phosphate level (25-100 mM) similar to that of native MV. Mineral formation by these LUVs was measured by 45Ca(2+) uptake and FTIR analysis following incubation in SCL.

Intracellular zinc fluxes associated with apoptosis in growth plate chondrocytes.

Matrix vesicles released by epiphyseal growth plate chondrocytes are known to contain a significant quantity of labile Zn(2+). Zonal analysis of chicken metatarsal bones showed that the resting/proliferative region of the growth plate contained high levels of Zn(2+) with significantly lower levels in the hypertrophic cartilage suggesting a loss of cellular Zn(2+) as the chondrocytes mature. Intracellular labile Zn(2+) was measured in primary cultures of growth plate chondrocytes by assay with the fluorescent Zn-chelator toluenesulfonamidoquinoline (TSQ) and imaged by multi-photon laser scanning microscopy (MPLSM) with the TSQ derivative zinquin.

Discovery of sonic hedgehog expression in postnatal growth plate chondrocytes: differential regulation of sonic and Indian hedgehog by retinoic acid.

Sonic hedgehog (Shh) is a key signal protein in early embryological patterning of limb bud development. Its analog, Indian hedgehog (Ihh), primarily expressed during early cartilage development in prehypertrophic chondrocytes, regulates proliferation and suppresses terminal differentiation of postnatal growth plate (GP) chondrocytes. We report here for the first time that both Shh and Ihh mRNA are expressed in the GP of rapidly growing 6-week-old broiler-strain chickens.

Transport of inorganic phosphate in primary cultures of chondrocytes isolated from the tibial growth plate of normal adolescent chickens.

This report describes Pi transport activity in chondrocytes isolated from the growth plate (GP) of normal adolescent chickens grown in primary cell culture. Our recent work showed that Pi transport in matrix vesicles (MV) isolated from normal GP cartilage was not strictly Na+-dependent, whereas previously characterized Pi transport from rachitic GP cartilage MV was. This Na+-dependent Pi transporter (NaPiT), a member of the Type III Glvr-1 gene family, is expressed only transiently during early differentiation of GP cartilage, is enhanced by Pi-deficiency, and is most active at pH 6.

Changes in phospholipid extractability and composition accompany mineralization of chicken growth plate cartilage matrix vesicles.

Matrix vesicles are lipid bilayer-enclosed structures that initiate extracellular mineral formation. Little attention has been given to how newly formed mineral interacts with the lipid constituents and then emerges from the lumen. To explore whether specific lipids bind to the incipient mineral and if breakdown of the membrane is involved, we analyzed changes in lipid composition and extractability during vesicle-induced calcification.