Fig 2: mTOR inhibition prevents inflammation and production of MMPs but not medial thickening or induction of matricellular proteins.Apoe–/– mice were infused with AngII with or without rapamycin (Rapa) treatment, and the suprarenal abdominal aortas were analyzed at 7 days. (A) Biomechanical testing of AngII (n = 4) or AngII plus Rapa (n = 5) treated aortas for circumferential (circ) and axial stretch, stress, and material stiffness and (B) vasoconstriction responses to KCl and phenylephrine (PE) assessed by reduction of normalized outer diameter. (C) Lumen, media, and adventitia areas of suprarenal abdominal aortas without (n = 9) or with (n = 4–5) hematomas. (D) Immunostains for F4/80+ cells in intima (I), media (M), adventitia (A), and perivascular tissue (P) of aorta, scale bar: 50 μm. (E) Number of F4/80+ macrophages (M) per high power field (HPF) in vascular wall layers of aortas without hematomas (n = 6–7). (F) Number of SMCs per cross section (x-sec) of aortas without hematomas (n = 8). (G) Forward scatter area (FSC‑A) indicative of cell size of enzymatically isolated SMA+ SMCs (n = 8). (H) Quantitative RT-PCR for Col1a1, Col3a1, Spp1, Mmp2, Mmp3, Mmp14, Thbs1, Tnc, and Ccn2 transcript levels at 1 or 7 days (n = 4). Individual data shown, bars represent mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, unpaired, 2-tailed t test (A, F, and G) and 2-way ANOVA with Sidak’s multiple-comparison test (C, E, and H) for AngII + Rapa versus AngII.

Fig 3: Rapamycin-insensitive CTGF inhibits SMC adhesion to exogenous ECM.(A) Apoe–/– mice were infused with saline or AngII with or without rapamycin (Rapa) treatment for 7 days and the suprarenal abdominal aortas were analyzed by Western blot for CTGF, thrombospondin-1 (TSP1), tenascin-C (TNC), phospho-S6 (p-S6), and S6. (B) Densitometry of protein expression relative to HSP90 or (C) phospho-S6 expression relative to S6; expression normalized to peak levels with AngII treatment alone, (n = 4). (D) Colorimetric assay for number of murine aortic SMCs adherent to fibronectin-coated plates after 1 hour following cell pretreatment with vehicle, AngII at 100 nM, and/or rapamycin at 100 ng/mL for 45 minutes (n = 9–10, pooled from 3 experiments); OD405 readings normalized to vehicle-treated controls. (E) Similar fibronectin adhesion assay of SMCs pretreated with CTGF, thrombospondin-1, or tenascin-C at various doses for 45 minutes (n = 5–10, pooled from 3 experiments). (F) Colorimetric assay for number of SMCs adherent to plates coated with CTGF, thrombospondin-1, or tenascin-C at various concentrations (in the absence of fibronectin) after 1 hour (n = 2). (G) CTGF adhesion assay of SMCs pretreated with blocking antibody to integrin α5 (Itga5 Ab) or isotype-matched control antibody (Ctrl Ab) for 45 minutes (n = 10–11, pooled from 3 experiments). (H) Fibronectin adhesion assay of human aortic SMCs from 3 individuals pretreated with CTGF at various doses for 45 minutes (n = 4, shown separately for each subject). Individual data shown, bars represent mean ± SEM or lines represent nonlinear regression fitting by least-squares regression, **P < 0.01, ***P < 0.001, unpaired, 2-tailed t test (G), 1‑way ANOVA with Tukey’s multiple-comparison test (C–E and H), and 2‑way ANOVA with Sidak’s multiple-comparison test (B).

Fig 4: ECM-driven 3D invasion does not correlate with effects on 2D cell migration. (a) Representative images of spheroids made from 231-GFP cells embedded in media, Collagen I, Fibronectin, Tenascin C, or Collagen IV gels for 5 days. The scale bar is 200 μm. (b) Quantification of fold change in 231-GFP spheroid area on day 5 relative to day 1. Data pooled from at least five biological replicates, with three technical triplicate per experiment. ***p < 0.001 by one-way ANOVA and Dunn's multiple comparison test. (c) Correlation between mean fold change in spheroid area and 2D cell migration speed for 231-GFP cells. Correlation characterized by r2, p value and Q2. (d) Representative images of spheroids made from 468-GFP cells embedded in media, Collagen I, Fibronectin, Tenascin C, or Collagen IV gels for 5 days. Scale bar is 200 μm. (e) Quantification of fold change in 468-GFP spheroid area on day 5 relative to day 1. Data pooled from at least four biological replicates, with three technical triplicate per experiment. ***p < 0.001 by one-way ANOVA and Dunn's multiple comparison test. (f) Correlation between mean fold change in spheroid area and 2D cell migration speed for 468-GFP. Correlation characterized by r2, p value, and Q2.

Fig 5: ECM-driven effects on 2D cell migration speed do not correlate with effects on persistence. (a) Representative roseplots for MDA-MB-231 cells plated on glass, Collagen I, Fibronectin, Tenascin C, or Collagen IV for 16 h and imaged every 10 min. Each line represents an individual cell. Axis length is 500 μm. Quantification of cell migration speed (μm/min) (b) and persistence (c). Results show entire distribution, no ECM (n = 128 cells), Collagen I (n = 45 cells), Fibronectin (n = 91 cells), Tenascin C (n = 25 cells), or Collagen IV (n = 39 cells). Correlation between 2D persistence and 2D cell migration speed (d) and between cell area (μm2) and 2D cell migration speed (e). Correlation characterized by r2, p value, and Q2. (f) Representative roseplots for MDA-MB-468 cells plated on glass, Collagen I, Fibronectin, Tenascin C, or Collagen IV for 16 h and imaged every 10 min. Each line represents an individual cell. The axis length is 500 μm. Quantification of cell migration speed (μm/min) (g) and persistence (h). Results show entire distribution, no ECM (n = 220 cells), Collagen I (n = 68 cells), Fibronectin (n = 126 cells), Tenascin C (n = 24 cells), or Collagen IV (n = 63 cells). Correlation between 2D persistence and 2D cell migration speed (i) and between the cell area and 2D cell migration speed (j). Correlation characterized by r2, p value, and Q2. Significance determined by one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.005, ns is not significant.