(a) Exposure of VEC to PTH
VEC were seeded in 6-well plates at a density of 150,000 cells/well and cultured in Cascade Biologics Medium 200 (Gibco, Waltham, MA, USA) with 10% FBS and antibiotics. On the second day after passage, the cells were incubated with 10−9 M PTH (Parthyroid Hormone Fragment 1-34, P3796, Merck, Darmstadt, Germany) for 48 h or 7 days. Every two days the medium was replaced with PTH-containing fresh culture medium. The 10−9 M concentration was chosen because it was previously reported to affect aortic endothelial cells [30 (link)]. Moreover, in our initial tests aiming to find the appropriate PTH concentration for VEC (since a variety of PTH concentrations are mentioned in the literature, depending on cell type), this concentration (10−9 M) significantly increased ROS (Supplementary Figure S4 ).
(b) Exposure of VIC to VEC secretome
VIC were seeded in 6-well plates at a density of 150,000 cells/well and cultured in DMEM medium with 10% FBS with antibiotics.
The secretome (culture medium) was collected from cultured VEC (control) or PTH-exposed VEC, as described above. VIC were then incubated for 7 days with the VEC secretome and investigated for protein expression of osteogenic molecules using western blot or ELISA assays.
VEC were seeded in 6-well plates at a density of 150,000 cells/well and cultured in Cascade Biologics Medium 200 (Gibco, Waltham, MA, USA) with 10% FBS and antibiotics. On the second day after passage, the cells were incubated with 10−9 M PTH (Parthyroid Hormone Fragment 1-34, P3796, Merck, Darmstadt, Germany) for 48 h or 7 days. Every two days the medium was replaced with PTH-containing fresh culture medium. The 10−9 M concentration was chosen because it was previously reported to affect aortic endothelial cells [30 (link)]. Moreover, in our initial tests aiming to find the appropriate PTH concentration for VEC (since a variety of PTH concentrations are mentioned in the literature, depending on cell type), this concentration (10−9 M) significantly increased ROS (
(b) Exposure of VIC to VEC secretome
VIC were seeded in 6-well plates at a density of 150,000 cells/well and cultured in DMEM medium with 10% FBS with antibiotics.
The secretome (culture medium) was collected from cultured VEC (control) or PTH-exposed VEC, as described above. VIC were then incubated for 7 days with the VEC secretome and investigated for protein expression of osteogenic molecules using western blot or ELISA assays.