Nitric oxide inhibition increases aortic wall matrix metalloproteinase-9 expression.
OBJECTIVE
Nitric oxide (NO) may mediate vessel wall remodeling by regulating expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). This study tested the hypothesis that nitric oxide synthase (NOS) inhibition in whole aortic wall causes increases in cytokine-stimulated MMP and TIMP expression.


METHODS
Cultured infrarenal aortic segments from Sprague-Dawley rats were exposed to increasing concentrations (0, 0.1, 0.5, 1, and 5 mM; n = 6 per concentration) of N(G)-monomethyl-l-arginine (L-NMMA), a known inhibitor of NOS. This was in the presence of 2 ng/ml of interleukin-1beta, a known inducer of NOS, MMP, and TIMP expression. Media nitrate and nitrite (NO(x)) were measured at 72 h using the Saville method. Media MMP activity was measured using gelatin zymography. MMP-2 and -9 protein and mRNA levels were determined by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). TIMP activity and mRNA levels were evaluated by reverse zymography and RT-PCR. Data were analyzed using ANOVA.


RESULTS
Increasing concentrations of L-NMMA produced a dose-dependent decrease in NO(x) (2214 +/- 405 to 347 +/- 37 ng/mg, P < 0.001). Zymography demonstrated a dose-dependent increase in 92-kDa MMP (pro-MMP-9) activity (P < 0.001) with corresponding increases in pro-MMP-9 protein (P = 0.03) and mRNA levels (P = 0.004). While there was a dose-dependent increase in 72-kDa MMP (pro-MMP-2) activity (P = 0.001), pro-MMP-2 protein and mRNA levels were unchanged. Reverse zymography demonstrated a dose-dependent increase in 29-kDa TIMP-1 activity (P = 0.01), but there was no change in TIMP-1 mRNA levels.


CONCLUSIONS
NOS inhibition in ex vivo aortic tissue causes a dose-dependent increase in MMP-9 expression and activity. It is speculated that deficiencies of NO in vivo alter MMP and TIMP homeostasis, favoring matrix degradation.
Eagleton
Matthew J
MJ
Jobst Vascular Research Laboratories, University of Michigan Medical School, Ann Arbor, Michigan, 48109-0329, USA. eagleton@med.umich.edu
Peterson
David A
DA
Sullivan
Vita V
VV
Roelofs
Karen J
KJ
Ford
John A
JA
Stanley
James C
JC
Upchurch
Gilbert R
GR
D000818:Animals; D001011:Aorta / Q000201:enzymology*; D046508:Culture Techniques; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D018093:Gelatinases / Q000737:chemistry / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D009569:Nitric Oxide / Q000037:antagonists & inhibitors*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052250:Nitric Oxide Synthase Type III; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D019323:omega-N-Methylarginine / Q000008:administration & dosage / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D018093:Gelatinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9