Department of Physiology and Pharmacology, School of Medicine, West Virginia University, Morgantown, West Virginia.
Am J Physiol Heart Circ Physiol. 2013 Nov 15;305(10):H1484-93. doi: 10.1152/ajpheart.00382.2013. Epub 2013 Sep 16.
Exogenously applied caveolin-1 scaffolding domain (CAV) has been shown to inhibit inflammatory mediator-induced nitric oxide (NO) production and NO-mediated increases in microvessel permeability. However, the effect of CAV on endothelial basal NO that prevents leukocyte adhesion remains unknown. This study aims to investigate the roles of exogenously applied CAV in endothelial basal NO production, leukocyte adhesion, and adhesion-induced changes in microvessel permeability. Experiments were conducted in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). NO was quantified with fluorescence imaging in DAF-2-loaded vessels. Perfusing venules with CAV inhibited basal NO production without affecting basal Lp. Resuming blood flow in CAV-perfused vessels significantly increased leukocyte adhesion. The firmly adherent leukocytes altered neither basal Lp nor adherens junction integrity. Increases in Lp occurred only upon formyl-Met-Leu-Phe application that induces release of reactive oxygen species from the adherent leukocytes. The application of NO synthase inhibitor showed similar results to CAV, and NO donor abolished CAV-mediated leukocyte adhesion. Immunofluorescence staining showed increases in binding of ICAM-1 to an adhesion-blocking antibody concurrent with a Src-dependent ICAM-1 phosphorylation following CAV perfusion. Pre-perfusing vessels with anti-ICAM-1 blocking antibody or a Src kinase inhibitor attenuated CAV-induced leukocyte adhesion. These results indicate that the application of CAV, in addition to preventing excessive NO-mediated permeability increases, also causes reduction of basal NO and promotes ICAM-1-mediated leukocyte adhesion through Src activation-mediated ICAM-1 phosphorylation. CAV-induced leukocyte adhesion was uncoupled from leukocyte oxidative burst and microvessel barrier function, unless in the presence of a secondary stimulation.
外源性添加的 caveolin-1 支架结构域（CAV）已被证明可抑制炎症介质诱导的一氧化氮（NO）产生和 NO 介导的微血管通透性增加。然而，CAV 对防止白细胞黏附的内皮基础 NO 的影响尚不清楚。本研究旨在探讨外源性添加的 CAV 在内皮基础 NO 产生、白细胞黏附以及黏附诱导的微血管通透性变化中的作用。实验在单独灌注的大鼠肠系膜小静脉中进行。微血管通透性通过测量水力传导率（Lp）来确定。在加载 DAF-2 的血管中用荧光成像来定量 NO。用 CAV 灌注小静脉会抑制基础 NO 的产生，而不影响基础 Lp。在 CAV 灌注的血管中恢复血流会显著增加白细胞黏附。牢固黏附的白细胞既不改变基础 Lp 也不改变黏附连接的完整性。仅在应用诱导白细胞释放活性氧物质的甲酰基-Met-Leu-Phe 时才会发生 Lp 增加。NO 合酶抑制剂的应用与 CAV 相似，NO 供体可消除 CAV 介导的白细胞黏附。免疫荧光染色显示，在用 CAV 灌注后，ICAM-1 与一种黏附阻断抗体的结合增加，同时 Src 依赖性的 ICAM-1 磷酸化。在用抗 ICAM-1 阻断抗体或 Src 激酶抑制剂预灌注血管后，可减弱 CAV 诱导的白细胞黏附。这些结果表明，CAV 的应用除了防止过度的 NO 介导的通透性增加外，还会导致基础 NO 的减少，并通过 Src 激活介导的 ICAM-1 磷酸化促进 ICAM-1 介导的白细胞黏附。CAV 诱导的白细胞黏附与白细胞氧化爆发和微血管屏障功能脱耦联，除非存在二次刺激。
