Steven Idell

Coagulation, fibrinolysis, and fibrin deposition in acute lung injury.

ObjectivesTo review: a) the role of extravascular fibrin deposition in the pathogenesis of acute lung injury; b) the abnormalities in the coagulation and fibrinolysis pathways that promote fibrin deposition in the acutely injured lung; and c) the pathways that contribute to the regulation of the fibrinolytic system via the lung epithelium, including newly recognized posttranscriptional and urokinase-dependent pathways. Another objective was to determine how novel anticoagulant or fibrinolytic strategies may be used to protect against acute inflammation or accelerated fibrosis in acute lung injury. Data SourcesPublished medical literature. Data SummaryAlveolar fibrin deposition is characteristic of diverse forms of acute lung injury. Intravascular thrombosis or disseminated intravascular coagulation can also occur in the acutely injured lung. Extravascular fibrin deposition promotes lung dysfunction and the acute inflammatory response. In addition, transitional fibrin in the alveolar compartment undergoes remodeling leading to accelerated pulmonary fibrosis similar to the events associated with wound healing, or desmoplasia associated with solid neoplasms. In acute lung injury, alveolar fibrin deposition is potentiated by consistent changes in endogenous coagulation and fibrinolytic pathways. Procoagulant activity is increased in conjunction with depression of fibrinolytic activity in the alveolar compartment. Initiation of the procoagulant response occurs as a result of local overexpression of tissue factor associated with factor VII. Depression of fibrinolytic activity occurs as a result of inhibition of urokinase plasminogen activator (uPA) by plasminogen activators, or series inhibition of plasmin by antiplasmins. Locally increased amplification of plasminogen activator inhibitor-1 (PAI-1) is largely responsible for this fibrinolytic defect. Newly described pathways by which lung epithelial cells regulate expression of uPA, its receptor uPAR, and PAI-1 at the posttranscriptional level have been identified. These pathways operate by cis-trans interactions between mRNA binding proteins; regulatory sequences within these mRNAs control their stability. The regulatory mechanisms seem to involve multiple protein–mRNA interactions, and the phosphorylation state of the proteins appears to determine whether complex formation of, or dissociation from, the regulatory sequences occurs. uPA is capable of inducing its own expression in lung epithelial cells as well as that of uPAR and PAI-1—the effects involve posttranscriptional regulatory components. These and related observations have led to the implementation of anticoagulant or fibrinolytic strategies to protect the lung against acute lung injury. The success of new fibrinolytic strategies to block pleural loculation suggests that a similar approach might be used to prevent accelerated pulmonary fibrosis, which can occur in association with many forms of acute lung injury. ConclusionsDisordered coagulation and fibrinolysis promote extravascular fibrin deposition in acute lung injury. It is this deposition that characterizes acute lung injury and repair. Expression of uPA, uPAR, and PAI-1 by the lung epithelium, as well as the ability of uPA to induce other components of the fibrinolytic system, involves posttranscriptional regulation. These pathways may contribute to disordered fibrin turnover in the injured lung. The success of anticoagulant or fibrinolytic strategies designed to reverse the abnormalities of local fibrin turnover in acute lung injury supports the inference that abnormalities of coagulation, fibrinolysis, and fibrin deposition have a critical role in the pathogenesis of acute lung injury.

Urokinase-Type Plasminogen Activator Potentiates Lipopolysaccharide-Induced Neutrophil Activation

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. Although increased circulating levels of uPA are present in endotoxemia and sepsis, conditions in which activated neutrophils contribute to the development of acute organ dysfunction, the ability of uPA to participate directly in LPS-induced neutrophil activation has not been examined. In the present experiments, we show that uPA can enhance activation of neutrophils exposed to submaximal stimulatory doses of LPS. In particular, uPA increased LPS-induced activation of intracellular signaling pathways, including Akt and c-Jun N-terminal kinase, nuclear translocation of the transcriptional regulatory factor NF-κB, and expression of proinflammatory cytokines, including IL-1β, macrophage-inflammatory protein-2, and TNF-α. There was no effect of uPA on LPS-induced activation of p38 mitogen-activated protein kinase in neutrophils. Transgenic mice unable to produce uPA (uPA−/−) were protected from endotoxemia-induced lung injury, as determined by development of lung edema, pulmonary neutrophil accumulation, lung IL-1β, macrophage-inflammatory protein-2, and TNF-α cytokine levels. These results demonstrate that uPA can potentiate LPS-induced neutrophil responses and also suggest that such effects are sufficiently important in vivo to play a major contributory role in neutrophil-mediated inflammatory responses, such as the development of acute lung injury.

GM-CSF in the Lung Protects against Lethal Influenza Infection

RATIONALE Alveolar macrophages contribute to host defenses against influenza in animal models. Enhancing alveolar macrophage function may contribute to protection against influenza. OBJECTIVES To determine if increased expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the lung increases resistance to influenza. METHODS Wild-type mice and transgenic mice that expressed GM-CSF in the lung were infected with influenza virus, and lung pathology, weight loss, and mortality were measured. We also administered GM-CSF to the lungs of wild-type mice that were infected with influenza virus. MEASUREMENTS AND MAIN RESULTS Wild-type mice all died after infection with different strains of influenza virus, but all transgenic mice expressing GM-CSF in the lungs survived. The latter also had greatly reduced weight loss and lung injury, and showed histologic evidence of a rapid host inflammatory response that controlled infection. The resistance of transgenic mice to influenza was abrogated by elimination of alveolar phagocytes, but not by depletion of T cells, B cells, or neutrophils. Transgenic mice had far more alveolar macrophages than did wild-type mice, and they were more resistant to influenza-induced apoptosis. Delivery of intranasal GM-CSF to wild-type mice also conferred resistance to influenza. CONCLUSIONS GM-CSF confers resistance to influenza by enhancing innate immune mechanisms that depend on alveolar macrophages. Pulmonary delivery of this cytokine has the potential to reduce the morbidity and mortality due to influenza virus.

Pathogenesis of pleural fibrosis

Abstract:  Pleural fibrosis resembles fibrosis in other tissues and can be defined as an excessive deposition of matrix components that results in the destruction of normal pleural tissue architecture and compromised function. Pleural fibrosis may be the consequence of an organised haemorrhagic effusion, tuberculous effusion, empyema or asbestos‐related pleurisy and can manifest itself as discrete localised lesions (pleural plaques) or diffuse pleural thickening and fibrosis. Although the pathogenesis is unknown, it is likely that the complex interactions between resident and inflammatory cells, profibrotic mediators and coagulation, and fibrinolytic pathways are integral to pleural remodelling and fibrosis. It is generally considered that the primary target cell for pleural fibrosis is the subpleural fibroblast. However, increasing evidence suggests that mesothelial cells may also play a significant role in the pathogenesis of this condition, both by initiating inflammatory responses and producing matrix components. A greater understanding of the interactions between pleural and inflammatory cells, cytokines and growth factors, and blood derived proteins is required before adequate therapies can be developed to prevent pleural fibrosis from occurring.

Endothelium and disordered fibrin turnover in the injured lung: Newly recognized pathways

ObjectivesTo review derangements of pathways of fibrin turnover that promote pathologic fibrin deposition in the acute respiratory distress syndrome and to review the contribution of the endothelium and parenchymal lung cells to the derangements. In addition, to review how these pathways can be exploited in specific clinical circumstances, including sepsis and acute lung injury. Lastly, to review newly recognized posttranscriptional and urokinase-dependent pathways by which the fibrinolytic system is regulated in the lung. Data SourcesMedical literature published in English from 1966 to present. Data SummaryLocal abnormalities of fibrin turnover in the injured lung recapitulate the systemic changes observed in sepsis. In both circumstances, the procoagulant response is increased, whereas fibrinolytic activity is concurrently depressed. The increased procoagulant activity is related to tissue factor associated with factor VII/VIIa. Fibrinolytic activity in the vasculature is mainly attributable to tissue plasminogen activator, whereas extravascular fibrinolytic activity in the lung is mainly attributable to urokinase plasminogen activator (uPA). Depressed fibrinolytic activity is in large part attributable to plasminogen activator inhibitor-1. In sepsis, activated protein C is also deficient, potentiating the inflammatory response, coagulopathy, and depressed fibrinolysis. Recombinant human activated protein C (drotrecogin alfa [activated]) was successful as an intervention for sepsis in a recent phase 3 clinical trial (PROWESS). Recently, novel posttranscriptional pathways that regulate expression of uPA, its receptor (uPAR), and plasminogen activator inhibitor-1 have been identified. The responsible mechanisms involve cis-trans interactions between newly recognized messenger RNA (mRNA) binding sequences and mRNA binding proteins. A 51 nucleotide mRNA binding sequence within the coding region of uPAR mRNA interacts with a novel 50-kDa mRNA binding protein to destabilize the message. Sequences within the 3′ untranslated region of uPA or plasminogen activator inhibitor-1 mRNA interact with 30- and 60-kDa proteins, respectively, to regulate message stability. All of these pathways operate in lung epithelial cells, and endothelial cells regulate uPA expression through a similar pathway. In addition, uPA itself is capable of inducing expression of other components of the fibrinolytic system, including uPAR. This observation defines another feedback loop that could amplify local fibrinolysis and other uPA- or uPAR-mediated cellular responses, including cellular proteolysis, proliferation, and directed cellular migration. ConclusionsNovel posttranscriptional pathways regulate expression of uPA, uPAR, and plasminogen activator inhibitor-1. uPA itself is capable of inducing other components of the fibrinolytic system. Some or all of these newly recognized pathways are operative in endothelial and parenchymal lung cells and may influence disordered fibrin turnover in the injured lung.

Neutrophil α-Defensins Cause Lung Injury by Disrupting the Capillary–Epithelial Barrier

RATIONALE The involvement of neutrophil activation in the sentinel, potentially reversible, events in the pathogenesis of acute lung injury (ALI) is only partially understood. alpha-Defensins are the most abundant proteins secreted by activated human neutrophils, but their contribution to ALI in mouse models is hindered by their absence from murine neutrophils and the inability to study their effects in isolation in other species. OBJECTIVES To study the role of alpha-defensins in the pathogenesis of ALI in a clinically relevant setting using mice transgenic for polymorphonuclear leukocyte expression of alpha-defensins. METHODS Transgenic mice expressing polymorphonuclear leukocyte alpha-defensins were generated. ALI was induced by acid aspiration. Pulmonary vascular permeability was studied in vivo using labeled dextran and fibrin deposition. The role of the low-density lipoprotein-related receptor (LRP) in permeability was examined. MEASUREMENTS AND MAIN RESULTS Acid aspiration induced neutrophil migration and release of alpha-defensins into lung parenchyma and airways. ALI was more severe in alpha-defensin-expressing mice than in wild-type mice, as determined by inspection, influx of neutrophils into the interstitial space and airways, histological evidence of epithelial injury, interstitial edema, extravascular fibrin deposition, impaired oxygenation, and reduced survival. Within 4 hours of insult, alpha-defensin-expressing mice showed greater disruption of capillary-epithelial barrier function and ALI that was attenuated by systemic or intratracheal administration of specific inhibitors of the LRP. CONCLUSIONS alpha-Defensins mediate ALI through LRP-mediated loss of capillary-epithelial barrier function, suggesting a potential new approach to intervention.

δ ENaC: a novel divergent amiloride-inhibitable sodium channel

The fourth subunit of the epithelial sodium channel, termed delta subunit (δ ENaC), was cloned in human and monkey. Increasing evidence shows that this unique subunit and its splice variants exhibit biophysical and pharmacological properties that are divergent from those of α ENaC channels. The widespread distribution of epithelial sodium channels in both epithelial and nonepithelial tissues implies a range of physiological functions. The altered expression of SCNN1D is associated with numerous pathological conditions. Genetic studies link SCNN1D deficiency with rare genetic diseases with developmental and functional disorders in the brain, heart, and respiratory systems. Here, we review the progress of research on δ ENaC in genomics, biophysics, proteomics, physiology, pharmacology, and clinical medicine.

The fibrinolytic system and the regulation of lung epithelial cell proteolysis, signaling, and cellular viability

The urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) are key components of the fibrinolytic system and are expressed by lung epithelial cells. uPA, uPAR, and PAI-1 have been strongly implicated in the pathogenesis of acute lung injury (ALI) and pulmonary fibrosis. Recently, it has become clear that regulation of uPA, uPAR, and PAI-1 occurs at the posttranscriptional level of mRNA stability in lung epithelial cells. uPA further mediates its own expression in these cells as well as that of uPAR and PAI-1 through induction of changes in mRNA stability. In addition, uPA-mediated signaling controls the expression of the tumor suppressor protein p53 in lung epithelial cells at the posttranslational level. p53 has recently been shown to be a trans-acting uPA, uPAR, and PAI-1 mRNA-binding protein that regulates the stability of these mRNAs. It is now clear that signaling initiated by uPA mediates dose-dependent regulation of lung epithelial cell apoptosis and likewise involves changes in p53, uPA, uPAR, and PAI-1 expression. These findings demonstrate that the uPA-uPAR-PAI-1 system of lung epithelial cells mediates a broad repertoire of responses that encompass but extend well beyond traditional fibrinolysis, involve newly recognized interactions with p53 that influence the viability of the lung epithelium, and are thereby implicated in the pathogenesis of ALI and its repair.

Urokinase Induces Expression of Its Own Receptor in Beas2B Lung Epithelial Cells

Interaction between the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) localizes cellular proteolysis and promotes cellular proliferation and migration. The interaction between uPA and uPAR at the surface of epithelial cells thereby contributes to the pathogenesis of lung inflammation and neoplasia. In this study, we sought to determine if uPA itself alters uPAR expression by lung epithelial cells. uPA enhanced uPAR expression as well as 125I-uPA binding in Beas2B lung epithelial cells in a time- and concentration-dependent manner. The uPA-mediated induction of uPAR is not accomplished through its receptor and requires enzymatic activity. The low molecular weight fragment of uPA, lacking the receptor binding domain, was as potent as intact two-chain uPA in inducing expression of uPAR at the cell surface. Plasmin, the end product of plasminogen activation, did not alter uPA-mediated uPAR expression. Induction of uPAR by uPA represents a novel pathway by which epithelial cells can regulate uPAR-dependent cellular responses that may contribute to stromal remodeling in lung injury or neoplasia.

The kringle domain of urokinase-type plasminogen activator potentiates LPS-induced neutrophil activation through interaction with αVβ3 integrins

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. In addition, uPA has been shown to have proinflammatory properties, particularly in potentiating lipopolysaccharide (LPS)‐induced neutrophil responses. To explore the mechanisms by which uPA exerts these effects, we examined the ability of specific uPA domains to increase cytokine expression in murine and human neutrophils stimulated with LPS. Whereas the addition of intact uPA to neutrophils cultured with LPS increased mRNA and protein levels of interleukin‐1β, macrophage‐inflammatory protein‐2, and tumor necrosis factor α, deletion of the kringle domain (KD) from uPA resulted in loss of these potentiating effects. Addition of purified uPA KD to LPS‐stimulated neutrophils increased cytokine expression to a degree comparable with that produced by single‐chain uPA. Inclusion of the arginine‐glycine‐aspartic but not the arginine‐glycine‐glutamic peptide to neutrophil cultures blocked uPA kringle‐induced potentiation of proinflammatory responses, demonstrating that interactions between the KD and integrins were involved. Antibodies to αV or β3 integrins or to the combination of αVβ3 prevented uPA kringle‐induced enhancement of expression of proinflammatory cytokines and also of adhesion of neutrophils to the uPA KD. These results demonstrate that the KD of uPA, through interaction with αVβ3 integrins, potentiates neutrophil activation.