Effect of GHK treatment on collagen contraction by fibroblasts from former smokers with COPD. (a) Representative immunofluorescent images of distal lung fibroblasts from former smokers with and without COPD treated with GHK (10 nM), TGFβ1 (10 ng/ml), or media control for 48 h and stained with phalloidin to localize the actin cytoskeleton (red), integrin-β1antibody (green) and DAPI to localize nuclei (blue). (b) Representative images of collagen I gel bioassays at 24 h after being seeded with distal lung fibroblasts from former smokers with and without COPD previously treated with GHK, TGFβ1, or media control for 48 h. (c) The percentage of collagen I contraction was significantly decreased in fibroblasts derived from former smokers with COPD compared to former smokers without COPD (P < 0.05) but was significantly increased with addition of TGFβ1 or GHK (P < 0.01). (d) Representative enface Z-stack slices of three-dimensional reconstructed collagen I gel bioassays demonstrating actin in fibroblasts (green, phalloidin) and second harmonic signal originating from collagen fibrils (purple, 414 nM). Fibroblasts from former smokers with COPD were unable to efficiently remodel collagen into fibrils. However, this intrinsic defect was restored with treatment of TGFβ1 or GHK.