Journal:

Article Title: Myofibroblast Differentiation Is Induced in Keratinocyte-Fibroblast Co-Cultures and Is Antagonistically Regulated by Endogenous Transforming Growth Factor-? and Interleukin-1

doi:

Figure Lengend Snippet: Top: Inhibition of endogenous TGF-β represses α-SMA induction in co-cultured iHDFs. iHDFs were cultured in the presence (+) or absence (−) of HaCaT cells for 7 days. A neutralizing monoclonal antibody against the TGF-β isoforms 1, 2, and 3 (10 μg/ml, left two lanes) or plain culture medium (0.5% FCS, right two lanes) were added to the co-cultures. Medium was changed every 2 days. α-SMA expression was assessed by Western blotting and the MOPC 21-isotype control antibody had no effect in HaCaT-iHDFs or iHDF control cultures (data not shown). A Coomassie-stained parallel gel is shown at the bottom. A representative blot is shown out of three independent experiments. Data represent the average ± SEM of three independent experiments. α-SMA levels in co-cultures were set at 100%. The neutralizing TGF-β antibody inhibited α-SMA induction significantly but not completely in co-cultures.

Article Snippet: Human platelet-derived and recombinant TGF-β1, IL-1 receptor antagonist, IL-1α, and the neutralizing mouse anti-TGF-β1, -2, -3 (clone 1D11) monoclonal antibody were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany).

Techniques: Inhibition, Cell Culture, Expressing, Western Blot, Control, Staining

Journal:

Article Title: Myofibroblast Differentiation Is Induced in Keratinocyte-Fibroblast Co-Cultures and Is Antagonistically Regulated by Endogenous Transforming Growth Factor-? and Interleukin-1

doi:

Figure Lengend Snippet: Induction of latent TGF-β production and TGF-β activation in HaCaT-HDF co-cultures. A total of 105 cells were plated, at various ratios of HDF to HaCaT cells, into 24-well plates. The relative cell percentages are indicated at the left. Cells had direct cell-to-cell contact (top) or were separated by transwell inserts (bottom). Conditioned media (serum-free DMEM containing 0.1% pyrogen-poor BSA) were collected after 48 hours and analyzed for total (after heat activation, middle column) and active (right column) TGF-β activity using the PAI/L bioassay. Data represent the mean value ± SEM of two to six experiments each analyzed in triplicate. Efficient latent TGF-β activation only occurred in co-cultures and required direct cell-to-cell contact.

Article Snippet: Human platelet-derived and recombinant TGF-β1, IL-1 receptor antagonist, IL-1α, and the neutralizing mouse anti-TGF-β1, -2, -3 (clone 1D11) monoclonal antibody were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany).

Techniques: Activation Assay, Activity Assay, Bioassay

Journal:

Article Title: Myofibroblast Differentiation Is Induced in Keratinocyte-Fibroblast Co-Cultures and Is Antagonistically Regulated by Endogenous Transforming Growth Factor-? and Interleukin-1

doi:

Figure Lengend Snippet: Keratinocyte-derived IL-1 blocks TGF-β-mediated induction of α-SMA in fibroblast monocultures. IL-1α was added to fibroblast monocultures (0.2 ng/ml) stimulated with different concentrations of TGF-β1. Medium was changed every 2 days. α-SMA expression was assessed by Western blotting. Co-stimulation of IL-1 and TGF-β blocks TGF-β-mediated α-SMA induction, suggesting interference with TGF-β-mediated transcription. Values represent the average ± SD of three independent experiments. α-SMA levels in co-cultures were set at 100%.

Article Snippet: Human platelet-derived and recombinant TGF-β1, IL-1 receptor antagonist, IL-1α, and the neutralizing mouse anti-TGF-β1, -2, -3 (clone 1D11) monoclonal antibody were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany).

Techniques: Derivative Assay, Expressing, Western Blot

Journal:

Article Title: Myofibroblast Differentiation Is Induced in Keratinocyte-Fibroblast Co-Cultures and Is Antagonistically Regulated by Endogenous Transforming Growth Factor-? and Interleukin-1

doi:

Figure Lengend Snippet: Proposed model for the fibroblast phenotype in the wound environment. A balance of proinflammatory and TGF-β stimuli governs the phenotype of fibroblasts in tissue repair. Fibroblasts initially respond to PMNs, monocytes/macrophages, and keratinocyte-derived IL-1 with activation of NF-κB and subsequently with an up-regulation of IL-1-responsive genes such as KGF, IL-6, and GM-CSF. Although TGF-β activity is present from platelets and activated keratinocyte-fibroblast interactions, NF-κB activation seems to interfere with TGF-β signaling. Later, when the wound environment becomes less proinflammatory dominated, the fibroblasts will respond to TGF-β and differentiate into myofibroblasts as seen by their induction of α-SMA, ECM molecules, and matrix-modifying enzymes. Dominant pathways are shown by black arrows. Pathways that are blocked or do not prevail are indicated by gray arrows.

Article Snippet: Human platelet-derived and recombinant TGF-β1, IL-1 receptor antagonist, IL-1α, and the neutralizing mouse anti-TGF-β1, -2, -3 (clone 1D11) monoclonal antibody were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany).

Techniques: Derivative Assay, Activation Assay, Activity Assay

Journal:

Article Title: Myofibroblast Differentiation Is Induced in Keratinocyte-Fibroblast Co-Cultures and Is Antagonistically Regulated by Endogenous Transforming Growth Factor-? and Interleukin-1

doi:

Figure Lengend Snippet: Continued

Article Snippet: Human platelet-derived and recombinant TGF-β1, IL-1 receptor antagonist, IL-1α, and the neutralizing mouse anti-TGF-β1, -2, -3 (clone 1D11) monoclonal antibody were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany).

Techniques: