Department of Medicine, University of Arizona Health Sciences, Tucson, AZ, United States of America.
Department of Anesthesiology. University of California Los Angeles, Los Angeles, CA, United States of America.
Biochim Biophys Acta Mol Basis Dis. 2022 Dec 1;1868(12):166562. doi: 10.1016/j.bbadis.2022.166562. Epub 2022 Sep 27.
The loss of vascular integrity is a cardinal feature of acute inflammatory responses evoked by activation of the TLR4 inflammatory cascade. Utilizing in vitro and in vivo models of inflammatory lung injury, we explored TLR4-mediated dysregulated signaling that results in the loss of endothelial cell (EC) barrier integrity and vascular permeability, focusing on Dock1 and Elmo1 complexes that are intimately involved in regulation of Rac1 GTPase activity, a well recognized modulator of vascular integrity. Marked reductions in Dock1 and Elmo1 expression was observed in lung tissues (porcine, rat, mouse) exposed to TLR4 ligand-mediated acute inflammatory lung injury (LPS, eNAMPT) in combination with injurious mechanical ventilation. Lung tissue levels of Dock1 and Elmo1 were preserved in animals receiving an eNAMPT-neutralizing mAb in conjunction with highly significant decreases in alveolar edema and lung injury severity, consistent with Dock1/Elmo1 as pathologic TLR4 targets directly involved in inflammation-mediated loss of vascular barrier integrity. In vitro studies determined that pharmacologic inhibition of Dock1-mediated activation of Rac1 (TBOPP) significantly exacerbated TLR4 agonist-induced EC barrier dysfunction (LPS, eNAMPT) and attenuated increases in EC barrier integrity elicited by barrier-enhancing ligands of the S1P1 receptor (sphingosine-1-phosphate, Tysiponate). The EC barrier-disrupting influence of Dock1 inhibition on S1PR1 barrier regulation occurred in concert with: 1) suppressed formation of EC barrier-enhancing lamellipodia, 2) altered nmMLCK-mediated MLC2 phosphorylation, and 3) upregulation of NOX4 expression and increased ROS. These studies indicate that Dock1 is essential for maintaining EC junctional integrity and is a critical target in TLR4-mediated inflammatory lung injury.
血管完整性的丧失是 TLR4 炎症级联激活引起的急性炎症反应的主要特征。利用炎症性肺损伤的体外和体内模型，我们研究了 TLR4 介导的失调信号，导致内皮细胞 (EC) 屏障完整性和血管通透性丧失，重点关注 Dock1 和 Elmo1 复合物，它们密切参与调节 Rac1 GTPase 活性，这是血管完整性的公认调节剂。在 TLR4 配体介导的急性炎症性肺损伤（LPS、eNAMPT）与损伤性机械通气联合作用下，暴露于其中的猪、大鼠和小鼠肺组织中观察到 Dock1 和 Elmo1 的表达明显减少。在接受 eNAMPT 中和单克隆抗体的动物中，肺组织中的 Dock1 和 Elmo1 水平得到保留，肺泡水肿和肺损伤严重程度显著降低，这与 Dock1/Elmo1 作为直接参与炎症介导的血管屏障完整性丧失的病理 TLR4 靶标一致。体外研究确定，Dock1 介导的 Rac1 激活的药理学抑制（TBOPP）显著加剧 TLR4 激动剂诱导的 EC 屏障功能障碍（LPS、eNAMPT），并减弱 S1P1 受体（鞘氨醇-1-磷酸、Tysiponate）的屏障增强配体引起的 EC 屏障完整性增加。Dock1 抑制对 S1PR1 屏障调节的 EC 屏障破坏作用与以下因素同时发生：1）抑制形成 EC 屏障增强的片状伪足，2）改变 nmMLCK 介导的 MLC2 磷酸化，3）NOX4 表达上调和 ROS 增加。这些研究表明，Dock1 对于维持 EC 连接完整性是必不可少的，并且是 TLR4 介导的炎症性肺损伤的关键靶标。
