Fig 2: Stiffness-dependent regulation of exocytosis. High substrate stiffness triggers a mechanosignaling cascade via ß1 integrin and RhoA that increases plasma membrane tension. In response, both caveolae formation and vesicle trafficking increase in order to buffer the high membrane tension. This, in turn, promotes the exocytosis of TIMP-1, preventing ECM degradation and maintaining the stiff environment.

Fig 3: Hypotonic medium-induced plasma membrane tension affects TIMP-1 trafficking in hepatic stellate cells (HSCs). (A) TIRF images of HSCs on glass transfected with CellLight Plasma Membrane-RFP, TIMP-1 GFP, and colocalized vesicles containing TIMP-1 for exocytosis analysis. (B) Representative TIRF images for TIMP-1 containing vesicles for isotonic and hypotonic medium used for quantification of TIMP-1 vesicle lifetime. Each individual panel in (B) represents 10 s. (C) Representative TIRF time-lapse kymographs of HSC TIMP-1 vesicles in (i) isotonic and (ii) hypotonic conditions. TIMP-1 (D) vesicle lifetime, (E) vesicle area, and (F) vesicle number per µm2 for the cells in control isotonic, control hypotonic, siRNA scramble isotonic, siRNA scramble hypotonic, caveolin-1 siRNA hypotonic, ß1 integrin blocking hypotonic, ß3 integrin blocking hypotonic, and dynamin-2 siRNA hypotonic conditions. Scale bars represent (A) 20 µm, (B) 500 nm, and (C) 10 µm. Scatter dot and histogram bars represent mean ± s.e.m; dots represent individual vesicles (D, E) or cells (F). Data are representative of n = 3 experimental replicates. Markers denote significant difference from control isotonic condition by one way ANOVA with Dunnett’s post hoc test, ** 0.001 < p < 0.01, *** 0.0001 < p < 0.001, **** p < 0.0001.

Fig 4: 3D self-assembling peptide gels of high fibrosis-mimicking stiffness trigger upregulation of caveolin-1 and TIMP-1. (A) Average storage modulus (G') across 0.1–10% strain sweep and (B) elastic modulus of PeptiGels calculated as E = 2 × G' (1 + ?) where, ? = Poisson’s ratio of 0.5. Histogram bars represent mean ± s.e.m; dots represent individual data points. (C) Representative epifluorescent images of hepatic stellate cells caveolin-1 and TIMP-1 immunostaining of formalin fixed, paraffin embedded soft (4 kPa, gamma 2), and stiff (10 kPa, alpha 2) 3D MBG PeptiGels; protein of interest (red), alpha smooth muscle actin (green) and nuclei (blue), scale bar is 20 µm. TIMP-1 (D) and caveolin-1 (E) immunofluorescence staining for panel (C). TIMP-1 n = 13 and 11, caveolin-1 n = 14 and 18 for gamma 2 and alpha 2 respectively. Mean ± s.e.m. Markers denote significant difference between gamma 2 and alpha 2 by t test, ** 0.001 < p < 0.01, **** p < 0.0001.

Fig 5: Caveolin-1 and TIMP-1 are upregulated in cirrhosis and HCC. (A) Representative epifluorescent images of human tissue caveolin-1 and TIMP-1 immunostaining of healthy, cirrhotic, and HCC (hepatocellular carcinoma) liver; protein of interest (red), alpha smooth muscle actin (green) and nuclei (blue); scale bar is 20 µm. Quantification of TIMP-1 (B), and caveolin-1 (C) immunofluorescence staining for panel (A) (n = number of patients: 5 healthy, 21 cirrhosis, 19 HCC stage I and II, 26 HCC stage III A and B, 7 HCC stage III C and IV A). Whiskers on box and whiskers graphs represent minimum and maximum. Markers denote significant difference from healthy condition by one-way ANOVA with Dunnett’s post hoc test, n.s.: not significant, ** 0.001 < p < 0.01, *** 0.0001 < p < 0.001, **** p < 0.0001.