Fibrin Turnover in Lung Inflammation and Neoplasia

STEVEN IDELL, ANDREW P. MAZAR, PETER BITTERMAN, SURESH MOHLA, and ANDREA L. HARABIN

Department of Medical Specialties, University of Texas Health Center at Tyler, Tyler, Texas; Attenuon, LLC, San Diego, California; Department of Medicine, University of Minnesota, Minneapolis, Minnesota; Division of Cancer Biology, National Cancer Institute; and Division of Lung Diseases, National Heart, Lung and Blood Institute, Bethesda, Maryland

	ARTICLE

TOP ARTICLE REFERENCES

This workshop was convened to review the latest findings from ongoing research in the field, to identify gaps, and to suggest research directions that might rapidly increase understanding of the relationship of fibrin turnover to injury and neoplasia in the lung and pleura. Opportunities to expedite translation of promising findings from basic and preclinical work to the bedside were considered and interventions likely to provide clinical benefit were evaluated and prioritized. This report draws on presentations of the participants, using material they provided, and the recommendations as synthesized by the organizers and discussants.

It is now clear that extravascular fibrin deposition characterizes most forms of acute and chronic lung injury (1). A progression of increased vascular permeability, formation of a transitional fibrin gel, and remodeling and organization of the fibrinous neomatrix are common to the pathogenesis of lung inflammation and neoplasia, including tumors of the lung and pleural space (2). Of particular interest, florid alveolar and interstitial fibrin is found in the acute respiratory distress syndrome (ARDS) (3). Alveolar fibrin deposition can likewise be found in other forms of lung injury including interstitial lung diseases (1, 4) and in exudative pleuritis (1). In parenchymal lung or pleural disease and neoplasia, a transitional fibrin neomatrix constitutes part of the acute inflammatory response and appears to initiate a sequence of events that leads to increased vascular permeability, tissue remodeling, and ultimate fibrosis (see online data supplement, Figure 1) (7).

Disordered coagulation or fibrinolysis can influence or be influenced by tissue inflammation and remodeling in a variety of ways. For example, tissue factor expression is stimulated by a variety of cytokines that are elaborated in lung or pleural inflammation and neoplastic diseases (2). Proteases and products of coagulation and fibrinolysis also interact with other inflammatory pathways, including the complement and kinin systems, to amplify the local inflammatory response and increase vascular permeability (5, 8, 9). In the alveolar space, fibrin(ogen) derivatives can impair surfactant function (10) and potentiate lung dysfunction and remodeling in acute or chronic lung injury. Fibrin and its products can influence migration of macrophages and fibroblasts (11, 12). Plasmin also activates transforming growth factor  (TGF-) and promotes fibrotic repair, in part via TGF--mediated induction of plasminogen activator inhibitor 1 (PAI-1) (13).

A rapidly evolving body of work has elucidated mechanisms by which fibrin turnover is altered in lung injury and neoplasia (14) (see online data supplement, Figure 2). Intravascular as well as extravascular coagulation is associated with acute lung injury (5, 15). In the normal lung, tissue factor is expressed by alveolar macrophages, the lung epithelium, and lung fibroblasts (20, 21). Tissue factor expression is increased in lung and pleural injuries, initiating coagulation via the extrinsic activation pathway. Coagulation substrates move into the injured alveolar space or pleural cavity, potentiating thrombin generation and fibrin formation. This scenario is consistently observed in ARDS, other forms of acute lung injury (22), interstitial lung diseases (25), organizing pleuritis (26), and lung and pleural neoplasia (2, 7).

Disordered fibrinolysis often occurs in association with increased procoagulant activity in these same clinical situations. For example, locally depressed fibrinolysis occurs in the lungs in ARDS and is attributable to inhibition by plasminogen activators (PAI), mainly PAI-1, and downstream by antiplasmins (27). This "defect" in expression of fibrinolytic activity occurs with concurrently increased tissue factor-related procoagulant activity and promotes local fibrin deposition (22, 23). Similar derangements are observed in the interstitial lung diseases (23) and inflammatory pleuritis (6, 25, 26). Responses to particulates including silica and asbestos appear to induce complex changes in the fibrinolytic cascade, including induction of urokinase plasminogen activator (uPA) or its receptor (uPAR) (30). Abnormalities of fibrinolytic pathways have similarly been associated with progression of neoplasms (34- 37). Overexpression of uPA, uPAR, and PAI-1 has been related to tumor progression and prognosis (35). Disordered expression of these proteins has been related to neovascularization, tumor invasiveness, cellular signaling and adhesion, and cellular proliferation (34, 38).

Formation and Dissolution of Transitional Extravascular Fibrin in Lung Injury and Neoplasia

Molecules involved in the formation and dissolution of transitional fibrin appear to be involved in the progression of lung remodeling that occurs in lung injury and malignancy (see Figure 2). Multiple potential interventional targets exist within each pathway and many of these targets are proteases or cofactors for protease activity. Many of these proteases have pleiotropic activities and mediate both extracellular proteolysis as well as intracellular signaling effects in target cells in the lung. Examples of novel fibrinolytic activities for the matrix metalloproteases (MMPs) have opened yet another potential avenue for intervention (39). The critical role of cell surfaces in the regulation of coagulation and fibrinolysis is clear. However, the precise role of these pathways in fibrin turnover in lung injury and neoplasia remains to be elucidated. Events in cell signaling and effector cross-talk and synergy in events required for cell motility in inflammation, angiogenesis, and tumor cell invasion and metastasis also require further clarification.

Coagulation Reactions and Induction of Diverse Cellular Responses in the Lung

Parenchymal lung cells, including fibroblasts and epithelial and endothelial cells, all play a role in modulating coagulation. Mesothelial cells likewise exhibit this capacity in the pleural compartment. Tissue factor (TF) is an integral plasma membrane protein expressed by endothelial cells, macrophages, and some tumor cells. TF initiates the extrinsic pathway of coagulation by acting as a cofactor for the activation of Factor VII to VIIa (40). The Factor VIIa-TF complex can activate Factor X to Xa either directly or indirectly through the activation of Factor IXa, which then activates Factor X in the presence of Factor VIII, a cofactor (see Figure 2). Direct activation of Factor Xa may not be an efficient process in vivo because of rapid quenching of the Factor VIIa-FactorXa-TF complex by tissue factor pathway inhibitor (TFPI). This leads to internalization and association of the complex with caveolae. Factor Xa catalyzes the conversion of prothrombin to thrombin, leading to fibrin formation. In regions of tissue damage, endothelial cell TF activates thrombin, resulting in local fibrin generation. This procoagulant response occurs in most lung or pleural injuries. Fibrinogen and fibrin both provide adhesion sites for inflammatory cells that are recruited to the site of tissue damage. Inflammatory cells secrete cytokines that further activate endothelial cells inducing migration, proliferation, endothelial repair, and angiogenesis in response to injury (41).

Thrombin can also initiate fibrinolysis, a process facilitated through its interaction with its receptor, thrombomodulin (42). In addition to these cell surface activities, durable effects on cell phenotype can also be initiated through the activation of the thrombin receptor. Thrombomodulin is a G protein-coupled receptor, of the protease-activated receptor (PAR) family, and may modulate differentiation and chemotaxis in macrophages, platelets, and endothelial and smooth muscle cells. These interactions may be of importance in lung remodeling after injury or desmoplasia associated with solid neoplasms. Other receptors for coagulation proteins (such as EPR-1, the receptor for Factor Xa) may also be important in the inflammatory response. Data also suggest that thrombin can stimulate the release and/or expression of MMPs, emphasizing a novel role for these enzymes in fibrin turnover (42). Interactions between MMPs and pathways of fibrin turnover may influence lung remodeling and deserve further study.

Interventions that Selectively Target Coagulation Pathways to Block Lung Injury

While anticoagulant molecules have been used to block tissue injury or morbidity in preclinical models, it is clear that such anticoagulants are not equal in potential to prevent tissue injury. Because of secondary cellular responses associated with the activation of certain coagulation proteins, the point of intervention (e.g., the enzyme target) may be critical to achieving the desired therapeutic effect. For example, direct inhibition of either TF or thrombin (strategies that could independently inhibit fibrin formation) could lead to markedly different effects in vivo. In primate models of sepsis, complete suppression of fibrin formation did not influence animal mortality, indicating that mortality was not due to microthrombus formation but rather to other downstream effects that occurred in response to the activation of certain coagulation enzymes (43). Novel nematode anticoagulant proteins (NAP5 and NAPc2) that inhibited either Factor Xa (NAP5) or the TF-VIIa-Xa complex improved survival in the experimental models (44- 46), emphasizing the importance of targeting the correct step in the coagulation cascade. Recombinant NAPc2 is currently in Phase II trials for sepsis and additional data are required to fully evaluate its potential when used in this context.

Responses in the injured lung involve a complex interplay between the regulation of fibrin matrix deposition and the lung-specific lipoprotein surfactant. Pulmonary surfactant is incorporated into polymerizing fibrin and is thereby inactivated. This interaction leads to a loss of surfactant ability to reduce alveolar surface tension (10) and inactivation of the pulmonary surfactant system has been observed in models of acute lung injury and pneumonia (47). Incorporation of surfactant into fibrin also affects the properties of the fibrin matrix, which makes it less sensitive to fibrinolysis (48). In a bleomycin-induced model of pulmonary fibrosis, aerosolized heparin during the early ARDS-like phase (Days 2-12), or urokinase during late injury (Days 14-24) prevented pulmonary fibrosis to a great extent (49).

New approaches for the local administration of anticoagulants are being developed for the treatment of fibrin deposition in the lung. Chan and colleagues (50) developed a heparin-antithrombin III (ATIII) conjugate that prevents de novo fibrin formation. Intratracheal administration of this conjugate resulted in its retention within the lung, with no evidence of systemic distribution of the heparin-ATIII complex (50). Whether treatment with this conjugate is effective in animal models of acute or fibrotic lung disease is not yet known. The potential problems of drug delivery and optimal delivery locally to the lung will need to be addressed.

The theme of step-specific inhibition of coagulation has been addressed in a baboon model of ARDS that is produced through the intravenous infusion of live Escherichia coli. In preliminary studies, either TFPI or site-inactivated Factor VII (FFR-VIIa) prevented lung dysfunction in this model and inhibited renal parenchymal fibrin deposition and intravascular coagulopathy (51). Pulmonary gas exchange was improved and postmortem analysis showed there was significant attenuation of edema in animals treated with either anticoagulant compared with controls. The molecular bases for the lung protection attributable to either agent have yet to be characterized. Thus, inhibition of the initiation of the extrinsic pathway of coagulation appears to be a reasonable interventional strategy for the prevention of acute lung injury. It is not yet known how these agents would protect against established lung injury or lung injury caused by other, nonseptic causes. Experiments to address these questions are essential to define the scope of the applicability of these selected agents to different forms of acute lung injury.

Regulation of uPA-dependent Fibrinolysis: Novel Modes of Regulation

Advances have been made to improve our understanding about how key components of the fibrinolytic system are regulated. For example, expression of the uPA, uPAR and PAI-1 genes is regulated at the posttranscriptional level (52). These pathways have been demonstrated in cultured lung epithelial cells, fibroblasts, and in pleural mesothelial or malignant mesothelioma cells, indicating that control at the posttranscriptional level could influence expression of uPA-dependent fibrinolytic activity in the context of either parenchymal lung or pleural disease. Posttranscriptional regulation of uPA involves an interaction between a 30-kDa uPA mRNA-binding protein and a 66-nucleotide (nt) 3' untranslated region (UTR) sequence of uPA mRNA (52). Posttranscriptional regulation of uPAR mRNA involves the interaction of a 51-nt coding region fragment with a 50-kDa mRNA-binding protein (53). The interaction of uPA and uPAR mRNA-binding proteins with their respective mRNAs appears to destabilize uPA and uPAR mRNA, respectively. PAI-1 mRNA expression, on the other hand, involves the interaction of a specific 60-kDa mRNA-binding protein with a sequence within the PAI-1 mRNA 3' UTR (54). At this time, the regulatory function of this mRNA-binding protein or its binding sequence is unknown. The role of these regulatory pathways in the pathogenesis of either lung injury or neoplasia remains to be studied.

uPA-uPAR System: Newly Defined Linkages to Cellular Signaling and Adhesion

Signaling functions of components of the fibrinolytic system remain to be elucidated in detail but appear to be regulated at multiple levels. Dissolution of the transitional fibrin matrix could alter integrin ligation of fibrin and fibrinogen, affecting signaling through integrins such as CD11b/CD18, CD11c/CD18, and _v3. Fibrin degradation products are proinflammatory and may mediate inflammatory cell recruitment or activation through these integrins. Signaling cascades mediated by uPAR have also been described and these may be initiated either by uPAR aggregation (in the absence of binding by uPA) or by uPAR occupancy by uPA (38). uPAR has been colocalized with integrins as well as with Src tyrosine kinases, which suggests that these proximate associations could promote cellular signaling. The potential regulatory role of such interactions remains to be clarified. uPAR aggregation triggered by cross-linking antibodies leads to increased intracellular Ca²⁺ concentrations independent of uPA binding in a model system using U937 cells (55). However, ligation with uPA may activate other signaling pathways. Thus, regulation of cellular responses to molecules involved in fibrin dissolution is complex and remains to be fully characterized.

Studies demonstrate that uPAR-dependent signaling is contingent on the structural elements involved in uPAR-integrin interactions. uPAR-transfected 293 cells have been shown to bind to fibrinogen in a Mac-1 (CD11b/CD18 ₂ integrin)-dependent fashion independent of uPAR receptor occupancy. A peptide derived from phage display peptide 25 (M25) was able to block this interaction (56) and had some homology to loop W4 of Mac-1. A region homologous to M25 was also identified in integrin _M, also a fibrinogen-binding integrin. The homologous peptide from _M was synthesized and demonstrated to inhibit the binding of uPAR-transfected 293 cells to fibrinogen more potently than peptide 25. M25 was also demonstrated to bind to uPAR directly. Homology searches of other integrins identified similar peptide regions in ₃ (in the W4 loop) and in ₅ (in a non-W4 region). A common motif for binding to uPAR may exist in multiple integrin types and may regulate the interaction of uPAR with various integrins in response to various stimuli. Thus, the ligation state of uPAR (with either uPA or one of a set of integrin partners) may be central to cell signaling through this pathway.

Fibrinolysins as Interventional Agents: Potential Applications for Lung Injury or Cancer

Newly developed fibrinolysins have the potential to prevent lung injury or neoplastic spread by disrupting deposition of an extravascular, transitional fibrin matrix. A novel plasminogen activator that consists of the zymogen form of urokinase plasminogen activator (scuPA) complexed with the soluble form of its receptor (suPAR) is an example of this new genre. The scuPA-suPAR complex is relatively resistant to inactivation by plasminogen activator inhibitors (PAIs), which may contribute to its greater potency in lysing fibrin clots in vitro (57). These in vitro observations suggest that scuPA-suPAR may be especially useful in forms of lung or pleural injury that are enriched in PAI-1 as a result of inflammation. The activity of the scuPA-suPAR complex was tested in vivo in a new model of pulmonary embolism. Homogeneous suspensions of ¹²⁵I-labeled microemboli (ME) were injected into the tail veins of mice. Greater than 50% of the injected ME lodged in the lungs after 10 min. ME totally resolved in wild-type mice within 5 h, but the clot burden was 10 times higher in uPA^-/- and tissue plasminogen activator homozygous negative (tPA^-/- ) mice at this time. scuPA, infused over the period of 1 h, rescued the phenotype of the uPA^-/- mouse completely. Infusion of equivalent amounts of the scuPA-suPAR complex increased the extent of lysis 2-fold compared with scuPA alone. The lungs of the mice studied in this model are not likely not to be as rich in PAI-1 as would be anticipated after acute lung injury leading to ARDS or in exudative pleural disease. Administration of scuPA-suPAR may be even more advantageous in these settings.

Urokinase has also been implicated in the control of vascular contractility. Single-chain uPA exerts anticontractile activity, whereas two-chain uPA (tcuPA) exerts the opposite effect. The epitope responsible for anticontractile activity was identified as being within the connecting region (amino acids 136-143) of uPA. A peptide, termed A6 (Ac-KPSSPPEE-Am), corresponding to this sequence, reverses the procontractile effect of phenylephrine on aortic smooth muscle contraction. Urokinase-derived peptides also regulate vascular smooth muscle contraction in vitro and in vivo (58).

The plasminogen-activating activity of scuPA was maintained when it was bound to its cell surface receptor (59). The cell surface is therefore capable of exerting an important role in plasminogen activation by scuPA in the absence of conversion to tcuPA. Cell surface-bound scuPA plasminogen activation is refractory to inactivation by all PAIs (PAI-1, PAI-2, PAI-3, or protein C inhibitor). Interaction between uPA and uPAR accelerates removal of fibrin from the pulmonary alveolar space (60). In addition, studies currently being conducted show that fibrin-bound single-chain tissue plasminogen activator (sctPA) may behave analogously to cell surface-bound scuPA, where the fibrin surface mimicks the effect of the cell surface for sctPA. Localization of sctPA and scuPA to surfaces increases their catalytic efficiency while protecting these enzymes from PAI inactivation. The utility of these agents in lung or pleural injury and in neoplastic processes remains to be evaluated in preclinical trials.

Organization and Remodeling of Transitional Fibrin in Lung Injury and Neoplasia: Links to Angiogenesis and Cellular Adhesion

A conceptual bridge exists between the biochemistry of coagulation and fibrinolysis and biological processes regulated by fibrin (2, 7). A key step in the genesis of the fibrin gel is the transfer of substrates, enzymes, and cofactors from the vascular to the tissue compartment. Vascular endothelial cell growth factor (VEGF) facilitates this process. VEGF is expressed during physiological and pathological angiogenesis. Through the interaction of tissue factor with Factor VII, extrinsic coagulation is activated in regions of plasma leak, leading to formation of a cross-linked fibrin gel. While native extracellular matrix is a relatively poor substrate for angiogenesis, the fibrin gel is highly proangiogenic, providing ligands for _V3 integrins and other extracellular matrix receptors on endothelial cells, fibroblasts, and monocytes. Expression of these molecules promotes migration and attachment of the cells, facilitating remodeling of the extracellular matrix.

Provisional Fibrin-rich Matrix and Signaling Functions that Sustain the Inflammatory Response

Work has demonstrated that extracellular ligands, including those involved in pathways of fibrin turnover, exert their effects via specific transduction pathways. For example, it is now clear that transcriptional activation of interleukin 1 (IL-1) by fibrinogen in a monocytic cell line operates through NF-B in an AP-1-dependent CRE-inhibitable manner (61). The key target cells and receptors that participate in this regulatory pathway remain to be defined. This theme is also supported by in vitro evidence implicating uPA as a central mediator in the process by which leukocytes degrade matrix proteins and traverse tissue planes during inflammation. Receptors for uPA (uPAR) are found on the surface of monocytes and neutrophils. Clusters of uPAR localize to the leading edge of migrating cells and uPAR is required for monocyte and neutrophil chemotaxis (62). The ramifications have been examined in transgenic mice in vivo, leading to significantly increased understanding of the physiological function of the uPA-uPAR system. While uPA-deficient mice recruit inflammatory cells as well as wild-type mice when the stimulus is nonspecific (e.g., thioglycolate), markedly different results are obtained when the inciting agent is the fungal pathogen Cryptococcus neoformans (63). By 3 wk postinoculation there are significantly fewer pulmonary inflammatory cells in uPA-deficient animals compared with their wild-type counterparts. Furthermore, there is a significant survival advantage conferred by the presence of uPA. Similar results are seen when Pneumocystis carinii is used as the infectious challenge. Challenging uPAR-null mice with Pseudomonas aeruginosa led to decreased neutrophil recruitment, a process requiring a ₂ integrin-mediated function (64). These studies clearly implicate the uPA-uPAR system in host recruitment and activation of immune and inflammatory cells.

Mechanisms by Which the Fibrin Gel Regulates Fibroblast and Endothelial Cell Viability in Injury and Repair

Evidence indicates that endothelial cell and fibroblast viability is regulated at the transcriptional, translational, and posttranslational levels (65). Considerable attention has been focused on transcriptional regulation via nuclear regulatory proteins, such as NF-B, and through posttranslational pathways culminating in phosphorylation or dephosphorylation of Bcl family proteins. Less attention has been given to translational control of cell viability. A large proportion of survival signaling by defined peptide growth factor ligands is interpreted through Ras leading to activation of phosphatidylinositol 3-kinase (PI3 kinase) and Akt kinase. Downstream of Atk lies the transcriptional regulatory pathway mediated by NF-B and the posttranslational pathway mediated by Bcl family peptides. Akt is also known to enhance translation initiation, activating FRAP/mTOR kinase, which, in turn, results in phosphorylation of the translational repressor eIF-4E-binding protein 1 (4E-BP1). While this pathway has been examined in considerable detail in terms of its ability to influence peptide growth factor signaling, little work has been done to elucidate survival signals mediated by ligation of integrins by provisional matrix proteins of the fibrin gel. Integrin ligation is known to influence translation initiation. Both collagen, via ₂₁, and fibrinogen, by _v3, lead to activation of FRAP/mTOR kinase, which activates translation by inhibiting the translational repressor 4E-BP1 and activating ribosomal p70S6 kinase (69). Of note, there is now evidence that links activation of Ras with translationally mediated survival signaling. Endothelial cell survival signaling is also a major determinant of remodeling of the transitional fibrin matrix.

In addition, interactions of endothelial cells with extracellular matrix influence endothelial cell survival. Data indicate that extracellular ATP and adenosine trigger endothelial apoptosis. These molecules act by inhibiting S-adenylhomocysteine hydrolase and methyltransferases (70). Accumulation of adenosine-homocysteine leads to disruption of focal contacts and loss of focal adhesion kinase (FAK) kinase activity. This is followed by caspase-mediated degradation of the focal adhesion complex proteins. The mechanism by which the accumulated metabolites lead to disruption of focal adhesion contacts remains to be elucidated.

Central Role of PAI-1 in the Pathogenesis of Acute and Chronic Lung Diseases

Data show that, in acute and chronic fibrotic lung diseases, increased levels of PAI-1 inhibit the degradation of fibrin in the alveolar space. Mice with deletions of the PAI-1 gene develop less fibrosis after bleomycin challenge. In contrast, mice constitutively expressing a PAI-1 transgene develop more fibrosis after bleomycin-induced lung injury (71). Mice genetically deficient in uPA or tPA also demonstrate increased pulmonary fibrosis after bleomycin-induced lung injury (C. Swaisgood, Cleveland Clinic Foundation, Cleveland, OH; personal communication). Further study of this phenomenon, using an adenoviral vector to transfer human uPA on Day 21 after bleomycin challenge, revealed marked attenuation of the fibrotic response, suggesting that augmentation of uPA is a viable therapeutic strategy to prevent pulmonary fibrosis (72).

Modulation of the uPA System as a Potential Therapeutic Strategy for Solid Cancers

A range of clinical and basic research studies implicates uPA in tumor invasion and metastases (34). The design of in vivo studies must take into account significant species specificity of the uPA-uPAR system. However, studies using a rat model have been conducted to examine the role of this system in the progression of prostate and breast cancer. Rat prostate cancer cells (Dunning R3227) overexpressing uPA (Mat-Ly-Lu-uPA) have been inoculated into syngeneic rats. In this model, skeletal metastases arose, closely emulating the findings in human prostate cancer. Treated animals developed time-limited paralysis much earlier than controls given wild-type cancer cells (73). Similarly, treatment of animals with rat breast cancer cells overexpressing uPAR resulted in large tumors that metastasized (74).

On the basis of the strong linkage of the uPA-uPAR system to tumor progression, therapy using a synthetic active site inhibitor of uPA was tested in prostate cancer and in breast cancer in the rat model of tumor progression (34). There was a dose-dependent inhibition of tumor growth and metastases in prostate cancer. In breast cancer, similar effects were observed and synergy was seen between the uPA inhibitor and the antimitogenic agent tamoxifen (Tam). These studies provide further support for the premise that overexpression of uPA or uPAR is associated with tumor progression. Inhibition of the proteolytic function of uPA or blockage of uPAR is promising as a therapeutic strategy alone or in combination with currently available antitumor agents (75). More recently a synthetic peptide corresponding to the non-receptor-binding domain of uPA (amino acid residues 136-143) was shown to block breast cancer progression by inhibiting tumor angiogenesis and to promote breast cancer cell apoptosis (76).

Other inhibitors of fibrinolytic pathways may contribute to disordered fibrin turnover in lung injury or neoplasia. For example, ₂-macroglobulin (₂-M), a glycoprotein inhibitor of proteinases, including plasminogen activators, appears to be involved in lung injury and repair. A receptor for ₂-M is termed the ₂-M receptor/low density lipoprotein receptor- related protein (LRP), which can interact with ligands other then ₂-M, including complexes of uPA with PAI-1 (77). LRP also mediates the internalization of uPA-PAI-1 complexes initially bound to uPAR (77). Patients with ARDS have significant amounts of ₂-M in their lung lining fluids (78). Because of its ability to interact with various cytokines, the uPA- uPAR system, and growth factors, ₂-M could influence remodeling in lung injury or neoplasia. This possibility requires further investigation.

Final Recommendations

Recommendations were developed by the committee to prioritize research into the role of fibrin turnover in the development of acute and chronic lung injuries and neoplasia and to facilitate the development of new treatments targeting this process.

1. Animal models are needed that functionally and temporally reproduce the progression of events in lung injury and repair. The models should include clinically relevant interventions, such as mechanical ventilation, hyperoxia, and exposure to therapeutic agents.

2. Novel animal models should be developed in which genes can be selectively manipulated at the organ, tissue, or cellular level in these diseases.

3. Reliable biomarkers that reflect the key events in the pathogenesis of lung injury, repair, and neoplasia are needed. These will include contemporary screening techniques such as DNA arrays to detect genes related to disease progression and outcome.

4. The links between the fibrinolytic and coagulation systems and cellular motility, proliferation, apoptosis, and connective tissue synthesis need to be identified in lung injury and neoplasia. Areas that need attention are as follows: the mechanisms by which transitional fibrin is organized or resolved; cell-to-cell interactions that regulate, or are regulated by, the coagulation and fibrinolytic systems; the mechanisms by which cells regulate remodeling of the extracellular matrix; and signals from the matrix that govern cellular motility, proliferation, viability, and differentiation.

5. A number of agents should be studied in models of lung injury or neoplastic spread. These include agents that target fibrin turnover and are in current clinical use for other applications, including heparin fragments or analogs, coumarin derivatives, hirudins and novel antithrombins or combination therapy. Fibrinolysins (scuPA-suPAR, uPA, and tPA) as well as agents that inhibit additional fibrin formation (ATIII, NAPc2, and NAP5) should be studied. Optimal methods of drug delivery need to be defined, including aerosols.

6. Preclinical evidence suggesting efficacy of blockade of the extrinsic coagulation pathway in septic ARDS is encouraging and should be pursued and confirmed in models of lung injury from other (nonseptic) risk factors.

7. The contribution of changes in vascular permeability to the control of fibrin production in lung injury and neoplasia should be elucidated. Studies of the mechanisms of vascular egress of coagulation and fibrinolytic proteins and cellular elements and the mechanisms by which fibrin polymerization is regulated are needed. Components of the fibrin gel and extracellular matrix receptors that support angiogenesis need to be identified.

8. Fibrin turnover should be fully characterized in pulmonary hypertension and in vascular remodeling.

9. Preclinical studies of fibrinolytic interventions should be pursued in models of organizing pleuritis.

	Footnotes

Correspondence and requests for reprints should be addressed to Andrea L. Harabin, Ph.D., Division of Lung Diseases, National Heart, Lung, and Blood Institute, 6701 Rockledge Drive, Bethesda, MD 20892-7952. E-mail: harabin{at}nih.gov

(Received in original form May 31, 2000 and in revised form September 25, 2000).

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