Supplemental Digital Content 3 Cell culture
Vascular SMCs were isolated from the thoracic aortas of WT or KO mice by enzymatic dissociation using the previously reported method (5) with slight modifications. Briefly, harvested aorta was incubated in Hank’s solution supplemented with 1.3 mg/mL type 1 collagenase and 0.3 U/mL type 1 elastase (Sigma, St. Louis, MO) for 40 minutes at 37°C. The adventitia was gently removed, and then the aorta was further incubated in Hank’s solution supplemented with 2 mg/mL type 1 collagenase and 3.0 U/mL type 1 elastase for 1 hour at 37°C.
Isolated SMCs were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/mL), streptomycin (50 mg/mL) and kanamycin (100 mg/mL) at 37°C in a humidified atmosphere of 5% CO2 in air. To identify SMCs (6, 7), adherent cells at passage 5 were detached by 0.25% Trypsin-EDTA Solution (Sigma), fixed in 4% paraformaldehyde and permeabilized with 0.1% saponin (Sigma). After blocking with 5% normal donkey serum, cells were incubated with the rabbit-anti SMA mAb (Epitomics, Burlingame, CA), followed by a secondary antibody, Alexa Fluor 488 donkey anti- rabbit IgG (Invitrogen, San Diego, CA). Fluorescence-activated cell sorting analysis was carried out with an EPICS XL-MCL flow cytometer (Beckman Coulter, Fullerton, CA) and data were analyzed with EXPO 32 MultiCOMP Software (Beckman Coulter). The purity of the SMCs at passage 5 was >95% and used for in vitro experiments.
For the culture of CD45+ leukocytes and SMLCs, BM cells were harvested by flushing tibiae, femora and humeri in WT or KO mice, and mononuclear cells (MNCs) were isolated using Lympholyte (Cedarlane, Ontario, Canada). CD45+ leukocytes were separated from the
MNCs using the MACS separation system (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s instructions. For SMLC culture, MNCs were cultured at a density of 5 x 106 per fibronectin-coated dish in DMEM supplemented with 10 ng/mL platelet-derived growth factor (PDGF)-BB (PeproTech, Rocky Hill, NJ) and 50 ng/mL basic fibroblast growth factor (bFGF, PeproTech). The culture medium was changed twice per week, and nonadherent cells were removed. At 14 days, cells were cultured in DMEM without PDGF-BB and bFGF. As described above, the identification of SMLCs was performed by fluorescence-activated cell sorting analysis (6, 7). The purity of the SMLCs at passage 5 was >95% and used for in vitro experiments.