Fig 2: Identification, mathematical modeling, and computational simulation of the bi-modal VSMC fate response to ECM stiffnessAsynchronous mouse VSMCs were cultured on stiff and soft collagen-coated hydrogels with 20% FBS for 24 h.(A) The incubation was performed in the presence of EdU, and the fraction of EdU-positive nuclei was determined relative to Hoechst-stained nuclei. Results show mean + SD; n = 3.(B) Fixed cells were immunostained for SMA, SMMHC, and TAGLN; n ˜ 110–160 cells per condition.(C) Model used to capture the dual VSMC fate response to increased ECM stiffness with “c” and “p” representing the contractile and proliferative states, respectively.(D) Selected phase portraits of the VSMC states predicted at the indicated ECM stiffness. The black squares in the phase portraits at 0.1 and 2 kPa depict the position of the null state predicted for VSMCs on soft substrates, and the black circles at 25 and 50 kPa depict the positions of the predicted contractile (solid line) and proliferative (dashed line) end states seen in VSMCs on stiff substrates.

Fig 3: SMC and stem cell marker expression in A10, A7r5 and MVSCs. A. Representative immunoblots for SMC differentiation markers (SM-MHC, SMA and CNN1) and neural stem cell markers (Sox10, Sox17, S100ß in lysates from quiesced cells grown in normal DMEM media supplemented with FBS for 3 d. The levels of SM-MHC (Sm1, Sm2 and non-muscle MHC), SMA, CNN1, Sox10, Sox17 and S100ß were determined using specific antisera against these antigens. Data are representative of blots with similar results. Equal loading was confirmed by Ponceau S staining of the membranes and by measuring the constitutive ß-actin gene. B. Immunocytochemical staining of c-Kit and Flt-1 in MVSCs, rSMC, A7r5 and A10 cells. Quiesced cells were grown in normal DMEM media supplemented with FBS for 3 d before the cells were stained for c-kit and flt-1. C. Representative flow cytometry analysis of Flt-1 and c-Kit in A7r5, A10 and MVSCs using antibodies against Flt-1 and c-Kit. Open curves represent negative control samples; red filled curves represent samples stained with antibodies.

Fig 4: Selective upregulation of CArG-dependent genes by myocardin.Embryoid bodies were treated with no virus, Ad-LacZ or Ad-Myo from day 10 to day 28 and then harvested for RNA. SMC marker expression for a range of CArG-dependent and non-dependent genes was measured using real time RT-PCR and is normalised by three housekeeping genes and then presented relative to no virus controls. RT-PCR data represent means from at least three independent experiments. Error bars represent s.e.m. SMaA and SMMHC expression levels in response to myocardin were significantly higher than the other genes and thus the precise levels are depicted by numbers above the black bars (± s.e.m.).

Fig 5: Timing of viral transduction reveals an early role in SMC induction for myocardin.Embryoid bodies were transduced with Ad-LacZ or Ad-Myo early (day 10 alone or days 10 & 14), late (days 18 & 23) or throughout differentiation (days 10, 14, 18 &13). Late delivery of Ad-Myo reduced number of SMaA+ cells (A) (*p<0.05 by ANOVA and Tukey HSD) but had no significant effect on SMMHC+ cell numbers (B). Values represent mean cell proportions from three independent experiments (± s.e.m.). *p<0.05.