Steven Idell

University of Texas Health Science Center at Tyler, Tyler, Texas, United States

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Publications (140)700.08 Total impact

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    ABSTRACT: Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of the extrinsic coagulation cascade and its expression is reported to be relatively stable. Various pathophysiologic agents have been shown to influence TFPI activity either by regulating its expression or modifying the protein. It is not clear how TFPI activity is regulated in normal physiology or in injury. Because thrombin and TFPI are both locally elaborated in pleural injury, we sought to determine if thrombin could regulate TFPI in human pleural mesothelial cells (HPMCs). Thrombin significantly decreased TFPI mRNA and protein levels by greater than 70%. Further, thrombin-mediated down-regulation of TFPI promoted factor X activation by HPMCs. The ability of thrombin to significantly decrease TFPI mRNA and protein levels was maintained at nanomolar concentrations. Protease activated receptor (PAR)-1, a mediator of thrombin signaling, is detectable in the mesothelium in human and murine pleural injury. PAR-1 silencing blocked thrombin-mediated decrements of TFPI in HPMCs. Thrombin activates PI3K/Akt and NFκB signaling in HPMCs. Inhibition of PI3K (by PX-866) and NFκB (by SN50) prevented thrombin-mediated TFPI mRNA and protein down-regulation. These are the first studies to demonstrate that thrombin decreases TFPI expression in HPMCs. Our findings demonstrate a novel mechanism by which thrombin regulates TFPI expression in PMCs and promotes an unrestricted procoagulant response. Further, they suggest that interactions between PI3K and NFκB signaling pathways are linked in HPMCs and control TFPI expression. These findings raise the possibility that targeting of this pathway could limit the ability of the mesothelium to support extravascular fibrin deposition and organization associated with pleural injury.
    American Journal of Respiratory Cell and Molecular Biology 10/2014; · 4.15 Impact Factor
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    ABSTRACT: Epithelial sodium channels (ENaC) govern transepithelial salt and fluid homeostasis. ENaC contributes to polarization, apoptosis, epithelial-mesenchymal transformation etc. Fibrinolytic proteases play a crucial role in virtually all of these processes and are elaborated by the airway epithelium. We hypothesized that urokinase-like plasminogen activator (uPA) regulates ENaC function in airway epithelial cells and tested that possibility in primary murine tracheal epithelial cells (MTE). Both basal and cAMP-activated Na(+) flow through ENaC were significantly reduced in monolayers of uPA-deficient cells. The reduction in ENaC activity was further confirmed in basolateral membrane permeabilized cells. A decrease in the Na(+)/K(+)-ATPase activity in the basolateral membrane could contribute to the attenuation of ENaC function in intact monolayer cells. Dysfunctional fluid resolution was seen in uPA-disrupted cells. Administration of uPA and plasmin partially restores ENaC activity and fluid re-absorption by MTEs. ERK1/2, but not Akt phosphorylation was observed in the cells and lungs of uPA-deficient mice. On the other hand, cleavage of γ ENaC is significantly depressed in the lungs of uPA knockout mice vs those of wild type controls. Expression of caspase 8, however, did not differ between wild type and uPA(-/-) mice. In addition, uPA deficiency did not alter transepithelial resistance. Taken together, the mechanisms for the regulation of ENaC by uPA in MTEs include augmentation of Na(+)/K(+)-ATPase, proteolysis, and restriction of ERK1/2 phosphorylation. We demonstrate for the first time that ENaC may serve as a downstream signaling target by which uPA controls the biophysical profiles of airway fluid and epithelial function.
    American journal of physiology. Lung cellular and molecular physiology. 08/2014;
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    ABSTRACT: Introduction: Endogenous active plasminogen activator inhibitor 1 (PAI-1) was targeted in vivo with monoclonal antibodies (mAbs) that redirect its reaction with proteinases to the substrate branch. mAbs were used as an adjunct to prourokinase (scuPA) intrapleural fibrinolytic therapy (IPFT) of tetracycline-induced pleural injury in rabbits. Methods: Outcomes of scuPA IPFT (0.25 or 0.0625 mg/kg) with 0.5 mg/kg of mouse IgG or mAbs (MA-33H1F7 and MA-8H9D4) were assessed at 24h. Pleural fluid (PF) was collected at 0, 10, 20, 40 min and 24h after IPFT and analyzed for plasminogen activating (PA), urokinase (uPA), fibrinolytic activities, levels of total plasmin/plasminogen, α-macroglobulin (αM), mAbs/IgG antigens, free active uPA and αM/uPA complexes. Results:Anti-PAI-1 mAbs, but not mouse IgG, delivered with an 8-fold reduction in the minimal effective dose of scuPA (from 0.5 to 0.0625 mg/kg), improved the outcome of IPFT (p<0.05). mAbs and IgG were detectable in PFs at 24h. Compared to identical doses of scuPA alone or with IgG, treatment with scuPA and anti-PAI-1 mAbs generated higher PF uPA amidolytic and PA activities, faster formation of αM/uPA complexes, and slower uPA inactivation. However, PAI-1 targeting did not significantly affect intrapleural fibrinolytic activity or levels of total plasmin/plasminogen and αM antigens. Conclusions: Targeting PAI-1 did not induce bleeding and rendered otherwise ineffective doses of scuPA able to improve outcome in tetracycline-induced pleural injury. PAI-1 neutralizing mAbs improved IPFT by increasing the durability of intrapleural PA activity. These results suggest a novel, well-tolerated IPFT strategy that is tractable for clinical development.
    American Journal of Respiratory Cell and Molecular Biology 08/2014; · 4.15 Impact Factor
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    ABSTRACT: ABSTRACT Receptor tyrosine kinases, including the epidermal growth factor receptors (EGFR), are able to activate the mitogen activated protein kinases (MAPK) via several adaptor proteins and protein kinases such as Raf. EGFR can be activated by a variety of extracellular stimuli including neutrophil elastase, but we are aware of no report as to whether Pseudomonas aeruginosa bacterium produced elastase (PE) could elicit such signaling through EGFR activation. We sought to test the inference that PE modulates inflammatory responses in human lung fibroblasts and that the process occurs by activation of the EGFR/MAPK pathways. We utilized interleukin (IL)-8 cytokine expression as a pathway specific end-point measure of fibroblasts inflammatory response to PE. Western blot analysis was performed to detect phosphorylation of EGFR and signal transduction intermediates. Northern blot, real time polymerase chain reaction (PCR), and ELISA methods were utilized to determine cytokine gene expression levels. We found that PE induces phosphorylation of the EGFR and the extracellular signal-regulated proteins (ERK1/2) of the MAPK pathway, and nuclear translocation of transcription factor kappa B (NF-ĸB). Furthermore, enzymatically active PE enhances IL-8 mRNA and protein secretion. Pretreatment of the cells with specific inhibitors of EGFR, MEK, and NF-ĸB markedly attenuated the PE-induced signal proteins phosphorylation and IL-8 gene expression and protein secretion. Collectively, the data show that PE produced by Pseudomonas aeruginosa can modulate lung inflammation by exploiting the EGFR/ERK signaling cascades and enhancing IL-8 production in the lungs via NF-ĸB activation.
    Microbiology 01/2014; · 3.06 Impact Factor
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    ABSTRACT: Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD) are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 5 (AQP5) proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI) and II (ATII)-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3) was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.
    PLoS ONE 01/2014; 9(10):e109725. · 3.53 Impact Factor
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    ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases (PKD1, PKD2 and PKD3) were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease.
    PLoS ONE 01/2014; 9(7):e101983. · 3.53 Impact Factor
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    ABSTRACT: Local derangements of fibrin turnover and plasminogen activator inhibitor-1 (PAI-1) have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black-bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic (Tg) mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes and compliance were comparable to WT. CBB-injury in PAI-1-/- mice significantly increased visceral pleural thickness (P<0.001) and lung compliance (P<0.01) while decreasing lung volumes. Collagen, α-SMA, and tissue factor were increased in the thickened visceral pleura of PAI-1-/- mice. Colocalization of α-SMA and calretinin within pleural mesothelial cells (PMCs) was increased in CBB injured PAI-1-/- mice. Thrombin, FXa, plasmin and urokinase induced meso-mesenchymal transition (MesoMT), TF expression and activity in primary human PMCs. In PAI-1-/- mice, D-dimer and thrombin-antithrombin complex concentrations were increased in pleural lavage fluids. The results demonstrate that PAI-1 regulates CBB-induced pleural injury severity via unrestricted fibrinolysis and cross-talk with coagulation proteases. While overexpression of PAI-1 augments intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction.
    American Journal of Respiratory Cell and Molecular Biology 09/2013; · 4.15 Impact Factor
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    ABSTRACT: Rationale: Intrapleural processing of prourokinase (scuPA) in tetracycline (TCN)-induced pleural injury in rabbits was evaluated to better understand the mechanisms governing successful scuPA-based intrapleural fibrinolytic therapy (IPFT); capable of clearing pleural adhesions in this model. Methods: Pleural fluid (PF) was withdrawn 0-80 min and 24 h after IPFT with scuPA (0-0.5 mg/kg) and activities of free urokinase (uPA), plasminogen activator inhibitor 1 (PAI-1) and uPA complexed with α-macroglobulin (αM) were assessed. Similar analyses were performed using PFs from patients with empyema, parapneumonic, and malignant pleural effusions. Results: The peak of uPA activity (5-40 min) reciprocally correlated with the dose of intrapleural scuPA. Endogenous active PAI-1 (10-20 nM) decreased the rate of intrapleural scuPA activation. The slow step of intrapleural inactivation of free uPA (t1/2(β)=40±10 min) was dose-independent and 6.7-fold slower than in blood. Up to 260±70 nM of αM/uPA formed in vivo (kass=580±60 M(-1)s(-1)). αM/uPA and products of its degradation contributed to durable intrapleural plasminogen activation up to 24 h after IPFT. Active PAI-1, active α2M, and α2M/uPA found in empyema, pneumonia, and malignant PFs demonstrate the capacity to support similar mechanisms in humans. Conclusion: Intrapleural scuPA processing differs from that in the bloodstream and includes (i) dose-dependent control of scuPA activation by endogenous active PAI-1; (ii) two-step inactivation of free uPA with simultaneous formation of αM/uPA; (iii) slow intrapleural degradation of αM/uPA releasing active free uPA. This mechanism offers potential clinically relevant advantages that may enhance the bioavailability of intrapleural scuPA and may mitigate the risk of bleeding complications.
    AJP Lung Cellular and Molecular Physiology 08/2013; · 3.52 Impact Factor
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    ABSTRACT: Plasminogen activator inhibitor 1 (PAI-1) levels are elevated in a number of life threatening conditions and often correlate with unfavorable outcomes. Spontaneous inactivation due to active to latent transition limits PAI-1 activity in vivo. While endogenous vitronectin (Vn) stabilizes PAI-1 by 1.5-2.0 fold, further stabilization occurs in a "Molecular Sandwich" Complex (MSC) where a ligand that restricts the exposed reactive center loop is bound to PAI-1/Vn. The effects of S195A two chain urokinase (tcuPA) and Vn on inactivation of wild type (wt) glycosylated (Gl-PAI-1), non-glycosylated (rPAI-1) and Q123K PAI-1 (lacks Vn binding) were studied. S195A tcuPA decreased the rate constant (kL) for spontaneous inactivation at 37°C for rPAI-1, Q123K, and Gl-PAI-1 by 6.7, 3.4, and 7.8 fold, respectively, and with both S195A tcuPA and Vn by 66.7, 5.5, and 103.3 fold. Analysis of the temperature dependences of kL revealed a synergistic increase in the Gibbs free activation energy for spontaneous inactivation of wt Gl-PAI-1 and rPAI-1 in MSC from 99.8 and 96.1 to 111.3 and 107.0 kJ/mol, due to an increase in the activation enthalpy and a decrease in the activation entropy, respectively. Anti-PAI-1 mAbs (MA-56A7C10, MA-42A2F6, MA-44E4) competing with proteinase also stabilize PAI-1/Vn. The rate of inhibition of target proteinases by MSCs, with stoichiometry close to unity, was limited by the dissociation (k=10(-4)10(-3) s(-1)) of S195A tcuPA or mAb. The stabilization of PAI-1 in MSCs in vivo may potentiate uncontrolled thrombosis or extravascular fibrin deposition, suggesting a new paradigm for using PAI-1 inhibitors and novel potential targets for therapy.
    Biochemistry 06/2013; · 3.38 Impact Factor
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    ABSTRACT: Alveolar type II epithelial cell (ATII) apoptosis and proliferation of mesenchymal cells are the hallmarks of idiopathic pulmonary fibrosis, a devastating disease of unknown cause characterized by alveolar epithelial injury and progressive fibrosis. We used a mouse model of bleomycin (BLM)-induced lung injury to understand the involvement of p53-mediated changes in urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) levels in the regulation of alveolar epithelial injury. We found marked induction of p53 in ATII cells from mice exposed to BLM. Transgenic mice expressing transcriptionally inactive dominant negative p53 in ATII cells showed augmented apoptosis, whereas those deficient in p53 resisted BLM-induced ATII cell apoptosis. Inhibition of p53 transcription failed to suppress PAI-1 or induce uPA mRNA in BLM-treated ATII cells. ATII cells from mice with BLM injury showed augmented binding of p53 to uPA, uPA receptor (uPAR), and PAI-1 mRNA. p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions neither interfered with p53 DNA binding activity nor p53-mediated promoter transactivation. However, increased expression of p53 binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions in ATII cells suppressed PAI-1 and induced uPA after BLM treatment, leading to inhibition of ATII cell apoptosis and pulmonary fibrosis. Our findings indicate that disruption of p53-fibrinolytic system cross talk may serve as a novel intervention strategy to prevent lung injury and pulmonary fibrosis.
    American Journal Of Pathology 05/2013; · 4.60 Impact Factor
  • Torry Tucker, Steven Idell
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    ABSTRACT: Lung and pleural injuries are characterized by inflammation, fibrinous transitional matrix deposition, and ultimate scarification. The accumulation of extravascular fibrin is due to concurrently increased local coagulation and decreased fibrinolysis, the latter mainly as a result of increased plasminogen activator inhibitor-1 (PAI-1) expression. Therapeutic targeting of disordered fibrin turnover has long been used for the treatment of pleural disease. Intrapleural fibrinolytic therapy has been found to be variably effective in clinical trials, which likely reflects empiric dosing that does not account for the wide variation in pleural fluid PAI-1 levels in individual patients. The incidence of empyema is increasing, providing a strong rationale to identify more effective, nonsurgical treatment to improve pleural drainage and patient outcomes. Therapeutics designed to resist inhibition by PAI-1 are in development for the treatment of pleural loculation and impaired drainage. The efficacy and safety of these strategies remains to be proven in clinical trial testing. Fibrinolytic therapy administered via the airway has also been proposed for the treatment of acute lung injury, but this approach has not been rigorously validated and is not part of routine clinical management at this time. Challenges to airway delivery of fibrinolysins relate to bioavailability, distribution, and dosing of the interventional agents.
    Seminars in Thrombosis and Hemostasis 03/2013; · 4.22 Impact Factor
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    ABSTRACT: The level of active urokinase (uPA) is decreased in lung fluids of patients with acute lung injury / acute respiratory distress syndrome (ALI/ARDS) whereas α(2)-macroglobulin (α(2)-M), a plasma proteinase inhibitor, is a major component of these fluids. Since there have been reports describing the ability of α(2)-M to form complexes with uPA in vitro, we hypothesized that α(2)-M may interact with uPA in the lung to modulate its biological activity. Pulmonary edema fluids and lung tissues from patients with ALI/ARDS were evaluated for the presence of uPA associated with α(2)-M. Complexes between α(2)-M and uPA were detected in alveolar edema fluids as well as in lungs of patients with ALI/ARDS where they were located mainly in close proximity to epithelial cells. While uPA bound to α(2)-M retains its amidolytic activity towards low molecular weight substrates, it is not inhibited by its main physiological inhibitor, plasminogen activator inhibitor 1. We also investigated functional consequences of formation of complexes between uPA and α(2)-M in vitro. We found that when α(2)-M:uPA complexes were added to cultures of human bronchial epithelial cell (BEAS-2B), activation of nuclear factor κB as well as production of interleukin-6 and -8 was substantially suppressed compared to the addition of uPA alone. Our findings indicate for the first time that the function of uPA in patients with ALI/ARDS may be modulated by α(2)-M and that the effects may include the regulation of the fibrinolytic and signaling activities of uPA.
    AJP Lung Cellular and Molecular Physiology 10/2012; · 3.52 Impact Factor
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    ABSTRACT: 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 non-epithelial tissues implies a range of physiological functions. The altered expression of SCNN1D is associated with numerous pathologic 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.
    AJP Lung Cellular and Molecular Physiology 09/2012; · 3.52 Impact Factor
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    ABSTRACT: The low-density lipoprotein receptor-related protein 1 (LRP-1) binds and can internalize a diverse group of ligands, including members of the fibrinolytic pathway, urokinase plasminogen activator (uPA), and its receptor, uPAR. In this study, we characterized the role of LRP-1 in uPAR processing, collagen synthesis, proteolysis, and migration in pleural mesothelial cells (PMCs). When PMCs were treated with the proinflammatory cytokines TNF-α and IL-1β, LRP-1 significantly decreased at the mRNA and protein levels (70 and 90%, respectively; P < 0.05). Consequently, uPA-mediated uPAR internalization was reduced by 80% in the presence of TNF-α or IL-1β (P < 0.05). In parallel studies, LRP-1 neutralization with receptor-associated protein (RAP) significantly reduced uPA-dependent uPAR internalization and increased uPAR stability in PMCs. LRP-1-deficient cells demonstrated increased uPAR t(1/2) versus LRP-1-expressing PMCs. uPA enzymatic activity was also increased in LRP-1-deficient and neutralized cells, and RAP potentiated uPA-dependent migration in PMCs. Collagen expression in PMCs was also induced by uPA, and the effect was potentiated in RAP-treated cells. These studies indicate that TNF-α and IL-1β regulate LRP-1 in PMCs and that LRP-1 thereby contributes to a range of pathophysiologically relevant responses of these cells.
    American Journal of Respiratory Cell and Molecular Biology 02/2012; 46(2):196-206. · 4.15 Impact Factor
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    ABSTRACT: Aging increases susceptibility to infection, in part because thymic involution culminates in reduced naïve T-lymphocyte output. Thymic epithelial cells (TECs) are critical to ensure normal maturation of thymocytes and production of peripheral T cells. The forkhead-class transcription factor, encoded by FoxN1, regulates development, differentiation, and function of TECs, both in the prenatal and postnatal thymus. We recently showed that expression of FoxN1, by keratin 14 (K14)-expressing epithelial cells is essential for maintenance of thymic medullary architecture, and deletion of FoxN1 in K14 promoter-driven TECs inhibited development of mature TECs and reduced the number of total thymocytes. These findings are reminiscent of changes observed during normal thymic aging. In the current report, we compared the effects of K14-driven FoxN1 deletion on peripheral T cell function in response to influenza virus infection with those associated with normal aging in a mouse model. FoxN1-deleted mice had reduced numbers of peripheral CD62L+CD44- naïve T-cells. In addition, during influenza infection, these animals had reduced antigen-specific CD8+ T-cell and IgG responses to influenza virus, combined with increased lung injury, weight loss and mortality. These findings paralleled those observed in aged wild type mice, providing the first evidence that K14-mediated FoxN1 deletion causes changes in T-cell function that mimic those in aging during an immune response to challenge with an infectious agent.
    PLoS ONE 01/2012; 7(4):e34681. · 3.53 Impact Factor
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    ABSTRACT: Urokinase-type plasminogen activator (uPA) is expressed by lung epithelial cells and regulates fibrin turnover and epithelial cell viability. PMA, LPS, and TNF-alpha, as well as uPA itself, induce uPA expression in lung epithelial cells. PMA, LPS, and TNF-alpha induce uPA expression through increased synthesis as well as stabilization of uPA mRNA, while uPA increases its own expression solely through uPA mRNA stabilization. The mechanism by which lung epithelial cells regulate uPA expression at the level of mRNA stability is unclear. To elucidate this process, we sought to characterize protein-uPA mRNA interactions that regulate uPA expression. Regulation of uPA at the level of mRNA stability involves the interaction of a ~40 kDa cytoplasmic-nuclear shuttling protein with a 66 nt uPA mRNA 3'UTR sequence. We purified the uPA mRNA 3'UTR binding protein and identified it as ribonucleotide reductase M2 (RRM2). We expressed recombinant RRM2 and confirmed its interaction with a specific 66 nt uPA 3'UTR sequence. Immunoprecipitation of cell lysates with anti-RRM2 antibody and RT-PCR for uPA mRNA confirmed that RRM2 binds to uPA mRNA. Treatment of Beas2B cells with uPA or LPS attenuated RRM2-endogenous uPA mRNA interactions, while overexpression of RRM2 inhibited uPA protein and mRNA expression through destabilization of uPA mRNA. LPS exposure of lung epithelial cells translocates RRM2 from the cytoplasm to the nucleus in a time-dependent manner, leading to stabilization of uPA mRNA. This newly recognized pathway could influence uPA expression and a broad range of uPA-dependent functions in lung epithelial cells in the context of lung inflammation and repair.
    Biochemistry 12/2011; 51(1):205-13. · 3.38 Impact Factor
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    ABSTRACT: The increased levels of extracellular DNA found in a number of disorders involving dysregulation of the fibrinolytic system may affect interactions between fibrinolytic enzymes and inhibitors. Double-stranded (ds) DNA and oligonucleotides bind tissue-(tPA) and urokinase (uPA)-type plasminogen activators, plasmin, and plasminogen with submicromolar affinity. The binding of enzymes to DNA was detected by EMSA, steady-state, and stopped-flow fluorimetry. The interaction of dsDNA/oligonucleotides with tPA and uPA includes a fast bimolecular step, followed by two monomolecular steps, likely indicating slow conformational changes in the enzyme. DNA (0.1-5.0 μg/ml), but not RNA, potentiates the activation of Glu- and Lys-plasminogen by tPA and uPA by 480- and 70-fold and 10.7- and 17-fold, respectively, via a template mechanism similar to that known for fibrin. However, unlike fibrin, dsDNA/oligonucleotides moderately affect the reaction between plasmin and α(2)-antiplasmin and accelerate the inactivation of tPA and two chain uPA by plasminogen activator inhibitor-1 (PAI-1), which is potentiated by vitronectin. dsDNA (0.1-1.0 μg/ml) does not affect the rate of fibrinolysis by plasmin but increases by 4-5-fold the rate of fibrinolysis by Glu-plasminogen/plasminogen activator. The presence of α(2)-antiplasmin abolishes the potentiation of fibrinolysis by dsDNA. At higher concentrations (1.0-20 μg/ml), dsDNA competes for plasmin with fibrin and decreases the rate of fibrinolysis. dsDNA/oligonucleotides incorporated into a fibrin film also inhibit fibrinolysis. Thus, extracellular DNA at physiological concentrations may potentiate fibrinolysis by stimulating fibrin-independent plasminogen activation. Conversely, DNA could inhibit fibrinolysis by increasing the susceptibility of fibrinolytic enzymes to serpins.
    Journal of Biological Chemistry 12/2011; 286(49):41949-62. · 4.65 Impact Factor
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    ABSTRACT: Alveolar type II (ATII) cell apoptosis and depressed fibrinolysis that promotes alveolar fibrin deposition are associated with acute lung injury (ALI) and the development of pulmonary fibrosis (PF). We therefore sought to determine whether p53-mediated inhibition of urokinase-type plasminogen activator (uPA) and induction of plasminogen activator inhibitor-1 (PAI-1) contribute to ATII cell apoptosis that precedes the development of PF. We also sought to determine whether caveolin-1 scaffolding domain peptide (CSP) reverses these changes to protect against ALI and PF. Tissues as well as isolated ATII cells from the lungs of wild-type (WT) mice with BLM injury show increased apoptosis, p53, and PAI-1, and reciprocal suppression of uPA and uPA receptor (uPAR) protein expression. Treatment of WT mice with CSP reverses these effects and protects ATII cells against bleomycin (BLM)-induced apoptosis whereas CSP fails to attenuate ATII cell apoptosis or decrease p53 or PAI-1 in uPA-deficient mice. These mice demonstrate more severe PF. Thus p53 is increased and inhibits expression of uPA and uPAR while increasing PAI-1, changes that promote ATII cell apoptosis in mice with BLM-induced ALI. We show that CSP, an intervention targeting this pathway, protects the lung epithelium from apoptosis and prevents PF in BLM-induced lung injury via uPA-mediated inhibition of p53 and PAI-1.
    AJP Lung Cellular and Molecular Physiology 12/2011; 302(5):L463-73. · 3.52 Impact Factor
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    ABSTRACT: Malignant pleural mesothelioma (MPM) is a rare cancer that is refractory to current treatments. It is characterized by robust transitional fibrin deposition that is in part promoted by tumor cells. MPM cells express tissue factor (TF) and its inhibitor; tissue factor pathway inhibitor (TFPI), but their contribution to the pathogenesis of MPM has been unclear. We found that REN MPM cells fail to express TFPI. Based on the tumor growth promoting properties of TF, we hypothesized that stable transfection of TFPI into REN MPM cells would decrease their aggressiveness. We tested our hypothesis using in vitro, in vivo and ex vivo analyses. TFPI knock-in decreased proliferation, invasion and TF activity of REN cells in vitro. REN TFPI knock-in cells, empty vector and naïve controls were next injected intrapleurally in nude mice. TFPI expression significantly decreased tissue invasion, inflammation, fibrin and collagen deposition associated with tumor tissues, pleural effusions, and tumor burden. In ex vivo analyses, REN cells were cultured from the harvested tumors. TFPI over-expression was maintained in cells propagated from the TFPI knock-in tumors and attenuated activation of Factor X and tumor cell invasiveness. These analyses demonstrate that TFPI reduces the aggressiveness of MPM in vitro, in vivo and ex vivo and that the effect involves inhibition of TF procoagulant activity. These observations suggest the possibility that the TF-TFPI interaction represents a novel therapeutic target for the treatment of MPM.
    American Journal of Respiratory Cell and Molecular Biology 08/2011; · 4.15 Impact Factor
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    ABSTRACT: Malignant pleural mesothelioma (MPM) is a rare cancer that is refractory to current treatments. It is characterized by a robust deposition of transitional fibrin that is in part promoted by tumor cells. MPM cells express tissue factor (TF) and the tissue factor pathway inhibitor (TFPI), but their contribution to the pathogenesis of MPM has been unclear. We found that REN MPM cells fail to express TFPI. Based on the tumor growth-promoting properties of TF, we hypothesized that the stable transfection of TFPI into REN MPM cells would decrease their aggressiveness. We tested our hypothesis using in vitro, in vivo, and ex vivo analyses. TFPI knock-in decreased the proliferation, invasion, and TF activity of REN cells in vitro. REN TFPI knock-in cells, empty vector, and naive control cells were next injected intrapleurally into nude mice. The expression of TFPI significantly decreased tissue invasion, inflammation, and the deposition of fibrin and collagen associated with tumor tissue, pleural effusions, and tumor burden. In ex vivo analyses, REN cells were cultured from harvested tumors. The overexpression of TFPI was maintained in cells propagated from TFPI knock-in tumors, and attenuated the activation of Factor X and the invasiveness of tumor cells. These analyses demonstrate that TFPI reduces the aggressiveness of MPM in vitro and in vivo, and that its effect involves the inhibition of TF procoagulant activity. These observations suggest that the interactions of TF and TFPI represent a novel therapeutic target in the treatment of MPM.
    American Journal of Respiratory Cell and Molecular Biology 08/2011; 46(2):173-9. · 4.15 Impact Factor

Publication Stats

2k Citations
700.08 Total Impact Points

Institutions

  • 1988–2014
    • University of Texas Health Science Center at Tyler
      Tyler, Texas, United States
    • Temple University
      • Sol Sherry Thrombosis Research Center (SSTRC)
      Philadelphia, PA, United States
  • 2011
    • University of Texas at Tyler
      Tyler, Texas, United States
    • University of Pennsylvania
      • Department of Pathology and Laboratory Medicine
      Philadelphia, PA, United States
  • 2007–2011
    • University of Toledo
      • Department of Pharmacology
      Toledo, OH, United States
    • Austin Peay State University
      Clarksville, Tennessee, United States
  • 2010
    • University of California, San Francisco
      • Division of Hospital Medicine
      San Francisco, CA, United States
  • 2009
    • Northwestern University
      • Chemistry of Life Processes Institute
      Evanston, Illinois, United States
  • 2004
    • University of Western Australia
      Perth City, Western Australia, Australia
  • 2003
    • University of Colorado
      • Division of Pulmonary Sciences and Critical Care Medicine
      Denver, CO, United States
  • 2000
    • Washington University in St. Louis
      San Luis, Missouri, United States
  • 1995
    • Medical University of South Carolina
      • Department of Medicine
      Charleston, SC, United States
  • 1992
    • University of Maryland, Baltimore
      • Department of Pediatrics
      Baltimore, MD, United States