Sandro Lepidi, MD, Richard D. Kenagy, PhD, Elaine W

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Presentation transcript:

MMP9 production by human monocyte-derived macrophages is decreased on polymerized type I collagen Sandro Lepidi, MD, Richard D. Kenagy, PhD, Elaine W. Raines, MSa, Ernest S. Chiu, MDa, Alan Chait, MDb, Russell Ross, PhDa, Alexander W. Clowes, MD Journal of Vascular Surgery Volume 34, Issue 6, Pages 1111-1118 (December 2001) DOI: 10.1067/mva.2001.119401 Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 1 A, Gelatin zymography of medium conditioned by monocyte/macrophages. Cells were isolated by method 1 and cultured on plastic in autologous serum plus or minus 10 ng/mL PMA. Medium was changed to serum-free medium plus or minus 10 ng/mL PMA at the indicated time and conditioned for 24 hours before performing zymography. The representative zymogram shown is consistent with a replicate experiment and published results.8,15B, The effect of 10 ng/mL of either PDGF-BB, IL1β, TNFα, or PMA on MMP9 synthesis by macrophages isolated by method 1 and cultured on plastic in autologous serum for 7 days with 50 μCi/mL 35S-methionine for the last 48 hours. MMP9 was radioimmunoprecipitated, quantitated by means of PhosphorImaging analysis, and expressed as fold of control values. N = 4-5. *P <.05 versus control. Journal of Vascular Surgery 2001 34, 1111-1118DOI: (10.1067/mva.2001.119401) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 2 Synthesis of MMP9 by monocytes isolated by method 1 and cultured for 7 days on plastic or polymerized collagen gels with 50 μCi/mL 35S-methionine for the last 48 hours. *P <.05 versus plastic; †P <.05 versus polymerized collagen, n = 4. Journal of Vascular Surgery 2001 34, 1111-1118DOI: (10.1067/mva.2001.119401) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 3 Phase-contrast microscopy of human monocyte-derived macrophages cultured 7 days on a thick layer of polymerized collagen or on plastic with 20% autologous serum (magnification, 200×). Journal of Vascular Surgery 2001 34, 1111-1118DOI: (10.1067/mva.2001.119401) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 4 Degradation of 125I Acetyl-LDL by monocyte/macrophages cultured on polymeric collagen (closed circles) or plastic (open circles) in RPMI and 20% autologous serum for 7 days. Data (mean ± SEM; n = 3) are expressed as a percent of maximal degradation, which averaged (± SEM) 946 ± 108 ng 125I-AcLDL/24 hours/0.3·106 cells. Journal of Vascular Surgery 2001 34, 1111-1118DOI: (10.1067/mva.2001.119401) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 5 A, Scanning electron micrographs of monocytes isolated by means of method 2 and seeded on either monomer (top panel) or polymerized (bottom panel) collagen for 2 hours. The few filaments observed in the monomer preparation are polymerized collagen. The bar indicates 1 micron. B, Phase-contrast microscopy of monocytes isolated by means of method 2 that were cultured in suspension on Hydron for 5 days, then seeded onto thin layers of either monomer or polymerized collagen (phase-contrast at 200×). Journal of Vascular Surgery 2001 34, 1111-1118DOI: (10.1067/mva.2001.119401) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 6 Effect of polymerized and monomer collagen on MMP9 synthesis by monocyte/macrophages differentiated in suspension (method 2) with 10% PDS and M-CSF. After 5 days, cells were seeded onto thin layers of monomer or polymerized collagen or maintained in suspension on Hydron for 27 hours with 50 μCi/mL 35S-methionine. MMP9 was radioimmunoprecipitated, quantitated by means of PhosphorImaging analysis, and expressed as a percent of the value of Hydron controls (mean ± SEM; n = 5). *P <.05 versus Hydron. Journal of Vascular Surgery 2001 34, 1111-1118DOI: (10.1067/mva.2001.119401) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 7 A, Dose-response study of the effect of PDGF-BB, PDGF-AA, IL1β, and TNFα on MMP9 synthesis by macrophages (isolated by means of method 1) cultured on a thick layer of polymerized collagen. Cells were cultured with 20% autologous serum for 7 days. During the last 48 hours, test factors and 35S Methionine were added. Data from PhosphoImager analysis are expressed as fold of control values (n = 3-6, except for 0.1 ng/mL IL1β and 0.001 ng/mL TNFα, which are n = 2). B, PDGF-BB, IL1β, and TNFα were given alone and in combination (all at 10 ng/mL) to cells as described in A. The data are expressed as fold over control (n = 8). *P <.05 versus control. Journal of Vascular Surgery 2001 34, 1111-1118DOI: (10.1067/mva.2001.119401) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions