Plasmin Triggers Rapid Contraction and Degradation of Fibroblast-Populated Collagen Lattices George D. Pins, Maura E. Collins-Pavao, Livingston Van De.

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Plasmin Triggers Rapid Contraction and Degradation of Fibroblast-Populated Collagen Lattices George D. Pins, Maura E. Collins-Pavao, Livingston Van De Water, Martin L. Yarmush, Jeffrey R. Morgan Journal of Investigative Dermatology Volume 114, Issue 4, Pages 647-653 (April 2000) DOI: 10.1046/j.1523-1747.2000.00858.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Plasmin enhances the contraction of collagen lattices seeded with dermal fibroblasts. (a) A typical time course of contraction of fibroblast-populated collagen lattices with varying concentrations of plasmin. Plasmin was added 48 h after casting. (b) Appearance of contracted lattices 24 h after the addition of plasmin. Control lattices are approximately 17–20 mm in diameter. Journal of Investigative Dermatology 2000 114, 647-653DOI: (10.1046/j.1523-1747.2000.00858.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Serum concentration and time of plasmin addition affect the ability of dermal fibroblasts to contract collagen lattices. Typical time courses of contraction for collagen lattices cast with medium containing a final concentration of 1% (a, b, c), 5% (d, e, f), or 10% FBS (g, h, i). At indicated time points, different concentrations of plasmin were added to the lattice medium. Journal of Investigative Dermatology 2000 114, 647-653DOI: (10.1046/j.1523-1747.2000.00858.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Plasmin-mediated degradation of collagen lattices. Collagen lattices were made with 3H labeled type I collagen, treated with plasmin or conditioned medium, and analyzed for radioactivity released into the medium. In (a), the total amount of radioactivity in the medium was measured after fibroblast-populated collagen lattices were treated with plasmin for 24 h. In (b), the total radioactivity in the medium was analyzed after acellular lattices were treated for 24 h with conditioned medium from fibroblast-populated collagen lattices. All lattice experiments were conducted in duplicate or triplicate. Error bars represent standard deviations. Journal of Investigative Dermatology 2000 114, 647-653DOI: (10.1046/j.1523-1747.2000.00858.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Plasmin cleaves MMP-1 secreted by fibroblasts. Aliquots of DMEM with 0.3% FBS treated with 0.48 U per ml (1000 nM) of plasmin (Cont) and conditioned medium samples (CM) from fibroblast-populated collagen lattices treated with 0.0 or 0.48 U per ml (1000 nM) of plasmin were analyzed by Western blot using antibodies against MMP-1. Arrows indicate the location of the MMP-1 bands (52, 40, and 23 kDa). Journal of Investigative Dermatology 2000 114, 647-653DOI: (10.1046/j.1523-1747.2000.00858.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Aprotinin and TIMP-1 inhibit enhanced contraction of collagen lattices. Appearance of collagen lattices 24 h after simultaneous treatment with different concentrations of plasmin and (a) aprotinin [0.0 μg per well (–) or 10 μg per well (3.1–3.9 μM) (+)] or (b) TIMP-1. Control lattices are approximately 7–10 mm in diameter. Journal of Investigative Dermatology 2000 114, 647-653DOI: (10.1046/j.1523-1747.2000.00858.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 TIMP-1 inhibits degradation of collagen lattices. Lattices were made with radiolabeled collagen, treated with plasmin and TIMP-1 or conditioned medium, and analyzed for radioactivity released into the medium. (a) The total amount of radioactivity in the medium of fibroblast-populated collagen lattices was measured after treating them with indicated concentrations of plasmin and TIMP-1 for 24 h. In (b), collagen lattices lacking cells were treated with conditioned medium from fibroblast-populated collagen lattices containing different concentrations of plasmin and TIMP-1, and radioactivity released into the lattice medium was measured after 24h. All lattice experiments were conducted in triplicate. Error bars represent standard deviations. (An asterisk * indicates a significant difference between groups, p ≤ 0.05, one-tailed Student's t test.) Journal of Investigative Dermatology 2000 114, 647-653DOI: (10.1046/j.1523-1747.2000.00858.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions