Changes in ECM composition and structure during myofibroblast differentiation. (A) Granulation tissue sections of splinted and nonsplinted wounds from female Wistar rats were stained for α-SMA (blue), ED-A FN (red), and LTBP-1 (green). Normal rat skin is compared with 3-d-old nonsplinted wounds. Additionally, the arrowhead indicates the region of granulation tissue that is compared from splinted and nonsplinted wounds at day 3, 6, and 9. Insets show LTBP-1 only. Bars, 10 µm. (B) hDfs (control) were differentiated into hDMfs using 2 ng/ml TGF-β1 for 5 d in passage 1 (+TGF-β1) and used at passage 4 after 6 d of growth. Cells were labeled for α-SMA and F-actin and for LTBP-1, ED-A FN, and nuclei (blue). Bars, 50 µm. (C) Expression of ED-A FN, LTBP-1, vinculin, and α-SMA was determined by Western blotting of cells grown without (control [con]) and with TGF-β1 and quantified as a percentage of control normalized to vimentin as a loading control. (D) The mean density of fibril events per image field was quantified from 14 LTBP-1–stained images per three independent experiments. (E and F) Active TGF-β1 was measured by directly co-culturing fibroblastic cells with TMLC reporter cells either after inducing cell contraction with thrombin (E) or without inducing cell contraction (F, baseline). (G) Levels of active TGF-β1 are presented as a percentage of total TGF-β1 determined from heat-activated ECMs and cells. Graphs show mean values and SDs from at least three independent experiments (*, P ≤ 0.05; two-tailed paired t test). a.u., arbitrary unit.