TY - JOUR
T1 - Nitric oxide inhibition increases matrix metalloproteinase-9 expression by rat aortic smooth muscle cells in vitro
AU - Upchurch, Gilbert R.
AU - Ford, John W.
AU - Weiss, Steven J.
AU - Knipp, Brian S.
AU - Peterson, David A.
AU - Thompson, Robert W.
AU - Eagleton, Matthew J.
AU - Broady, Autumn J.
AU - Proctor, Mary C.
AU - Stanley, James C.
N1 - Funding Information:
Supported by the Jobst Foundation, an American Surgical Association Foundation Fellowship (Dr Upchurch), and the William and Joanne Price Research Fund.
PY - 2001/7
Y1 - 2001/7
N2 - 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]-1β, 50 ng/mL interferon-γ, and 30 μg/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-1β 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 dose-dependent 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-1β-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.
AB - 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]-1β, 50 ng/mL interferon-γ, and 30 μg/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-1β 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 dose-dependent 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-1β-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.
UR - https://www.scopus.com/pages/publications/0035406154
U2 - 10.1067/mva.2001.115598
DO - 10.1067/mva.2001.115598
M3 - Article
C2 - 11436078
AN - SCOPUS:0035406154
SN - 0741-5214
VL - 34
SP - 76
EP - 83
JO - Journal of Vascular Surgery
JF - Journal of Vascular Surgery
IS - 1
ER -