Zamorano Patricia, Marín Natalie, Córdova Francisco, Aguilar Alejandra, Meininger Cynthia, Boric Mauricio P, Golenhofen Nikola, Contreras Jorge E, Sarmiento José, Durán Walter N, Sánchez Fabiola A
Instituto de Inmunología, Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile.
Department of Medical Physiology, Texas A&M Health Science Center, Temple, Texas.
Am J Physiol Heart Circ Physiol. 2017 Jul 1;313(1):H66-H71. doi: 10.1152/ajpheart.00135.2017. Epub 2017 May 19.
We tested the hypothesis that platelet-activating factor (PAF) induces -nitrosylation of vasodilator-stimulated phosphoprotein (VASP) as a mechanism to reduce microvascular endothelial barrier integrity and stimulate hyperpermeability. PAF elevated -nitrosylation of VASP above baseline levels in different endothelial cells and caused hyperpermeability. To ascertain the importance of endothelial nitric oxide synthase (eNOS) subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced -nitrosylation of VASP in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Reconstitution of VASP knockout myocardial endothelial cells with cysteine mutants of VASP demonstrated that -nitrosylation of cysteine 64 is associated with PAF-induced hyperpermeability. We propose that regulation of VASP contributes to endothelial cell barrier integrity and to the onset of hyperpermeability. -nitrosylation of VASP inhibits its function in barrier integrity and leads to endothelial monolayer hyperpermeability in response to PAF, a representative proinflammatory agonist. Here, we demonstrate that -nitrosylation of vasodilator-stimulated phosphoprotein (VASP) on C64 is a mechanism for the onset of platelet-activating factor-induced hyperpermeability. Our results reveal a dual role of VASP in endothelial permeability. In addition to its well-documented function in barrier integrity, we show that -nitrosylation of VASP contributes to the onset of endothelial permeability.
血小板活化因子（PAF）诱导血管舒张刺激磷蛋白（VASP）的亚硝基化，以此作为降低微血管内皮屏障完整性并刺激高通透性的一种机制。PAF使不同内皮细胞中VASP的亚硝基化水平升高至基线水平以上，并导致高通透性。为了确定内皮型一氧化氮合酶（eNOS）亚细胞定位在此过程中的重要性，我们使用了转染了胞质型eNOS（GFP-eNOS-G2A）和质膜型eNOS（GFP-eNOS-CAAX）的ECV-304细胞。PAF在具有胞质型eNOS的细胞中诱导VASP的亚硝基化，但在eNOS锚定在细胞膜上的细胞中则不然。用VASP的半胱氨酸突变体重构VASP基因敲除的心肌内皮细胞表明，半胱氨酸64的亚硝基化与PAF诱导的高通透性有关。我们提出，VASP的调节有助于内皮细胞屏障完整性以及高通透性的发生。VASP的亚硝基化抑制其在屏障完整性中的功能，并导致内皮单层对代表性促炎激动剂PAF产生高通透性。在此，我们证明血管舒张刺激磷蛋白（VASP）第64位半胱氨酸的亚硝基化是血小板活化因子诱导高通透性发生的一种机制。我们的结果揭示了VASP在内皮通透性中的双重作用。除了其在屏障完整性方面有充分记录的功能外，我们还表明VASP的亚硝基化有助于内皮通透性的发生。
