Nitric oxide inhibition increases matrix metalloproteinase-9 expression by rat aortic smooth muscle cells in vitro.
OBJECTIVE
The hypothesis to be tested was that diminished bioavailable nitric oxide (NO) affects matrix metalloproteinase (MMP) expression and activation in vascular smooth muscle cells (SMCs).


METHODS
Cultivated rat aortic SMCs (RA-SMCs) were exposed to increasing concentrations of L-N-monomethyl arginine (L-NMMA), a nonselective inhibitor of NO synthase, in the presence of proinflammatory cytokines (50 ng/mL interleukin [IL]-1beta, 50 ng/mL interferon-gamma, and 30 microg/mL lipopolysaccharide). Nitrite and nitrate, two of the final end products of NO metabolism, were measured in media collected at 48 hours with the use of the Saville assay (n = 4). MMP activity was measured with 1% gelatin zymography (n = 4). In separate experiments in which 2 ng/mL of IL-1beta and L-NMMA was used, MMP protein and messenger RNA (mRNA) levels were determined with Western blot analysis (n = 3) and semiquantitative reverse transcriptase-polymerase chain reaction (n = 3), respectively. Data were analyzed with nonparametric analysis of variance.


RESULTS
Increasing concentrations of the NO synthase inhibitor L-NMMA caused a dose-dependent decrease (P <.05) in nitrite and nitrate production by RA-SMCs after cytokine exposure. Zymography documented an early dosedependent increase (P <.05 compared with cytokines alone) in 92-kd MMP activity, with no significant changes in 72-kd MMP activity after treatment with L-NMMA (P >.05 compared with cytokines alone). Reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that the addition of L-NMMA to IL-1beta-stimulated RA-SMCs led to significant increases in MMP-9 mRNA (n = 3, P <.01 for 1.0 mmol/L L-NMMA) and MMP-9 protein levels (n = 3, P <.05), respectively. No differences in MMP-2 mRNA or protein levels were demonstrated.


CONCLUSIONS
Inhibition of cytokine-induced NO expression in RA-SMCs is associated with a selective, dose-dependent increase in MMP-9 expression and synthesis. These findings suggest that alterations in local NO synthesis may influence MMP-9-dependent vessel wall damage.
Upchurch
G R
GR
Jobst Vascular Research Laboratories, Section of Vascular Surgery, Department of Surgery, University of Michigan Medical School, USA. riversu@umich.edu
Ford
J W
JW
Weiss
S J
SJ
Knipp
B S
BS
Peterson
D A
DA
Thompson
R W
RW
Eagleton
M J
MJ
Broady
A J
AJ
Proctor
M C
MC
Stanley
J C
JC
D000818:Animals; D001011:Aorta / Q000166:cytology; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D009569:Nitric Oxide / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019323:omega-N-Methylarginine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D020780:Matrix Metalloproteinase 9