1. Fig. 1. Matrix stiffness sensitizes myofibroblasts to apoptosis induced by inhibition of their mechanotransduction pathways.
Matrix stiffness sensitizes myofibroblasts to apoptosis induced by inhibition of their mechanotransduction pathways. (A) Schematic diagram of atomic force microscopy (AFM) for microindentation on skin tissues. AFM was applied to map local elastic properties of thin slices of fresh mouse skin samples, harvested after 28 daily subcutaneous (sc) injections of either saline or bleomycin (BLM). (B) Representative hematoxylin and eosin (H&E)–stained images from 28-day saline- versus bleomycin-treated mice. Scale bars, 100 μm. n = 6 for all groups. (C) Representative elastographs from AFM microindentation of tissue stiffness in normal and fibrotic skin. The color bar indicates Young’s modulus, which is quantified in (D). Maps were made from tissue in the respective regions of interest (boxes) identified in (B). Data are means ± SD of stiffness measurements pooled from five animals each for normal and fibrotic groups in two independent bleomycin injection experiments. P value was determined by Student’s t test. (E and F) Effect of matrix stiffness on α-smooth muscle actin (α-SMA) protein expression as assessed by immunofluorescence. Human dermal fibroblasts (HDFs) were cultured on collagen-coated polyacrylamide hydrogels that recapitulated the stiffness of normal (4 kPa) (E) or densely fibrotic (50 kPa) (F) skin for 24 hours. Myofibroblasts were identified by staining for α-SMA (green). Fibroblasts were costained with phalloidin (red) to visualize F-actin and 4′,6-diamidino-2-phenylindole (DAPI; blue) to visualize nuclei. Scale bars, 50 μm. (G to L) Effect of matrix stiffness on the susceptibility of primary HDFs to apoptosis induced by inhibition of mechanotransduction pathways. HDFs were cultured on collagen-coated polyacrylamide hydrogels that recapitulated the stiffness of normal (4 kPa) or densely fibrotic (50 kPa) skin for 24 hours and treated with or without the agents indicated for an additional 48 hours. Apoptosis was assessed by annexin V staining. Data are means ± SD from three independent experiments. P value was determined by Student’s t test.
David Lagares et al., Sci Transl Med 2017;9:eaal3765
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