Targeting the PAI-1 Mechanism with a Small Peptide Increases the Efficacy of Alteplase in a Rabbit Model of Chronic Empyema.

The incidence of empyema is increasing and associated with a mortality rate of 20% in patients older than 65 years. Since 30% of patients with advanced empyema have contraindications to surgical treatment, novel, low-dose, pharmacological treatments are needed. A Streptococcus pneumoniae-induced rabbit model of chronic empyema recapitulates the progression, loculation, fibrotic repair, and pleural thickening of human disease. Treatment with single chain (sc) urokinase (scuPA) or tissue type (sctPA) plasminogen activators in doses 1.0-4.0 mg/kg were only partially effective in this model. Docking Site Peptide (DSP; 8.0 mg/kg), which decreased the dose of sctPA for successful fibrinolytic therapy in acute empyema model did not improve efficacy in combination with 2.0 mg/kg scuPA or sctPA. However, a two-fold increase in either sctPA or DSP (4.0 and 8.0 mg/kg or 2.0 and 16.0 mg/kg sctPA and DSP, respectively) resulted in 100% effective outcome. Thus, DSP-based Plasminogen Activator Inhibitor 1-Targeted Fibrinolytic Therapy (PAI-1-TFT) of chronic infectious pleural injury in rabbits increases the efficacy of alteplase rendering ineffective doses of sctPA effective. PAI-1-TFT represents a novel, well-tolerated treatment of empyema that is amenable to clinical introduction. The chronic empyema model recapitulates increased resistance of advanced human empyema to fibrinolytic therapy, thus allowing for studies of muti-injection treatments.

A Novel Rabbit Model of Retained Hemothorax with Pleural Organization.

Retained hemothorax (RH) is a commonly encountered and potentially severe complication of intrapleural bleeding that can organize with lung restriction. Early surgical intervention and intrapleural fibrinolytic therapy have been advocated. However, the lack of a reliable, cost-effective model amenable to interventional testing has hampered our understanding of the role of pharmacological interventions in RH management. Here, we report the development of a new RH model in rabbits. RH was induced by sequential administration of up to three doses of recalcified citrated homologous rabbit donor blood plus thrombin via a chest tube. RH at 4, 7, and 10 days post-induction (RH4, RH7, and RH10, respectively) was characterized by clot retention, intrapleural organization, and increased pleural rind, similar to that of clinical RH. Clinical imaging techniques such as ultrasonography and computed tomography (CT) revealed the dynamic formation and resorption of intrapleural clots over time and the resulting lung restriction. RH7 and RH10 were evaluated in young (3 mo) animals of both sexes. The RH7 recapitulated the most clinically relevant RH attributes; therefore, we used this model further to evaluate the effect of age on RH development. Sanguineous pleural fluids (PFs) in the model were generally small and variably detected among different models. The rabbit model PFs exhibited a proinflammatory response reminiscent of human hemothorax PFs. Overall, RH7 results in the consistent formation of durable intrapleural clots, pleural adhesions, pleural thickening, and lung restriction. Protracted chest tube placement over 7 d was achieved, enabling direct intrapleural access for sampling and treatment. The model, particularly RH7, is amenable to testing new intrapleural pharmacologic interventions, including iterations of currently used empirically dosed agents or new candidates designed to safely and more effectively clear RH.

Precision targeting of the plasminogen activator inhibitor-1 mechanism increases efficacy of fibrinolytic therapy in empyema.

Plasminogen activator inhibitor-1 (PAI-1) is an endogenous irreversible inhibitor of tissue-type (tPA) and urokinase (uPA) plasminogen activators. PAI-1-targeted fibrinolytic therapy (PAI-1-TFT) is designed to decrease the therapeutic dose of tPA and uPA, attenuating the risk of bleeding and other complications. Docking site peptide (DSP) mimics the part of the PAI-1 reactive center loop that interacts with plasminogen activators, thereby affecting the PAI-1 mechanism. We used DSP for PAI-1-TFT in two rabbit models: chemically induced pleural injury and Streptococcus pneumoniae induced empyema. These models feature different levels of inflammation and PAI-1 expression. PAI-1-TFT with DSP (2.0 mg/kg) converted ineffective doses of single chain (sc) tPA (72.5 µg/kg) and scuPA (62.5 µg/kg) into effective ones in chemically induced pleural injury. DSP (2.0 mg/kg) was ineffective in S. pneumoniae empyema, where the level of PAI-1 is an order of magnitude higher. DSP dose escalation to 8.0 mg/kg resulted in effective PAI-1-TFT with 0.25 mg/kg sctPA (1/8th of the effective dose of sctPA alone) in empyema. There was no increase in the efficacy of scuPA. PAI-1-TFT with DSP increases the efficacy of fibrinolytic therapy up to 8-fold in chemically induced (sctPA and scuPA) and infectious (sctPA) pleural injury in rabbits. PAI-1 is a valid molecular target in our model of S. pneumoniae empyema in rabbits, which closely recapitulates key characteristics of empyema in humans. Low-dose PAI-1-TFT is a novel interventional strategy that offers the potential to improve fibrinolytic therapy for empyema in clinical practice.

From Bedside to the Bench-A Call for Novel Approaches to Prognostic Evaluation and Treatment of Empyema.

Empyema, a severe complication of pneumonia, trauma, and surgery is characterized by fibrinopurulent effusions and loculations that can result in lung restriction and resistance to drainage. For decades, efforts have been focused on finding a universal treatment that could be applied to all patients with practice recommendations varying between intrapleural fibrinolytic therapy (IPFT) and surgical drainage. However, despite medical advances, the incidence of empyema has increased, suggesting a gap in our understanding of the pathophysiology of this disease and insufficient crosstalk between clinical practice and preclinical research, which slows the development of innovative, personalized therapies. The recent trend towards less invasive treatments in advanced stage empyema opens new opportunities for pharmacological interventions. Its remarkable efficacy in pediatric empyema makes IPFT the first line treatment. Unfortunately, treatment approaches used in pediatrics cannot be extrapolated to empyema in adults, where there is a high level of failure in IPFT when treating advanced stage disease. The risk of bleeding complications and lack of effective low dose IPFT for patients with contraindications to surgery (up to 30%) promote a debate regarding the choice of fibrinolysin, its dosage and schedule. These challenges, which together with a lack of point of care diagnostics to personalize treatment of empyema, contribute to high (up to 20%) mortality in empyema in adults and should be addressed preclinically using validated animal models. Modern preclinical studies are delivering innovative solutions for evaluation and treatment of empyema in clinical practice: low dose, targeted treatments, novel biomarkers to predict IPFT success or failure, novel delivery methods such as encapsulating fibrinolysin in echogenic liposomal carriers to increase the half-life of plasminogen activator. Translational research focused on understanding the pathophysiological mechanisms that control 1) the transition from acute to advanced-stage, chronic empyema, and 2) differences in outcomes of IPFT between pediatric and adult patients, will identify new molecular targets in empyema. We believe that seamless bidirectional communication between those working at the bedside and the bench would result in novel personalized approaches to improve pharmacological treatment outcomes, thus widening the window for use of IPFT in adult patients with advanced stage empyema.

Perfluorochemical-facilitated plasminogen activator delivery to the airways: A novel treatment for inhalational smoke-induced acute lung injury.

BACKGROUND: Effective clinical management of airway clot and fibrinous cast formation of severe inhalational smoke-induced acute lung injury (ISALI) is lacking. Aerosolized delivery of tissue plasminogen activator (tPA) is confounded by airway bleeding; single-chain urokinase plasminogen activator (scuPA) moderated this adverse effect and supported transient improvement in gas exchange and lung mechanics. However, neither aerosolized plasminogen activator (PA) yielded durable improvements in physiologic responses or reduction in cast burden. Here, we hypothesized that perfluorochemical (PFC) liquids would facilitate PA distribution and sustain improvements in physiologic outcomes in ISALI. METHODS: Spontaneously breathing adult sheep (n = 36) received anesthesia and analgesia and were instrumented, exposed to cotton smoke inhalation, and supported by mechanical ventilation for 48 h. Groups (n = 6/group) were studied without supplemental treatment, or, starting 4 h post injury, they received intratracheal low volume (8 mL) PFC liquid alone or a dose range of tPA/PFC or scuPA/PFC suspensions (4 or 8 mg in 8 mL PFC) every 8 h. Outcomes were evaluated by sequential measurements of cardiopulmonary parameters, lung histomorphology, and biochemical analyses of bronchoalveolar lavage fluid. RESULTS: Dose-response and PA-type comparisons of outcomes demonstrated sustained superiority with low-volume PFC suspensions of scuPA over tPA or PFC alone, favoring the highest dose of scuPA/PFC suspension over lower doses, without airway bleeding. CONCLUSIONS: We propose that this improved profile over previously reported aerosolized delivery is likely related to improved dose distribution. Sustained salutary responses to scuPA/PFC suspension delivery in this translational model are encouraging and support the possibility that the observed outcomes could be of clinical importance.

Molecular mechanism of two nanobodies that inhibit PAI-1 activity reveals a modulation at distinct stages of the PAI-1/plasminogen activator interaction.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasminogen activators (PAs) tissue-type PA (tPA) and urokinase-type PA (uPA) plays a crucial role in many (patho)physiological processes (e.g., cardiovascular disease, tissue fibrosis) as well as in many age-related pathologies. Therefore, much effort has been put into the development of small molecule or antibody-based PAI-1 inhibitors. OBJECTIVE: To elucidate the molecular mechanism of nanobody-induced PAI-1 inhibition. METHODS AND RESULTS: Here we present the first crystal structures of PAI-1 in complex with two neutralizing nanobodies (Nbs). These structures, together with biochemical and biophysical characterization, reveal that Nb VHH-2g-42 (Nb42) interferes with the initial PAI-1/PA complex formation, whereas VHH-2w-64 (Nb64) redirects the PAI-1/PA interaction to PAI-1 deactivation and regeneration of active PA. Furthermore, whereas vitronectin does not have an impact on the inhibitory effect of Nb42, it strongly potentiates the inhibitory effect of Nb64, which may contribute to a strong inhibitory potential of Nb64 in vivo. CONCLUSIONS: These findings illuminate the molecular mechanisms of PAI-1 inhibition. Nb42 and Nb64 can be used as starting points to engineer further improved antibody-based PAI-1 inhibitors or guide the rational design of small molecule inhibitors to treat a wide range of PAI-1-related pathophysiological conditions.

Increased expression of plasminogen activator inhibitor-1 (PAI-1) is associated with depression and depressive phenotype in C57Bl/6J mice.

Plasminogen activator inhibitor 1 (PAI-1), which is elevated in numerous disease states, has been implicated as a stress-related protein involved in the pathogenesis of depression. We measured PAI-1 in the plasma of healthy and depressed individuals and assessed plasminogen activator (PA) expression and regulation by PAI-1 in cultured normal human astrocytes (NHA). Elevated plasma PAI-1 levels were found in depressed patients. Brain tissues from depressed individuals also showed stronger expression of hippocampal PAI-1 by confocal imaging in comparison to healthy individuals. Using a lipopolysaccharide-induced inflammatory model of depression in mice, we measured PAI-1 in murine plasma and brain, by ELISA and immunohistochemistry, respectively. Similar elevations were seen in plasma but not in brain homogenates of mice exposed to LPS. We further correlated the findings with depressive behavior. Ex vivo experiments with NHA treated with proinflammatory cytokines implicated in the pathogenesis of depression showed increased PAI-1 expression. Furthermore, these studies suggest that urokinase-type plasminogen activator may serve as an astrocyte PA reservoir, able to promote cleavage of brain-derived neurotrophic factor (BDNF) during stress or inflammation. In summary, our findings confirm that derangements of PAI-1 variably occur in the brain in association with the depressive phenotype. These derangements may impede the availability of active, mature (m)BDNF and thereby promote a depressive phenotype.

Phase 1 trial of intrapleural LTI-01; single chain urokinase in complicated parapneumonic effusions or empyema.

BACKGROUND: Current dosing of intrapleural fibrinolytic therapy (IPFT) in adults with complicated parapneumonic effusion (CPE) / empyema is empiric, as dose-escalation trials have not previously been conducted. We hypothesized that LTI-01 (scuPA), which is relatively resistant to PA inhibitor-1 (PAI-1), would be well-tolerated. METHODS: This was an open-label, dose-escalation trial of LTI-01 IPFT at 50,000-800,000 IU daily for up to 3 days in adults with loculated CPE/empyema and failed pleural drainage. The primary objective was to evaluate safety and tolerability, and secondary objectives included assessments of processing and bioactivity of scuPA in blood and pleural fluid (PF), and early efficacy. RESULTS: LTI-01 was well tolerated with no bleeding, treatment-emergent adverse events or surgical referrals (n=14 subjects). uPA antigen increased in PFs at 3 hours after LTI-01 (p<0.01) but not in plasma. PF saturated active PAI-1, generated PAI-1-resistant bioactive complexes, increased PA and fibrinolytic activities and D-dimers. There was no systemic fibrinogenolysis, nor increments in plasma D-dimer. Decreased pleural opacities occurred in all but one subject. Both subjects receiving 800,000 IU required two doses to relieve pleural sepsis, with two other subjects similarly responding at lower doses. CONCLUSION: LTI-01 IPFT was well-tolerated at these doses with no safety concerns. Bioactivity of LTI-01 IPFT was confirmed, limited to PFs where its processing simulated that previously reported in preclinical studies. Preliminary efficacy signals including reduction of pleural opacity were observed.

Nebulization of Single-Chain Tissue-Type and Single-Chain Urokinase Plasminogen Activator for Treatment of Inhalational Smoke-Induced Acute Lung Injury.

Single-chain tissue-type plasminogen activator (sctPA) and single-chain urokinase plasminogen activator (scuPA) have attracted interest as enzymes for the treatment of inhalational smoke-induced acute lung injury (ISALI). In this study, the pulmonary delivery of commercial human sctPA and lyophilized scuPA and their reconstituted solution forms were demonstrated using vibrating mesh nebulizers (Aeroneb® Pro (active) and EZ Breathe® (passive)). Both the Aeroneb® Pro and EZ Breathe® vibrating mesh nebulizers produced atomized droplets of protein solution of similar size of less than about 5 µm, which is appropriate for pulmonary delivery. Enzymatic activities of scuPA and of sctPA were determined after nebulization and both remained stable (88.0% and 93.9%). Additionally, the enzymatic activities of sctPA and tcuPA were not significantly affected by excipients, lyophilization or reconstitution conditions. The results of these studies support further development of inhaled formulations of fibrinolysins for delivery to the lungs following smoke-induced acute pulmonary injury.

Lack of durable protection against cotton smoke-induced acute lung injury in sheep by nebulized single chain urokinase plasminogen activator or tissue plasminogen activator.

BACKGROUND: Airway fibrin casts are clinically important complications of severe inhalational smoke-induced acute lung injury (ISIALI) for which reliable evidence-based therapy is lacking. Nebulized anticoagulants or a tissue plasminogen activator; tPA, has been advocated, but airway bleeding is a known and lethal potential complication. We posited that nebulized delivery of single chain urokinase plasminogen activator, scuPA, is well-tolerated and improves physiologic outcomes in ISIALI. To test this hypothesis, we nebulized scuPA or tPA and delivered these agents every 4 h to sheep with cotton smoke induced ISIALI that were ventilated by either adaptive pressure ventilation/controlled mandatory ventilation (APVcmv; Group 1, n = 14) or synchronized controlled mandatory ventilation (SCMV)/limited suctioning; Group 2, n = 32). Physiologic readouts of acute lung injury included arterial blood gas analyses, PaO2/FiO2 ratios, peak and plateau airway pressures, lung resistance and static lung compliance. Lung injury was further assessed by histologic scoring. Biochemical analyses included determination of antigenic and enzymographic uPA and tPA levels, plasminogen activator and plasminogen activator inhibitor-1 activities and D-dimer in bronchoalveolar lavage (BAL). Plasma levels of uPA, tPA antigens, D-dimers and α-macroglobulin-uPA complex levels were also assessed. RESULTS: In Group 1, tPA at the 2 mg dose was ineffective, but at 4 mg tPA or scuPA, the PaO2/FiO2 ratios, peak/plateau pressures improved during evolving injury (p < 0.01) without significant differences at 48 h. To improve delivery of the interventions, the experiments were repeated in Group 2 with limited suctioning/SCMV, which generally increased PAs in (BAL). In Group 2, tPA was ineffective, but scuPA (4 or 8 mg) improved physiologic outcomes (p < 0.01) and plateau pressures remained lower at 48 h. Airway bleeding occurred at 8 mg tPA. BAL plasminogen activator (PA) levels positively correlated with physiologic outcomes at 48 h. CONCLUSIONS: Physiologic outcomes improved in sheep in which better delivery of the PAs occurred. The benefits of nebulized scuPA were achieved without airway bleeding associated with tPA, but were transient and largely abrogated at 48 h, in part attributable to the progression and severity of ISIALI.

Fibrin turnover and pleural organization: bench to bedside.

Recent studies have shed new light on the role of the fibrinolytic system in the pathogenesis of pleural organization, including the mechanisms by which the system regulates mesenchymal transition of mesothelial cells and how that process affects outcomes of pleural injury. The key contribution of plasminogen activator inhibitor-1 to the outcomes of pleural injury is now better understood as is its role in the regulation of intrapleural fibrinolytic therapy. In addition, the mechanisms by which fibrinolysins are processed after intrapleural administration have now been elucidated, informing new candidate diagnostics and therapeutics for pleural loculation and failed drainage. The emergence of new potential interventional targets offers the potential for the development of new and more effective therapeutic candidates.

Formulation for a novel inhaled peptide therapeutic for idiopathic pulmonary fibrosis.

A caveolin-1 scaffolding domain, CSP7, is a newly developed peptide for the treatment of idiopathic pulmonary fibrosis. To develop a CSP7 formulation for further use we have obtained, characterized and compared a number of lyophilized formulations of CSP7 trifluoroacetate with DPBS and in combination with excipients (mannitol and lactose at molar ratios 1:5, 70 and 140). CSP7 trifluoroacetate was stable (>95%) in solution at 5 and 25 °C for up to 48 h and tolerated at least 5 freeze/thaw cycles. Lyophilized cakes of CSP7 trifluoroacetate with excipients were stable (>96%) for up to 4 weeks at room temperature (RT), and retained more than 98% of the CSP7 trifluoroacetate in the solution at 8 h after reconstitution at RT. The lyophilized CSP7 formulations were stable for up to 10 months at 5 °C protected from moisture. Exposure of the lyophilized cakes of CSP7 to 75% relative humidity (RH) resulted in an increase in the absorbed moisture, promoted crystallization of the excipients and induced reversible formation of CSP7 aggregates. Increased molar ratio of mannitol slightly affected formation of the aggregates. In contrast, lactose significantly decreased (up to 20 times) aggregate formation with apparent saturation at the molar ratio of 1:70. The possible mechanisms of stabilization of CSP7 trifluoroacetate in solid state by lactose include physical state of the bulking agent and the interactions between lactose and CSP7 trifluoroacetate (e.g. formation of a Schiff base with the N-terminal amino group of CSP7). Finally, CSP7 trifluoroacetate exhibited excellent stability during nebulization of formulations containing mannitol or lactose.

Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits.

Elevated active plasminogen activator inhibitor-1 (PAI-1) has an adverse effect on the outcomes of intrapleural fibrinolytic therapy (IPFT) in tetracycline-induced pleural injury in rabbits. To enhance IPFT with prourokinase (scuPA), two mechanistically distinct approaches to targeting PAI-1 were tested: slowing its reaction with urokinase (uPA) and monoclonal antibody (mAb)-mediated PAI-1 inactivation. Removing positively charged residues at the "PAI-1 docking site" (179RHRGGS184→179AAAAAA184) of uPA results in a 60-fold decrease in the rate of inhibition by PAI-1. Mutant prourokinase (0.0625-0.5 mg/kg; n = 12) showed efficacy comparable to wild-type scuPA and did not change IPFT outcomes ( P > 0.05). Notably, the rate of PAI-1-independent intrapleural inactivation of mutant uPA was 2 times higher ( P < 0.05) than that of the wild-type enzyme. Trapping PAI-1 in a "molecular sandwich"-type complex with catalytically inactive two-chain urokinase with Ser195Ala substitution (S195A-tcuPA; 0.1 and 0.5 mg/kg) did not improve the efficacy of IPFT with scuPA (0.0625-0.5 mg/kg; n = 11). IPFT failed in the presence of MA-56A7C10 (0.5 mg/kg; n = 2), which forms a stable intrapleural molecular sandwich complex, allowing active PAI-1 to accumulate by blocking its transition to a latent form. In contrast, inactivation of PAI-1 by accelerating the active-to-latent transition mediated by mAb MA-33B8 (0.5 mg/kg; n = 2) improved the efficacy of IPFT with scuPA (0.25 mg/kg). Thus, under conditions of slow (4-8 h) fibrinolysis in tetracycline-induced pleural injury in rabbits, only the inactivation of PAI-1, but not a decrease in the rate of its reaction with uPA, enhances IPFT. Therefore the rate of fibrinolysis, which varies in different pathologic states, could affect the selection of PAI-1 inhibitors to enhance fibrinolytic therapy.

Precision-guided, Personalized Intrapleural Fibrinolytic Therapy for Empyema and Complicated Parapneumonic Pleural Effusions: The Case for the Fibrinolytic Potential.

Complicated pleural effusions and empyema with loculation and failed drainage are common clinical problems. In adults, intrapleural fibrinolytic therapy is commonly used with variable results and therapy remains empiric. Despite the intrapleural use of various plasminogen activators; fibrinolysins, for about sixty years, there is no clear consensus about which agent is most effective. Emerging evidence demonstrates that intrapleural administration of plasminogen activators is subject to rapid inhibition by plasminogen activator inhibitor-1 and that processing of fibrinolysins is importantly influenced by other factors including the levels and quality of pleural fluid DNA. Current therapy for loculation that accompanies pleural infections also includes surgery, which is invasive and for which patient selection can be problematic. Most of the clinical literature published to date has used flat dosing of intrapleural fibrinolytic therapy in all subjects but little is known about how that strategy influences the processing of the administered fibrinolysin or how this influences outcomes. We developed a new test of pleural fluids ex vivo, which is called the Fibrinolytic Potential or FP, in which a dose of a fibrinolysin is added to pleural fluids ex vivo after which the fibrinolytic activity is measured and normalized to baseline levels. Testing in preclinical and clinical empyema fluids reveals a wide range of responses, indicating that individual patients will likely respond differently to flat dosing of fibrinolysins. The test remains under development but is envisioned as a guide for dosing of these agents, representing a novel candidate approach to personalization of intrapleural fibrinolytic therapy.

Organizing empyema induced in mice by Streptococcus pneumoniae: effects of plasminogen activator inhibitor-1 deficiency.

BACKGROUND: Pleural infection affects about 65,000 patients annually in the US and UK. In this and other forms of pleural injury, mesothelial cells (PMCs) undergo a process called mesothelial (Meso) mesenchymal transition (MT), by which PMCs acquire a profibrogenic phenotype with increased expression of α-smooth muscle actin (α-SMA) and matrix proteins. MesoMT thereby contributes to pleural organization with fibrosis and lung restriction. Current murine empyema models are characterized by early mortality, limiting analysis of the pathogenesis of pleural organization and mechanisms that promote MesoMT after infection. METHODS: A new murine empyema model was generated in C57BL/6 J mice by intrapleural delivery of Streptococcus pneumoniae (D39, 3 × 10(7)-5 × 10(9) cfu) to enable use of genetically manipulated animals. CT-scanning and pulmonary function tests were used to characterize the physiologic consequences of organizing empyema. Histology, immunohistochemistry, and immunofluorescence were used to assess pleural injury. ELISA, cytokine array and western analyses were used to assess pleural fluid mediators and markers of MesoMT in primary PMCs. RESULTS: Induction of empyema was done through intranasal or intrapleural delivery of S. pneumoniae. Intranasal delivery impaired lung compliance (p < 0.05) and reduced lung volume (p < 0.05) by 7 days, but failed to reliably induce empyema and was characterized by unacceptable mortality. Intrapleural delivery of S. pneumoniae induced empyema by 24 h with lung restriction and development of pleural fibrosis which persisted for up to 14 days. Markers of MesoMT were increased in the visceral pleura of S. pneumoniae infected mice. KC, IL-17A, MIP-1ß, MCP-1, PGE2 and plasmin activity were increased in pleural lavage of infected mice at 7 days. PAI-1(-/-) mice died within 4 days, had increased pleural inflammation and higher PGE2 levels than WT mice. PGE2 was induced in primary PMCs by uPA and plasmin and induced markers of MesoMT. CONCLUSION: To our knowledge, this is the first murine model of subacute, organizing empyema. The model can be used to identify factors that, like PAI-1 deficiency, alter outcomes and dissect their contribution to pleural organization, rind formation and lung restriction.

Dose dependency of outcomes of intrapleural fibrinolytic therapy in new rabbit empyema models.

The incidence of empyema (EMP) is increasing worldwide; EMP generally occurs with pleural loculation and impaired drainage is often treated with intrapleural fibrinolytic therapy (IPFT) or surgery. A number of IPFT options are used clinically with empiric dosing and variable outcomes in adults. To evaluate mechanisms governing intrapleural fibrinolysis and disease outcomes, models of Pasteurella multocida and Streptococcus pneumoniae were generated in rabbits and the animals were treated with either human tissue (tPA) plasminogen activator or prourokinase (scuPA). Rabbit EMP was characterized by the development of pleural adhesions detectable by chest ultrasonography and fibrinous coating of the pleura. Similar to human EMP, rabbits with EMP accumulated sizable, 20- to 40-ml fibrinopurulent pleural effusions associated with extensive intrapleural organization, significantly increased pleural thickness, suppression of fibrinolytic and plasminogen-activating activities, and accumulation of high levels of plasminogen activator inhibitor 1, plasminogen, and extracellular DNA. IPFT with tPA (0.145 mg/kg) or scuPA (0.5 mg/kg) was ineffective in rabbit EMP (n = 9 and 3 for P. multocida and S. pneumoniae, respectively); 2 mg/kg tPA or scuPA IPFT (n = 5) effectively cleared S. pneumoniae-induced EMP collections in 24 h with no bleeding observed. Although intrapleural fibrinolytic activity for up to 40 min after IPFT was similar for effective and ineffective doses of fibrinolysin, it was lower for tPA than for scuPA treatments. These results demonstrate similarities between rabbit and human EMP, the importance of pleural fluid PAI-1 activity, and levels of plasminogen in the regulation of intrapleural fibrinolysis and illustrate the dose dependency of IPFT outcomes in EMP.

The time course of resolution of adhesions during fibrinolytic therapy in tetracycline-induced pleural injury in rabbits.

The time required for the effective clearance of pleural adhesions/organization after intrapleural fibrinolytic therapy (IPFT) is unknown. Chest ultrasonography and computed tomography (CT) were used to assess the efficacy of IPFT in a rabbit model of tetracycline-induced pleural injury, treated with single-chain (sc) urokinase plasminogen activators (scuPAs) or tissue PAs (sctPA). IPFT with sctPA (0.145 mg/kg; n = 10) and scuPA (0.5 mg/kg; n = 12) was monitored by serial ultrasonography alone (n = 12) or alongside CT scanning (n = 10). IPFT efficacy was assessed with gross lung injury scores (GLIS) and ultrasonography scores (USS). Pleural fluids withdrawn at 0-240 min and 24 h after IPFT were assayed for PA and fibrinolytic activities, α-macroglobulin/fibrinolysin complexes, and active PA inhibitor 1 (PAI-1). scuPA and sctPA generated comparable steady-state fibrinolytic activities by 20 min. PA activity in the scuPA group decreased slower than the sctPA group (kobs = 0.016 and 0.042 min(-1)). Significant amounts of bioactive uPA/α-macroglobulin (but not tPA; P < 0.05) complexes accumulated at 0-40 min after IPFT. Despite the differences in intrapleural processing, IPFT with either fibrinolysin was effective (GLIS ≤ 10) in animals imaged with ultrasonography only. USS correlated well with postmortem GLIS (r(2) = 0.85) and confirmed relatively slow intrapleural fibrinolysis after IPFT, which coincided with effective clearance of adhesions/organization at 4-8 h. CT scanning was associated with less effective (GLIS > 10) IPFT and higher levels of active PAI-1 at 24 h following therapy. We concluded that intrapleural fibrinolysis in tetracycline-induced pleural injury in rabbits is relatively slow (4-8 h). In CT-scanned animals, elevated PAI-1 activity (possibly radiation induced) reduced the efficacy of IPFT, buttressing the major impact of active PAI-1 on IPFT outcomes.

Plasminogen activator inhibitor-1 suppresses profibrotic responses in fibroblasts from fibrotic lungs.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive interstitial scarification. A hallmark morphological lesion is the accumulation of myofibroblasts or fibrotic lung fibroblasts (FL-fibroblasts) in areas called fibroblastic foci. We previously demonstrated that the expression of both urokinase-type plasminogen activator (uPA) and the uPA receptor are elevated in FL-fibroblasts from the lungs of patients with IPF. FL-fibroblasts isolated from human IPF lungs and from mice with bleomycin-induced pulmonary fibrosis showed an increased rate of proliferation compared with normal lung fibroblasts (NL-fibroblasts) derived from histologically "normal" lung. Basal expression of plasminogen activator inhibitor-1 (PAI-1) in human and murine FL-fibroblasts was reduced, whereas collagen-I and α-smooth muscle actin were markedly elevated. Conversely, alveolar type II epithelial cells surrounding the fibrotic foci in situ, as well as those isolated from IPF lungs, showed increased activation of caspase-3 and PAI-1 with a parallel reduction in uPA expression. Transduction of an adenovirus PAI-1 cDNA construct (Ad-PAI-1) suppressed expression of uPA and collagen-I and attenuated proliferation in FL-fibroblasts. On the contrary, inhibition of basal PAI-1 in NL-fibroblasts increased collagen-I and α-smooth muscle actin. Fibroblasts isolated from PAI-1-deficient mice without lung injury also showed increased collagen-I and uPA. These changes were associated with increased Akt/phosphatase and tensin homolog proliferation/survival signals in FL-fibroblasts, which were reversed by transduction with Ad-PAI-1. This study defines a new role of PAI-1 in the control of fibroblast activation and expansion and its role in the pathogenesis of fibrosing lung disease and, in particular, IPF.

Targeting of plasminogen activator inhibitor 1 improves fibrinolytic therapy for tetracycline-induced pleural injury in rabbits.

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 (single-chain [sc] urokinase [uPA]) intrapleural fibrinolytic therapy (IPFT) of tetracycline-induced pleural injury in rabbits. 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 24 hours. Pleural fluid (PF) was collected at 0, 10, 20, and 40 minutes and 24 hours after IPFT and analyzed for plasminogen activating (PA), uPA, fibrinolytic activities, levels of total plasmin/plasminogen, α-macroglobulin (αM), mAbs/IgG antigens, free active uPA, and αM/uPA complexes. Anti-PAI-1 mAbs, but not mouse IgG, delivered with an eightfold 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 24 hours. Compared with 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. Targeting PAI-1 did not induce bleeding, and rendered otherwise ineffective doses of scuPA able to improve outcomes 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.

Elucidation of final steps of the marineosins biosynthetic pathway through identification and characterization of the corresponding gene cluster.

The marine Streptomyces sp. CNQ-617 produces two diastereomers, marineosins A and B. These are structurally related to alkyl prodiginines, but with a more complex cyclization and an unusual spiroaminal skeleton. We report the identification of the mar biosynthetic gene cluster and demonstrate production of marineosins through heterologous expression in a S. venezuelae host named JND2. The mar cluster shares the same gene organization and has high homology to the genes of the red cluster (which directs the biosynthesis of undecylprodiginine) but contains an additional gene, named marA. Replacement of marA in the JND2 strain leads to the accumulation of premarineosin, which is identical to marineosin with the exception that the middle pyrrole (Ring B) has not been reduced. The final step of the marineosin pathway is thus a MarA catalyzed reduction of this ring. Replacement of marG (a homologue of redG that directs undecylprodiginine cyclization to give streptorubin B) in the JND2 strain leads to the loss of all spiroaminal products and the accumulation of 23-hydroxyundecylprodiginine and a shunt product, 23-ketoundecylprodiginine. MarG thus catalyzes the penultimate step of the marineosin pathway catalyzing conversion of 23-hydroxyundecylprodiginine to premarineosin. The preceding steps of the biosynthetic marineosin pathway likely mirror that in the red-directed biosynthetic process, with the exception of the introduction of the hydroxyl functionality required for spiroaminal formation. This work presents the first experimentally supported scheme for biosynthesis of marineosin and provides a new biologically active molecule, premarineosin.