Single chain urokinase (LTI-01) intrapleural enzymatic therapy (IET) was safe and promising in a phase 1 clinical trial to overcome failed drainage in patients with pleural infection. The LTI-01-2001 phase 2a trial was a randomized, double-blind, placebo-controlled, multi-center, dose-ranging study in hospitalized subjects with infected, non-draining pleural effusions. LTI-01, in doses of 400,000, 800,000 or 1,200,000 Units, or placebo was administered intrapleurally once daily for up to 3 days. The primary efficacy endpoint was incidence of treatment failure within 7 days of starting study medication. Treatment failure was defined as requiring alternative pleural therapy irrespective of subsequent treatment. Pleural opacification was a secondary endpoint and was assessed by CT imaging of the change in opacified area expressed as a percentage of the ipsilateral hemithorax (relative change) or absolute change in pleural opacification volume expressed in liters. 40/43 enrolled patients received LTI-01 or placebo due to constraints of the COVID-19 pandemic. There was no significant difference in incidence of treatment failure between the LTI-01 and placebo groups (OR 1.04, 95

Background: Pharmacological treatment under conditions of slow fibrinolysis/thrombolysis requires the targeted delivery of plasminogen-activating activity. Echogenic liposomal formulations (regular TELIP) of single-chain tissue plasminogen activator (sctPA), while possessing high affinity to fibrin, contain free/loosely bound sctPA. We hypothesized that removal of free sctPA, which competes with liposomes and plasmin for fibrin, enhances unique features of the TELIP. Methods: Optimized and regular TELIP were assessed for the distribution of active sctPA (loosely bound, tightly bound, encapsulated), stability, binding to fibrin, initiating fibrinolysis in vitro and ex vivo using a battery of biochemical methods. Results: One milligram of the regular TELIP consists of 2.0–5.0 × 109 echogenic liposomes (700–900 nm diameter). Non-specifically bound sctPA readily dissociates at the physiological ionic strength and pH. While up to 60% of sctPA in the regular TELIP is loosely bound with 6–15% encapsulated, and the rest is tightly bound to the liposomes; in the optimized TELIP, more than 80% of active sctPA is tightly bound with up to 40% of encapsulated. The latter is protected from high-molecular-weight ligands and could be released by an ultrasound pulse. Optimized TELIP shows low competition with plasmin for fibrin and effectively supports fibrinolysis in vitro and ex vivo. The optimized TELIP with maximal load of sctPA 3% (w/w) retains integrity at 37 °C for 5 h in vitro and up to 2 h ex vivo. Conclusions: The optimized TELIP is stable in vitro and ex vivo, does not interfere with fibrinolysis and retains a high level of encapsulated sctPA delivered precisely to the thrombus/fibrin clot.

Malignant pleural effusion (MPE) can lead to pleural organization with loculation and impaired drainage. This condition is becoming increasingly more common due to advancements in cancer therapy and extended patient survival. Factors such as repeated thoracentesis through an indwelling pleural catheter (IPC), intrapleural bleeding, and tumor progression contribute to MPE organization. Loculated MPE causes breathlessness and reduced quality of life, and current therapies, including intrapleural fibrinolytic or enzymatic therapy (IPFT/IET), have limitations in efficacy and safety. Identifying new therapeutic targets is crucial for improving treatment outcomes. Research is needed to understand the role of profibrogenic factors in pleural neoplasia, their regulation, and their impact on different stages of pleural organization. The development of a rabbit model of organizing MPE could provide insights into underlying mechanisms and novel interventions. Comparative studies of pleural tissues and effusions from MPE patients and other forms of pleural organization may reveal valuable information. Cellular and molecular profiling, assessment of biomarkers, and personalized IPFT dosing are potential areas of investigation. Suppression of PAI-1 activity and the role of hyaluronic acid in malignant mesothelioma are also important research directions. Understanding the profibrogenic capacity of pleural mesothelial cells undergoing mesenchymal transition (MesoMT) and identifying key contributors and effectors involved in this process are essential for developing effective treatments for loculated MPE.

Lung fibrosis progression is closely associated with elevated levels of PAI-1 (Plasminogen Activator Inhibitor-1), a critical inhibitor of ECM (Extracellular Matrix) protein degradation that exacerbates fibrotic remodeling. Our previous study using confocal microscopy identified a close association between KIF5 and PAI-1-containing vesicles on microtubules. Knockdown of either CLSTN2 or KIF5c significantly reduced PAI-1 secretion from HPMCs and decreased the proximity signals between PAI-1 and KIF5c. Additionally, live imaging and kymograph analysis revealed that PAI-1 and KIF5c move together with directional manner along microtubules, indicating a collaborative role for CLSTN2 and KIF5c in PAI-1 containing vesicular transport to facilitate PAI-1 secretion to drive fibrosis progression. We further conducted immunofluorescent analyses on pleural tissue sections from CBB-treated and control mice. Using anti-CLSTN2 and anti-α-SMA antibodies, respectively, we found a marked increase in CLSTN2 expression along with α-SMA, a key fibrosis marker. Fluorescence in situ Hybridization (FISH) further revealed that CLSTN2 mRNA levels are substantially higher in the CBB-treated lung tissue in pleura compared to controls. These findings support the idea that myocardin-dependent CLSTN2 up-regulation facilitates the development of pleural fibrosis. To elucidate the mechanism of CLSTN2-dependent activation of PAI-1 transport, we studied the interaction among KIF5c, kinesin light chain (KLC), and CLSTN2 using the respective isolated proteins. We demonstrated that CLSTN2 associates with KIF5c through its binding to KLC. These results indicate that CLSTN2 functions as a linker to promote the association of KIF5 to PAI-1 containing vesicles. It is plausible that CLSTN2 may function as a regulator of KIF5 motor activity thus facilitating PAI-1 transport in addition to the function as a linker. Collectively, these results highlight the critical role of myocardin, CLSTN2 and KIF5c in modulating PAI-1 vesicular transport and secretion, thus playing a role as a key driver in the development of pleural fibrosis.

Background: Effective management of infectious pleural injury requires that a sustained, low-level activation of plasminogen be produced throughout the sites of fibrin deposition. We hypothesized that a double-membrane echogenic liposome (DM-TELIP) formulation of single-chain tissue plasminogen activator (sctPA, alteplase) will increase total encapsulation of the plasminogen activator, protecting it from plasminogen activator inhibitor-1 (PAI-1) until its release with ultrasound treatment. We also hypothesized that introduction of L-valine into the formulation would enhance sctPA encapsulation and storage stability. Methods: DM-Val-TELIP was produced by creating a single shell of lipids that was hydrated with a solution containing L-valine and sctPA to perform a partial encapsulation. After a freeze-thaw period, the second shell was created to complete the encapsulation procedure, followed by, lyophilization in the presence of D-mannitol. The DM-Val-TELIP was characterized via echogenicity, enzymatic activity capacity, tPA distribution (loosely bound, tightly bound and true encapsulation), storage stability, surface charge (Zeta Potential), and particle size. Results. DM-Val-TELIP exhibited: a median diameter of 875-896 nm; loading efficiency of 79.7 µg sctPA/mg lipid (99.5% total), of which 94.8% was tightly associated/encapsulated with the liposomes. The Zeta potential ranged from -53.4 to -69.7mV. All measured characteristics were preserved after one month's storage at 4°C. Activity and tPA distribution were assessed for both DM- and DM-Val-TELIP preparations and compared to single membrane TELIP. Conclusions. Optimized DM-TELIP, with and without L-valine, are stable echogenic formulations suitable for preclinical testing. DM-TELIP may represent a novel adjunct for fibrinolytic therapy that encapsulates more sctPA than previous TELIP formulations. The efficacy of DM-Val-TELIP alone and in combination with transthoracic ultrasound to release the internalized product will now be tested in a rabbit model of an acute infectious pleural injury.

Smooth muscle cell (SMC) differentiation plays a crucial role in angiogenesis and vasculogenesis during embryonic development. The underlying mechanisms controlling SMC differentiation, especially progenitor-specific regulation, however, remain largely unclear. In this study, we identified bromodomain-containing protein 4 (BRD4) as a novel regulator for SMC differentiation. Transforming growth factor-β (TGF-β) induces BRD4 expression in the initial phase of SMC differentiation of pluripotent murine 10T1/2 cells. BRD4 was found critical in mediating TGF-β-induced SMC differentiation. Knockdown of BRD4 with siRNA suppressed TGF-β-induced expression of SMC markers including α-SMA and SM22α. In addition, the BRD4 inhibitor JQ1 and degraders ARV-825 and dBET1 suppressed TGF-β-induced SMC marker gene expression. BRD4 regulates SMC differentiation by activating SMC marker gene transcription. BRD4 mediated SMC differentiation is independent of the phosphorylation of Smad2/3. Instead, BRD4 mediated TAZ expression induced by TGF-β. Consistent with the function of TAZ, the inhibition of BRD4 reduced nuclear retention of Smad3, thereby impairing Smad3 mediated SMC gene transcription. Myocardin is an important transcriptional modulator for SMC markers. Interestingly, the knockdown of BRD4 also attenuated the induction of myocardin due to TGF-β in 10T1/2 cells. Taken together, this study demonstrates that BRD4 is a novel modulator for SMC differentiation from mesenchymal progenitor cells through the regulation of TAZ and myocardin.

Intrapleural fibrinolytic therapy (IPFT), also known as intrapleural enzymatic therapy (IET), has been utilized for decades to treat pleural infections by expediting drainage in patients with pleural organization. The successful MIST2 trial demonstrated that IPFT improves pleural opacification, reduces hospital stays, and decreases short-term surgical referrals. Despite significant progress, gaps remain in identification of the optimal fibrinolytic agents, dosing, and safety improvements. IPFT is generally recommended for patients with loculation and failed pleural drainage, with a consensus panel advocating for combined tissue plasminogen activator (tPA) and DNase therapy. How each agent may affect the activity or function of the other in the combination remains unclear. While IPFT can reduce the need for surgical intervention, there are relatively few comparative clinical trials to guide initial therapy. Emerging low-dose IPFT treatment approaches may benefit patients who are poor surgical candidates. Personalized IPFT candidate approaches, such as the Fibrinolytic Potential Assay (FPA), could refine dosing and improve outcomes. Additionally, biomarkers like pleural fluid PAI-1 and suPAR concentrations may predict clinical outcomes and guide treatment. New therapeutic agents, including PAI-1 inhibiting peptides and mesothelial profibrogenic targets, are under investigation to enhance IPFT efficacy. These advances hold promise for improving the management of pleural infections and other forms of pleural organization.

Pleural conditions causing exudative effusions (empyema or complicated parapneumonia) can result in pathological pleural organization leading to pleural fibrosis (PF). Pleural mesothelial cells (PMCs) undergo mesenchymal transition (MesoMT) and acquire a profibrotic phenotype characterized by increased expression of ACTA2; collagen type I (Col-1); and phenotypic changes, including elongation, stress fiber formation, and contraction. Using RNA-sequencing analysis, we identified Tuftelin-1 (Tuft1) as a novel potential target. Although prior studies have shown that Tuft1 expression is associated with aggressive cellular phenotypes, its role in PF is unknown. Our prior studies show that inhibition of PI3K/Akt, mTORC2, or GSK-3β blocks MesoMT. In this study, we build on previous findings and suggest that Tuft1 plays a key role in promoting MesoMT. In human PMCs, various mediators that induce MesoMT result in upregulation of Tuft1 expression. Furthermore, we also found that Tuft1 was increased in human pleuritis tissues and in murine models of PF compared with normal lung. In our studies, TGF-β-mediated increase in Tuft1 was blocked by the GSK-3β inhibitor 9-ING-41. Knockdown of Tuft1 in vitro blocked TGF-β-mediated MesoMT. Conversely, Tuft1 overexpression induced mTORC2 signaling and promoted MesoMT in the absence of TGF-β. In vivo analyses showed that mesothelial cell-specific Tuft1 knockout mice (Tuft1PMC-/-) were protected from Streptococcus pneumoniae-mediated pleural injury. Histological analysis showed that pleural thickening and profibrotic markers were significantly reduced in Tuft1PMC-/- mice compared with wild-type control animals. These studies strongly support therapeutic targeting of Tuft1 as a novel means to mitigate PF.

The incidence of empyema has continued to rise over the past several decades, and mortality rates remain up to 20-27% among patients >65 years old with comorbidities. Up to 30% of adult patients with empyema are poor candidates for both surgical treatment and conventional fibrinolytic therapy. Plasminogen activator inhibitor 1 (PAI-1) inhibits fibrinolysis, promoting pleural fibrosis and lung restriction. We hypothesized that targeting PAI-1 would result in a low-dose pharmacological treatment for empyema, improving patient survival. This hypothesis was tested in rabbit models of chemically-induced and Streptococcus pneumoniae infectious pleural injury. Anti-PAI-1 monoclonal antibodies (mAbs) and a short Docking Site Peptide (DSP), which affect the PAI-1 mechanism differently, were first tested in a rabbit model of chemically-induced pleural injury. Modulating different steps of the PAI-1 mechanism resulted in an up to eight-fold increase in the efficacy of exogenous fibrinolysins. DSP, subsequently tested in a rabbit model of empyema, increased the efficacy of sctPA in both early- and advanced-stage empyema by at least eight and four-fold, respectively. While small-molecule PAI-1 inhibitors are under investigation, there is currently no FDA-approved PAI-1-targeted treatment. We believe that DSP and phage-display selected peptides that compete with mAbs for PAI-1 will be well-tolerated, tractable in clinical settings, and address both pleural fibrosis and thickening with minimal off-target effects. The results support our hypothesis and objective to develop a novel, cost-effective, low-dose pharmacological intervention for empyema suitable for patients who are at elevated risk for surgery or conventional fibrinolytic therapy.

Severe pleural space inflammation associated with exudative pleural effusions leads to the development of pleural fibrosis (PF). Pathological tissue remodeling in PF is associated with profibrotic changes in the pleural mesothelium and neoangiogenesis within the fibrotic region. However, the factors that promote these processes remain poorly understood. This study investigates the role of extracellular vesicles (EVs) in the development and progression of PF, focusing on mesothelial-to-mesenchymal transition and neoangiogenesis. Human pleural mesothelial cells (HPMCs) were treated with coagulation proteases FXa (factor Xa) and thrombin, and EV production was quantified using nanoparticle tracking analysis. The functional relevance of these EVs was assessed by evaluating their ability to promote a profibrotic phenotype in HPMCs and induce tube formation in endothelial cells. FXa and thrombin treatments significantly increased EV generation from HPMCs via PAR (protease-activated receptor)-mediated cell signaling. Our studies showed that these EVs primed HPMCs toward a profibrotic phenotype and enhanced tube formation in endothelial cells. Further investigations in preclinical mouse models of PF revealed elevated EV levels in pleural fluids from injury-induced mice, compared with saline control mice. In clinical specimens, exudative pleural effusions from patients with empyema and parapneumonic effusions exhibited significantly elevated EV numbers compared with transudative effusions from patients with congestive heart failure. More importantly, EVs isolated from exudative effusions promoted a profibrotic phenotype in naive HPMCs and enhanced tube formation similar to the effects observed with FXa-and thrombin-generated EVs. These findings offer new insights into PF pathogenesis by identifying EVs as previously unknown contributors that modulate mesothelial-tomesenchymal transition and neoangiogenesis.

Early detection and monitoring of pleural disease is critical in guiding appropriate treatment decisions. In the United States, chest radiography and computed tomography (CT) are the most employed imaging techniques to assess pleural disease for diagnosis and management. However, repeated imaging using these modalities exposes patients to cumulative ionizing radiation. In contrast, chest ultrasonography offers a safer, cost-effective, and portable alternative that could be of advantage to pediatric, rural, or resource-limited hospital settings. In addition, ultrasonography brings great utility to preclinical research where advanced imaging is limited. In this study, we developed and validated an ultrasound-based tool that enabled pleural effusion and clot volume quantification in our preclinical models of disease (early-stage empyema, advanced-stage empyema, and retained hemothorax). Four methods were tested for estimating pleural effusion and of the two of these most accurate methods were used to quantify clot volume. Across three animal models of pleural disease, both the Predicted Pleural Effusion and Clot Volume (mL) were closest to the actual volume (gold standard, mL) when area volumetry (pleural effusion or clot) was conducted in the coronal view of the pleural space of the subjects. Lastly, ultrasonographic imaging was able to distinguish the difference in clot dissolution (Predicted Clot Volume, mL) between subjects treated with vehicle control or fibrinolytic therapy, providing evidence of its utility in our preclinical model. These findings support the potential applications of these volumetric tools for preclinical and potential clinical use.

Progressive lung scarring due to persistent pleural organization often results in pleural fibrosis (PF). This process affects patients with complicated parapneumonic pleural effusions, empyema, and other pleural diseases prone to loculation. In PF, pleural mesothelial cells undergo mesomesenchymal transition (MesoMT) to become profibrotic, characterized by increased expression of α-smooth muscle actin (α-SMA) and matrix proteins, including collagen (Col)-1. In our previous study, we showed that blocking PI3K/Akt signaling inhibits MesoMT induction in human pleural mesothelial cells (HPMCs). However, the downstream signaling pathways leading to MesoMT induction remain obscure. Here, we investigated the role of mammalian target of rapamycin (mTOR) complexes (mTORC1/2) in MesoMT induction. Our studies show that activation of the downstream mediator mTORC1/2 complex is likewise a critical component of MesoMT. Specific targeting of mTORC1/2 complex using pharmacological inhibitors, such as INK128 and AZD8055, significantly inhibited TGF-β-induced MesoMT markers in HPMCs. We further identified mTORC2/Rictor complex as the principal contributor to MesoMT progression induced by TGF-β. Knockdown of Rictor, but not Raptor, attenuated TGF-β induced MesoMT in these cells. In these studies, we further show that concomitant activation of the SGK1/NDRG1 signaling cascade is essential for inducing MesoMT. Targeting SGK1 and NDRG1 with siRNA and small molecular inhibitors attenuated TGF-β-induced MesoMT in HPMCs. Additionally, preclinical studies in our Streptococcus pneumoniae mediated mouse model of PF showed that inhibition of mTORC1/2 with INK128 significantly attenuated the progression of PF in sub-acute and chronic injury. In conclusion, our studies demonstrate that mTORC2/Rictor-mediated activation of SGK1/NDRG1 are critical for MesoMT induction, and targeting this pathway could inhibit or even reverse the progression of MesoMT and PF.