Abstract：
BACKGROUND: Currently, clinical indicators for evaluating endothelial permeability in sepsis are unavailable. Endothelium-derived extracellular vesicles (EDEVs) are emerging as biomarkers of endothelial injury. Platelet endothelial cell adhesion molecule (PECAM) and vascular endothelial (VE)-cadherin are constitutively expressed endothelial intercellular adhesion molecules that regulate intercellular adhesion and permeability. Herein, we investigated the possible association between EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) and endothelial permeability and sepsis severity.
METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor alpha (TNF-α) directly or after pretreatment with permeability-modifying reagents such as angiopoietin-1, prostacyclin, or vascular endothelial growth factor (VEGF) to alter TNF-α-induced endothelial hyperpermeability. Endothelial permeability was measured using the dextran assay or transendothelial electrical resistance. Additionally, a prospective cross-sectional observational study was conducted to analyze circulating EDEV levels in patients with sepsis. EDEVs were examined in HUVEC culture supernatants or patient plasma (nonsepsis, n = 30; sepsis, n = 30; septic shock, n = 42) using flow cytometry. The Wilcoxon rank-sum test was used for comparisons between 2 groups. Comparisons among 3 or more groups were performed using the Steel-Dwass test. Spearman\'s test was used for correlation analysis. Statistical significance was set at P < .05.
RESULTS: TNF-α stimulation of HUVECs significantly increased EDEV release and endothelial permeability. Pretreatment with angiopoietin-1 or prostacyclin suppressed the TNF-α-induced increase in endothelial permeability and inhibited the release of PECAM+ and VE-cadherin+ EDEVs. In contrast, pretreatment with VEGF increased TNF-α-induced endothelial permeability and the release of PECAM+ and VE-cadherin+ EDEVs. However, pretreatment with permeability-modifying reagents did not affect the release of EDEVs expressing inflammatory stimulus-inducible endothelial adhesion molecules such as E-selectin, intracellular adhesion molecule-1, or vascular cell adhesion molecule-1. The number of PECAM+ EDEVs on admission in the septic-shock group (232 [124, 590]/μL) was significantly higher (P = .043) than that in the sepsis group (138 [77,267]/μL), with an average treatment effect of 98/μL (95% confidence interval [CI], 2-270/μL), and the number of VE-cadherin+ EDEVs in the septic-shock group (173 [76,339]/μL) was also significantly higher (P = .004) than that in the sepsis group (81 [42,159]/μL), with an average treatment effect (ATE) of 79/μL (95% CI, 19-171/μL); these EDEV levels remained elevated until day 5.
CONCLUSIONS: EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) may reflect increased endothelial permeability and could be valuable diagnostic and prognostic markers for sepsis.
METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor alpha (TNF-α) directly or after pretreatment with permeability-modifying reagents such as angiopoietin-1, prostacyclin, or vascular endothelial growth factor (VEGF) to alter TNF-α-induced endothelial hyperpermeability. Endothelial permeability was measured using the dextran assay or transendothelial electrical resistance. Additionally, a prospective cross-sectional observational study was conducted to analyze circulating EDEV levels in patients with sepsis. EDEVs were examined in HUVEC culture supernatants or patient plasma (nonsepsis, n = 30; sepsis, n = 30; septic shock, n = 42) using flow cytometry. The Wilcoxon rank-sum test was used for comparisons between 2 groups. Comparisons among 3 or more groups were performed using the Steel-Dwass test. Spearman\'s test was used for correlation analysis. Statistical significance was set at P < .05.
RESULTS: TNF-α stimulation of HUVECs significantly increased EDEV release and endothelial permeability. Pretreatment with angiopoietin-1 or prostacyclin suppressed the TNF-α-induced increase in endothelial permeability and inhibited the release of PECAM+ and VE-cadherin+ EDEVs. In contrast, pretreatment with VEGF increased TNF-α-induced endothelial permeability and the release of PECAM+ and VE-cadherin+ EDEVs. However, pretreatment with permeability-modifying reagents did not affect the release of EDEVs expressing inflammatory stimulus-inducible endothelial adhesion molecules such as E-selectin, intracellular adhesion molecule-1, or vascular cell adhesion molecule-1. The number of PECAM+ EDEVs on admission in the septic-shock group (232 [124, 590]/μL) was significantly higher (P = .043) than that in the sepsis group (138 [77,267]/μL), with an average treatment effect of 98/μL (95% confidence interval [CI], 2-270/μL), and the number of VE-cadherin+ EDEVs in the septic-shock group (173 [76,339]/μL) was also significantly higher (P = .004) than that in the sepsis group (81 [42,159]/μL), with an average treatment effect (ATE) of 79/μL (95% CI, 19-171/μL); these EDEV levels remained elevated until day 5.
CONCLUSIONS: EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) may reflect increased endothelial permeability and could be valuable diagnostic and prognostic markers for sepsis.
摘要：
背景：目前，目前尚无评价脓毒症患者内皮通透性的临床指标.内皮衍生的细胞外囊泡（EDEV）正在成为内皮损伤的生物标志物。血小板内皮细胞粘附分子（PECAM）和血管内皮（VE）-钙黏着蛋白是组成型表达的内皮细胞间粘附分子，可调节细胞间的粘附和通透性。在这里，我们研究了表达细胞间粘附分子(PECAM+或VE-cadherin+EDEV)的EDEV与内皮通透性和脓毒症严重程度之间的可能关联.
方法：用肿瘤坏死因子-α（TNF-α）直接刺激人脐静脉内皮细胞（HUVECs），或用渗透性修饰试剂如血管生成素-1，前列环素，或血管内皮生长因子（VEGF）改变TNF-α诱导的内皮通透性过高。使用葡聚糖测定法或跨内皮电阻测量内皮通透性。此外,我们进行了一项前瞻性横断面观察性研究,分析脓毒症患者的循环EDEV水平.在HUVEC培养上清液或患者血浆中检查EDEV（非脓毒症，n=30；败血症，n=30；感染性休克，n=42)使用流式细胞术。两组间比较采用Wilcoxon秩和检验。使用Steel-Dwass测试进行3组或更多组之间的比较。采用Spearman检验进行相关性分析。统计学显著性设定为P<0.05。
结果：TNF-α刺激HUVECs可显着增加EDEV释放和内皮通透性。血管生成素-1或前列环素预处理抑制了TNF-α诱导的内皮通透性增加，并抑制了PECAM和VE-cadherinEDEV的释放。相比之下,VEGF预处理可增加TNF-α诱导的内皮通透性以及PECAM和VE-cadherinEDEVs的释放。然而,用渗透性修饰试剂预处理不影响表达炎症刺激诱导的内皮粘附分子如E-选择素的EDEV的释放,细胞内粘附分子-1或血管细胞粘附分子-1。感染性休克组入院时PECAM+EDEV的数量(232[124,590]/μL)明显高于脓毒症组(138[77,267]/μL)(P=0.043),平均治疗效果为98/μL(95%置信区间[CI]，2-270/μL),感染性休克组VE-cadherin+EDEV的数量(173[76,339]/μL)也显著高于脓毒症组(81[42,159]/μL)(P=.004),平均治疗效果（ATE）为79/μL（95％CI，19-171/μL）；这些EDEV水平一直升高到第5天。
结论：表达细胞间粘附分子的EDEV（PECAM+或VE-cadherin+EDEV）可能反映内皮通透性增加，可能是脓毒症的有价值的诊断和预后标志物。
方法：用肿瘤坏死因子-α（TNF-α）直接刺激人脐静脉内皮细胞（HUVECs），或用渗透性修饰试剂如血管生成素-1，前列环素，或血管内皮生长因子（VEGF）改变TNF-α诱导的内皮通透性过高。使用葡聚糖测定法或跨内皮电阻测量内皮通透性。此外,我们进行了一项前瞻性横断面观察性研究,分析脓毒症患者的循环EDEV水平.在HUVEC培养上清液或患者血浆中检查EDEV（非脓毒症，n=30；败血症，n=30；感染性休克，n=42)使用流式细胞术。两组间比较采用Wilcoxon秩和检验。使用Steel-Dwass测试进行3组或更多组之间的比较。采用Spearman检验进行相关性分析。统计学显著性设定为P<0.05。
结果：TNF-α刺激HUVECs可显着增加EDEV释放和内皮通透性。血管生成素-1或前列环素预处理抑制了TNF-α诱导的内皮通透性增加，并抑制了PECAM和VE-cadherinEDEV的释放。相比之下,VEGF预处理可增加TNF-α诱导的内皮通透性以及PECAM和VE-cadherinEDEVs的释放。然而,用渗透性修饰试剂预处理不影响表达炎症刺激诱导的内皮粘附分子如E-选择素的EDEV的释放,细胞内粘附分子-1或血管细胞粘附分子-1。感染性休克组入院时PECAM+EDEV的数量(232[124,590]/μL)明显高于脓毒症组(138[77,267]/μL)(P=0.043),平均治疗效果为98/μL(95%置信区间[CI]，2-270/μL),感染性休克组VE-cadherin+EDEV的数量(173[76,339]/μL)也显著高于脓毒症组(81[42,159]/μL)(P=.004),平均治疗效果（ATE）为79/μL（95％CI，19-171/μL）；这些EDEV水平一直升高到第5天。
结论：表达细胞间粘附分子的EDEV（PECAM+或VE-cadherin+EDEV）可能反映内皮通透性增加，可能是脓毒症的有价值的诊断和预后标志物。
