Abstract
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. Isolated matrix vesicles were incubated in synthetic cartilage lymph to induce mineral formation. At various times, samples of the lipids were taken for analysis, extracted both before and after demineralization to remove deposited mineral. Phosphatidylserine and phosphatidylinositol both rapidly disappeared from extracts made before decalcification, indicating rapid degradation. However, extracts made after demineralization revealed that phosphatidylserine had become complexed with newly forming mineral. Concomitantly, its levels actually increased, apparently by base-exchange with phosphatidylethanolamine. Though partially complexed with the mineral, phosphatidylinositol was nevertheless rapidly broken down. Sphingomyelin and phosphatidylethanolamine also underwent rapid breakdown, but phosphatidylcholine was degraded more slowly, all accompanied by a buildup of free fatty acids. The data indicate that phosphatidylserine forms complexes that accompany mineral formation, while degradation of other membrane phospholipids apparently enables egress of crystalline mineral from the vesicle lumen.
Highlights
Matrix vesicles (MV)1 are extracellular microstructures released by calcifying cells that initiate mineral formation in newly forming bone (1– 4)
Our findings reveal that extensive phospholipid degradation occurred during MV calcification, and this was accompanied by a concomitant rise in the amount of free fatty acids (FFA) apparently released by phospholipases present in the vesicles
There was a dramatic increase in the levels of PS at time points when MV calcification was in rapid progress
Summary
Isolation of Matrix Vesicles—Large batches of collagenase-released matrix vesicles (CRMV) were isolated from the metatarsal growth plate cartilage of 6Ϫ to 8-week-old broiler strain chickens using previously described methods (14). The pellet was resuspended as a stock containing 5.0 mg of vesicle protein/ml in SCL modified to have one-half the normal level of. At each time point (0, 2, 4, 6, and 24 h) for lipid analysis a 110-ml sample was centrifuged at 100,000 ϫ g for 60 min to sediment the CRMV. For all studies reported here, after the initial lipid extraction, the CRMV pellets were demineralized with 0.5 M sodium salt EDTA for 20 min at room temperature and sedimented by centrifugation at 3,000 rpm for 12 min. 11.6 pounds per square inch, was used as the carrier at a flow rate of 2.02 standard liters per min in the ELS detector, which was operated at 72 °C
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