Induction of calcification by serum depletion in cell culture: a model for focal calcification in aortas related to atherosclerosis.
BACKGROUND
Since aortic calcification has been shown to initiate in the lower zone of well-thickened plaques (LZP) adjacent to the aortic media of rabbits fed supplemental cholesterol diets, a restricted supply of serum to vascular cells could play a role in vascular calcification. This study was designed to use a cell culture model to support this hypothesis.


RESULTS
Rabbit aortic smooth muscle cells were grown to confluence in a culture media containing 10 % fetal bovine serum (FBS). The confluent cells were then exposed to the media for 2 hrs with or without serum at a Ca x P ion product range of 4.5-9.4 mM2. In contrast to the cells cultured in the presence of FBS, confluent cells in its absence displayed marked mineral-positive alizarin red staining and infrared absorption of mineral phosphate. A kinetic parameter C1/2 was used to designate the concentration of serum or its protein constituents needed to reduce the deposition of Ca and P by half. The C1/2 for FBS and rabbit serum was 0.04-0.07 % The C1/2 value for rabbit serum proteins was 13.5 mug/ml corresponding to the protein concentration in 0.06 % of serum. This C1/2 was markedly smaller than 86.2 mug/ml for bovine serum albumin present in 0.37 % serum (p < 0.05). Serum depletion also caused marked membrane translocation as evidenced through a specific apoptosis dye uptake by cells. The proteomic analysis of calcifying vesicles, which can be released by serum depletion, revealed several calcification-related proteins.


CONCLUSIONS
The aortic smooth muscle cell culture model suggests that serum depletion may play a role in the initiation of aortic calcification. The serum exhibits remarkable ability to inhibit cell-mediated calcification.
Hsu
Howard H
HH
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160, USA. hhsu@kumc.edu
Artigues
Antonio
A
Villar
Maria T
MT
D000818:Animals; D001011:Aorta / Q000473:pathology*; D017209:Apoptosis; D050197:Atherosclerosis / Q000473:pathology*; D002114:Calcinosis / Q000175:diagnosis* / Q000473:pathology*; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D016895:Culture Media, Serum-Free / Q000378:metabolism; D004195:Disease Models, Animal; D006801:Humans; D040901:Proteomics / Q000379:methods; D011817:Rabbits
D016895:Culture Media, Serum-Free; D002784:Cholesterol; D002118:Calcium