Fig. 4: MMP13 activity in HGPS-iPSC SMCs cultured under flow shear stress.

a Volcano plot representing differentially expressed genes in HGPS-iPSC-SMC cultured under flow conditions at day 0 and 4. Each point represents one of 53,617 genes. 26 and 31 genes were upregulated (red; fold change ≥ 3; p < 0.001) and downregulated (yellow; fold change ≤ 3; p < 0.001), respectively. Graph shows qRT-PCR validation for 16 genes with fold changes >3. Fold change was between days 0 and 4. Gene expression was normalized by the housekeeping gene GAPDH. Results are mean ± SEM, n = 4 technical replicates from a pool of three independent experiments. Statistical analyses were performed by a two-tailed unpaired Student’s t test. b Schematic representation of the experimental protocol used. c Effect of HGPS-iPSC SMC or N-iPSC SMCs conditioned media (in both cases obtained after 4 days under flow conditions) on hVSMCs cultured under flow conditions. n = 1–5 images examined over three independent experiments. d Quantification of MMP13 activity (cell culture media) by ELISA. Cells were analyzed at days 0 and 4 under flow. Fluorescence signal was normalized by cell number. n = 3 independent experiments. Statistical analyses were performed by a two-tailed unpaired Student’s t test. e Effect of MMP13 or BB94 inhibition in HGPS-iPSC SMC detachment. The number of cells was evaluated after 7 and 12 days under arterial flow and was normalized by the number of cells present at day 0. n = 3–5 images examined over three independent experiments. Statistical analyses were performed by one-way ANOVA followed by Newman–Keuls’s post test. f MMP13 knockdown by siRNA in HGPS-iPSC SMCs. MMP13 mRNA transcripts were quantified by qRT-PCR and normalized by GAPDH. Mean ± SEM (n = 4 technical replicates from a pool of three independent experiments). Statistical analyses were performed by one-way ANOVA followed by Newman–Keuls’s post test. g Number of cells per microfluidic area during culture under flow shear conditions normalized by the number of cells in control experimental groups (i.e., cells transfected with control siRNA). n = 7 independent experiments for day 7 and n = 6 independent experiments for day 10. h Percentage of progerin-positive cells after 7 days under flow conditions with SmGM-2 media supplemented or not with MMP13 inhibitor. n = 1–5 images examined over three independent experiments. Statistical analyses were performed by a two-tailed unpaired Student’s t test. i Activity of alkaline phosphatase in HGPS-iPSCs-SMC normalized by cell number per mm², in cells cultured 4 days under flow conditions. Cells were treated or not with MMP13 inhibitor. n = 3 independent experiments. In graphs a–h, results are mean ± SEM. *, **, ***, **** denote statistical significance (p < 0.05, p < 0.01, p < 0.001, p < 0.0001).