Transcriptome of human aortic smooth muscle cells is dependent on mechanical stretch and substrate coating
Transcriptome of human aortic smooth muscle cells is dependent on mechanical stretch and substrate coating
Human aortic SMCs (ATCC: PCS-100-012) cultured in smooth muscle cell growth medium (SMC-GM) containing 5% fetal bovine serum (FBS), bFGF [2 ng/mL], EGF [0.5 ng/mL], insulin [5 µg/mL], gentamicin [50 µg/mL], and amphothericin B [50 ng/mL] (Provitro, 211-0601) at 37 ºC and 5% CO2. Only cells in passages 4-6 were used for the experiments. Cells were seeded onto BioFlex 6-well plates precoated with COL1 (Flexcell, BF-3001C), or coated with FIB or LAM in house, and treated with one of the growth protocols. SMCs from each condition were harvested and resuspended as single-cell suspensions in PBS. For plastic labware, plates were coated with collagen-I (COL1) from rat tail (20 µg/mL, Corning, 354236) diluted in 0.1% acetic acid for >20 min at room temperature (RT) and washed once with phosphate-buffered saline. Fibronectin (FIB) (10 µg/mL, Sigma, F0895-5MG) was diluted in Hank's Buffer Salt Solution and incubated at RT for 20 min and air-dried. Laminin (LAM) (20 µg/mL, Corning, 354232) was diluted in serum-free SMC-GM and incubated at 37 ºC overnight. For BioFlex plates, we used precoated COL1 plates (Flexcell, BF-3001C) and in-house FIB- or LAM-coated untreated BioFlex plates (Flexcell, BF-3001U), following the same protocol as for plastic labware. At ~80% confluency, cells were exposed to either static conditions, or 10% or 15% equibiaxial stretch for 6 h. The Flexcell tension system (FX-5000 T) applied a sinusoidal waveform stretch at a frequency of 1Hz. RNA was isolated using the NucleoSpin RNA Plus Kit (Macherey-Nagel, 790984). RNA quantity and quality were measured with Nanodrop One spectrophotometer.   All RNA samples passed quality control (Agilent 4200 electrophoresis system). Non-stranded DNA libraries were prepared from polyA-selected mRNA in BGI Genomics (Copenhagen, Denmark). 