In Vitro Modeling of Matrix Vesicle Nucleation

Abstract

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. Constructed by the addition of Ca 2؉ to PS, emulsions prepared in an intracellular phosphate buffer matched in ionic composition to the intracellular fluid of growth plate chondrocytes, these biomimetic PS-CPLX nucleators had little nucleational activity. However, incorporation of Anx-A5 transformed them into potent nucleators, with significantly greater activity than those made from ACP without PS. The ability of Anx-A5 to enhance the nucleation and growth of mineral appears to stem from its ability to form twodimensional crystalline arrays on PS-containing monolayers. However, some stimulatory effect also may result from its ability to exclude Mg 2؉ and HCO 3 ؊ from nucleation sites. Comparing the various annexins for their ability to activate PS-CPLX nucleation yields the following: avian cartilage Anx-A5 > human placental Anx-A5 > avian liver Anx-A5 > avian cartilage Anx-A6 Ͼ Ͼ cartilage Anx-A2. The stimulatory effect of human placental Anx-A5 and avian cartilage Anx-A6 depended on the presence of PS, since in its absence they either had no effect or actually inhibited the nucleation activity of ACP. Anx-A2 did not significantly enhance mineralization. Matrix vesicles (MVs)2 are extracellular, lipid bilayer-enclosed microstructures released by cells that initiate mineral formation in newly forming bone (1-4). MVs have also been found to initiate ectopic calcification in calcific tendonitis, apatite deposition osteoarthritis, cardiac valve calcification, and atherosclerotic lesions (5-7). MVs isolated from growth plate cartilage by gentle proteolytic digestion and trituration of growth plate tissue (8) retain the ability to induce mineral formation when incubated in synthetic cartilage lymph (SCL) (9). They have been shown to be enriched in several phospholipids, especially phosphatidylserine (PS) (10, 11), a lipid with high affinity for Ca 2ϩ (12,13 MVs contain a detergent-stable core that can induce mineral formation (22, 23). The activity of this nucleational core is inhibited by Zn 2ϩ and is destroyed by treatment with pH 6 citrate buffer. Fourier-transform infrared, NMR, and SDS-PAGE characterization (8,(23)(24)(25) has shown that the nucleational core contains three main components: 1) amorphous calcium phosphate (ACP), 2) membrane-associated CPLX, and, of special interest here, 3) the annexins, lipid-dependent Ca 2ϩ -binding proteins that are especially rich in MVs (8,[25][26][27][28].To elucidate the roles of these components...

Mineralization of Annexin-5-containing Lipid-Calcium-Phosphate Complexes

“…We found that all of the tested purified native human (Anx-H5) and avian annexin-A5 (Anx-A5) samples strongly stimulated mineral formation by PS-CPLX (45). For this study, we used both native chicken liver Anx-A5 and native human placental Anx-H5, which had comparable activity.…”

“…Using a variety of analytical methods (17,18,41,42), this core has been shown to contain three main components as follows: 1) amorphous calcium phosphate (ACP); 2) membrane-associated CPLXs; and 3) annexins-A5, -A6, and -A2. Recently we documented the remarkable enhancement that annexin-A5 has on mineral formation by biomimetic nucleational complexes (45, 46).…”

“…MVs interact with both the matrix collagens (9 -11) and proteoglycans (12,13) in the extracellular matrix. The interaction between MVs and the matrix collagens has been shown to be mediated by annexin 5 (14 -16), the major protein in MV (17)(18)(19)(20)(21)(22). MV are enriched in phosphatidylserine (PS) (23,24), a lipid that has high affinity for Ca 2ϩ (25,26) and is able to form complexes with both Ca 2ϩ and P i (27).…”

“…Using a variety of analytical methods (17,18,41,42), this core has been shown to contain three main components as follows: 1) amorphous calcium phosphate (ACP); 2) membrane-associated CPLXs; and 3) annexins-A5, -A6, and -A2. These lipid-dependent Ca 2ϩ -binding proteins are unusually rich in MV (17)(18)(19)(20)(21)(22) and are often associated with PS in apoptotic bodies (43). However, what particularly distinguishes MV from apoptotic bodies is their content of significant amounts of Ca 2ϩ and P i (34,44).…”

“…However, what particularly distinguishes MV from apoptotic bodies is their content of significant amounts of Ca 2ϩ and P i (34,44). These lipid-dependent Ca 2ϩ -binding proteins are unusually rich in MV (17)(18)(19)(20)(21)(22) but are not typically associated with PS in apoptotic bodies (43). Recently we documented the remarkable enhancement that annexin-A5 has on mineral formation by biomimetic nucleational complexes (45,46).…”