Introduction 1 2 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 We used an in vitro model of vascular calcification by neonatal rat VSMCs, isolated by outgrowth from aortic explants. The purpose of this investigation was to identify the factors, and their concentrations, that are mandatory to induce calcification of aortic VSMCs in vitro. Since calcium is such a major component of advanced atherosclerotic lesions, we furthermore studied whether the CA amlodipine and the statin atorvastatin, alone and in combination, affected calcification of aortic VSMCs in vitro. Materials and methods Cell culture Vascular smooth muscle cells (VSMCs) were obtained from segments of aortas explanted from 2-day old Wistar rats. Aortic segments were obtained aseptically and cut open longitudinally. The endothelium was removed by gently rubbing the luminal side of the aortas over the surface of a tissue culture dish (Falcon). Subsequently, the aortas were placed, lumen side down, on the bottom of a tissue culture flask (Greiner), and allowed to adhere for approximately 3 h. Then tissues were immersed in growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies), penicillin (100 U/ml), and streptomycin (100 μg/ml) (both supplied by BioWhittaker Europe). Seven days later the aortic tissues were removed, and the VSMCs that had grown out were detached by trypsinization. The detached cells were resuspended in growth medium and seeded in tissue culture flasks (Greiner), 6 or 12-well plates or on glass cover slips. Immunocytochemistry Subconfluent cultures at early passage were examined for the presence of α-smooth muscle actin, SM myosin heavy chain and calponin using specific antibodies (anti-SM actin clone 1A4 (1:400), Sigma; anti-SM myosin heavy chain (1:100), SanverTech; anti-calponin (1:10,000), Sigma). After fixation in 1% formalin in PBS on ice for 30 min, cells were permeabilized with 0.1% Triton X-100 for 30 min, and subsequently incubated with the primary antibody for several hours at 4°C. Secondary antibodies were conjugated with fluorescein isothiocyanate (FITC) or Cy3 (Sigma). To identify cell nuclei, the cells were also stained with Hoechst 33342 (10 μg/ml; Molecular Probes). Immunofluorescent images were obtained using a fluorescence microscope (Nikon Eclipse) equipped with 20×, 40×, and 100× objectives and a digital camera (Nikon DXM1200). In vitro calcification of VSMCs 21 2 2 In another series of experiments, calcification medium was supplemented with amlodipine (0.01–1 μmol/l), atorvastatin (2–50 μmol/l), or a combination of these drugs. The CA amlodipine was dissolved in absolute ethanol at a concentration of 1 mmol/l. This stock solution was to be diluted at least 1:1,000 in culture medium. Atorvastatin, an inhibitor of HMG-CoA reductase, was dissolved in absolute ethanol at a concentration of 10 mmol/l. This stock solution was to be diluted at least 1:200 in culture medium. Cells treated with calcification medium without added drugs were used as controls. Appropriate amounts of solvent (ethanol) were added to these controls. Addition of ethanol at these levels did not affect cell growth. After 3 weeks of incubation, calcification was quantified. Assessment of calcium deposition o Proliferation assay 3 4 Detection of apoptosis To identify apoptotic cells by assessment of condensation of nuclear chromatin, VSMC cultures were stained with Hoechst 33342 (10 μg/ml; Molecular Probes) for 10 min in the dark. Immunofluorescent images were obtained using a fluorescence microscope (Nikon Eclipse) equipped with 20x, 40x, and 100x objectives and a digital camera (Nikon DXM1200). In 6 frames per treatment, an average of 250 nuclei per frame were assessed for the presence of apoptosis. Statistical analysis P Results 1 Fig. 1 A B C D  2+ 2+ 2+ 2+ 2 P 2+ Fig. 2 A 2 B 2+ o n P P P  Calcium deposition and dependence of extracellular phosphate concentration 21 P 2 Calcium deposition and dependence of extracellular dexamethasone concentration 3 Fig. 3 A B o n P P  Calcium deposition and dependence of extracellular ascorbic acid concentration 3 P Effects of amlodipine on in vitro VSMC calcification 4 Fig. 4  (A B C o n P P  Effects of atorvastatin on in vitro VSMC calcification 4 P P Effect of a combination of CA and statin on in vitro VSMC calcification 4 P P P Effects of amlodipine, atorvastatin, and their combination on VSMC proliferation P 6 P P 5 P 5 Fig. 5 A B P P  Effects of amlodipine, atorvastatin, and their combination on VSMC apoptosis P 6 Fig. 6 A B C D  VSMCs treated with the combination of 0.1 μmol/l amlodipine and 10 μmol/l atorvastatin for 72 h had a number of apoptotic nuclei that did not significantly differ from those observed in 10 μmol/l atorvastatin only, 0.1 μmol/l amlodipine only, and VSMCs incubated in calcification medium in the absence of these drugs. Discussion P P P P P 22 8 23 24 i 25 26 2 21 27 28 29 30 17 18 31 32 33 3 2 34 34 35 36 37 38 19 20 39 2