Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation.
Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation.
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DOI：
10.3389/fphys.2017.01124
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发表时间：
2017
影响因子：
4
通讯作者：
He P
中科院分区：
文献类型：
--
作者：
Gao F;Lucke-Wold BP;Li X;Logsdon AF;Xu LC;Xu S;LaPenna KB;Wang H;Talukder MAH;Siedlecki CA;Huber JD;Rosen CL;He P
一氧化氮(NO)是一种已知的抗黏附分子，可阻止血小板聚集和白细胞与内皮细胞(ECs)的黏附。其作用机制主要是通过调节白细胞表面的黏附分子和调节血小板的黏附特性来实现的。我们先前在大鼠小静脉上进行的研究发现，EC基础NO合成的减少导致EC ICAM-1介导的白细胞在10-30分钟内牢固黏附。这种快速反应发生在白细胞上的黏附分子没有改变的情况下，也与ICAM-1介导的白细胞黏附的经典模式相反，后者需要蛋白质合成，并在刺激后数小时发生。本研究的目的是探讨在完整的微血管中观察到的基础NO诱导的EC介导的白细胞快速黏附减少的潜在机制。以培养的人脐静脉内皮细胞为材料，采用细胞分级和免疫印迹方法检测不同细胞区域ICAM-1的相对表达水平及其活化状态。用免疫沉淀法测定ICAM-1在非变性蛋白中的粘附性，以评估ICAM-1与其抑制性抗体mAb1A29和抗总ICAM-1抗体结合的变化。用原子力显微镜(AFM)检测EC ICAM-1对活细胞的黏附强度。结果表明，小窝蛋白-1支架结构域或一氧化氮合酶抑制剂L作用30min可显著降低EC基础NO水平，在未改变细胞间黏附分子-1的表达和分布的情况下，显著增加细胞间黏附分子-1的磷酸化水平及其与mAb1A29的结合。Src抑制剂PP1可抑制NO还原诱导的ICAM-1磷酸化和粘附性结合的增加。AFM检测到AP-CAV处理的内皮细胞与mAb1A29包被的探针之间的结合力显著增加。这些结果表明，EC基础NO的减少导致ICAM-1黏附结合的快速增加，这种结合是通过Src介导的磷酸化实现的，而不需要从头合成和转位蛋白质。本研究提示NO依赖的EC膜ICAM-1构象变化可能是体内观察到依赖ICAM-1的白细胞快速黏附的机制。这一新的机制洞察为更好地理解在循环系统中遇到基础NO减少的许多疾病条件下EC/白细胞相互作用介导的血管炎症提供了更好的理解。
Nitric oxide (NO) is a known anti-adhesive molecule that prevents platelet aggregation and leukocyte adhesion to endothelial cells (ECs). The mechanism has been attributed to its role in the regulation of adhesion molecules on leukocytes and the adhesive properties of platelets. Our previous study conducted in rat venules found that reduction of EC basal NO synthesis caused EC ICAM-1-mediated firm adhesion of leukocytes within 10–30 min. This quick response occurred in the absence of alterations of adhesion molecules on leukocytes and also opposes the classical pattern of ICAM-1-mediated leukocyte adhesion that requires protein synthesis and occurs hours after stimulation. The objective of this study is to investigate the underlying mechanisms of reduced basal NO-induced EC-mediated rapid leukocyte adhesion observed in intact microvessels. The relative levels of ICAM-1 at different cell regions and their activation status were determined with cellular fractionation and western blot using cultured human umbilical vein ECs. ICAM-1 adhesiveness was determined by immunoprecipitation in non-denatured proteins to assess the changes in ICAM-1 binding to its inhibitory antibody, mAb1A29, and antibody against total ICAM-1 with and without NO reduction. The adhesion strength of EC ICAM-1 was assessed by atomic force microscopy (AFM) on live cells. Results showed that reduction of EC basal NO caused by the application of caveolin-1 scaffolding domain (AP-CAV) or NOS inhibitor, L-NMMA, for 30 min significantly increased phosphorylated ICAM-1 and its binding to mAb1A29 in the absence of altered ICAM-1 expression and its distribution at subcellular regions. The Src inhibitor, PP1, inhibited NO reduction-induced increases in ICAM-1 phosphorylation and adhesive binding. AFM detected significant increases in the binding force between AP-CAV-treated ECs and mAb1A29-coated probes. These results demonstrated that reduced EC basal NO lead to a rapid increase in ICAM-1 adhesive binding via Src-mediated phosphorylation without de novo protein synthesis and translocation. This study suggests that a NO-dependent conformational change of constitutive EC membrane ICAM-1 might be the mechanism of rapid ICAM-1 dependent leukocyte adhesion observed in vivo. This new mechanistic insight provides a better understanding of EC/leukocyte interaction-mediated vascular inflammation under many disease conditions that encounter reduced basal NO in the circulation system.
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影响因子：
29
作者：
Fernández-Hernando C;Yu J;Suárez Y;Rahner C;Dávalos A;Lasunción MA;Sessa WC
通讯作者：
Sessa WC
影响因子：
20.1
作者：
MA, XL;WEYRICH, AS;LEFER, AM
通讯作者：
LEFER, AM
影响因子：
82.9
作者：
Bucci, M;Gratton, JP;Sessa, WC
通讯作者：
Sessa, WC
影响因子：
4.5
作者：
Fleming, Ingrid
通讯作者：
Fleming, Ingrid
DOI：
10.1073/pnas.0407224102
发表时间：
2005-01-18
影响因子：
11.1
作者：
Bernatchez, PN;Bauer, PM;Sessa, WC
通讯作者：
Sessa, WC