Smgm 2

VSMCs migration was evaluated using a modified Boyden chamber assay, as described previously [17] (link), [18] (link). Briefly, isolated VSMCs were detached mechanically by using a cell scraper, harvested by means of centrifugation, resuspended in 300 µL of the medium for each cell, and counted. Dulbecco's modified Eagle medium (DMEM, Sigma) was used for murine VSMCs, and smooth muscle growth medium-2 (SmGM-2, Lonza) was used for human aortic VSMCs. The 1×10⁵ VSMCs were placed in the upper chamber of a modified Boydenchamber (FluroBlock, Becton Dickinson Biosciences). The chamber was placed in a 24-well culture dish containing each medium for control and each medium with or without Ang II (40 µmol/L) and/or SNP (40 µmol/L). After 24 hours of incubation at 37°C, the lower side of the filter was washed with PBS and fixed with 2% paraformaldehyde. For quantification of cells that had migrated, cell nuclei were stained with 4′,6-diamino-phenylidole (DAPI, Sigma). Migrated cells in the lower chamber were counted manually in the 3 random high-power fields. Each experiment was performed in triplicate.

Primary normal human lung fibroblasts (LFs) were purchased from Lonza (CC-2512) and cultured in FGM-2 (Lonza CC-3132). Cells from three donors were used in these studies: #33652 (56 y.o., male), #29132 (19 y.o., female), and #41684 (37 y.o., male). Passages 3–6 were used for experiments. Primary human pulmonary artery smooth muscle cells were purchased from Lonza (CC-2581) and cultured in SmGM-2 (Lonza CC-3182). Cells from three donors were used in these studies: #30020 (64 y.o., male), #27662 (35 y.o., male), #26698 (51 y.o., male), #19828 (51 y.o., male) and #45518 (56 y.o., female). Passages 4–7 were used for experiments. Cell authentication was performed by the vendor. Cells were maintained in a standard tissue culture incubator in 5% CO₂ at 37 °C.

HASMCs were seeded onto 96-well plates (1 × 10⁴ cells/100 µL/well) and incubated at 37 °C in 5% CO₂ for 24 h in SmGM-2 (Lonza). Cells were further incubated for 48 h with the indicated concentrations of legumain, with renewal of each medium. Then, 10 μL of WST-8 solution (Cell Count Reagent SF; Nacalai Tesque, Kyoto, Japan) was added to each well [2 (link),5 (link),7 (link),10 (link),30 (link),31 (link),41 (link),42 (link),43 (link),44 (link),45 (link)]. After 1 h of incubation, the amount of formazan product was determined by measuring the absorbance at 450 nm using a Sunrise Remote R™ microplate reader (Tecan, Kawasaki, Japan) [2 (link),5 (link),7 (link),10 (link),30 (link),31 (link),41 (link),42 (link),43 (link),44 (link),45 (link)].

HUVECs and GFP-HUVECs were used between passages 4 to 7. HUVECs, GFP-HUVECs, and HMEC-1 cells were grown in complete MCDB131® medium (Life Technologies, Rockville, MD) supplemented with 20% fetal bovine serum (FBS), endothelial cell growth supplement, 2mmol/l glutamine, heparin, and gentamicin and incubated at 37°C and 5% CO₂. Primary human lung fibroblasts were cultured in MEM medium (Life Technologies, Rockville, MD) supplemented with 10% FBS and antibiotics. HAVICs, generated as previously described (Yu et al., 2017 (link)), were used at passages 3–5 and cultured in DMEM medium supplemented with 10% FBS, glutamine, and antibiotics. HASMCs were cultured in smooth muscle cell growth medium (SmGM™ 2; Lonza, Basel, Switzerland) supplemented with 5% FBS, insulin, FGF-B, gentamycin, amphotericin, and epithelial growth factor (EGF).