Mitchell A. Olman

Changes in procoagulant and fibrinolytic gene expression during bleomycin-induced lung injury in the mouse.

Bleomycin-induced lung injury is an established murine model of human pulmonary fibrosis. Although procoagulant molecules (e.g., tissue factor [TF]) and fibrinolytic components (e.g., urokinase [u-PA] and type 1 plasminogen activator inhibitor [PAI-1]) have been detected in alveolar fluid from injured lungs, the origin of these molecules remains unknown. We therefore examined the expression of procoagulant and fibrinolytic components in relation to the distribution of parenchymal fibrin in bleomycin-injured lungs. Extravascular fibrin localized to the alveolar and extracellular matrix in injured lung tissue. Injured lung tissue extracts contained elevated levels of PAI-1 activity and decreased levels of u-PA activity. Whole lung PAI-1 and TF mRNAs were dramatically induced by lung injury. In situ hybridization of injured lungs revealed that PAI-1, u-PA, and TF mRNAs were induced within the fibrin-rich fibroproliferative lesions, primarily in fibroblast-like and macrophagelike cells, respectively, while TF mRNA was also induced in perilesional alveolar cells. Taken together, these observations suggest that the induction of PAI-1 and TF gene expression plays and important role in the formation and persistence of extracellular fibrin in bleomycin injured murine lungs.

Pulmonary Edema Fluid from Patients with Early Lung Injury Stimulates Fibroblast Proliferation through IL-1β-Induced IL-6 Expression

Although the fibroproliferative response to lung injury occurs with a high frequency in patients with clinical acute lung injury, the mechanisms that initiate this response are largely unknown. This study was undertaken first to identify fibroblast mitogenic factors in pulmonary edema fluid, and second to examine the human lung fibroblast’s gene expression profile in response to pulmonary edema fluid. The edema fluid obtained from patients with early lung injury has an eightfold higher concentration of IL-1β and a twofold greater IL-1β-dependent mitogenic effect than does fluid obtained from control patients with hydrostatic pulmonary edema. Furthermore, fibroblasts responded to acute lung injury patient-derived edema fluid through production of soluble mediators that possess an autocrine mitogenic effect. Gene array analysis reveals that acute lung injury edema fluid induces several inflammation-modulating and proliferation-related genes in fibroblasts, whose inductions are similarly dependent on bioactive IL-1β. Most notably, the 20-fold induction of IL-6 mRNA and protein was completely blocked by IL-1 receptor antagonist. The combined addition of IL-1β and IL-6 was mitogenic, and the proliferative response to conditioned medium from IL-1β-exposed cells was blocked by antagonistically acting Abs to IL-6 or to gp130. These novel findings indicate that soluble IL-1β bioactivity and autocrine IL-1β-dependent IL-6 up-regulation are critical initiators of fibroblast activation and proliferation and that they likely play a role in the fibroproliferative response seen in human acute lung injury.

Endogenous fibrinolytic system in chronic large-vessel thromboembolic pulmonary hypertension.

BackgroundChronic thromboembolic pulmonary hypertension (CTEPH) is a disorder characterized by pulmonary arterial hypertension as a consequence of organized thrombotic material in the central pulmonary arteries. Incomplete resolution of acute pulmonary emboli is believed to be pathogenically important; however, the mechanism for poor thrombus dissolution remains to be explained. We undertook this study to assess the major determinants of plasma fibrinolysis in patients with CTEPH (n = 32) Methods and ResultsImmunological and functional levels of tissue-type plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) were quantified in platelet-poor plasma (PPP) from patients with CTEPH as well as age-matched controls. Although basal PPP t-PA antigen levels (CTEPH mean, 29.5 ng/ml; control mean, 2.7 ng/ml) and PAI-1 antigen levels (CTEPH mean, 55.8 ng/ml; control mean, 21.0 ng/ml) were higher in the CTEPH group, no between-group differences were detected in the enzymatic activities of these two molecules. The CTEPH group demonstrated a greater rise in t-PA antigen (CTEPH mean rise, 53.0 ng/ml; control mean rise, 5.6 ng/ml) and PA activity (CTEPH mean rise, 10.5 IU/ml; control mean rise, 1.2 IU/ml) than controls in response to an experimentally induced venous occlusion. Immunoprecipitation and fibrin autography of PPP from two patients with markedly elevated basal t-PA antigen levels demonstrate that the t-PA antigen was present in PPP primarily in complex with PAI-1. ConclusionsAlthough abnormalities of the fibrinolytic system were detected, neither a high resting plasma PAI-1 activity nor a blunted response of t-PA to venous occlusion can be invoked as an etiology for CTEPH.

Expression of type 1 plasminogen activator inhibitor in chronic pulmonary thromboemboli.

BACKGROUNDnChronic thromboembolic pulmonary hypertension is the result of nonresolving pulmonary emboli that lead to chronic obstruction of the central pulmonary arteries.nnnMETHODS AND RESULTSnTo determine if the failure to lyse pulmonary thromboemboli is caused by the local expression of the primary inhibitor of tissue-type plasminogen activator (type 1 plasminogen activator inhibitor, PAI-1), levels of PAI-1 antigen and mRNA were analyzed by immunohistochemistry and in situ hybridization in specimens harvested from a series of patients during pulmonary thromboendarterectomies. Red, fibrin-rich thrombi within the thromboendarterectomy specimens were lined with a single layer of endothelial cells exhibiting high levels of PAI-1 antigen. Quantitation of the in situ hybridization signal revealed that a significant increase in PAI-1 mRNA was present in the endothelial cells lining the fresh thrombi in comparison to the signal present in the endothelial cells from noninvolved areas of patients pulmonary arteries (n = 16, P < .001). In contrast, tissue-type plasminogen activator antigen levels were low in all samples. Yellowish-white thrombi were composed of smooth muscle cells and endothelial cells in numerous vessels that stained prominently for PAI-1 antigen. Both types of cells within the highly organized tissues also exhibited elevated PAI-1 mRNA levels in comparison to patient pulmonary artery specimens that were free of thrombus (n = 16, P < .02).nnnCONCLUSIONSnThe prevalence of PAI-1 expression within pulmonary thromboemboli suggests that this inhibitory may play a role in the stabilization of vascular thrombi.

Parallel analysis of tissue-type plasminogen activator and type 1 plasminogen activator inhibitor in plasma and endothelial cells derived from patients with chronic pulmonary thromboemboli.

BackgroundChronic thromboembolic pulmonary hyper-tension is the result of nonresolving pulmonary emboli that lead to chronic obstruction of the central pulmonary arteries. Methods and ResultsTo determine if the failure to lyse pulmonary thromboemboli is caused by an abnormality in the endothelial cell (EC)-associated fibrinolytic system, conditions were established to culture ECs from patient main pulmonary arteries during surgical pulmonary thromboendarterectomies and to analyze the conditioned media for levels of tissue-type plasminogen activator (TPA) and type 1 plasminogen activator inhibitor (PAI-1). Our data indicate that the levels of TPA antigen and PAI-1 activity in media conditioned by primary ECs harvested from areas free of thrombus were not significantly different between patients with chronic thromboemboli and organ donors. In 13 consecutive patients, no correlation was obtained in either the TPA antigen or PAI-1 activity level in a patients plasma and the respective levels in media conditioned by the patients pulmonary ECs. Moreover, patient pulmonary arterial ECs were observed to increase the secretion of TPA and PAI-1 in response to thrombin in a fashion similar to donor pulmonary artery ECs. ConclusionsThe data suggest that an inherent EC-mediated fibrinolytic imbalance is not a generalized phenomenon observed in pulmonary arteries of patients with chronic pulmonary thromboemboli.

Chronic pulmonary thromboembolism in dogs treated with tranexamic acid.

BackgroundMany questions remain regarding the pathogenesis, natural history, diagnosis, and treatment of chronic thromboembolic pulmonary hypertension in patients. To answer such questions, we developed an animal model of this disorder. The brisk thrombolytic response of canines to acute embolism has, previously, prevented the establishment of such a model. Methods and ResultsThe fibrinolytic inhibitor tranexamic acid was given orally to canines before, and for intervals after, pulmonary emboli were released from venous thrombi formed in vivo in femoral veins or the inferior vena cava. Preliminary studies disclosed that embolic residuals from femoral vein thrombi were not sufficient to cause significant, persistent pulmonary hypertension. With repetitive, larger thrombi embolized from the inferior vena cava, however, persistent pulmonary hypertension was achieved in most animals. ConclusionsResolution of emboli in the canine can be inhibited by tranexamic acid. As in humans, a spectrum of embolic residuals is encountered, and the perfusion lung scan consistently underestimates the extent of embolic residuals. Studies of this animal model continue.

Factors contributing to increased vascular fibrinolytic activity in mongrel dogs.

BackgroundNumerous investigators have observed that pulmonary emboli are rapidly lysed in a canine model system. This study was undertaken to delineate the unique mechanism that accounts for the rapid dissolution of pulmonary emboli in mongrel dogs Methods and ResultsCanine plasminogen activator (PA) activity (2.6±1.1 IU/mL acidified platelet-poor plasma [PPP], <0.3 IU/mL acidified whole blood serum [WBSJ, mean±SD; n=6) and PA inhibitor activity (6.1±2.6 U/mL PPP, 35.4±7.8 U/mL WBS; n=6) were determined in standard plasminogen-based chromogenic assays. Analysis of canine PPP, WBS, platelet lysates, and primary canine endothelial cell (EC) cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography revealed a plasminogen-dependent lytic zone at 45-kd relative molecular mass that was shown to be related to urokinase-type PA (u-PA) by its selective inhibition through amiloride. Analysis of canine platelets on standard 125I fibrin plate assays revealed a net fibrinolytic activity. In a clot lysis assay system, canine platelets were able to stimulate fibrinolysis when layered on the outside of fibrin clots containing autologous PPP. Moreover, net fibrinolytic activity of primary canine pulmonary artery endothelial cells was higher than the activities expressed by canine aortic or carotid artery endothelial cells. ConclusionRapid lysis of pulmonary emboli in mongrel dogs appears to be a result of 1) the high u-PA activity in canine PPP and 2) the predominant association of u-PA activity with canine platelets and canine pulmonary artery endothelial cells. (Circulation 1993;87:1990-2000)

Urokinase-type Plasminogen Activator Receptor (uPAR) Ligation Induces a Raft-localized Integrin Signaling Switch That Mediates the Hypermotile Phenotype of Fibrotic Fibroblasts

Background: Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) overexpress the urokinase-type plasminogen activator receptor (uPAR) and are hypermotile. Results: uPAR ligation increases fibroblast motility by localizing α5β1 integrin-Fyn signaling complexes to lipid rafts. Conclusion: The hypermotile phenotype of IPF fibroblasts is due to lipid raft-localized uPAR-integrin-Fyn signaling complexes. Significance: These unique lipid raft signals may be therapeutic targets for IPF. The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Urokinase receptor mediates lung fibroblast attachment and migration toward provisional matrix proteins through interaction with multiple integrins.

Fibroblasts from patients with pulmonary fibrosis express higher levels of the receptor for urokinase, and the extent of fibrosis in some animal models exhibits a dependence on the urokinase receptor. Recent observations have identified the urokinase receptor as a trans-interacting receptor with consequences on signaling and cell responses that vary depending on its interacting partner, the relative levels of expression, and the state of cellular transformation. We undertook this study to define the urokinase-type plasminogen activator cellular receptor (u-PAR)-integrin interactions and to determine the functional consequences of such interactions on normal human lung fibroblast attachment and migration. u-PAR colocalizes in lammelipodia/filopodia with relevant integrins that mediate fibroblast attachment and spreading on the provisional matrix proteins vitronectin, fibronectin, and collagens. Inhibitory antibody studies have revealed that human lung fibroblasts utilize alpha(v)beta(5) to attach to vitronectin, predominantly alpha(5)beta(1) (and alpha(v)beta(3)) to attach to fibronectin, and alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) to attach to collagen. Blocking studies with alpha-integrin subunit decoy peptides and u-PAR neutralizing antibodies indicate that u-PAR modulates the integrin-mediated attachment to purified provisional matrix proteins, to anti-integrin antibodies, or to fibroproliferative lesions from fibrotic lungs. Furthermore, these decoy peptides blunt fibroblast spreading and migration. We show that u-PAR can interact with multiple alpha-integrins but with a preference for alpha(3). Taken together, these data demonstrate that u-PAR may interact with multiple integrins in normal human lung fibroblasts thereby promoting attachment, spreading, and migration. Modulation of fibroblast invasion would be expected to lead to amelioration of fibroproliferative diseases of the lung.

Reply: A Placebo-controlled Randomized Trial of Warfarin in Idiopathic Pulmonary Fibrosis: A Hidden Subgroup?

Idiopathic Pulmonary Fibrosis: Clinically Meaningful Primary Endpoints in Phase 3 Clinical Trials 3 Hot on the breath: Mortality as a primary end-point in IPF treatment trials: the best is the enemy of the good 4 Idiopathic Pulmonary Fibrosis: Lung Function is a Clinically Meaningful Endpoint for Phase 3 Trials 4 III. About Sarcoidosis 5 A Novel Sarcoidosis Risk Locus for Europeans on Chromosome 11q13.1 5 Sarcoidosis-related Mortality in the United States from 1988 to 2007 5 Inflammatory activity assessment by F18 FDG-PET/CT in persistent symptomatic sarcoidosis 6 Development of a sarcoidosis murine lung granuloma model using Mycobacterium superoxide dismutase A peptide. 6