Neuronal Wiskott-Aldrich syndrome protein regulates Pseudomonas aeruginosa-induced lung vascular permeability through the modulation of actin cytoskeletal dynamics.
神经元型 Wiskott-Aldrich 综合征蛋白通过调节肌动蛋白细胞骨架动力学调节铜绿假单胞菌诱导的肺血管通透性。
- 作者列表："Che P","Wagener BM","Zhao X","Brandon AP","Evans CA","Cai GQ","Zhao R","Xu ZX","Han X","Pittet JF","Ding Q
:Pulmonary edema associated with increased vascular permeability is a severe complication of Pseudomonas (P.) aeruginosa-induced acute lung injury. The mechanisms underlying P aeruginosa-induced vascular permeability are not well understood. In the present study, we investigated the role of neuronal Wiskott Aldrich syndrome protein (N-WASP) in modulating P aeruginosa-induced vascular permeability. Using lung microvascular endothelial and alveolar epithelial cells, we demonstrated that N-WASP downregulation attenuated P aeruginosa-induced actin stress fiber formation and prevented paracellular permeability. P aeruginosa-induced dissociation between VE-cadherin and β-catenin, but increased association between N-WASP and VE-cadherin, suggesting a role for N-WASP in promoting P aeruginosa-induced adherens junction rupture. P aeruginosa increased N-WASP-Y256 phosphorylation, which required the activation of Rho GTPase and focal adhesion kinase. Increased N-WASP-Y256 phosphorylation promotes N-WASP and integrin αVβ6 association as well as TGF-β-mediated permeability across alveolar epithelial cells. Inhibition of N-WASP-Y256 phosphorylation by N-WASP-Y256F overexpression blocked N-WASP effects in P aeruginosa-induced actin stress fiber formation and increased paracellular permeability. In vivo, N-WASP knockdown attenuated the development of pulmonary edema and improved survival in a mouse model of P aeruginosa pneumonia. Together, our data demonstrate that N-WASP plays an essential role in P aeruginosa-induced vascular permeability and pulmonary edema through the modulation of actin cytoskeleton dynamics.
: 与血管通透性增加相关的肺水肿是铜绿假单胞菌引起的急性肺损伤的严重并发症。铜绿假单胞菌诱导血管通透性的机制尚不十分清楚。在本研究中，我们研究了神经元型 Wiskott Aldrich 综合征蛋白 (N-WASP) 在调节铜绿假单胞菌诱导的血管通透性中的作用。使用肺微血管内皮细胞和肺泡上皮细胞，我们证明了 N-WASP 下调减弱了铜绿假单胞菌诱导的肌动蛋白应力纤维形成，并阻止了细胞旁通透性。铜绿假单胞菌诱导 VE-cadherin 和 β-catenin 解离，但 N-WASP 和 VE-cadherin 之间的关联增加, 提示 N-WASP 在促进铜绿假单胞菌诱导的粘连连接破裂中的作用。铜绿假单胞菌增加 N-WASP-Y256 磷酸化，这需要 Rho gtp 酶和黏着斑激酶的激活。N-WASP-Y256 磷酸化增加促进 N-WASP 和整合素 α v β 6 的结合以及 TGF-β 介导的跨肺泡上皮细胞的通透性。N-WASP-Y256 过表达抑制 N-WASP-Y256F 磷酸化可阻断 N-WASP 在铜绿假单胞菌诱导的肌动蛋白应力纤维形成中的作用，并增加细胞旁通透性。在体内，N-WASP 敲除可减弱肺水肿的发展，改善铜绿假单胞菌肺炎小鼠模型的存活率。总之，我们的数据证明 N-WASP 通过调节肌动蛋白细胞骨架动力学在铜绿假单胞菌诱导的血管通透性和肺水肿中发挥重要作用。
METHODS:BACKGROUND AND PURPOSE:A critical role for sphingosine kinase/sphingosine-1-phosphate (S1P) pathway in the control of airway function has been demonstrated in respiratory diseases. Here, we address S1P contribution in a mouse model of mild chronic obstructive pulmonary disease (COPD). EXPERIMENTAL APPROACH:C57BL/6J mice have been exposed to room air or cigarette smoke up to 11 months and killed at different time points. Functional and molecular studies have been performed. KEY RESULTS:Cigarette smoke caused emphysematous changes throughout the lung parenchyma coupled to a progressive collagen deposition in both peribronchiolar and peribronchial areas. The high and low airways showed an increased reactivity to cholinergic stimulation and α-smooth muscle actin overexpression. Similarly, an increase in airway reactivity and lung resistances following S1P challenge occurred in smoking mice. A high expression of S1P, Sph-K2 , and S1P receptors (S1P2 and S1P3 ) has been detected in the lung of smoking mice. Sphingosine kinases inhibition reversed the increased cholinergic response in airways of smoking mice. CONCLUSIONS AND IMPLICATIONS:S1P signalling up-regulation follows the disease progression in smoking mice and is involved in the development of airway hyperresponsiveness. Our study defines a therapeutic potential for S1P inhibitors in management of airways hyperresponsiveness associated to emphysema in smokers with both asthma and COPD.
METHODS::The interim results from this 90-day multi-dose, inhalation toxicology study with life-time post-exposure observation has shown an important fundamental difference in persistence and pathological response in the lung between brake dust derived from brake-pads manufactured with chrysotile, TiO2 or chrysotile alone in comparison to the amphiboles, crocidolite and amosite asbestos. In the brake dust exposure groups no significant pathological response was observed at any time. Slight macrophage accumulation of particles was noted. Wagner-scores, were from 1 to 2 (1 = air-control group) and were similar to the TiO2 group. Chrysotile being biodegradable, shows a weakening of its matrix and breaking into short fibers & particles that can be cleared by alveolar macrophages and continued dissolution. In the chrysotile exposure groups, particle laden macrophage accumulation was noted leading to a slight interstitial inflammatory response (Wagner-score 1-3). There was no peribronchiolar inflammation and occasional very slight interstitial fibrosis. The histopathology and the confocal analyses clearly differentiate the pathological response from amphibole asbestos, crocidolite and amosite, compared to that from the brake dust and chrysotile. Both crocidolite and amosite induced persistent inflammation, microgranulomas, and fibrosis (Wagner-scores 4), which persisted through the post exposure period. The confocal microscopy of the lung and snap-frozen chestwalls quantified the extensive inflammatory response and collagen development in the lung and on the visceral and parietal surfaces. The interim results reported here, provide a clear basis for differentiating the effects from brake dust exposure from those following amphibole asbestos exposure. The subsequent results through life-time post-exposure will follow.
METHODS::The respiratory tract is lined by a pseudo-stratified epithelium from the nose to terminal bronchioles. This first line of defense of the lung against external stress includes five main cell types: basal, suprabasal, club, goblet and multiciliated cells, as well as rare cells such as ionocytes, neuroendocrine and tuft/brush cells. At homeostasis, this epithelium self-renews at low rate but is able of fast regeneration upon damage. Airway epithelial cell lineages during regeneration have been investigated in the mouse by genetic labeling, mainly after injuring the epithelium with noxious agents. From these approaches, basal cells have been identified as progenitors of club, goblet and multiciliated cells, but also of ionocytes and neuroendocrine cells. Single-cell RNA sequencing, coupled to lineage inference algorithms, has independently allowed the establishment of comprehensive pictures of cell lineage relationships in both mouse and human. In line with genetic tracing experiments in mouse trachea, studies using single-cell RNA sequencing (RNAseq) have shown that basal cells first differentiate into club cells, which in turn mature into goblet cells or differentiate into multiciliated cells. In the human airway epithelium, single-cell RNAseq has identified novel intermediate populations such as deuterosomal cells, 'hybrid' mucous-multiciliated cells and progenitors of rare cells. Novel differentiation dynamics, such as a transition from goblet to multiciliated cells have also been discovered. The future of cell lineage relationships in the respiratory tract now resides in the combination of genetic labeling approaches with single-cell RNAseq to establish, in a definitive manner, the hallmarks of cellular lineages in normal and pathological situations.