Isolation of calcifiable vesicles from aortas of rabbits fed with high cholesterol diets

Advanced arterial wall calcification in atherosclerosis imposes a serious r upturing effect on the aorta. However, the mechanism of dystrophic calcific ation linked to hyperlipidemia, that causes atherosclerosis remains unknown . Emerging morphological and biochemical evidence reveals that calcifiable vesicles may have a role in plaque calcification. To determine whether a hi gh cholesterol diet can induce arterial calcification and produce or activa te calcifiable vesicles in aortas, a rabbit model was used. After 2 months of daily high lipid feeding (supplemented with 2% cholesterol and 6% peanut oil), typical atherosclerotic lesions developed. However, the mineral, if present in aortas, was insufficient to be detected by Fourier transform-inf rared spectroscopy (FT-IR) or alizarin red staining, indicative of a non-ca lcifying stage of atherosclerosis. Small segments of thoracic aortas were d igested in a crude collagenase solution to release calcifiable vesicles. Ve sicles were also isolated from normal aortas as control to consider the pos sibility that membrane vesicles may be produced by crude collagenase digest ion, which could cause the degradation of some cells. Calcifiable vesicles were precipitated at 300 000 x g after subcellular particles were removed b y centrifugation at 30 000 x g. Calcifiability of isolated vesicles was the n tested using calcifying media containing physiological levels of Ca2+ and Pi and 1 mM ATP. Electron microscopic observations showed that the isolate d vesicles were heterogeneous in size and shape and capable of depositing e lectron dense particles. Fourier transform infrared spectroscopic analysis of the deposited particles revealed the presence of an amorphous mineral ph ase. The spectroscopic mineral to matrix ratios, related to the amount of m ineralization, indicated that vesicles from cholesterol-fed rabbits produce d more minerals than control vesicles obtained from the normal aortas. Aliz arin red staining for mineral further demonstrated substantially higher cal cifiability of the experimental vesicles. A 3-5 h exposure of the vesicles to calcifying media caused significant deposition of Ca-45 and (32)Pi in a vesicle protein-concentration dependent manner. Similar to previously repor ted observations with human atherosclerotic aorta vesicles, rabbit vesicles were enriched in ATP-hydrolyzing enzymes including Mg2+ - or Ca2+ -ATPase and NTP pyrophosphohydrolase that are implicated in normal and pathological calcification. Altogether, these observations suggest that accumulation of the released calcifiable vesicles, as a result of high cholesterol diets, may have a role in dystrophic calcification in hyperlipidemia-related ather osclerosis. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.