Department of Medicine, Division of Cardiology, Emory University, 101 Woodruff Circle, WMB 308a, Atlanta, GA 30322, USA.
Wallace H. Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, 313 Ferst Dr NW, Atlanta, GA 30332.
Cardiovasc Res. 2022 Aug 24;118(11):2506-2518. doi: 10.1093/cvr/cvab295.
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.
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.
Poldip2 is a potent regulator of endothelial dysfunction during sepsis-induced lung injury, and its endothelium-specific inhibition may provide clinical benefit.
脓毒症诱导的肺损伤与显著的发病率和死亡率相关。先前，我们发现聚合酶 δ 相互作用蛋白 2（Poldip2）的杂合缺失可防止脓毒症诱导的肺损伤。由于内皮屏障破坏被认为是脓毒症诱导的肺损伤的主要机制，我们试图确定观察到的保护作用是否专门归因于内皮 Poldip2 减少的影响。
内皮特异性 Poldip2 敲除小鼠（EC-/-）及其野生型同窝仔鼠（EC+/+）被注射盐水或脂多糖（18mg/kg）以模拟脓毒症诱导的肺损伤。在注射后 18 小时，处死小鼠并收集支气管肺泡灌洗液（BAL）和肺组织以评估白细胞浸润。与 Poldip2 EC+/+ 小鼠相比，Poldip2 EC-/- 小鼠的 BAL 中的肺白细胞浸润减少（0.21 ± 0.9×106 与 1.29 ± 1.8×106 细胞/mL）和肺组织中（12.7 ± 1.8% 与 23 ± 3.7% 中性粒细胞占总细胞数的比例）。对肺组织的 qPCR 分析显示，炎症基因表达（TNFα 2.23 ± 0.39 与 4.15 ± 0.5 倍，IκBα 4.32 ± 1.53 与 8.97 ± 1.59 倍）、中性粒细胞趋化因子基因表达（CXCL1 68.8 ± 29.6 与 147 ± 25.7 倍，CXCL2 65 ± 25.6 与 215 ± 27.3 倍）和内皮激活标志物（VCAM1 1.25 ± 0.25 与 3.8 ± 0.38 倍）的诱导在 Poldip2 EC-/- 中明显减弱与 Poldip2 EC+/+ 肺相比。使用人肺微血管内皮细胞的体外模型评估了 Poldip2 敲低对内皮激活和通透性的影响。TNFα 诱导的内皮通透性和 VE-钙粘蛋白破坏在用 siRNA 介导的 Poldip2 敲低时显著降低（通透性为 5 ± 0.5 与 17.5 ± 3 倍，VE-钙粘蛋白破坏比例为 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 活性降低相关（0.94 ± 0.05 与 1.29 ± 0.01 的相对 RhoA 活性），并伴有活性-RhoA 染色向细胞中心的重新分布。
Poldip2 是脓毒症诱导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。
