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Elastolysis Induces Collagenolysis in a Gingival Lamina Propria Model

¹ Laboratoire Interface Biomatériaux/Tissus Hôtes, INSERM ERM 0203, Institut "Biomolécules" (IFR53), Faculté d’Odontologie, Université de Reims Champagne-Ardenne, 1 rue Maréchal Juin, 51095 Reims Cedex, France; and
² Laboratoire de Biochimie et Biologie Moléculaire, CNRS UMR 6198, Institut "Biomolécules" (IFR53), Faculté de Médecine, Université de Reims Champagne-Ardenne, 51 rue Cognacq Jay, 51095 Reims Cedex, France

View larger version (28K): [in this window] [in a new window]   Figure 1. Effects of elastin peptides on MMP expression. Human gingival fibroblasts, at confluence, were treated with 100 µg/mL kappa-elastin (kE) in serum-free medium, and urokinase-plasminogen activator (uPA), MMPs, and TIMPs expressions were analyzed by RTPCR (A). C, control (i.e., cells cultured in the absence of kE). Variations in MMP-1, MMP-3, and MMP-2 levels (B) were obtained from Western blotting (MMP-1,-3) and gelatin zymography (MMP-2); they were quantified by image analysis (Vilbert-Loumat, France) and are representative of separate experiments with 6 different fibroblast strains. Bars indicate standard deviations. Significantly different from control: *p < 0.03; **p < 0.005. S-Gal expression by human gingival fibroblasts mediates MMP-3 up-regulation (C). The presence of the S-Gal elastin receptor in gingival fibroblasts (2) was identified by Western blotting (CI) with an antibody directed against the V14 peptide, corresponding to the spliced βGAL region; dermal fibroblast (1) plasma membranes were similarly analyzed. (CII) The influences of VGVAPG (200 µg/mL), (VGVAPG)3 (200 µg/mL), and kE (100 µg/mL) on MMP-3 expression were compared; control (100%) refers to fibroblasts cultured in the absence of peptides. (CIII) Effects of lactose (50 µmol/L), vβ3 blocking antibody (ABvβ3; 10 µg/mL), and U0126 (10 nmol/L, an ERK1/2 inhibitor) on kE-mediated MMP-3 up-regulation (100%). Data presented are means from experiments performed in triplicate from 3 different cell strains.

View larger version (43K): [in this window] [in a new window]   Figure 2. Triggering of the plasmin/MMP-3/MMP-1 proteolytic cascade and collagenolysis in elastin peptides (100 µg/mL)-treated floating gingival-fibroblast-populated collagen lattices (FCL) following 4 days of culture. FCL was prepared with 2.5% serum. Plasmin activity (A) was determined with S2251 as substrate, and MMP-3 (B), MMP-1 (C), TIMP-1 (E), and TIMP-2 (F) were analyzed by Western blotting; TIMPs were also identified by gelatin reverse zymography (D). Collagen degradation was quantified by hydroxyproline assay (G). The numbers 1, 2, 3, and 4 refer to control, FCL cultured in the presence of 100 µg/mL kE, FCL cultured in the presence of plasminogen (30 µg/mL), and FCL cultured in the presence of both kE and plasminogen, respectively. Variations are representative of separate experiments performed in triplicate with 4 different cell strains. Bars indicate standard deviations. Significantly different from control: O, p < 0.05; OO, p < 0.01; OOO, p < 0.001.

View larger version (45K): [in this window] [in a new window]   Figure 3. Elastin peptides induced collagenolysis in detached gingival-fibroblast-populated collagen lattices following 4 days of culture. (A) Collagen degradation was determined similarly as for FCLs in Fig. 2. The numbers 1, 2, 3, and 4 refer to control, FCL cultured in the presence of 100 µg/mL kE, FCL cultured in the presence of plasminogen (30 µg/mL), FCL cultured in the presence of both kE and plasminogen, respectively. Variations are representative of separate experiments performed in triplicate with 4 different cell strains. Bars indicate standard deviations. Significantly different from control: O, p < 0.05; OOO, p < 0.001. (B) Lattice morphology was examined by scanning electron microscopy. By day 4, gingival fibroblasts—whatever the conditions, i.e., in the presence or absence of plasminogen (30 µg/mL), or the presence or absence of elastin peptides (100 µg/mL)—possessed a typical elongated morphology (dark star) exhibiting a parallel orientation to the surfaces of lattices (not shown). The presence of pericellular areas of lysed matrix (V) were observed essentially in lattices containing both elastin and plasminogen, suggesting localized collagenolysis (G).

Journal of Dental Research, Vol. 85, No. 8, 745-750 (2006)
DOI: 10.1177/154405910608500811


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