Figure 1

TGFβ induces dermal fibroblast Nox4 expression and promotes transdifferentiation to myofibroblast. (a) Immunofluorescence detection of αSMA+ myofibroblasts in skin biopsies taken from burn patients, 12–48 months after injury, at the site of HTS formation and from the adjacent NBS. Positive staining for αSMA is in red and counterstaining for nuclei is in blue. Note that myofibroblasts have almost completely disappeared from the dermis 48 months after injury. There is some positive staining of glandular smooth muscle cells and vascular smooth muscle cells with anti-αSMA antibody in NBS and HTS sections, which serves as an internal positive control. Scale bar represents 100 microns. (b) IHC for TGFβ in HTS and NBS tissue sections from burn patients. Positive staining for TGFβ is brown and counterstaining for nuclei is blue. Most of the positive staining for TGFβ was observed in HTS biopsies at 12–24 months in the same location as the highest amount of αSMA+ staining. Scale bar represents 100 microns. IHC was performed on up to three tissue sections from a patient at each time point. Representative images are shown. (c) hDFs were stimulated with TGFβ (10 ng/ml) for 0–48 h, and changes in gene expression for SM22α, Nox4, fibronectin and Col1α1 were analyzed via quantitative real-time PCR. Gene expression was normalized to DNA polymerase β mRNA. Data are presented as mean±S.E.M. This experiment was repeated three times. *P<0.05 versus 0 h. (d) hDF cells were seeded on coverslips, pretreated with 10 μM ALK5i LY2157299 before being stimulated with TGFβ (10 ng/ml) for up to 48 h. Immunostaining for αSMA (green) and staining for filamentous actin using AlexaFluor-568-conjugated phalloidin (red) was performed. αSMA+ cells (green or yellow fibers) were considered to be myofibroblasts. Scale bar represents 100 microns. This experiment was repeated twice and similar results were observed. (e) hDF cells were pretreated with vehicle or 10 μM ALK5i and then stimulated with TGFβ (10 ng/ml). Whole-cell extracts were collected after 24 h and western blotting analysis was performed to analyze de novo αSMA production and phospho-Smad2/3. GAPDH and Smad2/3 served as loading controls. Experiment was repeated twice and similar results were obtained