Isolation of calcifiable vesicles from human atherosclerotic aortas

Abstract

Advanced mineralization can cause brittleness of aortic walls with decreased elasticity thereby causing the wall to rupture. Although the precise mechanisms of dystrophic calcification remain unknown, morphological evidence reveals the presence of mineral-associated vesicles in the lesions and defective bioprosthetic valves. In an attempt to demonstrate the calcifiability of the vesicles, small segments of human atherosclerotic aortas with calcified lesions were removed at autopsy and then digested in a crude collagenase solution to release vesicles. A differential centrifugation was then used to isolate calcifiable vesicles, which was precipitated at 300 000× g for 20 min. An exposure of the vesicles to a calcifying medium containing physiologic levels of Ca 2+, Pi, and 1 mM ATP caused Ca deposition in a vesicle protein-concentration dependent manner. The calcifiability of the vesicles was further demonstrated by electron microscopy. Fourier transform spectroscopic analysis of the deposited mineral revealed the presence of a hydroxyapatite phase, closely resembling the native form of mineral in atherosclerotic plaques. In addition, calcifiable vesicles were enriched in ATP-hydrolyzing enzymes including Mg 2+- or Ca 2+-ATPase and NTP pyrophosphohydrolase that may be involved in normal and pathological calcification. Triton X-100 at 0.01% abolished 80% of both ATPase activity and ATP-initiated calcification. A comparison of vesicles isolated from non-atherosclerotic and atherosclerotic aortas indicated that atherosclerotic vesicles tended to have higher calcifiability. These observations suggest that the calcifiable vesicles play a part in dystrophic calcification of aortas in atherosclerosis.