TAK1 is essential for endothelial barrier maintenance and repair after lung vascular injury

TGF–β-activated kinase 1 (TAK1) plays crucial roles in innate and adaptive immune responses and is required for embryonic vascular development. However, TAK1’s role in regulating vascular barrier integrity is not well defined. Here we show that endothelial TAK1 kinase function is required to maintain and repair the injured lung endothelial barrier. We observed that inhibition of TAK1 with 5Z-7-oxozeaenol markedly reduced expression of β-catenin (β-cat) and VE-cadherin at endothelial adherens junctions and augmented protease-activated receptor-1 (PAR-1)- or toll-like receptor-4 (TLR-4)-induced increases in lung vascular permeability. In inducible endothelial cell (EC)-restricted TAK1 knockout (TAK1^i∆EC) mice, we observed that the lung endothelial barrier was compromised and in addition, TAK1^i∆EC mice exhibited heightened sensitivity to septic shock. Consistent with these findings, we observed dramatically reduced β-cat expression in lung ECs of TAK1^i∆EC mice. Further, either inhibition or knockdown of TAK1 blocked PAR-1- or TLR-4-induced inactivation of glycogen synthase kinase 3β (GSK3β), which in turn increased phosphorylation, ubiquitylation, and degradation of β-cat in ECs to destabilize the endothelial barrier. Importantly, we showed that TAK1 inactivates GSK3β through AKT activation in ECs. Thus our findings in this study point to the potential of targeting the TAK1-AKT-GSK3β axis as a therapeutic approach to treat uncontrolled lung vascular leak during sepsis.     

Hyperoxia mediates acute lung injury and increased lethality in murine Legionella pneumonia: the role of apoptosis

Legionella pneumophila is a major cause of life-threatening pneumonia, which is characterized by a high incidence of acute lung injury and resultant severe hypoxemia. Mechanical ventilation using high oxygen concentrations is often required in the treatment of patients with L. pneumophila pneumonia. Unfortunately, oxygen itself may propagate various forms of tissue damage, including acute lung injury. The effect of hyperoxia as a cofactor in the course of L. pneumophila pneumonia is poorly understood. In this study, we show that exposure to hyperoxic conditions during the evolution of pneumonia results in a marked increase in lethality in mice with Legionella pneumonia. The enhanced lethality was associated with an increase in lung permeability, but not changes in either lung bacterial burden or leukocyte accumulation. Interestingly, accelerated apoptosis as evidenced by assessment of histone-DNA fragments and caspase-3 activity were noted in the infected lungs of mice exposed to hyperoxia. TUNEL staining of infected lung sections demonstrated increased apoptosis in hyperoxic mice, predominantly in macrophages and alveolar epithelial cells. In vitro exposure of primary murine alveolar epithelial cells to Legionella in conjunction with hyperoxia accelerated apoptosis and loss of barrier function. Fas-deficient mice demonstrated partial resistance to the lethal effects of Legionella infection induced by hyperoxia, which was associated with attenuated apoptosis in the lung. These results demonstrate that hyperoxia serves as an important cofactor for the development of acute lung injury and lethality in L. pneumophila pneumonia. Exaggerated apoptosis, in part through Fas-mediated signaling, may accelerate hyperoxia-induced acute lung injury in Legionella pneumonia.     

Simvastatin attenuates vascular leak and inflammation in murine inflammatory lung injury

Therapies to limit the life-threatening vascular leak observed in patients with acute lung injury (ALI) are currently lacking. We explored the effect of simvastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor that mediates endothelial cell barrier protection in vitro, in a murine inflammatory model of ALI. C57BL/6J mice were treated with simvastatin (5 or 20 mg/kg body wt via intraperitoneal injection) 24 h before and again concomitantly with intratracheally administered LPS (2 microg/g body wt). Inflammatory indexes [bronchoalveolar lavage (BAL) myeloperoxidase activity and total neutrophil counts assessed at 24 h with histological confirmation] were markedly increased after LPS alone but significantly reduced in mice that also received simvastatin (20 mg/kg; approximately 35-60% reduction). Simvastatin also decreased BAL albumin (approximately 50% reduction) and Evans blue albumin dye extravasation into lung tissue (100%) consistent with barrier protection. Finally, the sustained nature of simvastatin-mediated lung protection was assessed by analysis of simvastatin-induced gene expression (Affymetrix platform). LPS-mediated lung gene expression was significantly modulated by simvastatin within a number of gene ontologies (e.g., inflammation and immune response, NF-kappaB regulation) and with respect to individual genes implicated in the development or severity of ALI (e.g., IL-6, Toll-like receptor 4). Together, these findings confirm significant protection by simvastatin on LPS-induced lung vascular leak and inflammation and implicate a potential role for statins in the management of ALI.     

脂多糖致急性肺血管内皮屏障功能失调小鼠FLI-1蛋白表达的变化及其意义

目的: 观察感染性急性肺损伤(ALI)肺血管内皮屏障功能失调小鼠肺组织中弗里德白血病病毒插入位点1(FLI-1)蛋白表达的变化,以探讨FLI-1在感染性ALI肺血管内皮屏障功能失调发生发展中的意义。方法: SPF级雄性ICR小鼠60只,腹腔注射脂多糖(LPS,7.5 mg/kg)复制ALI模型,在给予LPS 0 h、12 h、24 h、48 h后,检测小鼠肺血管内皮屏障通透性和肺湿干重比,ELISA法检测肺泡灌洗液中TNF-α和IL-6含量,Western blot法检测肺组织FLI-1和Src酪氨酸激酶(SRC)蛋白的表达。结果: 与0 h组比较,12 h组、24 h组肺血管内皮屏障通透性分别升高74.3%和162.4%,而48 h组较24 h组降低27.0%(P均＜0.05);与0 h组比较,12 h组、24 h组肺湿干重比分别升高50.1%和122.9%,而48 h组较24 h组降低10.7%(P均＜0.05);与0 h组比较,12 h、24 h肺泡灌洗液IL-6和TNF-α含量均显著升高,而48 h肺泡灌洗液IL-6和TNF-α含量较24 h分别下降28.3%和21.6%(P均＜ 0.05);与0 h组比较,12 h组、24 h组肺组织FLI-1蛋白表达水平分别下调20.4%和56.9%,而48 h组较24 h组上调18.2%(P均＜0.05);与0 h组比较,12 h组、24 h组肺组织SRC蛋白表达水平分别上调76.8%和176.7%,而48 h组较24 h组下调33.4%(P均＜0.05);肺血管内皮屏障通透性与FLI-1蛋白表达水平呈显著负相关(r= -0.8992,P＜0.01),而与SRC蛋白表达水平呈显著正相关(r=0.8918,P＜0.01),肺组织FLI-1与SRC蛋白表达呈显著负相关(r=-0.8087,P=0.0014)。结论: FLI-1可能参与LPS诱导的急性肺损伤肺血管内皮屏障功能失常过程。     

Pathophysiology of pulmonary hypertension in acute lung injury

Acute lung injury (ALI) and acute respiratory distress syndrome are characterized by protein rich alveolar edema, reduced lung compliance, and acute severe hypoxemia. A degree of pulmonary hypertension (PH) is also characteristic, higher levels of which are associated with increased morbidity and mortality. The increase in right ventricular (RV) afterload causes RV dysfunction and failure in some patients, with associated adverse effects on oxygen delivery. Although the introduction of lung protective ventilation strategies has probably reduced the severity of PH in ALI, a recent invasive hemodynamic analysis suggests that even in the modern era, its presence remains clinically important. We therefore sought to summarize current knowledge of the pathophysiology of PH in ALI.     

CRIM1 is necessary for coronary vascular endothelial cell development and homeostasis

Endothelial cells form a critical component of the coronary vasculature, yet the factors regulating their development remain poorly defined. Here we reveal a novel role for the transmembrane protein CRIM1 in mediating cardiac endothelial cell development. In the absence of Crim1 in vivo, the coronary vasculature is malformed, the number of endothelial cells reduced, and the canonical BMP pathway dysregulated. Moreover, we reveal that CRIM1 can bind IGFs, and regulate IGF signalling within endothelial cells. Finally, loss of CRIM1 from human cardiac endothelial cells results in misregulation of endothelial genes, predicted by pathway analysis to be involved in an increased inflammatory response and cytolysis, reminiscent of endothelial cell dysfunction in cardiovascular disease pathogenesis. Collectively, these findings implicate CRIM1 in endothelial cell development and homeostasis in the coronary vasculature.     

Long pentraxin 3 in pulmonary infection and acute lung injury

Long pentraxin 3 (PTX3) is a newly discovered acute phase protein produced at the sites of infection and inflammation by tissue cells, macrophages, monocytes, and dendritic cells. PTX3 plays an important role in preventing infection of certain fungi, bacteria, and viruses in the lung. Recombinant PTX3 has been proposed as a potential antifungal molecule for therapy. However, under certain experimental conditions, such as intestinal ischemia-reperfusion, high volume mechanical ventilation, or severe bacterial infection, increased expression of PTX3 is associated with more severe lung injury. Therefore, it is necessary to further explore the sources of PTX3 in the lung and the regulatory mechanisms of its expression. It is also essential to further determine how PTX3 binds to pathogens, complement, and apoptotic cells, and to determine whether PTX3 has a specific receptor in targeted cells. These studies will provide insight into the pathological processes of pulmonary infection and acute lung injury and provide potential novel therapeutic strategies to control pulmonary infections without severe lung injury.     

Dopamine inhibits pulmonary edema through the VEGF-VEGFR2 axis in a murine model of acute lung injury

The neurotransmitter dopamine and its dopamine receptor D2 (D2DR) agonists are known to inhibit vascular permeability factor/vascular endothelial growth factor (VEGF)-mediated angiogenesis and vascular permeability. Lung injury is a clinical syndrome associated with increased microvascular permeability. However, the effects of dopamine on pulmonary edema, a phenomenon critical to the pathophysiology of both acute and chronic lung injuries, have yet to be established. Therefore, we sought to determine the potential therapeutic effects of dopamine in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Compared with sham-treated controls, pretreatment with dopamine (50 mg/kg body wt) ameliorated LPS-mediated edema formation and lowered myeloperoxidase activity, a measure of neutrophil infiltration. Moreover, dopamine significantly increased survival rates of LPS-treated mice, from 0-75%. Mechanistically, we found that dopamine acts through the VEGF-VEGFR2 axis to reduce pulmonary edema, as dopamine pretreatment in LPS-treated mice resulted in decreased serum VEGF, VEGFR2 phosphorylation, and endothelial nitric oxide synthase phosphorylation. We used D2DR knockout mice to confirm that dopamine acts through D2DR to block vascular permeability in our lung injury model. As expected, a D2DR agonist failed to reduce pulmonary edema in D2DR(-/-) mice. Taken together, our results suggest that dopamine acts through D2DR to inhibit pulmonary edema-associated vascular permeability, which is mediated through VEGF-VEGFR2 signaling and conveys protective effects in an ALI model.     

Neuregulin-1-human epidermal receptor-2 signaling is a central regulator of pulmonary epithelial permeability and acute lung injury

The mechanisms behind the loss of epithelial barrier function leading to alveolar flooding in acute lung injury (ALI) are incompletely understood. We hypothesized that the tyrosine kinase receptor human epidermal growth factor receptor-2 (HER2) would be activated in an inflammatory setting and participate in ALI. Interleukin-1β (IL-1β) exposure resulted in HER2 activation in human epithelial cells and markedly increased conductance across a monolayer of airway epithelial cells. Upon HER2 blockade, conductance changes were significantly decreased. Mechanistic studies revealed that HER2 trans-activation by IL-1β required a disintegrin and metalloprotease 17 (ADAM17)-dependent shedding of the ligand neuregulin-1 (NRG-1). In murine models of ALI, NRG-1-HER2 signaling was activated, and ADAM17 blockade resulted in decreased NRG-1 shedding, HER2 activation, and lung injury in vivo. Finally, NRG-1 was detectable and elevated in pulmonary edema fluid from patients with ALI. These results suggest that the ADAM17-NRG-1-HER2 axis modulates the alveolar epithelial barrier and contributes to the pathophysiology of ALI.     

Hypoxia-induced acute lung injury in murine models of sickle cell disease

Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human alpha/sickle beta-globin (halphabetaS) transgene (SCD mice) or are heterozygous for the normal murine beta-globin gene and express the halphabetaS transgene (mbeta+/-, halphabetaS+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing *NO generation and predisposing the lung to vaso-occlusion.     

Keratinocyte growth factor therapy in murine oleic acid-induced acute lung injury

Alveolar type II (ATII) cell proliferation and differentiation are important mechanisms in repair following injury to the alveolar epithelium. KGF is a potent ATII cell mitogen, which has been demonstrated to be protective in a number of animal models of lung injury. We have assessed the effect of recombinant human KGF (rhKGF) and liposome-mediated KGF gene delivery in vivo and evaluated the potential of KGF as a therapy for acute lung injury in mice. rhKGF was administered intratracheally in male BALB/c mice to assess dose response and time course of proliferation. SP-B immunohistochemistry demonstrated significant increases in ATII cell numbers at all rhKGF doses compared with control animals and peaked 2 days following administration of 10 mg/kg rhKGF. Protein therapy in general is very expensive, and gene therapy has been suggested as a cheaper alternative for many protein replacement therapies. We evaluated the effect of topical and systemic liposome-mediated KGF-gene delivery on ATII cell proliferation. SP-B immunohistochemistry showed only modest increases in ATII cell numbers following gene delivery, and these approaches were therefore not believed to be capable of reaching therapeutic levels. The effect of rhKGF was evaluated in a murine model of OA-induced lung injury. This model was found to be associated with significant alveolar damage leading to severe impairment of gas exchange and lung compliance. Pretreatment with rhKGF 2 days before intravenous OA challenge resulted in significant improvements in PO2, PCO2, and lung compliance. This study suggests the feasibility of KGF as a therapy for acute lung injury.     

Prickle1 is necessary for the caudal migration of murine facial branchiomotor neurons

Facial branchiomotor neurons (FBMs) of vertebrates typically develop in rhombomere 4 (r4), and in mammals and several other vertebrate taxa, migrate caudally into r6 and subsequently laterally and ventrally to the pial surface. How similar or dissimilar these migratory processes between species are at a molecular level remains unclear. In zebrafish and mouse, mutations in certain PCP genes disrupt normal caudal migration of FBMs. Zebrafish prickle1a (prickle-like 1a) and prickle1b, two orthologs of Prickle1, act non-cell-autonomously and cell-autonomously, respectively, to regulate FBM migration. Here, we show that, in Prickle1 ^C251X/C251X mice which have reduced Prickle1 expression, the caudal migration of FBMs is affected. Most FBM neurons do not migrate caudally along the floor plate. However, some neurons perform limited caudal migration such that the neurons eventually lie near the pial surface from r4 to anterior r6. FBMs in Prickle1 ^C251X/C251X mice survive until P0 and form an ectopic nucleus dorsal to the olivo-cochlear efferents of r4. Ror2, which modifies the PCP pathway in other systems, is expressed by the migrating mouse FBMs, but is not required for FBM caudal migration. Our results suggest that, in mice, Prickle1 is part of a molecular mechanism that regulates FBM caudal migration and separates the FBM and the olivo-cochlear efferents. This defective caudal migration of FBMs in Prickle1C251X mutants resembles Vangl2 mutant defects. In contrast to other developing systems that show similar defects in Prickle1, Wnt5a and Ror2, the latter two only have limited or no effect on FBM caudal migration.     

Adenovirus-mediated transfer of the SOCS-1 gene to mouse lung confers protection against hyperoxic acute lung injury

Suppressor of cytokine signaling-1 (SOCS-1) is a member of the suppressor of cytokine signaling family of proteins and an inhibitor of interleukin-6 (IL-6) signaling. SOCS-1 has been shown to protect cells from cellular damage and apoptosis induced by tumor necrosis factor (TNF), lipopolysaccharide (LPS), and interferon gamma (IL-γ). However, it is not known whether increased SOCS-1 is protective during pulmonary oxidative stress. Therefore, we hypothesized that increased SOCS-1 in the lungs of mice would be protective in the setting of hyperoxic lung injury. We administered SOCS-1 adenovirus (Ad-SOCS-1) intratracheally into the lungs and exposed the mice to 100% O₂. Mice infected with GFP adenovirus (Ad-GFP) were used as controls. Mice treated with Ad-SOCS-1 had enhanced survival in 100% oxygen compared to Ad-GFP-administered mice. After 3 days of hyperoxia, Ad-GFP mice were ill and tachypnic and died after 4 days. In contrast, all Ad-SOCS-1-treated mice survived for at least 6 days in hyperoxia and 80% survived beyond 7 days. Ad-SOCS-1 transfection protected mouse lungs from injury as indicated by lower lung wet/dry weight, alveolar–capillary protein leakage, reduced infiltration of inflammatory cells, and lower content of thiobarbituric acid-reactive substances in lung homogenate. Our results also indicated that Ad-SOCS-1 significantly inhibits hyperoxia-induced ASK-1 (apoptosis signal-regulating kinase 1) expression. Taken together, these findings show that increased expression of adenovirus-mediated SOCS-1 in the lungs of mice significantly protects against hyperoxic lung injury.     

Time course of lung parenchyma remodeling in pulmonary and extrapulmonary acute lung injury.

The aim of this study is to test the hypothesis that the early changes in lung mechanics and the amount of type III collagen fiber do not predict the evolution of lung parenchyma remodeling in pulmonary and extrapulmonary acute lung injury (ALI). For this purpose, we analyzed the time course of lung parenchyma remodeling in murine models of pulmonary and extrapulmonary ALI with similar degrees of mechanical compromise at the early phase of ALI. Lung histology (light and electron microscopy), the amount of elastic and collagen fibers in the alveolar septa, the expression of matrix metalloproteinase-9, and mechanical parameters (lung-resistive and viscoelastic pressures, and static elastance) were analyzed 24 h, 1, 3, and 8 wk after the induction of lung injury. In control (C) pulmonary (p) and extrapulmonary (exp) groups, saline was intratracheally (it; 0.05 ml) instilled and intraperitoneally (ip; 0.5 ml) injected, respectively. In ALIp and ALIexp groups, mice received Escherichia coli lipopolysaccharide (10 microg it and 125 microg ip, respectively). At 24 h, all mechanical and morphometrical parameters, as well as type III collagen fiber content, increased similarly in ALIp and ALIexp groups. In ALIexp, all mechanical and histological data returned to control values at 1 wk. However, in ALIp, static elastance returned to control values at 3 wk, whereas resistive and viscoelastic pressures, as well as type III collagen fibers and elastin, remained elevated until week 8. ALIp showed higher expression of matrix metalloproteinase-9 than ALIexp. In conclusion, insult in pulmonary epithelium yielded fibroelastogenesis, whereas mice with ALI induced by endothelial lesion developed only fibrosis that was repaired early in the course of lung injury. Furthermore, early functional and morphological changes did not predict lung parenchyma remodeling.     

Divergent roles of murine neutrophil chemokines in hemorrhage induced priming for acute lung injury

Neutrophil associated lung injury is identified with a variety of local and systemic priming insults. In vitro studies have shown that TNF-alpha mediated suppression of neutrophil apoptosis is due to the secretion of interleukin-8 (IL-8), a human chemokine shown to alter neutrophil chemotaxis. Our initial in vitro antibody neutralization studies with neutrophil chemotactic proteins, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2alpha (MIP-2alpha), mouse IL-8 homologues, indicate that MIP-2alpha but not KC appears to mediate TNF-alpha suppression of mouse neutrophil apoptosis. Therefore, we hypothesized that in vivo neutralization of KC or MIP-2alpha during an initial priming insult would produce differential effects on the extent of lung injury by restoring normal neutrophil apoptotic function. To assess this, mice were hemorrhaged followed with septic challenge at 24 h. Antibody against KC or MIP-2alpha or a nonspecific IgG was given during resuscitation immediately following hemorrhage. Anti-MIP-2alpha treatment resulted in a significant reduction in lung tissue IL-6 and myeloperoxidase levels. Percentage of neutrophil apoptosis increased significantly in the anti-KC group. Tissue and plasma KC and MIP-2alpha were reduced in their respective treatment groups. These data suggest that KC and MIP-2alpha differ in their mediation of neutrophil function (apoptosis and chemotaxis) and contribution to the pathogenesis of lung injury following hemorrhage subsequent to sepsis.