Aims Sepsis-induced lung injury is associated with significant morbidity and mortality. Previously, we showed that heterozygous deletion of polymerase δ-interacting protein 2 (Poldip2) was protective against sepsis-induced lung injury. Since endothelial barrier disruption is thought to be the main mechanism of sepsis-induced lung injury, we sought to determine if the observed protection was specifically due to the effect of reduced endothelial Poldip2. Methods and results Endothelial-specific Poldip2 knock-out mice (EC−/−) and their wild-type littermates (EC+/+) were injected with saline or lipopolysaccharide (18 mg/kg) to model sepsis-induced lung injury. At 18 h post-injection mice, were euthanized and bronchoalveolar lavage (BAL) fluid and lung tissue were collected to assess leucocyte infiltration. Poldip2 EC−/− mice showed reduced lung leucocyte infiltration in BAL (0.21 ± 0.9×106 vs. 1.29 ± 1.8×106 cells/mL) and lung tissue (12.7 ± 1.8 vs. 23 ± 3.7% neutrophils of total number of cells) compared to Poldip2 EC+/+ mice. qPCR analysis of the lung tissue revealed a significantly dampened induction of inflammatory gene expression (TNFα 2.23 ± 0.39 vs. 4.15 ± 0.5-fold, IκBα 4.32 ± 1.53 vs. 8.97 ± 1.59-fold), neutrophil chemoattractant gene expression (CXCL1 68.8 ± 29.6 vs. 147 ± 25.7-fold, CXCL2 65 ± 25.6 vs. 215 ± 27.3-fold) and a marker of endothelial activation (VCAM1 1.25 ± 0.25 vs. 3.8 ± 0.38-fold) in Poldip2 EC−/− compared to Poldip2 EC+/+ lungs. An in vitro model using human pulmonary microvascular endothelial cells was used to assess the effect of Poldip2 knock-down on endothelial activation and permeability. TNFα-induced endothelial permeability and VE-cadherin disruption were significantly reduced with siRNA-mediated knock-down of Poldip2 (5 ± 0.5 vs. 17.5 ± 3-fold for permeability, 1.5 ± 0.4 vs. 10.9 ± 1.3-fold for proportion of disrupted VE-cadherin). Poldip2 knock-down altered expression of Rho-GTPase-related genes, which correlated with reduced RhoA activation by TNFα (0.94 ± 0.05 vs. 1.29 ± 0.01 of relative RhoA activity) accompanied by redistribution of active-RhoA staining to the centre of the cell. Conclusion Poldip2 is a potent regulator of endothelial dysfunction during sepsis-induced lung injury, and its endothelium-specific inhibition may provide clinical benefit.

中文翻译：

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内皮 Poldip2 通过 Rho 通路激活调节脓毒症引起的肺损伤


目的 脓毒症引起的肺损伤与显着的发病率和死亡率相关。此前，我们发现聚合酶δ相互作用蛋白2（Poldip2）的杂合缺失可以预防脓毒症引起的肺损伤。由于内皮屏障破坏被认为是脓毒症引起的肺损伤的主要机制，因此我们试图确定观察到的保护作用是否是由于内皮 Poldip2 减少的影响而产生的。方法和结果内皮特异性 Poldip2 敲除小鼠 (EC−/−) 及其野生型同窝小鼠 (EC++/+) 注射生理盐水或脂多糖 (18 mg/kg)，以模拟脓毒症引起的肺损伤。注射后 18 小时，对小鼠实施安乐死，并收集支气管肺泡灌洗 (BAL) 液和肺组织以评估白细胞浸润。 Poldip2 EC−/− 小鼠显示 BAL 中肺白细胞浸润减少（0.21 ± 0.9×106 与 1.29 ± 1.8×106 细胞/mL）和肺组织（12.7 ± 1.8 与细胞总数的 23 ± 3.7% 中性粒细胞）与 Poldip2 EC+/+ 小鼠相比。肺组织的 qPCR 分析显示，炎症基因表达的诱导显着减弱（TNFα 2.23 ± 0.39 比 4.15 ± 0.5 倍，IκBα 4.32 ± 1.53 比 8.97 ± 1.59 倍）、中性粒细胞趋化基因表达（CXCL1 68.8 ± 29.6）与 Poldip2 EC+/ 相比，Poldip2 EC−/− 中的 CXCL2 比 147 ± 25.7 倍，CXCL2 65 ± 25.6 比 215 ± 27.3 倍）和内皮激活标志物（VCAM1 1.25 ± 0.25 比 3.8 ± 0.38 倍） + 肺。使用人肺微血管内皮细胞的体外模型来评估 Poldip2 敲低对内皮活化和通透性的影响。 siRNA 介导的 Poldip2 敲低可显着降低 TNFα 诱导的内皮通透性和 VE-钙粘蛋白破坏（5 ± 0.5 vs. 17.渗透性为 5 ± 3 倍，破坏的 VE-钙粘蛋白比例为 1.5 ± 0.4 与 10.9 ± 1.3 倍）。 Poldip2 敲低改变了 Rho-GTPase 相关基因的表达，这与 TNFα 引起的 RhoA 激活减少相关（相对 RhoA 活性为 0.94 ± 0.05 对比 1.29 ± 0.01），同时活性 RhoA 染色重新分布到细胞中心。结论 Poldip2 是脓毒症引起的肺损伤期间内皮功能障碍的有效调节剂，其内皮特异性抑制可能提供临床益处。