Nitric oxide inhibition increases matrix metalloproteinase–9 expression by rat aortic smooth muscle cells in vitro Gilbert R. Upchurch, MD, John W. Ford,

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Nitric oxide inhibition increases matrix metalloproteinase–9 expression by rat aortic smooth muscle cells in vitro Gilbert R. Upchurch, MD, John W. Ford, BA, Steven J. Weiss, MD, Brian S. Knipp, MA, David A. Peterson, BA, Robert W. Thompson, MD, Matthew J. Eagleton, MD, Autumn J. Broady, Mary C. Proctor, MS, James C. Stanley, MD Journal of Vascular Surgery Volume 34, Issue 1, Pages 76-83 (July 2001) DOI: 10.1067/mva.2001.115598 Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 1 Graphic quantitation of total NOx accompanying increasing L-NMMA concentration (4 experiments, repeated in duplicate). In presence of increasing doses of L-NMMA, NOx production decreases in a dose-dependent fashion (asterisk indicates P <.05 compared with cytokines alone). Results are expressed as nanogram of NO per milligram of protein, with each point representing mean ± SD. Journal of Vascular Surgery 2001 34, 76-83DOI: (10.1067/mva.2001.115598) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 2 A, Representative zymogram demonstrating that increasing concentrations of L-NMMA are associated with selective increase in 92-kd MMP activity, in early dose-dependent manner (4 experiments, repeated in duplicate). B, Graphic quantitation of 92-kd MMP and 72-kd MMP activity (expressed as percent control) accompanying increasing concentrations of L-NMMA (4 experiments, repeated in duplicate). In presence of increasing doses of L-NMMA, 92-kd MMP activity is increased in dose-dependent fashion (asterisk indicates P <.05 compared with cytokines alone). Significant changes in MMP-2 activity were not evident. Results are expressed as mean ± SD. Journal of Vascular Surgery 2001 34, 76-83DOI: (10.1067/mva.2001.115598) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 3 Zymogram demonstrating that in vitro treatment of RA-SMCs with L-NAME, a nonselective NOS inhibitor, in presence of cytokines is associated with dose-dependent increase in 92-kd MMP activity. Journal of Vascular Surgery 2001 34, 76-83DOI: (10.1067/mva.2001.115598) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 4 Representative Western blot (3 experiments) compared with known active MMP-9 standard demonstrating that IL-1β (2 ng/mL) is associated with appearance of a band consistent with pro–MMP-9 and addition of L-NMMA (5 mmol/L L-NMMA) to media containing IL-1β leads to fivefold increase (P <.05) in pro–MMP-9 protein levels. Journal of Vascular Surgery 2001 34, 76-83DOI: (10.1067/mva.2001.115598) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 5 Zymogram documenting that media from IL-1β stimulated SMCs treated with 3 mmol/L APMA converts pro–MMP-9 to active MMP-9, further characterizing the species that is effected by NOS inhibition as pro–MMP-9. Journal of Vascular Surgery 2001 34, 76-83DOI: (10.1067/mva.2001.115598) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 6 A, Representative RT-PCR (3 experiments) and (B) densitometry of PCR performed after 24 hours of experimental interventions demonstrating an increase in MMP-9 mRNA levels, after normalizing for β-actin, in SMCs treated with IL-1β (2 ng/mL) and L-NMMA (0.1 and 1.0 mmol/L, P <.01 for 1.0 mmol/L L-NMMA) compared with IL-1β alone. Journal of Vascular Surgery 2001 34, 76-83DOI: (10.1067/mva.2001.115598) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions