Fibrinolytic Pathways In Lung Injury And Repair

Steven Idell, Chairman
University Of Texas Hlth Ctr At Tyler

Grant 5P01HL076406-04 from National Heart, Lung, And Blood Institute, IRG: HLBP

Abstract: Abnormalities in the plasminogen activator (PA) pathways have been implicated in the pathogenesis of acute lung (All) and pleural injury. Recent interventional trials suggest that targeting these pathways can reduce mortality in sepsis and protect against acute lung or pleural injury. The Project Leaders of this PPG have developed evidence that these pathways can influence ALI and pleural injury through newly recognized mechanisms. However the pathogenic mechanisms that link the PA pathways to ALI and pleural injury are poorly understood and are likely to involve non-proteolytic signal-transducing pathways. Our thematic objective is to address this gap by defining novel mechanisms by which urokinase (uPA), its receptor (uPAR), other novel uPA receptors and its inhibitor PAI-1 influence the course of inflammation, remodeling of transitional matrix and accelerated fibrosis in ALI and pleural injury. In Project 1, pathways that regulate PAI-1 and uPAR expression by the.mesothelium at the posttranscriptional level will be defined and a novel fibrinolytic intervention to prevent pleural loculation will be further evaluated. Project 2 will elucidate novel posttranscriptional mechanisms by which uPA and uPAR are regulated by the lung epithelium. Project 3 will define novel pathways by which uPA interacts with cell surface signaling adapter molecules to regulate pulmonary vasoconstriction and lung edema after ALI and ascertain the role of defensin in the process. These interactive projects derive from active programs directed by experienced Project Leaders and are now oriented to our thematic objective. In vitro, in vivo and interventional methods will be used. This PPG will accelerate the acquisition of new, clinically relevant information that will hasten the development of better treatments for ALI and/or pleural injury

Keywords: fibrin, fibrinolysis, lung injury, regeneration clinical research

Project start date: 2005-09-01

Project end date: 2010-08-31

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Control Of Fibrin Turnover In Pleural Disease

Steven Idell, Chairman
University Of Texas Hlth Ctr At Tyler 11937 Us Highway 271 Tyler, Tx 75708

Grant 5P01HL076406-030001 from National Heart, Lung, And Blood Institute, IRG: HLBP

Abstract: Disordered fibrin turnover has been implicated in the pathogenesis of pleural inflammation and repair. We hypothesize that deranged regulation of plasminogen activator inhibitor-1 (PAI-1) and of the urokinase receptor (uPAR) in pleural mesothelial cells are critical determinants of locally impaired fibrinolysis and intrapleural remodeling after asbestos exposure or in fibrosing pleuritis. Mechanisms that regulate expression of these proteins in mesothelial cells are now poorly understood. We will extend work done in previous funding cycles to address these important gaps. We will achieve this objective in four closely integrated specific aims. In Aim 1, we will determine mechanisms that regulate the expression of PAI-1 and uPAR in mesothelial cells exposed to asbestos or mediators of fibrosing pleural injury. In Aims 2 and 3, we will elucidate mechanism(s) by which PAI-1 is regulated by pleural mesothelial cells at the posttranscriptional level and will determine how control at this level influences pathophysiologic responses of these cells. In Aim 4, we will further test a novel interventional approach; intrapleural administration of single-chain uPA (scuPA) to prevent pleural loculation. We will use our established rabbit models of tetracycline- or P. multocida induced pleural injury in these studies. To accomplish the work, we will use a wide range of molecular, biochemical and histologic techniques, all of which are well-established in our laboratory. These studies will foster better understanding of the role of the mesothelial cell in the regulation of the PAI-1-uPA-uPAR system and will increase our understanding of how these cells contribute to pleural remodeling after injury. The project will also facilitate the development of novel, clinically applicable non-surgical approaches to prevent intrapleural loculation and its associated morbidity.

Keywords: fibrin, fibrinolysis, lung disorder, pathologic process, plasminogen activator inhibitor, pleural cavity effusion, RNA binding protein, mesothelioma, posttranscriptional RNA processing, protein binding, protein biosynthesis, protein protein interaction, receptor expression, urokinase, cell line, human tissue, immunocytochemistry

Fibrinolytic Pathways In Lung Injury And Repair

Steven Idell, Chairman
University Of Texas Hlth Ctr At Tyler 11937 Us Highway 271 Tyler, Tx 75708

Grant 5P01HL076406-03 from National Heart, Lung, And Blood Institute, IRG: HLBP

Abstract: Abnormalities in the plasminogen activator (PA) pathways have been implicated in the pathogenesis of acute lung (All) and pleural injury. Recent interventional trials suggest that targeting these pathways can reduce mortality in sepsis and protect against acute lung or pleural injury. The Project Leaders of this PPG have developed evidence that these pathways can influence ALI and pleural injury through newly recognized mechanisms. However the pathogenic mechanisms that link the PA pathways to ALI and pleural injury are poorly understood and are likely to involve non-proteolytic signal-transducing pathways. Our thematic objective is to address this gap by defining novel mechanisms by which urokinase (uPA), its receptor (uPAR), other novel uPA receptors and its inhibitor PAI-1 influence the course of inflammation, remodeling of transitional matrix and accelerated fibrosis in ALI and pleural injury. In Project 1, pathways that regulate PAI-1 and uPAR expression by the.mesothelium at the posttranscriptional level will be defined and a novel fibrinolytic intervention to prevent pleural loculation will be further evaluated. Project 2 will elucidate novel posttranscriptional mechanisms by which uPA and uPAR are regulated by the lung epithelium. Project 3 will define novel pathways by which uPA interacts with cell surface signaling adapter molecules to regulate pulmonary vasoconstriction and lung edema after ALI and ascertain the role of defensin in the process. These interactive projects derive from active programs directed by experienced Project Leaders and are now oriented to our thematic objective. In vitro, in vivo and interventional methods will be used. This PPG will accelerate the acquisition of new, clinically relevant information that will hasten the development of better treatments for ALI and/or pleural injury.

Keywords: fibrin, fibrinolysis, lung injury, regeneration, clinical research

Project start date: 2005-09-01

Project end date: 2010-08-31

5P01HL076406-03 (2007): $1471629

5P01HL076406-02 (2006): $1476651

CONTROL OF FIBRIN TURNOVER IN PLEURAL DISEASE

Steven Idell, Chairman
University Of Texas Hlth Ctr At Tyler 11937 Us Highway 271 Tyler, Tx 75708

Grant 5R01HL045018-13 from National Heart, Lung, And Blood Institute, IRG: LBPA

Abstract: Disordered fibrin turnover has been implicated in the pathogenesis of pleural inflammation and repair. We hypothesize the mesothelial cell responses play a crucial role in these events. Derangements of the regulation of plasminogen activator inhibitor -1 (PAI-1) and the urokinase receptor (uPAR) by pleural mesothelial cells appear to be critical determinants of locally impaired fibrinolysis and intrapleural remodeling after asbestos exposure or other forms of fibrosing pleuritis. Asbestos and cytokines implicated in asbestos-related or other forms of fibrosing pleuritis alter local regulation of these molecules, but the mechanisms responsible for these responses are poorly understood at this time. Our studies address these important gaps in our understanding of mesothelial cell biology. We will determine mechanisms by which mesothelial cells regulate PAI-1 and uPAR expression in response to asbestos or other mediators of fibrosing pleural injury. Direct and indirect mechanisms by which asbestos influences expression of these molecules and uPA-mediated responses by mesthelial cells will be defined. Interactions of these pathways with procoagulant pathways of mesothelial cells will also be elucidated in vitro. An established rabbit model of tetracycline (TCN)-induced pleural injury will be used to define in vivo responses of the uPA-uPAR system in pleural mesothelial cells that contribute to locally disordered fibrinolysis and pleural remodeling. We have recently determined that PAI-1 as well as uPAR are regulated at the posttranscriptional level by mesothelial cells and will now determine the mechanisms by which these pathways respond to asbestos or other mediators of fibrosing pleuritis. The role of these pathways in asbestos- induced responses of mesothelial cells or in TCN-induced pleural injury will be determined. Lastly, we will use our preliminary data to develop novel interventional approaches to selectively prevent pleural fibrin deposition and pleural fibrosis. We will test the ability of these approaches to safely and effectively block pleural loculation and fibrosis in the rabbit model. To accomplish these goals, we will use an array of molecular, biochemical and immunohistochemical techniques, all of which are well-established in our laboratory. These studies will allow us to define the role of the mesothelial cell in the regulation of the uPA-uPAR system and to understand how these cells thereby contribute to pleural remodeling after injury. This work could identify better, clinically feasible therapeutic approaches to prevent pleural fibrosis.

Keywords: cellular pathology, fibrin, fibrinolysis, lung injury, pleural cavity, pleurisy, asbestos, cytokine, fibrinogen receptor, plasminogen activator inhibitor, receptor binding, receptor expression, respiratory epithelium, urokinase, wound healing, immunologic assay /test, laboratory rabbit, tissue /cell culture

Project start date: 1991-09-01

Project end date: 2005-08-31

5R01HL045018-13 (2004): $268124

5R01HL045018-12 (2003): $266140

5R01HL045018-10 (2001): $262344

2R01HL045018-09 (2000): $260528

5R01HL045018-08 (2000): $130059

5R01HL045018-07 (1999): $217969

MOLECULAR BIOLOGY AND PHYSIOLOGY OF HUMAN FACTOR VII

Steven Idell, Chairman
University Of Texas Hlth Ctr At Tyler 11937 Us Highway 271 Tyler, Tx 75708

Grant 5R01HL037770-07 from National Heart, Lung, And Blood Institute, IRG: HEM

Abstract: Cellular disruption resulting from trauma and cellular stimulation by a variety of biological agonists can initiate the expression of tissue factor, an obligatory integral membrane protein cofactor for factor VII activity, leading to hemostasis and thrombosis. Factor VII is the first zymogen or, in its activated form, factor VIIa is the first serine protease which in association with tissue factor and calcium form the extrinsic pathway macromolecular complex responsible for initiating blood coagulation. Substrates for this enzyme complex are factors IX and X, and factor VII can be activated by factors IXa, Xa or XIIa. Regulation of this initiation complex can be by the exposure and interaction of tissue factor to blood, the dynamics of factor VII and factor VIIa to compete with near equal affinity for binding to the cofactor and the inhibition of the formed complex by factor X(Xa) and a serum lipoprotein. Factor VIIa may also be inhibited by antithrombin III in the presence of heparin. Thus, the multiple interactions of factor VII implies that several regions on the surface of this molecule control its association, and provides the basis to study the structure function relationships of this protein with its activators, cofactor, substrates and inhibitors. Identification of these molecular loci on factor VII is the overall goal of this project. Protein-protein interactions require that the regions mediating the associations leading to specific function must be at the solvent accessible surface of the molecules. Further, antibody binding to antigen requires the antigenic determinant be exposed to the hydrated surface. We will undertake experiments to identify the structural regions on factor VII/VIIa with synthetic peptides corresponding to specific segments of the primary structure of factor VII. Physical and biochemical studies will used to measure peptide binding and inhibition of complex formation and function. Monoclonal antibodies raised to predetermined specificities will be used as an independent approach to assign an antigenic site with specific function. Definition of a structural abnormality on naturally occurring variants of factor VII will provide a third avenue of investigation. From these three independent approaches we should be able to assign specific loci on the topology of factor VII/VIIa which mediate its functional activities. Identification of sites of interaction may lead to the production of therapeutic agents (peptides and/or monoclonal antibodies) which can impede coagulation and regulate hemostasis.

Keywords: activation product, chemical structure function, coenzyme, enzyme structure, enzyme substrate, molecular biology, peptide hydrolase, zymogen, antigen, antithrombin III, binding protein, chemical binding, coagulation factor IX, coagulation factor VII, coagulation factor X, coagulation factor XII, enzyme complex, hemostasis, protease inhibitor, protein sequence, synthetic peptide, thromboplastin, bioassay, chromatography, human tissue, immunochemistry, laboratory mouse, laboratory rabbit, monoclonal antibody, travel

Project start date: 1986-05-01

Project end date: 1994-04-30

5R01HL037770-07 (1992): $102930

CONTROL OF FIBRIN TURNOVER IN PLEURAL DISEASE

Steven Idell, Chairman
University Of Texas Hlth Ctr At Tyler 11937 Us Highway 271 Tyler, Tx 75708

Grant 5R01HL045018-02 from National Heart, Lung, And Blood Institute, IRG: PTHA

Abstract: Adapted from  s .) Despite long standing recognition of the relation between pleural fibrin deposition and organization of pleural exudates, there is only limited information concerning the molecular basis of disordered fibrin turnover in pleural injury. Exudative pleural effusions in humans were found to be characterized by increased procoagulant and depressed fibrinolytic activities and pilot studies with cells from these pleural fluids suggest that the mesothelium plays a key role in fibrin deposition. Proposed studies will address the hypothesis that mesothelial cells contribute to abnormalities of fibrin turnover and promote fibrin deposition within the pleural space. The mechanisms and regulation of procoagulant and fibrinolytic activity in mesothelial cells and pleural fluids will be determined. These experiments will involve coagulation assays, functional and immunologic assays of plasminogen activators, characterization and assay of plasminogen activator inhibitors and antiplasmins. It was found that human pleural mesothelial cells assemble procoagulant complexes on their surface. The molecular mechanisms that regulate assembly of the extrinsic activation complex (tissue factor associated with factor VII/VIIa) and the prothrombinase complex (factor Xa associated with Va) at the mesothelial cell surface will be determined. Binding studies will determine binding parameters (Kd and number of sites), cation requirements and functional activity of both the extrinsic activation complex and the prothrombinase complex. Monoclonal antibodies to factors VII and X will be used to verify the number of molecules bound and to relate binding with functional activity. In addition, binding site specificity will be probed using a series of synthetic peptides that compete with factors VII and X. Because mesothelial cells have low functional fibrinolytic activity, and because they secrete high levels of plasminogen activator inhibitor (PAI-1), we suspect that PAI-1 from the mesothelium permits fibrin deposition. The mechanisms that regulate PAI-1 in cultures of human mesothelial cells will be determined. These experiments will focus on the expression of mRNA for PAI-1 and the duration of the message under various conditions, such as cellular interaction with cytokines and the extracellular matrix.

Keywords: blood coagulation, fibrin, fibrinolysis, lung disorder, pleural cavity effusion, body fluid, chemical binding, coagulation factor X, cytokine, extracellular matrix protein, mesoderm, messenger RNA, monoclonal antibody, pathology, plasminogen activator, prothrombin, SDS polyacrylamide gel electrophoresis, biopsy, human tissue, immunologic assay /test, oligonucleotide, polymerase chain reaction, tissue /cell culture

Project start date: 1991-09-01

Project end date: 1995-08-31

5R01HL045018-02 (1992): $169494

5R01HL045018-03 (1993): $178656

Fibrinolytic Pathways In Lung Injury And Repair

Steven Idell, Chairman
University Of Texas Hlth Ctr At Tyler

Grant 5P01HL076406-04 from National Heart, Lung, And Blood Institute, IRG: HLBP

Abstract: Abnormalities in the plasminogen activator (PA) pathways have been implicated in the pathogenesis of acute lung (All) and pleural injury. Recent interventional trials suggest that targeting these pathways can reduce mortality in sepsis and protect against acute lung or pleural injury. The Project Leaders of this PPG have developed evidence that these pathways can influence ALI and pleural injury through newly recognized mechanisms. However the pathogenic mechanisms that link the PA pathways to ALI and pleural injury are poorly understood and are likely to involve non-proteolytic signal-transducing pathways. Our thematic objective is to address this gap by defining novel mechanisms by which urokinase (uPA), its receptor (uPAR), other novel uPA receptors and its inhibitor PAI-1 influence the course of inflammation, remodeling of transitional matrix and accelerated fibrosis in ALI and pleural injury. In Project 1, pathways that regulate PAI-1 and uPAR expression by the.mesothelium at the posttranscriptional level will be defined and a novel fibrinolytic intervention to prevent pleural loculation will be further evaluated. Project 2 will elucidate novel posttranscriptional mechanisms by which uPA and uPAR are regulated by the lung epithelium. Project 3 will define novel pathways by which uPA interacts with cell surface signaling adapter molecules to regulate pulmonary vasoconstriction and lung edema after ALI and ascertain the role of defensin in the process. These interactive projects derive from active programs directed by experienced Project Leaders and are now oriented to our thematic objective. In vitro, in vivo and interventional methods will be used. This PPG will accelerate the acquisition of new, clinically relevant information that will hasten the development of better treatments for ALI and/or pleural injury

Keywords: fibrin, fibrinolysis, lung injury, regeneration clinical research

Project start date: 2005-09-01

Project end date: 2010-08-31

3P01HL076406-03S1 (2007): $77000

3P01HL076406-01A1S3 (2006): $8466

1P01HL076406-01A1 (2005): $1534853

3P01HL076406-01A1S1 (2005): $14025

CONTROL OF FIBRIN TURNOVER IN PLEURAL DISEASE

Steven Idell, Chairman
Medicineuniversity Of Texas Hlth Ctr At Tyler
11937 Us Highway 271
tyler, Tx 75708

Grant 2R01HL045018-05A1 from National Heart, Lung, And Blood Institute, IRG: LBPA

Project start date: 1991-09-01

Project end date: 2000-08-31

2R01HL045018-05A1 (1996): $201525

Expression and Regulation of Epithelial Na+ Channels by Nucleotides in Pleural Mesothelial Cells. Am J Respir Cell Mol Biol. 2008 Oct 16. [Epub ahead of print] PMID: 18927349

Shetty S, Padijnayayveetil J, Tucker T, Stankowska D, Idell S.

The Fibrinolytic System and the Regulation of Lung Epithelial Cell Proteolysis, Signaling and Cellular Viability. Am J Physiol Lung Cell Mol Physiol. 2008 Oct 3. [Epub ahead of print] PMID: 18836029

Idell S.

The pathogenesis of pleural space loculation and fibrosis. Curr Opin Pulm Med. 2008 Jul; 14( 4): 310-5. Review. PMID: 18520264

Shetty S, Shetty P, Idell S, Velusamy T, Bhandary YP, Shetty RS.

Regulation of plasminogen activator inhibitor-1 expression by tumor suppressor protein p53. J Biol Chem. 2008 Jul 11; 283( 28): 19570-80. Epub 2008 May 9. PMID: 18469003

Idell S, Azghani A, Chen S, Koenig K, Mazar A, Kodandapani L, Bdeir K, Cines D, Kulikovskaya I, Allen T.

Intrapleural low-molecular-weight urokinase or tissue plasminogen activator versus single-chain urokinase in tetracycline-induced pleural loculation in rabbits. Exp Lung Res. 2007 Oct-Nov; 33( 8-9): 419-40. PMID: 17994370

Idell S.

Expression profiling and disseminated intravascular coagulation: finding genes gone wild. Am J Respir Crit Care Med. 2007 Sep 15; 176( 6): 528-30. No abstract available. PMID: 17915353

Liu MC, Yasuda S, Idell S.

Sulfation of nitrotyrosine: biochemistry and functional implications. IUBMB Life. 2007 Oct; 59( 10): 622-7. Review. PMID: 17891604

Iakhiaev AV, Rezaie AR, Idell S.

Thrombomodulin-mediated catabolism of protein C by pleural mesothelial and vascular endothelial cells. Thromb Haemost. 2007 Sep; 98( 3): 627-34. PMID: 17849052

Shetty S, Velusamy T, Idell S, Shetty P, Mazar AP, Bhandary YP, Shetty RS.

Regulation of urokinase receptor expression by p53: novel role in stabilization of uPAR mRNA. Mol Cell Biol. 2007 Aug; 27( 16): 5607-18. Epub 2007 Jun 4. PMID: 17548471

Yasuda S, Idell S, Fu J, Carter G, Snow R, Liu MC.

Cigarette smoke toxicants as substrates and inhibitors for human cytosolic SULTs. Toxicol Appl Pharmacol. 2007 May 15; 221( 1): 13-20. Epub 2007 Feb 28. PMID: 17433394

Shetty S, Velusamy T, Idell S, Tang H, Shetty PK.

Regulation of urokinase receptor expression by protein tyrosine phosphatases. Am J Physiol Lung Cell Mol Physiol. 2007 Feb; 292( 2): L414-21. Epub 2006 Oct 6. PMID: 17028265

Yasuda S, Idell S, Liu MC.

Generation and release of nitrotyrosine O-sulfate by HepG2 human hepatoma cells upon SIN-1 stimulation: identification of SULT1A3 as the enzyme responsible. Biochem J. 2007 Jan 15; 401( 2): 497-503. PMID: 17002600

Idell S, Allen T, Chen S, Koenig K, Mazar A, Azghani A.

Intrapleural activation, processing, efficacy, and duration of protection of single-chain urokinase in evolving tetracycline-induced pleural injury in rabbits. Am J Physiol Lung Cell Mol Physiol. 2007 Jan; 292( 1): L25-32. Epub 2006 Sep 15. PMID: 16980376

Kwak SH, Wang XQ, He Q, Fang WF, Mitra S, Bdeir K, Ploplis VA, Xu Z, Idell S, Cines D, Abraham E.

Plasminogen activator inhibitor-1 potentiates LPS-induced neutrophil activation through a JNK-mediated pathway. Thromb Haemost. 2006 May; 95( 5): 829-35. PMID: 16676075

Iakhiaev A, Idell S.

Activation and degradation of protein C by primary rabbit pleural mesothelial cells. Lung. 2006 Mar-Apr; 184( 2): 81-8. PMID: 16622777

Welty-Wolf KE, Carraway MS, Ortel TL, Ghio AJ, Idell S, Egan J, Zhu X, Jiao JA, Wong HC, Piantadosi CA.

Blockade of tissue factor-factor X binding attenuates sepsis-induced respiratory and renal failure. Am J Physiol Lung Cell Mol Physiol. 2006 Jan; 290( 1): L21-31. Epub 2005 Aug 12. PMID: 16100288

Kwak SH, Mitra S, Bdeir K, Strassheim D, Park JS, Kim JY, Idell S, Cines D, Abraham E.

The kringle domain of urokinase-type plasminogen activator potentiates LPS-induced neutrophil activation through interaction with {alpha}V{beta}3 integrins. J Leukoc Biol. 2005 Oct; 78( 4): 937-45. Epub 2005 Jul 20. PMID: 16033814

Shetty S, Ganachari M, Liu MC, Azghani A, Muniyappa H, Idell S.

Regulation of urokinase receptor expression by phosphoglycerate kinase is independent of its catalytic activity. Am J Physiol Lung Cell Mol Physiol. 2005 Oct; 289( 4): L591-8. Epub 2005 Jun 10. PMID: 15951330

Iakhiaev A, Pendurthi U, Idell S.

Asbestos induces tissue factor in Beas-2B human lung bronchial epithelial cells in vitro. Lung. 2004; 182( 4): 251-64. PMID: 15636197

Mutsaers SE, Prele CM, Brody AR, Idell S.

Pathogenesis of pleural fibrosis. Respirology. 2004 Nov; 9( 4): 428-40. Review. PMID: 15612953

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