Figure 9

Effects of CPP removal from the medium on cultured vascular endothelial and smooth muscle cells. (a) CPP levels in the tissue culture medium. The CPP-containing medium was prepared by increasing the calcium (Ca) and phosphate (Pi) concentration to 5 mM and 7 mM, respectively. CPP levels in the medium were measured before (-) and after filtration through the control column (C) or the alendronate column (A). The data represent the mean ± S.D. N = 3 for each group, ***P < 0.0001 vs C by Student’s t-test. (b) Effects of CPP removal on cell viability of cultured human vascular endothelial cells (HUVEC). Endothelial cells were cultured in CPP-free medium (the regular growth medium containing 2 mM calcium [Ca] and 1 mM phosphate [Pi]), CPP-containing medium (same as the CPP-free medium except that the [Ca] and [Pi] were increased to 5 mM and 7 mM, respectively), or the flow-through fraction of these media from either the control column (C) or the alendronate column (A) for 72 or 96 h. The data represent the mean ± S.D. N = 4 for each group, ***P < 0.0001 by Student’s t-test. (c) Effects of CPP removal on calcification of cultured rat vascular smooth muscle cells (A7r5). Smooth muscle cells were cultured in CPP-free medium, CPP-containing medium, or the flow-through fraction of these media from either the control column (C) or the alendronate column (A) for 28 days and then stained with Alizarin red that binds to calcium. The average values of optical density (OD) at 570 nm of the cell extracts were indicated.