Zhang Fan, Zhang Yang, Li Pin-Lan
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, 410 N 12th, Richmond, VA 23298, USA.
Microvasc Res. 2009 Jun;78(1):45-50. doi: 10.1016/j.mvr.2009.03.006. Epub 2009 Apr 1.
Cathepsin L is a cysteine protease that can generate endogenous endostatin in vascular and epithelial basement membranes and importantly participates in a variety of pathophysiological processes. The present study was designed to determine whether this cathepsin L-derived endogenous endostatin alters endothelium-dependent vasodilator responses in coronary arteries via NAD(P)H oxidase activation. In isolated and perfused small bovine coronary arteries, administration of cathepsin L (200 ng/ml) markedly attenuated endothelium-dependent vasodilator responses to bradykinin or A23187 by 56.16+/-9.58% and 68.95+/-10.32%, respectively. This inhibitory effect of cathepsin L on endothelium-dependent vasodilator responses could be significantly reversed by pre-incubation of the arteries with O(2)(-) scavenger, Tiron, or neutralizing anti-endostatin antibody. By fluorescent ELISA assay, cathepsin L dose-dependently increased endostatin production in coronary arteries. In situ high-speed dual wavelength switching fluorescent microscopic imaging showed that cathepsin L decreased bradykinin- and A23187-induced NO levels in the intact endothelium, but it had no effect on Ca(2+) response to these vasodilators. This cathepsin L-induced reduction of NO was restored by the pretreatment of an anti-endostatin antibody. Electron spin resonance (ESR) analysis demonstrated that cathepsin L increased O(2)(-) production which could be markedly attenuated by the NAD(P)H oxidase inhibitors, apocynin or anti-endostatin antibody. It is concluded that endostatin could be endogenously produced in coronary arteries when cathepsin L is increased and that this cathepsin L-derived endostatin, if excessive, may result in endothelial dysfunction through enhanced production of O(2)(-) due to NAD(P)H oxidase activation.
组织蛋白酶L是一种半胱氨酸蛋白酶，可在血管和上皮基底膜中产生内源性内皮抑素，并重要地参与多种病理生理过程。本研究旨在确定这种源自组织蛋白酶L的内源性内皮抑素是否通过NAD(P)H氧化酶激活改变冠状动脉中内皮依赖性血管舒张反应。在离体灌注的小牛冠状动脉中，给予组织蛋白酶L（200 ng/ml）可使内皮依赖性血管舒张反应对缓激肽或A23187的反应分别显著减弱56.16±9.58%和68.95±10.32%。组织蛋白酶L对内皮依赖性血管舒张反应的这种抑制作用可通过用O(2)(-)清除剂替诺隆或中和性抗内皮抑素抗体预孵育动脉而显著逆转。通过荧光ELISA测定，组织蛋白酶L剂量依赖性地增加冠状动脉中内皮抑素的产生。原位高速双波长切换荧光显微镜成像显示，组织蛋白酶L降低了完整内皮中缓激肽和A23187诱导的NO水平，但对这些血管舒张剂的Ca(2+)反应没有影响。抗内皮抑素抗体预处理可恢复组织蛋白酶L诱导的NO减少。电子自旋共振（ESR）分析表明，组织蛋白酶L增加了O(2)(-)的产生，而NAD(P)H氧化酶抑制剂阿朴吗啡或抗内皮抑素抗体可显著减弱这种增加。结论是，当组织蛋白酶L增加时，冠状动脉中可内源性产生内皮抑素，并且这种源自组织蛋白酶L的内皮抑素如果过量，可能通过NAD(P)H氧化酶激活导致O(2)(-)产生增加而导致内皮功能障碍。
