Runx3 Regulates CD8+ T Cell Local Expansion and CD43 Glycosylation in Mice by H1N1 Influenza A Virus Infection.

We have recently reported that transcription factor Runx3 is required for pulmonary generation of CD8+ cytotoxic T lymphocytes (CTLs) that play a crucial role in the clearance of influenza A virus (IAV). To understand the underlying mechanisms, we determined the effects of Runx3 knockout (KO) on CD8+ T cell local expansion and phenotypes using an inducible general Runx3 KO mouse model. We found that in contrast to the lungs, Runx3 general KO promoted enlargement of lung-draining mediastinal lymph node (mLN) and enhanced CD8+ and CD4+ T cell expansion during H1N1 IAV infection. We further found that Runx3 deficiency greatly inhibited core 2 O-glycosylation of selectin ligand CD43 on activated CD8+ T cells but minimally affected the cell surface expression of CD43, activation markers (CD44 and CD69) and cell adhesion molecules (CD11a and CD54). Runx3 KO had a minor effect on lung effector CD8+ T cell death by IAV infection. Our findings indicate that Runx3 differently regulates CD8+ T cell expansion in mLNs and lungs by H1N1 IAV infection. Runx3 is required for CD43 core 2 O-glycosylation on activated CD8+ T cells, and the involved Runx3 signal pathway may mediate CD8+ T cell phenotype for pulmonary generation of CTLs.

Caveolin-1-derived peptide attenuates cigarette smoke-induced airway and alveolar epithelial injury.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease with no effective treatment that can reduce mortality or slow the disease progression. COPD is the third leading cause of global death and is characterized by airflow limitations due to chronic bronchitis and alveolar damage/emphysema. Chronic cigarette smoke (CS) exposure damages airway and alveolar epithelium and remains a major risk factor for the pathogenesis of COPD. We found that the expression of caveolin-1, a tumor suppressor protein; p53; and plasminogen activator inhibitor-1 (PAI-1), one of the downstream targets of p53, was markedly increased in airway epithelial cells (AECs) as well as in type II alveolar epithelial (AT2) cells from the lungs of patients with COPD or wild-type mice with CS-induced lung injury (CS-LI). Moreover, p53- and PAI-1-deficient mice resisted CS-LI. Furthermore, treatment of AECs, AT2 cells, or lung tissue slices from patients with COPD or mice with CS-LI with a seven amino acid caveolin-1 scaffolding domain peptide (CSP7) reduced mucus hypersecretion in AECs and improved AT2 cell viability. Notably, induction of PAI-1 expression via increased caveolin-1 and p53 contributed to mucous cell metaplasia and mucus hypersecretion in AECs, and reduced AT2 viability, due to increased senescence and apoptosis, which was abrogated by CSP7. In addition, treatment of wild-type mice having CS-LI with CSP7 by intraperitoneal injection or nebulization via airways attenuated mucus hypersecretion, alveolar injury, and significantly improved lung function. This study validates the potential therapeutic role of CSP7 for treating CS-LI and COPD. NEW & NOTEWORTHY Chronic cigarette smoke (CS) exposure remains a major risk factor for the pathogenesis of COPD, a debilitating disease with no effective treatment. Increased caveolin-1 mediated induction of p53 and downstream plasminogen activator inhibitor-1 (PAI-1) expression contributes to CS-induced airway mucus hypersecretion and alveolar wall damage. This is reversed by caveolin-1 scaffolding domain peptide (CSP7) in preclinical models, suggesting the therapeutic potential of CSP7 for treating CS-induced lung injury (CS-LI) and COPD.

Caveolin-1-Related Intervention for Fibrotic Lung Diseases.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease (ILD) for which there are no effective treatments. Lung transplantation is the only viable option for patients with end-stage PF but is only available to a minority of patients. Lung lesions in ILDs, including IPF, are characterized by alveolar epithelial cell (AEC) senescence and apoptosis and accumulation of activated myofibroblasts and/or fibrotic lung (fL) fibroblasts (fLfs). These composite populations of fLfs show a high rate of basal proliferation, resist apoptosis and senescence, and have increased migration and invasiveness. They also more readily deposit ECM proteins. These features eventuate in progressive destruction of alveolar architecture and loss of lung function in patients with PF. The identification of new, safer, and more effective therapy is therefore mandatory for patients with IPF or related ILDs. We found that increased caveolin-1 and tumor suppressor protein, p53 expression, and apoptosis in AECs occur prior to and then with the proliferation of fLfs in fibrotic lungs. AECs with elevated p53 typically undergo apoptosis. fLfs alternatively demonstrate strikingly low basal levels of caveolin-1 and p53, while mouse double minute 2 homolog (mdm2) levels and mdm2-mediated degradation of p53 protein are markedly increased. The disparities in the expression of p53 in injured AECs and fLfs appear to be due to increased basal expression of caveolin-1 in apoptotic AECs with a relative paucity of caveolin-1 and increased mdm2 in fLfs. Therefore, targeting caveolin-1 using a caveolin 1 scaffolding domain peptide, CSP7, represents a new and promising approach for patients with IPF, perhaps other forms of progressive ILD or even other forms of organ injury characterized by fibrotic repair. The mechanisms of action differ in the injured AECs and in fLfs, in which differential signaling enables the preservation of AEC viability with concurrent limitation of fLf expansion and collagen secretion. The findings in three models of PF indicate that lung scarring can be nearly abrogated by airway delivery of the peptide. Phase 1 clinical trial testing of this approach in healthy volunteers has been successfully completed; Phase 1b in IPF patients is soon to be initiated and, if successful, will be followed by phase 2 testing in short order. Apart from the treatment of IPF, this intervention may be applicable to other forms of tissue injury characterized by fibrotic repair.

Inducible general knockout of Runx3 profoundly reduces pulmonary cytotoxic CD8+ T cells with minimal effect on outcomes in mice following influenza infection.

Respiratory viruses pose a continuing and substantive threat to human health globally. Host innate and adaptive immune responses are the critical antiviral defense mechanisms to control viral replication and spread. The present study is designed to determine the role of transcription factor Runx3 in the host immune response to influenza A virus (IAV) infection. As Runx3 is required for embryonic development, we generated an inducible Runx3 global knockout (KO) mouse model and found that Runx3 KO in adult C57BL/6 mice minimally affected thymic function under normal conditions and survival was at least 250 days post Runx3 deletion. We applied the mouse model to IAV infection and found that Runx3 KO resulted in a huge reduction (>85%) in numbers of total and antigen-specific pulmonary CD8+ cytotoxic T cells during IAV infection, while it had a minor effect on pulmonary generation of CD4+ T cells. To our surprise, this general KO of Runx3 did not significantly alter viral clearance and animal survival following IAV infection. Interestingly, we found that Runx3 KO significantly increased the numbers of pulmonary innate immune cells such as macrophages and neutrophils and the production of pro-inflammatory cytokines during IAV infection. We further found that Runx3 was strongly detected in CCR2+ immune cells in IAV-infected mouse lungs and was induced in activated macrophages and dendritic cells (DCs). As pulmonary CD8+ cytotoxic T cells play a central role in the clearance of IAV, our findings suggest that Runx3 KO may enhance host innate immunity to compensate for the loss of pulmonary CD8+ cytotoxic T cells during IAV infection.

Caveolin-1 scaffolding domain peptide abrogates autophagy dysregulation in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and fatal form of interstitial lung disease. IPF is characterized by irreversible scarring of the lungs leading to lung function decline. Although the etiology remains poorly understood, dysregulated autophagy in alveolar-epithelial cells (AECs) together with interplay between apoptotic-AECs and proliferative-myofibroblasts have been strongly implicated in IPF pathogenesis. Recent studies have revealed that a caveolin-1-derived 7-mer peptide, CSP7, mitigates established PF at least in part by improving AEC viability. In the present study, we aimed to determine whether and how CSP7 regulates autophagy in fibrotic-lung AECs. We found that p53 and autophagic proteins were markedly upregulated in AECs from mice with single/multi-doses of bleomycin-or silica-induced PF. This was abolished following treatment of PF-mice with CSP7. Further, CSP7 abrogated silica- or bleomycin-induced p53 and autophagy proteins in AECs. Immunoprecipitation further revealed that CSP7 abolishes the interaction of caveolin-1 with LC3BII and p62 in AECs. AEC-specific p53-knockout mice resisted silica- or bleomycin-induced changes in autophagy proteins, or CSP7 treatment. Our findings provide a novel mechanism by which CSP7 inhibits dysregulated autophagy in injured AECs and mitigates existing PF. These results affirm the potential of CSP7 for treating established PF, including IPF and silicosis.

Caveolin-1-Derived Peptide Reduces ER Stress and Enhances Gelatinolytic Activity in IPF Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by an excess deposition of extracellular matrix in the pulmonary interstitium. Caveolin-1 scaffolding domain peptide (CSP) has been found to mitigate pulmonary fibrosis in several animal models. However, its pathophysiological role in IPF is obscure, and it remains critical to understand the mechanism by which CSP protects against pulmonary fibrosis. We first studied the delivery of CSP into cells and found that it is internalized and accumulated in the Endoplasmic Reticulum (ER). Furthermore, CSP reduced ER stress via suppression of inositol requiring enzyme1α (IRE1α) in transforming growth factor ß (TGFß)-treated human IPF lung fibroblasts (hIPF-Lfs). Moreover, we found that CSP enhanced the gelatinolytic activity of TGFß-treated hIPF-Lfs. The IRE1α inhibitor; 4µ8C also augmented the gelatinolytic activity of TGFß-treated hIPF-Lfs, supporting the concept that CSP induced inhibition of the IRE1α pathway. Furthermore, CSP significantly elevated expression of MMPs in TGFß-treated hIPF-Lfs, but conversely decreased the secretion of collagen 1. Similar results were observed in two preclinical murine models of PF, bleomycin (BLM)- and adenovirus expressing constitutively active TGFß (Ad-TGFß)-induced PF. Our findings provide new insights into the mechanism by which lung fibroblasts contribute to CSP dependent protection against lung fibrosis.

Caveolin-1 peptide regulates p53-microRNA-34a feedback in fibrotic lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a life-threatening disease resulting from dysregulated repair responses to lung injury. Excessive extracellular matrix deposition by expanding myofibroblasts and fibrotic lung fibroblasts (fLfs) has been implicated in the pathogenesis of PF, including IPF. We explored fLfs' microRNA-34a (miR-34a) expression from IPF tissues. Basal miR-34a levels were decreased with reduced binding of p53 to the promoter DNA and 3'UTR mRNA sequences. Overexpression of miR-34a in fLfs increased p53, PAI-1, and reduced pro-fibrogenic markers. The regulatory effects of miR-34a were altered by modifying the p53 expression. Precursor-miR-34a lung transduction reduced bleomycin-induced PF in wild-type mice. fLfs treated with caveolin-1 scaffolding domain peptide (CSP) or its fragment, CSP7, restored miR-34a, p53, and PAI-1. CSP/CSP7 reduced PDGFR-ß and pro-fibrogenic markers, which was abolished in fLfs following blockade of miR-34a expression. These peptides failed to resolve PF in mice lacking miR-34a in fLfs, indicating miR-34a-p53-feedback induction required for anti-fibrotic effects.

Interferon-γ Preferentially Promotes Necroptosis of Lung Epithelial Cells by Upregulating MLKL.

Necroptosis, a form of programmed lytic cell death, has emerged as a driving factor in the pathogenesis of acute lung injury (ALI). As ALI is often associated with a cytokine storm, we determined whether pro-inflammatory cytokines modulate the susceptibility of lung cells to necroptosis and which mediators dominate to control necroptosis. In this study, we pretreated/primed mouse primary lung epithelial and endothelial cells with various inflammatory mediators and assessed cell type-dependent responses to different necroptosis inducers and their underlying mechanisms. We found that interferon-γ (IFNγ) as low as 1 ng/mL preferentially promoted necroptosis and accelerated the release of damage-associated molecular patterns from primary alveolar and airway epithelial cells but not lung microvascular endothelial cells. Type-I IFNα was about fifty-fold less effective than IFNγ. Conversely, TNFα or agonists of Toll-like receptor-3 (TLR3), TLR4, TLR7 and TLR9 had a minor effect. The enhanced necroptosis in IFNγ-activated lung epithelial cells was dependent on IFNγ signaling and receptor-interacting protein kinase-3. We further showed that necroptosis effector mixed lineage kinase domain-like protein (MLKL) was predominantly induced by IFNγ, contributing to the enhanced necroptosis in lung epithelial cells. Collectively, our findings indicate that IFNγ is a potent enhancer of lung epithelial cell susceptibility to necroptosis.

Caveolin-1 scaffolding domain peptide regulates glucose metabolism in lung fibrosis.

Increased metabolism distinguishes myofibroblasts or fibrotic lung fibroblasts (fLfs) from the normal lung fibroblasts (nLfs). The mechanism of metabolic activation in fLfs has not been fully elucidated. Furthermore, the antifibrogenic effects of caveolin-1 scaffolding domain peptide CSP/CSP7 involving metabolic reprogramming in fLfs are unclear. We therefore analyzed lactate and succinate levels, as well as the expression of glycolytic enzymes and hypoxia inducible factor-1α (HIF-1α). Lactate and succinate levels, as well as the basal expression of glycolytic enzymes and HIF-1α, were increased in fLfs. These changes were reversed following restoration of p53 or its transcriptional target microRNA-34a (miR-34a) expression in fLfs. Conversely, inhibition of basal p53 or miR-34a increased glucose metabolism, glycolytic enzymes, and HIF-1α in nLfs. Treatment of fLfs or mice having bleomycin- or Ad-TGF-ß1-induced lung fibrosis with CSP/CSP7 reduced the expression of glycolytic enzymes and HIF-1α. Furthermore, inhibition of p53 or miR-34a abrogated CSP/CSP7-mediated restoration of glycolytic flux in fLfs in vitro and in mice with pulmonary fibrosis and lacking p53 or miR-34a expression in fibroblasts in vivo. Our data indicate that dysregulation of glucose metabolism in fLfs is causally linked to loss of basal expression of p53 and miR-34a. Treatment with CSP/CSP7 constrains aberrant glucose metabolism through restoration of p53 and miR-34a.

Caveolin-1-derived peptide limits development of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with a median 5-year survival of ~20%. Current U.S. Food and Drug Administration-approved pharmacotherapies slow progression of IPF, providing hope that even more effective treatments can be developed. Alveolar epithelial progenitor type II cell (AEC) apoptosis and proliferation, and accumulation of activated myofibroblasts or fibrotic lung fibroblasts (fLfs) contribute to the progression of IPF. Full-length caveolin-1 scaffolding domain peptide (CSP; amino acids 82 to 101 of Cav1: DGIWKASFTTFTVTKYWFYR) inhibits AEC apoptosis and fLf activation and expansion and attenuates PF in bleomycin (BLM)-induced lung injury in mice. Like full-length CSP, a seven-amino acid deletion fragment of CSP, CSP7 (FTTFTVT), demonstrated antifibrotic effects in murine models of lung fibrosis. When CSP7 was administered during the fibrotic phase in three preclinical models [single-dose BLM, repeated-dose BLM, and adenovirus expressing constitutively active transforming growth factor-ß1 (Ad-TGF-ß1)-induced established PF], CSP7 reduced extracellular matrix (ECM) markers characteristic of PF, increased AEC survival, and improved lung function. CSP7 is amenable to both systemic (intraperitoneal) or direct lung delivery in a nebulized or dry powder form. Furthermore, CSP7 treatment of end-stage human IPF lung tissue explants attenuated ECM production and promoted AEC survival. Ames testing for mutagenicity and in vitro human peripheral blood lymphocyte and in vivo mouse micronucleus transformation assays indicated that CSP7 is not carcinogenic. Together, these findings support the further development of CSP7 as an antifibrotic treatment for patients with IPF or other interstitial lung diseases.

HDAC6 inhibition blocks inflammatory signaling and caspase-1 activation in LPS-induced acute lung injury.

HDAC6 is a member of the class II histone deacetylase. HDAC6 inhibition possesses anti-inflammatory effects. However, the effects of HDAC6 inhibition in acute lung inflammation have not been studied. Here, we investigated the effects of a highly selective and potent HDAC6 inhibitor CAY10603 in LPS-induced acute inflammatory lung injury. We also conducted a series of experiments including immunoblotting, ELISA, and histological assays to explore the inflammatory signaling pathways modulated by the selective HDAC6 inhibition. We observed that HDAC6 activity was increased in the lung tissues after LPS challenge, which was associated with a decreased level of ɑ-tubulin acetylation in the lung tissues. HDAC6 inhibition by CAY10603 prevented LPS-induced ɑ-tubulin deacetylation in the lung tissues. HDAC6 inhibition also exhibited protective effects against LPS-induced acute lung inflammation, which was demonstrated by the reduced production of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 and decreased leukocyte infiltration. Furthermore, HDAC6 inhibition blocked the decrease of E-cadherin level and inhibited the increase of MMP9 expression in the lung tissues, which could prevent the destruction of the lung architecture in LPS-induced inflammatory injury. Given the important roles of NFĸB and inflammasome activation in inflammatory responses, we investigated their regulation by HDAC6 inhibition in LPS-induced lung injury. Our results showed that HDAC6 inhibition blocked the activation of NFĸB by inhibiting IĸB phosphorylation in LPS-induced acute lung injury, and LPS-induced-inflammasome activity was reduced by HDAC6 inhibition as demonstrated by the decreased IL-1ß and caspase-1 cleavage and activation. Collectively, our data suggest that selective HDAC6 inhibition suppresses inflammatory signaling pathways and alleviates LPS-induced acute lung inflammation.

p53 Expression in Lung Fibroblasts Is Linked to Mitigation of Fibrotic Lung Remodeling.

Idiopathic pulmonary fibrosis (IPF) is a debilitating, incurable, and life-threatening disease. A cardinal feature of the pathogenesis of IPF is excessive extracellular matrix deposition attributable to proliferation of activated fibrotic lung fibroblasts (fLfs). To assess the underlying mechanism, we analyzed the status of the tumor suppressor protein p53 in fLfs from the lungs of IPF patients or mice with bleomycin-induced established PF. We report that basal expression of p53 is markedly reduced in fLfs. Forced expression of caveolin-1 in fLfs increased basal p53 and reduced profibrogenic proteins, including collagen-1. Transduction of fLfs with adenovirus expressing p53 reduced expression of these proteins. Conversely, inhibition of baseline p53 in control lung fibroblasts from lung tissues increased profibrogenic protein expression. Lung transduction of adenovirus expressing p53 reduced bleomycin-induced PF in wild-type or caveolin-1-deficient mice. Furthermore, treatment of fLfs or fibrotic lung tissues with caveolin-1 scaffolding domain peptide (CSP) or its fragment, CSP7, restored p53 and reduced profibrogenic proteins. Treatment of wild-type mice with i.p. CSP or CSP7 resolved bleomycin-induced PF. These peptides failed to resolve PF in inducible conditional knockout mice lacking p53 in fLfs, indicating the induction of baseline fLf p53 as the basis of the antifibrotic effects.

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.

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.

Regulation of p53-mediated changes in the uPA-fibrinolytic system and in lung injury by loss of surfactant protein C expression in alveolar epithelial cells.

Pulmonary surfactant protein C (SP-C) expression by type II alveolar epithelial cells (AECs) is markedly reduced in diverse types of lung injuries and is often associated with AEC apoptosis. It is unclear whether loss of SP-C contributes to the increased p53 and urokinase-type plasminogen activator (uPA) system cross-talk and apoptosis of AECs. Therefore, we inhibited SP-C expression in human and murine AECs using lentivirus vector expressing shRNA and tested p53 and downstream changes in the uPA-fibrinolytic system. Inhibition of SP-C expression in AECs induced p53 and activated caspase-3, indicating AEC apoptosis. We also found that bleomycin or cigarette smoke exposure failed to inhibit SP-C expression or apoptosis in AECs in p53- and plasminogen activator inhibitor-1 (PAI-1)-deficient mice. Depletion of SP-C expression by lentiviral SP-C shRNA in PAI-1-deficient mice failed to induce p53 or apoptosis in AECs, whereas it increased both AEC p53 and apoptosis in wild-type and uPA-deficient mice. SP-C inhibition in AECs also increased in CXCL1 and CXCL2 and their receptor CXCR2 as well as ICAM-1 expression, which is indicative of a proinflammatory response. Overexpression of p53-binding 3'-UTR sequences in AECs inhibited PAI-1 induction while maintaining uPA and uPAR protein and mRNA expression. Furthermore, caveolin-1 expression and phosphorylation were increased in AECs, indicating an intricate link between caveolin-1 and Src kinase-mediated cell signaling and AEC apoptosis due to loss of SP-C expression through p53 and uPA system-mediated cross-talk. The role of uPA, PAI-1, and p53 in the regulation of AEC apoptosis after injury was also determined in knockout mice.

p53 and miR-34a Feedback Promotes Lung Epithelial Injury and Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. The pathogenesis of interstitial lung diseases, including its most common form, IPF, remains poorly understood. Alveolar epithelial cell (AEC) apoptosis, proliferation, and accumulation of myofibroblasts and extracellular matrix deposition results in progressive loss of lung function in IPF. We found induction of tumor suppressor protein, p53, and apoptosis with suppression of urokinase-type plasminogen activator (uPA) and the uPA receptor in AECs from the lungs of IPF patients, and in mice with bleomycin, cigarette smoke, silica, or sepsis-induced lung injury. Treatment with the caveolin-1 scaffolding domain peptide (CSP) reversed these effects. Consistent with induction of p53, AECs from IPF lungs or mice with diverse types of lung injuries showed increased p53 acetylation and miR-34a expression with reduction in Sirt1. This was significantly reduced after treatment of wild-type mice with CSP, and uPA-deficient mice were unresponsive. Bleomycin failed to induce miR-34a in p53- or plasminogen activator inhibitor-1 (PAI-1)-deficient mice. CSP-mediated inhibition of miR-34a restored Sirt1, suppressed p53 acetylation and apoptosis in injured AECs, and prevented pulmonary fibrosis (PF). AEC-specific suppression of miR-34a inhibited bleomycin-induced p53, PAI-1, and apoptosis and prevented PF, whereas overexpression of precursor-miR-34a increased p53, PAI-1, and apoptosis in AECs of mice unexposed to bleomycin. Our study validates p53-miR-34a feedback as a potential therapeutic target in PF.

Dasatinib-loaded albumin nanoparticles possess diminished endothelial cell barrier disruption and retain potent anti-leukemia cell activity.

Dasatinib (DAS), a second-generation tyrosine kinase inhibitor, is highly effective in treating chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. However, its clinical use is limited due to serious adverse effects. DAS can disrupt endothelial barrier integrity and increase endothelial permeability which may cause peripheral edema and pleural effusion. Albumin nanoparticles (NPs) as a drug carrier may serve as a useful tool for cell-selective drug delivery to reduce DAS-induced endothelial hyperpermeability and maintain endothelial barrier integrity. In this study, we reported that DAS-loaded NPs exhibited potent anti-leukemia efficacy as DAS alone. Importantly, albumin NPs as a drug carrier markedly reduced DAS-induced endothelial hyperpermeability by restraining the inhibition of Lyn kinase signaling pathway in endothelial cells. Therefore, albumin NPs could be a potential tool to improve anti-leukemia efficacy of DAS through its cell-selective effects.

Selective HDAC6 inhibition prevents TNF-α-induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema.

Lung endothelial damage contributes to the pathogenesis of acute lung injury. New strategies against lung endothelial barrier dysfunction may provide therapeutic benefits against lung vascular injury. Cell-cell junctions and microtubule cytoskeleton are basic components in maintaining endothelial barrier integrity. HDAC6, a deacetylase primarily localized in the cytoplasm, has been reported to modulate nonnuclear protein function through deacetylation. Both α-tubulin and ß-catenin are substrates for HDAC6. Here, we examined the effects of tubastatin A, a highly selective HDAC6 inhibitor, on TNF-α induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema. Selective HDAC6 inhibition by tubastatin A blocked TNF-α-induced lung endothelial cell hyperpermeability, which was associated with increased α-tubulin acetylation and microtubule stability. Tubastatin A pretreatment inhibited TNF-α-induced endothelial cell contraction and actin stress fiber formation with reduced myosin light chain phosphorylation. Selective HDAC6 inhibition by tubastatin A also induced ß-catenin acetylation in human lung endothelial cells, which was associated with increased membrane localization of ß-catenin and stabilization of adherens junctions. HDAC6 knockdown by small interfering RNA also prevented TNF-α-induced barrier dysfunction and increased α-tubulin and ß-catenin acetylation in endothelial cells. Furthermore, in a mouse model of endotoxemia, tubastatin A was able to prevent endotoxin-induced deacetylation of α-tubulin and ß-catenin in lung tissues, which was associated with reduced pulmonary edema. Collectively, our data indicate that selective HDAC6 inhibition by tubastatin A is a potent approach against lung endothelial barrier dysfunction.

p53- and PAI-1-mediated induction of C-X-C chemokines and CXCR2: importance in pulmonary inflammation due to cigarette smoke exposure.

We previously demonstrated that tumor suppressor protein p53 augments plasminogen activator inhibitor-1 (PAI-1) expression in alveolar epithelial cells (AECs) during chronic cigarette smoke (CS) exposure-induced lung injury. Chronic lung inflammation with elevated p53 and PAI-1 expression in AECs and increased susceptibility to and exacerbation of respiratory infections are all associated with chronic obstructive pulmonary disease (COPD). We recently demonstrated that preventing p53 from binding to the endogenous PAI-1 mRNA in AECs by either suppressing p53 expression or blockading p53 interactions with the PAI-1 mRNA mitigates apoptosis and lung injury. Within this context, we now show increased expression of the C-X-C chemokines (CXCL1 and CXCL2) and their receptor CXCR2, and the intercellular cellular adhesion molecule-1 (ICAM-1), in the lung tissues of patients with COPD. We also found a similar increase in lung tissues and AECs from wild-type (WT) mice exposed to passive CS for 20 wk and in primary AECs treated with CS extract in vitro. Interestingly, passive CS exposure of mice lacking either p53 or PAI-1 expression resisted an increase in CXCL1, CXCL2, CXCR2, and ICAM-1. Furthermore, inhibition of p53-mediated induction of PAI-1 expression by treatment of WT mice exposed to passive CS with caveolin-1 scaffolding domain peptide reduced CXCL1, CXCL2, and CXCR2 levels and lung inflammation. Our study reveals that p53-mediated induction of PAI-1 expression due to chronic CS exposure exacerbates lung inflammation through elaboration of CXCL1, CXCL2, and CXCR2. We further provide evidence that targeting this pathway mitigates lung injury associated with chronic CS exposure.