induction of calcification by serum depletion in cell culture: a model for focal calcification in aortas related to atherosclerosis

Abstract

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 × 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 C_1/2 was used to designate the concentration of serum or its protein constituents needed to reduce the deposition of Ca and P by half. The C_1/2 for FBS and rabbit serum was 0.04–0.07 % The C_1/2 value for rabbit serum proteins was 13.5 μg/ml corresponding to the protein concentration in 0.06 % of serum. This C_1/2 was markedly smaller than 86.2 μg/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.

Conclusion

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.