Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and “pearls of wisdom”/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.     

Protective role of p120-catenin in maintaining the integrity of adherens and tight junctions in ventilator-induced lung injury

Background

Ventilator-induced lung injury (VILI) is one of the most common complications for patients with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Although p120 is an important protein in the regulation of cell junctions, further mechanisms should be explored for prevention and treatment of VILI.

Methods

Mouse lung epithelial cells (MLE-12), which were transfected with p120 small interfering (si)RNA, p120 cDNA, wild-type E-cadherin juxtamembrane domain or a K83R mutant juxtamembrane domain (K83R-JMD), were subjected to 20 % cyclic stretches for 2 or 4 h. Furthermore, MLE-12 cells and mice, which were pretreated with the c-Src inhibitor PP₂ or RhoA inhibitor Y27632, underwent 20 % cyclic stretches or mechanical stretching, respectively. Moreover, wild-type C57BL/6 mice were transfected with p120 siRNA-liposome complexes before mechanical ventilation. Cell lysates and lung tissues were then analyzed to detect lung injury.

Results

cyclic stretches of 20 % actived c-Src, which induced degradation of E-cadherin, p120 and occludin. However, loss of p120 increased the degradation and endocytosis of E-cadherin. Immunoprecipitation and Immunofluorescence results showed a decrease in the association between p120 and E-cadherin, while gap formation increased in p120 siRNA and K83R-JMD groups after 20 % cyclic stretches. Loss of p120 also reduced the occludin level and decreased the association of occludin and ZO-1 by enhancing RhoA activity. However, the altered levels of occludin and E-cadherin were reversed by PP₂ or Y27632 treatments compared with the cyclic stretch group. Consistently, the expression, redistribution and disassociation of junction proteins were all restored in the p120 overexpression group after 20 % cyclic stretches. Moreover, the role of p120 in VILI was confirmed by increased wet/dry weigh ratio and enhanced production of cytokines (tumor necrosis factor-α and interleukin-six) in p120-depleted mice under mechanical ventilation.

Conclusions

p120 protected against VILI by regulating both adherens and tight junctions. p120 inhibited E-cadherin endocytosis by increasing the association between p120 and juxtamembrane domain of E-cadherin. Furthermore, p120 reduced the degradation of occludin by inhibiting RhoA activity. These findings illustrated further mechanisms of p120 in the prevention of VILI, especially for patients with ALI or ARDS.     

p190RhoGAP mediates protective effects of oxidized phospholipids in the models of ventilator-induced lung injury

Products resulting from oxidation of cell membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibit potent protective effects against lung endothelial cell (EC) barrier dysfunction caused by pathologically relevant mechanical forces and inflammatory agents. These effects were linked to enhancement of peripheral cytoskeleton and cell adhesion interactions mediated by small GTPase Rac and inhibition of Rho-mediated barrier-disruptive signaling. However, the mechanism of OxPAPC-induced, Rac-dependent Rho downregulation critical for vascular barrier protection remains unclear. This study tested the hypothesis that Rho negative regulator p190RhoGAP is essential for OxPAPC-induced lung barrier protection against ventilator-induced lung injury (VILI), and investigated potential mechanism of p190RhoGAP targeting to adherens junctions (AJ) via p120-catenin. OxPAPC induced peripheral translocation of p190RhoGAP, which was abolished by knockdown of Rac-specific guanine nucleotide exchange factors Tiam1 and Vav2. OxPAPC also induced Rac-dependent tyrosine phosphorylation and association of p190RhoGAP with AJ protein p120-catenin. siRNA-induced knockdown of p190RhoGAP attenuated protective effects of OxPAPC against EC barrier compromise induced by thrombin and pathologically relevant cyclic stretch (18% CS). In vivo, p190RhoGAP knockdown significantly attenuated protective effects of OxPAPC against ventilator-induced lung vascular leak, as detected by increased cell count and protein content in the bronchoalveolar lavage fluid, and tissue neutrophil accumulation in the lung. These results demonstrate for the first time a key role of p190RhoGAP for the vascular endothelial barrier protection in VILI.     

ALI/ARDS患者使用保护性通气策略对于肺部损伤的影响

急性肺损伤(ALI)和急性呼吸窘迫综合征(ARDS)是常见的临床综合征,绝大多数ALI/ARDS患者需机械通气治疗,机械通气在提供可接受的肺部气体交换的同时治疗基础疾病,但机械通气本身也会引起肺部损伤,即机械通气性肺损伤(VILI)。而通过调整机械通气参数的设置,使用保护性通气策略可显著减低ALI/ARDS患者机械通气性肺损伤程度,从而减少肺部感染,缩短机械通气时间和住院时间,降低28天死亡率,明显改善ALI/ARDS患者的生存质量,起到最大程度地肺保护作用。本文从气道平台压,通气容积,呼气末正压等几个不同通气参数方面分别进行综述,讨论ALI/ARDS患者机械通气时使用保护性通气策略对于肺部损伤的影响。     

Hydrogen inhalation reduced epithelial apoptosis in ventilator-induced lung injury via a mechanism involving nuclear factor-kappa B activation

We recently demonstrated the inhalation of hydrogen gas, a novel medical therapeutic gas, ameliorates ventilator-induced lung injury (VILI); however, the molecular mechanisms by which hydrogen ameliorates VILI remain unclear. Therefore, we investigated whether inhaled hydrogen gas modulates the nuclear factor-kappa B (NFκB) signaling pathway. VILI was generated in male C57BL6 mice by performing a tracheostomy and placing the mice on a mechanical ventilator (tidal volume of 30 ml/kg or 10 ml/kg without positive end-expiratory pressure). The ventilator delivered either 2% nitrogen or 2% hydrogen in balanced air. NFκB activation, as indicated by NFκB DNA binding, was detected by electrophoretic mobility shift assays and enzyme-linked immunosorbent assay. Hydrogen gas inhalation increased NFκB DNA binding after 1 h of ventilation and decreased NFκB DNA binding after 2 h of ventilation, as compared with controls. The early activation of NFκB during hydrogen treatment was correlated with elevated levels of the antiapoptotic protein Bcl-2 and decreased levels of Bax. Hydrogen inhalation increased oxygen tension, decreased lung edema, and decreased the expression of proinflammatory mediators. Chemical inhibition of early NFκB activation using SN50 reversed these protective effects. NFκB activation and an associated increase in the expression of Bcl-2 may contribute, in part, to the cytoprotective effects of hydrogen against apoptotic and inflammatory signaling pathway activation during VILI.     

Mining the key genes for ventilator-induced lung injury using co-expression network analysis

Mechanical ventilation is extensively adopted in general anesthesia and respiratory failure management, but it can also induce ventilator-induced lung injury (VILI). Therefore, it is of great urgency to explore the mechanisms involved in the VILI pathogenesis, which might contribute to its future prevention and treatment. Four microarray datasets from the GEO database were selected in our investigation, and were subjected to the Weighted Gene Co-Expression Network Analysis (WGCNA) to identify the VILI-correlated gene modules. The limma package in R software was used to identify the differentially expressed genes (DEGs) between the VILI and control groups. WGCNA was constructed by merging the {"type":"entrez-geo","attrs":{"text":"GSE9314","term_id":"9314"}}GSE9314, {"type":"entrez-geo","attrs":{"text":"GSE9368","term_id":"9368"}}GSE9368, {"type":"entrez-geo","attrs":{"text":"GSE11434","term_id":"11434"}}GSE11434 and {"type":"entrez-geo","attrs":{"text":"GSE11662","term_id":"11662"}}GSE11662 datasets. A total of 49 co-expression network modules were determined as associated with VILI. The intersected genes between hub genes screened from DEGs for VILI and those identified using WGCNA were as follows: Tlr2, Hmox1, Serpine1, Mmp9, Il6, Il1b, Ptgs2, Fos and Atf3, which were determined to be key genes for VILI. Those key genes were validated by {"type":"entrez-geo","attrs":{"text":"GSE86229","term_id":"86229"}}GSE86229 and quantitative PCR (qPCR) experiment to have significantly statistical difference in their expression between the VILI and control groups. In a nutshell, nine key genes with expression differences in VILI were screened by WGCNA by integrating multiple datasets.