Arginase blockade lessens endothelial dysfunction after thrombosis.
Arginase blockade lessens endothelial dysfunction after thrombosis.
复制标题
精氨酸酶阻断可减轻血栓形成后的内皮功能障碍。
DOI：
10.1016/j.jvs.2008.02.030
复制
发表时间：
2008
影响因子：
4.3
通讯作者：
Kashyap,VikramS
中科院分区：
文献类型：
--
作者：
Lewis,Chandani;Zhu,Weifei;Pavkov,MirceaL;Kinney,CorttrellM;Dicorleto,PaulE;Kashyap,VikramS
前言急性动脉血栓形成导致内皮功能障碍是由于一氧化氮生物活性降低。增加的精氨酸酶活性可以调节细胞内L-精氨酸水平，一氧化氮的底物。本研究的目的是确定凝血酶在细胞培养的内皮功能障碍中的作用，以及在血栓暴露的动脉血管扩张反应中的作用。通过免疫印迹和实时聚合酶链反应分析细胞提取物。成年雄性大鼠通过结扎夹结扎1小时进行肾下主动脉血栓形成。取离体肾主动脉环段，置于生理缓冲液中，用力传感器测量内皮依赖性舒张（EDR）和内皮非依赖性舒张（EIR）。在测量EDR之前，通过将肾下主动脉环段与精氨酸酶抑制剂孵育1小时来进行精氨酸酶阻断。全组织提取物也进行免疫印迹分析。EDR和EIR曲线用方差分析进行比较。在暴露于凝血酶（30 U/mL）的大鼠主动脉内皮细胞中，发现凝血酶-TSA对凝血酶-I信使水平的诱导为6.76 ± 1.4倍（P = 0.001），凝血酶-I蛋白水平增加2.1倍。与14个未形成血栓的正常节段（对照组）相比，8个暴露于血栓形成1小时的肾下主动脉环节段的EDR曲线减小。对照肾下主动脉环段的最大EDR（乙酰胆碱10− 5 M剂量）为108% ± 4.3%，而血栓形成的肾下主动脉环段为63% ± 6.2%（P <0.001）。暴露于动脉血栓形成导致肾下主动脉环段中β-淀粉酶I蛋白水平增加3.8倍。与未处理的血栓形成节段相比，9个肾下主动脉环节段与非特异性（二氟甲基鸟氨酸）和6个肾下主动脉环节段与特异性（[S]-[2-二羟硼乙基]-L-半胱氨酸-盐酸盐[BEC]）抑制剂预孵育1小时可显著增加最大EDR（104 ± 5.2，108 ± 7.6 vs 63% ± 6.2，P <0.001）。将二氟甲基鸟氨酸和BEC处理的肾下主动脉环段的EDR曲线叠加在对照EDR曲线上。所有组的EIR和去甲肾上腺素引起的血管收缩相似。结论暴露于凝血酶的内皮细胞的精氨酸酶I信使RNA和蛋白质水平增加。动脉血栓形成引起内皮功能障碍，而不影响平滑肌反应性。精氨酸酶阻断可导致动脉血管功能正常化。
INTRODUCTIONAcute arterial thrombosis causes endothelial dysfunction due to decreased nitric oxide bioactivity. Increased arginase activity may modulate intracellular L-arginine levels, the substrate for nitric oxide. The purpose of this study was to identify the role of arginase in endothelial dysfunction in cell culture and in the vasomotor response of arteries exposed to thrombus.METHODSRat aortic endothelial cells were exposed to thrombin at different time points. The cell extract was analyzed by immunoblotting and real-time polymerase chain reaction. Adult male rats underwent infrarenal aortic thrombosis by clip ligature for 1 hour. Infrarenal aortic ring segments were harvested and placed in physiologic buffer baths, and a force transducer was used to measure endothelial-dependent relaxation (EDR) and endothelial-independent relaxation (EIR). Arginase blockade was performed by incubating infrarenal aortic ring segments with arginase inhibitors for 1 hour before measuring EDR. Whole tissue extracts also underwent immunoblot analysis. The EDR and EIR curves were compared with analyses of variance.RESULTSA 6.76 ± 1.4-fold induction in arginase I message levels (P = .001) was found in rat aortic endothelial cells exposed to thrombin (30 U/mL), and arginase I protein levels increased 2.1 times. The eight infrarenal aortic ring segments exposed to thrombosis for 1 hour had diminished EDR curves compared with 14 nonthrombosed normal segments (controls). The maximum (± SEM) EDR (acetylcholine 10−5M dose) in control infrarenal aortic ring segments was 108% ± 4.3% compared with 63% ± 6.2% for thrombosed infrarenal aortic ring segments (P < .001). Exposure to arterial thrombosis resulted in a 3.8-times increase in arginase I protein levels in infrarenal aortic ring segments. Preincubation of nine infrarenal aortic ring segments with the nonspecific (difluoromethylornithine) and six with specific ([S]-[2-boronoethyl]-L-Cysteine-HCl [BEC]) arginase inhibitor for 1 hour significantly increased the maximum EDR compared with untreated thrombosed segments (104 ± 5.2, 108 ± 7.6 vs 63% ± 6.2, P < .001). EDR curves for difluoromethylornithine- and BEC-treated infrarenal aortic ring segments were superimposed on control EDR curves. The EIR and the vasoconstriction with norepinephrine for all groups were similar.CONCLUSIONSEndothelial cells exposed to thrombin have increased arginase I messenger RNA and protein levels. Arterial thrombosis causes endothelial dysfunction without affecting smooth muscle responsiveness. Arginase blockade can lead to normalization of arterial vasomotor function.