Eagleton Matthew J, Peterson David A, Sullivan Vita V, Roelofs Karen J, Ford John A, Stanley James C, Upchurch Gilbert R
Jobst Vascular Research Laboratories, University of Michigan Medical School, Ann Arbor, Michigan, 48109-0329, USA.
J Surg Res. 2002 May 1;104(1):15-21. doi: 10.1006/jsre.2002.6396.
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.
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.
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.
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.
一氧化氮（NO）可能通过调节基质金属蛋白酶（MMPs）和金属蛋白酶组织抑制剂（TIMPs）的表达来介导血管壁重塑。本研究检验了以下假设：全主动脉壁中一氧化氮合酶（NOS）受抑制会导致细胞因子刺激的MMP和TIMP表达增加。
将来自Sprague-Dawley大鼠的培养肾下腹主动脉段暴露于浓度递增（0、0.1、0.5、1和5 mM；每个浓度n = 6）的N(G)-单甲基-L-精氨酸（L-NMMA）中，L-NMMA是一种已知的NOS抑制剂。同时存在2 ng/ml的白细胞介素-1β，它是已知的NOS、MMP和TIMP表达诱导剂。在72小时时使用Saville方法测量培养基中的硝酸盐和亚硝酸盐（NO(x)）。使用明胶酶谱法测量培养基中的MMP活性。通过蛋白质印迹法和逆转录聚合酶链反应（RT-PCR）测定MMP-2和-9的蛋白质及mRNA水平。通过反向酶谱法和RT-PCR评估TIMP活性和mRNA水平。数据采用方差分析进行分析。
L-NMMA浓度增加导致NO(x)呈剂量依赖性降低（从2214±405降至347±37 ng/mg，P < 0.001）。酶谱分析显示92-kDa MMP（前MMP-9）活性呈剂量依赖性增加（P < 0.001），前MMP-9蛋白（P = 0.03）和mRNA水平相应增加（P = 0.004）。虽然72-kDa MMP（前MMP-2）活性呈剂量依赖性增加（P = 0.001），但前MMP-蛋白和mRNA水平未改变。反向酶谱分析显示29-kDa TIMP-1活性呈剂量依赖性增加（P = 0.01），但TIMP-1 mRNA水平无变化。
体外主动脉组织中NOS受抑制导致MMP-9表达和活性呈剂量依赖性增加。推测体内NO缺乏会改变MMP和TIMP的稳态，有利于基质降解。
