Human pulmonary endothelial cell permeability after exposure to LPS-stimulated leukocyte supernatants derived from patients with early sepsis
Human pulmonary endothelial cell permeability after exposure to LPS-stimulated leukocyte supernatants derived from patients with early sepsis
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DOI：
10.1152/ajplung.00286.2018
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发表时间：
2018-11-01
影响因子：
4.9
通讯作者：
Matthay, Michael A.
中科院分区：
文献类型：
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作者：
Leligdowicz, Aleksandra;Chun, Lauren F.;Matthay, Michael A.
全身免疫激活是脓毒症的标志，可导致内皮损伤和急性呼吸窘迫综合征（ARDS）。本研究的目的是利用原代人肺微血管内皮细胞（HPMEC）和电细胞-基质阻抗传感（ECIS）平台研究脓毒症介导的内皮通透性的异质性。去除血浆后，用培养基稀释 35 名早期脓毒症重症监护病房 (ICU) 患者的全血细胞成分，并用脂多糖 (LPS) 或对照培养基刺激。将所得上清液与接种在 ECIS 板上的 HPMEC 共培养，并连续测量抗性。阻力的降低意味着渗透性的增加。孵育后，分离 HPMEC，使用流式细胞术和免疫组织化学对细胞粘附蛋白进行定量，并使用定量 PCR 分析基因表达。暴露于 LPS 刺激的白细胞上清液后，发现内皮细胞通透性存在显着的异质性。 ICU 脓毒症患者分为以下三组之一：最小渗透性（9/35，26%）、中等渗透性（18/35，51%）和最大渗透性（8/35，23%）。最大通透性与细胞间粘附分子-1 蛋白和 mRNA 表达增加以及血管内皮钙粘蛋白 mRNA 表达减少相关。这些发现表明，肺内皮通透性的显着异质性是由早期脓毒症患者的 LPS 刺激白细胞上清液诱导的，并为诱导肺血管损伤的一些机制提供了见解。此外，这种 LPS 刺激的白细胞肺内皮通透性的体外模型可能是测试可减轻脓毒症早期内皮损伤的治疗药物的有用方法。
Systemic immune activation is the hallmark of sepsis, which can result in endothelial injury and the acute respiratory distress syndrome (ARDS). The aim of this study was to investigate heterogeneity in sepsis-mediated endothelial permeability using primary human pulmonary microvascular endothelial cells (HPMECs) and the electric cell-substrate impedance sensing (ECIS) platform. After plasma removal, cellular component of whole blood from 35 intensive care unit (ICU) patients with early sepsis was diluted with media and stimulated with either lipopolysaccharide (LPS) or control media. Resulting supernatants were cocultured with HPMECs seeded on ECIS plates, and resistance was continually measured. A decrease in resistance signified increased permeability. After incubation, HPMECs were detached and cell adhesion proteins were quantified using flow cytometry and immunohistochemistry, and gene expression was analyzed with quantitative PCR. Significant heterogeneity in endothelial permeability after exposure to supernatants of LPS-stimulated leukocytes was identified. ICU patients with sepsis stratified into one of the following three groups: minimal (9/35, 26%), intermediate (18/35, 51%), and maximal (8/35, 23%) permeability. Maximal permeability was associated with increased intercellular adhesion molecule-1 protein and mRNA expression and decreased vascular endothelial-cadherin mRNA expression. These findings indicate that substantial heterogeneity in pulmonary endothelial permeability is induced by supernatants of LPS-stimulated leukocytes derived from patients with early sepsis and provide insights into some of the mechanisms that induce lung vascular injury. In addition, this in vitro model of lung endothelial permeability from LPS-stimulated leukocytes may be a useful method for testing therapeutic agents that could mitigate endothelial injury in early sepsis.