Fig. 2

TGF-β1 expression in CTRL and PAD gastrocnemius and its relationship with collagen density and ABI. a Representative greyscale images of gastrocnemius microvessels positive for TGF-β1 labeling (arrows) were captured with a wide-field fluorescence microscope (10× objective). Unlabeled myofibers (blue line) appear grey against black background. Specimens collected from control subjects (CTRL) and PAD patients at Fontaine Stage II (claudication, PAD-II) and Stage IV (tissue loss, PAD-IV) disease were labeled with primary antibody specific for TGF-β1 and a fluorescent secondary antibody. Both the intensity and extent of TGF-β1 labeling were increased in the microvessels of PAD versus CTRL and PAD-IV versus PAD-II specimens. b TGF-β1 expression was determined by quantitative fluorescence microscopy and defined as the sum of the products of area and mean pixel intensity of all positive events per microscopic field, normalized to the total area of specimen in the same field. The relationships between normalized TGF-β1 expression and collagen density (c), and Ankle-Brachial Index (d) were determined by the Pearson correlation analysis. Data are presented as mean ± standard error of the mean and significance is denoted as *p < 0.05; **p < 0.01; ***p < 0.001