Haosmc

Human aortic smooth muscle cells (HAoSMC, PromoCell, Heidelberg, Germany) were grown in culture flaks using SMC medium 2 (PromoCell) to 80% confluency at 37°C in humidified air containing 5% CO₂. Passaging was performed with the detachment kit (PromoCell) at a ratio of 1:4. All HAoSMCs were studied between the fifth and ninth passage. 5x10⁴ cells per well in 1 mL medium were grown for 24 h before further tests. A cell proliferation kit (CFSE, PromoCell) was used to measure proliferation of HAoSMCs. Warmed sterile Dulbecco´s PBS (PAA Cell Culture Company, Cambridge, USA) mixed with 5 μM CFSE was added to 5x10⁴ HAoSMC/mL. Cell suspensions were added on cover slip glass in 24-well plates with or without a material disc positioned in the center of the wells. Cells on the surface of the material discs (surface contact) and cells periphery of the 24-well plates not in contact with the discs (no contact) were fixed with 4% paraformaldehyde (PFA) in PBS. HAoSMC apoptosis was determined using propidium iodide solution (BD Biosciences, USA) with staurosporine (PromoCell) used as positive control. HAoSMC nuclei were stained with DAPI mounting medium (Vectashield, Vector, USA). Proliferating cells in three identically-sized areas of the center (disc surface) and in three areas of the periphery of the 24-well plates (no contact) were counted using fluorescence microscopy (AxioVision).

To perform in vivo analysis, aortic biopsy was performed and aortic media specimen (collected after the resected biopsies were dissected) was obtained from each participant.
To perform in vitro analysis, human aortic smooth muscle cells (HAoSMC, PromoCell) were cultivated with medium 231 in an incubator (37°C, 5% CO₂).

Primary cultures of human aortic smooth muscle cells (HAoSMC) (PromoCell) were maintained in complete VSMC growth medium 2 as described previously [22 (link)]. For cyclic strain culture, cells were plated into 6-well collagen 1 coated Bioflex plates (Flexcell) and cultured under cyclic biaxial strain for up to 14 days using Flexcell FX–4000 unit. 7% stretch was chosen to model artery pulsatile wall stretch [32 (link)]. Frequency of 30 cycles/min allowed cells to remain attached to the Bioflex plate for 7 days. For calcification experiments, cells were incubated for 7 days with 2 and 5mmol/L Ca²⁺, 50μmol/L Gd³⁺ or with combination of 2mmol/L Ca²⁺ and 50μmol/L Gd³⁺. Alternatively, HAoSMC were treated with 2 and 5mmol/L Ca²⁺ in the presence of 10, 100, 1000nmol/L calcimimetic R563 or 1000nmol/L S568 (inactive enantiomer) (Amgen). Control group was treated with 1.1 mmol/L Ca²⁺. To facilitate mineralisation, 5mmol/L -glycerophosphate (-GP) was added to all calcification experiments.
Since pilot experiments (S1 Fig) had demonstrated HAoSMC phenotypic stability over at least 7 days, we used a 7 day time period for all cell culture experiments.