Fig 1: Collagen bundles thickness treated with tissue inhibitor of metalloproteinase-2 (TIMP-2). (A) Hematoxylin and eosin staining of ex vivo culture of keloid tissues. Photomicrographs were taken using an Olympus AX70 microscope and a DP72 digital camera for collagen bundle thickness. Scale bars in yellow = 100 µm. (B) Quantitative analysis of collagen bundles thickness. The thickness of individual collagen bundles at 100 sites/field were measured in 5 random fields in each sample using Lumina vision software. Data represent 6 independent experiments using skin explants from 6 different donors. Data are shown in arbitrary units. *P < 0.03.
Fig 2: The effects of tissue inhibitor of metalloproteinase-2 (TIMP-2) on collagen expression. Keloid fibroblasts (KFs) were exposed to the indicated concentration of TIMP-2 for 48 hours. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of Col1A2 and Col3A1 mRNA expression (n = 6). *P < 0.05.
Fig 3: The effects of tissue inhibitor of metalloproteinase-2 (TIMP-2) on collagen production. Levels of collagen type I protein (procollagen type I C-peptide; PIP) in culture supernatants conditioned by keloid fibroblasts (KFs) were measured using an enzyme-linked immunosorbent assay (ELISA, n=6). *P < 0.05.
Fig 4: The effect of cyclical uniaxial mechanical stretch on expression of tissue inhibitor of metalloproteinase-2 (TIMP-2), TIMP-1, matrix metalloproteinase-2 (MMP-2), MT1-MMP, and MMP-1 mRNA in keloid fibroblasts (KFs, n = 7). KFs were allowed to attach for 48 hours to the bottom of silicon chambers in 2 mL of culture medium. After the medium was changed, continuous uniaxial sinusoidal stretch (20%, 1 cycle/100 s) was applied at 37°C, 5% CO2 for 72 hours. In the control group, the culture medium was changed, and the silicon chambers were incubated for 72 hours as described above but without stretching. *P < 0.03.
Fig 5: Scheme for keloid pathogenesis and possible mechanism of treatment with tissue inhibitor of metalloproteinase-2 (TIMP-2). (A) Downregulation of TIMP-2 leads to the progression of keloids because of relative increase of MT1-MMP activity. MT1-MMP–regulated bioavailability of TGFß-1 and the increase of active TGFß-1 lead to collagen synthesis of fibroblasts and collagen deposition in keloid development. (B) Administration of TIMP-2 decreases progression of keloids by inhibiting MT1-MMP. Because activation of pro-TGFß-1 was inhibited as a result of relative decrease of MT1-MMP activity with TIMP-2 treatment, collagen synthesis was decreased by inactivation of fibroblasts, and the imbalance of MMP-1/TIMP-1 ratios in collagen deposition was improved in keloid development.
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