Therapeutic Targeting of Redox Signaling in Myofibroblast Differentiation and Age-Related Fibrotic Disease

Figure 1

In vitro modeling of fibroblast-to-myofibroblast differentiation. Fibroblast-to-myofibroblast differentiation is induced following incubation of fibroblasts for at least 24 hours with 1 ng/mL transforming growth factor beta 1 (TGFβ1) in RPMI-1640 media supplemented with 1% steroid hormone-depleted charcoal-treated bovine calf serum (ctBCS). Steroid hormone depletion is particularly important for fibroblasts derived from endocrine tissues such as the prostate, since androgens attenuate TGFβ signaling and vice versa [32]. Similar to commercial formulations of fibroblast growth media, the fibroblast phenotype of mock control cells is maintained with 1 ng/mL basic fibroblast growth factor (bFGF) and serves to inhibit differentiation-inducing stimuli (e.g., TGFβ) in serum. Myofibroblast differentiation can be monitored at the morphological level by phase contrast microscopy (a) and molecular level by Western blotting (b) or immunofluorescence (c). (a) Primary human dermal fibroblasts (top left) and prostatic fibroblasts (lower left) exhibit a typical thin, elongated, and light refractive phenotype, whereas upon differentiation with TGFβ1 for 24 hours dermal (top right) and prostatic myofibroblasts (lower right) display a flattened, less light refractive phenotype. (b) TGFβ1 induces the expression of myofibroblasts markers  𝛼-smooth muscle cell actin (𝛼-SMA) and insulin-like growth factor binding protein 3 (IGFBP3) [25] in primary human dermal (left) and prostatic (right) fibroblasts as determined by Western blotting. GAPDH served as loading control. (c) TGFβ1 treatment of primary human prostatic fibroblasts induces the expression of myofibroblast markers tenascin and filamentous  𝛼-SMA as determined by immunofluorescence and confocal laser scanning microscopy. Both myofibroblasts and fibroblasts express the mesenchymal marker vimentin. Nuclei were counterstained with SytoxGreen. (c) Adapted from [25], used with permission.