Monocyte-induced downregulation of nitric oxide synthase in cultured aortic endothelial cells.
Monocyte-induced downregulation of nitric oxide synthase in cultured aortic endothelial cells.
复制标题
在培养的主动脉内皮细胞中单核细胞诱导的一氧化氮合酶下调。
DOI：
10.1161/01.atv.16.9.1095
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Catravas,JD
中科院分区：
文献类型：
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作者：
Marczin,N;Antonov,A;Papapetropoulos,A;Munn,DH;Virmani,R;Kolodgie,FD;Gerrity,R;Catravas,JD
由于内皮依赖性血管舒张在动脉粥样硬化中发生改变，单核细胞/内皮相互作用的增强与早期动脉粥样硬化有关，我们通过估计培养内皮细胞释放的生物活性NO和组成型NO合成酶（ecNOS）水平来评估单核细胞对内皮型一氧化氮（NO）通路的影响。利用内皮细胞诱导的血管平滑肌（SM）细胞cGMP积累，通过短期生物测定法估计NO释放。SM细胞暴露于猪主动脉内皮细胞（PAECs）和人主动脉内皮细胞（HAECs）后，SM cGMP含量大幅增加；内皮细胞NOS抑制剂ng -硝基-l-精氨酸甲酯可阻止这种增加。PAECs和HAECs与单核细胞共培养的融合单层也可刺激SM - cGMP的形成；然而，在共培养时间（2至48小时）和单核细胞浓度（20至200×103per）依赖的方式下，这些培养物的NO释放减弱。单核细胞粘附的这种作用似乎对一氧化氮释放具有选择性，因为其他生化途径，如心房肽和异丙肾上腺素诱导的内皮细胞内环核苷酸积累，不会被单核细胞改变。单核细胞来源的细胞因子肿瘤坏死因子(TNF)-α和白细胞介素(IL)-1α模拟了粘附的单核细胞对NO释放的影响。此外，单核细胞的条件培养基中含有大量的这些细胞因子。条件培养基以及与内皮细胞物理分离的单核细胞减弱了NO的释放，提示可溶性因子可能介导了单核细胞的作用。IL-1β中和抗体完全阻止NO功能障碍对直接贴壁单核细胞的反应。超氧化物歧化酶、过氧化氢酶、4,5-二羟基-1,3-苯二磺酸（铁）和外源精氨酸未能改善NO的释放，这表明氧化应激诱导的NO失活或底物可用性有限并不是单核细胞抑制作用的主要原因。Western blot分析显示，单核细胞/内皮共培养以及单核细胞条件培养基或TNF-α处理的HAECs中，ecNOS的数量减少。因此，单核细胞与内皮细胞和单核细胞衍生的分泌产物的粘附下调稳态ecNOS水平，这一事件与生物活性NO的释放减弱有关。这一机制可能有助于减少早期动脉粥样硬化中内皮依赖性和no介导的血管舒张。
Since endothelium-dependent vasodilation is altered in atherosclerosis and enhanced monocyte/endothelial interactions are implicated in early atherosclerosis, we evaluated the effects of monocytes on the endothelial nitric oxide (NO) pathway by estimating release of biologically active NO from cultured endothelial cells and levels of constitutive NO synthase (ecNOS). NO release was estimated in a short-term bioassay using endothelial cell–induced cGMP accumulation in vascular smooth muscle (SM) cells. Exposure of SM cells to porcine aortic endothelial cells (PAECs) and human aortic endothelial cells (HAECs) produced large increases in SM cGMP content; this increase was prevented byNG-nitro-l-arginine methyl ester, the inhibitor of endothelial NOS. Confluent monolayers of PAECs and HAECs cocultured with monocytes also stimulated SM cGMP formation; however, NO release from these cultures was attenuated in a coculture time (2 to 48 hours)- and monocyte concentration (20 to 200×103per well)–dependent manner. This effect of monocyte adhesion appeared to be selective for NO release since other biochemical pathways, such as atriopeptin- and isoproterenol-induced cyclic nucleotide accumulation within the endothelial cells, were not altered by monocytes. The effects of adherent monocytes on NO release were mimicked by monocyte-derived cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1α. Furthermore, the conditioned medium of monocytes contained significant quantities of these cytokines. Conditioned medium, as well as monocytes physically separated from the endothelial cells, attenuated NO release, suggesting that soluble factors may mediate the effects of monocytes. An IL-1β neutralizing antibody fully prevented the NO dysfunction in response to directly adherent monocytes. Superoxide dismutase, catalase, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and exogenousl-arginine failed to improve NO release, suggesting that oxidant stress–induced inactivation of NO or limited substrate availability were not primarily responsible for the inhibiting effects of monocytes. Western blot analysis revealed reduced quantities of ecNOS in monocyte/endothelium cocultures, as well as in HAECs treated with monocyte-conditioned medium or TNF-α. Thus, adhesion of monocytes to endothelial cells and monocyte-derived secretory products downregulate steady state levels of ecNOS, an event associated with attenuated release of biologically active NO. This mechanism may potentially contribute to diminished endothelium-dependent and NO-mediated vasodilation in early atherosclerosis.