Emergence of rare and low abundant anaerobic gut Firmicutes is associated with a significant downfall of Klebsiella in human colon cancer.

Gut bacterial dysbiosis has been linked to several gastrointestinal diseases, including deadly colorectal cancer (CRC), a leading cause of mortality in cancer patients. However, perturbation in gut bacteriome during colon cancer (CC, devoid of colorectal malignancy) remains poorly explored. Here, 16S rRNA gene amplicon sequencing was carried out for fecal DNA samples targeted to hypervariable V3-V4 region by employing MiSeq platform to explore the gut bacterial community shift in CC patients. While alpha diversity indices predicted high species richness and diversity, beta diversity showed marked gut bacterial compositional dissimilarity in CC versus healthy controls (HC, n = 10 each). We observed a significant (p < 0.05, Wilcoxon Rank-Sum test) emergence of low-abundant anaerobic taxa, including Parvimonas and Peptostreptococcus, in addition to Subdoligranulum, Coprococcus, Holdemanella, Solobacterium, Bilophila, Blautia, Dorea, Moryella and several unidentified taxa, mainly affiliated to Firmicutes, in CC patients. In addition, we also traced the emergence of putative probiotic taxon Slackia, belonging to Actinomycetota, in CC patients. The emergence of anaerobic Firmicutes in CC is accompanied by a significant (p < 0.05) decline in the Klebsiella, as determined through linear discriminant analysis effect size (LEfSe) and heat tree analyses. Shifts in core microbiome and variation in network correlation were also witnessed. Taken together, this study highlighted a significant and consistent emergence of rare anaerobic Firmicutes suggesting possible anaerobiosis driving gut microbial community shift, which could be exploited in designing diagnostic and therapeutic tools targeted to CC.

7-Ethoxycoumarin rescued Caenorhabditis elegans from infection of COPD derived clinical isolate Pseudomonas aeruginosa through virulence and biofilm inhibition via targeting Rhl and Pqs quorum sensing systems.

Pseudomonas aeruginosa is an ambidextrous Gram-negative contagium with density convoluted network defined quorum sensing, which enables the persistent survival within the host environment, contributing to various lung related diseases including Chronic Obstructive Pulmonary Disease (COPD). It is clear that P. aeruginosa is a powerful, exquisite pathogen that has adopted a variety of virulence properties through quorum sensing (QS) regulated phenomenon and that it dominates both in the development and exacerbations of COPD. Interestingly, 7-Ethoxycoumarin (7-EC), a compound that adequately mimics QS signaling molecule of P. aeruginosa, was introduced as part of the process of developing novel ways to treat the severe exacerbations. The results showed that, introduction of 7-EC significantly decreased exopolysaccharide-mediated biofilm development of strains isolated from COPD sputum, as evidenced by SEM analysis. Furthermore, 7-EC was able to modulate a variety of virulence factors and motility without subjecting planktonic cells to any selection pressure. Bacterial invasion assay revealed the potential activity of the 7-EC in preventing the active entry to A549 cells without causing any damage to the cells and found functionally active in protecting the C. elegans from P. aeruginosa infection and being non-toxic to the worms. Docking analysis was further proved that 7-EC to be the potential anti-QS compound competing specifically with Rhl and Pqs Systems. Therefore, 7-EC in the utilisation against the P. aeruginosa based infections, may open an avenue for the futuristic mechanistic study in chronic respiratory diseases and a initiator for the development of non-antibiotic based antibacterial therapy.

Curcumin intervention during progressive fibrosis controls inflammatory cytokines and the fibrinolytic system in pulmonary fibrosis.

Persistent injuries and chronic inflammation paired with dysregulated healing process in the lungs leads to scarring and stiffening of the tissue leading to a condition called pulmonary fibrosis. There is no efficacious therapy against the condition because of the poorly understood pathophysiology of the disease. Curcumin is well known anti-inflammatory natural compound and is shown to have beneficial effects in many diseases. It is also reported to show antifibrotic activities in pulmonary fibrosis. There are evidences that fibrinolytic system plays a crucial role in the development of pulmonary fibrosis. We aimed to see whether curcumin could regulate inflammation and fibrinolysis in murine model of pulmonary fibrosis. We prepared BLM induced pulmonary fibrosis model by administering BLM at a dose of 2 mg/ kg bodyweight. Curcumin (75 mg/kg body wt) was instilled intraperitoneally on different time points. The effect of curcumin on inflammatory cytokines and fibrinolytic system was studied using molecular biology techniques like RT-PCR, western blot and immunohistochemistry/immunofluorescence. We observed that BLM brought changes in the expressions of components in the fibrinolytic system, i.e. BLM favoured fibrin deposition by increasing the expression of PAI-1 (plasminogen activator inhibitor) and decreasing the expression of uPA (Urokinase plasminogen activator) and uPAR (Urokinase plasminogen activator receptor). We also demonstrate that curcumin could restore the normal expression of fibrinolytic components, uPA, uPAR and PAI-1. Curcumin could also minimize the expression of key enzymes in tissue remodeling in pulmonary fibrosis, MMP-2 and MMP-9, which were elevated in the BLM treated group. Our data suggest that curcumin exerts an anti-inflammatory and antifibrotic effect in lungs. We highlight curcumin as a feasible adjuvant therapy option against pulmonary 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.

Relevance of Inflammatory Cytokine mRNA Expression of Tumour Necrosis Factor- Alpha (TNF α), Interleukin 17A (IL 17A) and Interleukin 6 (IL 6) in Indian Patients with Psoriasis.

Background: Psoriasis, a chronic, immune-mediated skin disorder, has systemic manifestations as well as an ample negative impact on the quality of life (QOL) of the patient. An abnormal proliferation of keratinocyte and dermal infiltration by immune cells is a characteristic feature. It involves components of both innate and adaptive immunity, and the interaction of T cells with macrophages. Keratinocytes and dendritic cells are mediated by the secreted cytokines. This study was taken up to look into changes at the molecular level that occur during the expression of three cytokines namely tumour necrosis factor-alpha (TNFα), interleukin 17A (IL-17A) and interleukin 6 (IL-6) in Indian patients with psoriasis. Methods: A case-control study was conducted with samples from 15 psoriasis vulgaris patients and 10 healthy control subjects. Clinical parameters were recorded. Blood samples were analysed for peripheral blood messenger ribonucleic acid (mRNA) expression of TNFα, IL-17A and IL-6 using real-time polymerase chain reaction (RT-PCR). Results: The mRNA expression of TNFα, IL-17A and IL-6 in psoriasis patients were increased as compared to that in normal subjects. Conclusions: The elevated levels of Interleukins indicates a systemic inflammatory process that is akin to the cutaneous inflammation. This study indicates that the targeted therapies against these cytokines are likely to be beneficial in Indian psoriasis patients.

Temporal Quantitative Phosphoproteomics Profiling of Interleukin-33 Signaling Network Reveals Unique Modulators of Monocyte Activation.

Interleukin-33 (IL-33), a member of the IL-1 superfamily cytokines, is an endogenous danger signal and a nuclear-associated cytokine. It is one of the essential mediators of both innate and adaptive immune responses. Aberrant IL-33 signaling has been demonstrated to play a defensive role against various infectious and inflammatory diseases. Although the signaling responses mediated by IL-33 have been previously reported, the temporal signaling dynamics are yet to be explored. To this end, we applied quantitative temporal phosphoproteomics analysis to elucidate pathways and proteins induced by IL-33 in THP-1 monocytes. Employing a TMT labeling-based quantitation and titanium dioxide (TiO2)-based phosphopeptide enrichment strategy followed by mass spectrometry analysis, we identified and quantified 9448 unique phosphopeptides corresponding to 3392 proteins that showed differential regulation. Of these, 171 protein kinases, 60 phosphatases and 178 transcription factors were regulated at different phases of IL-33 signaling. In addition to the confirmed activation of canonical signaling modules including MAPK, NFκB, PI3K/AKT modules, pathway analysis of the time-dependent phosphorylation dynamics revealed enrichment of several cellular processes, including leukocyte adhesion, response to reactive oxygen species, cell cycle checkpoints, DNA damage and repair pathways. The detailed quantitative phosphoproteomic map of IL-33 signaling will serve as a potentially useful resource to study its function in the context of inflammatory and pathological conditions.

Comparative protein profiling reveals the inhibitory role of curcumin on IL-17A mediated minichromosome maintenance (MCM) proteins as novel putative markers for acute lung injury in vivo.

The Pro-inflammatory cytokine, Interleukin 17A (IL-17A) plays a vital role in the pathogenesis of inflammatory-induced acute lung injury (ALI). But, the mechanisms of this pro-inflammatory cytokine in response to activation after replication stress are not yet known. Control on DNA replication (DR) is vital for maintaining genome stability. Minichromosome maintenance (MCM) proteins play essential roles in various cancers, but their involvement during ALI is not yet been discussed. The present study was carried out to assess the participation of IL-17A during replication stress and to evaluate the contribution of curcumin on this. Mass spectrometry-based proteomic approach has been used on mice lung tissues treated with IL-17A, as a prime mediator to cause injury and curcumin a natural polyphenol as an intervention. Several trends were identified from the proteomic subset which revealed that IL-17A induces expressions of proteins like MCM2, MCM3, and MCM6 along with other proteins involved in DR. Interestingly, curcumin was found in suppressing the expression levels of these proteins. This was also confirmed via validating LC-MS/MS data using appropriate molecular techniques. Pathway and gene ontology analysis were performed with DAVID GO databases. Apart from this, the present study also reports the unique contribution of curcumin in suppressing the mRNA levels of other MCMs like MCM4, MCM5, and MCM7 as well as of ORC1 and ORC2. Hence, the present study revolves around linking the replication stress by pro-inflammatory effects, highlighting the implications for ALI and therapies. This study, therefore, enhances our capacity to therapeutically target DR-specific proteins.

Inflammatory mediators in various molecular pathways involved in the development of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a type of interstitial lung disease (ILD) that is marked by scarring of lung tissue, ultimately leading to respiratory failure. The survival rate of IPF is disappointing and to date demonstrates a clinical quandary. The exact etiology of the disease remains under discussion. According to the recent hypothesis, inflammatory mediators cause severe damage to the alveolar epithelium leading to the impairment of the alveolar structure. The role of inflammation in the development of the IPF has been controversial for years. There are two schools of thought regarding the role of inflammation. One group of researchers claims that cell death and fibroblast dysfunction are the primary causes and inflammation is just a secondary cause of IPF. The other group claims inflammation to be the primary cause. Studies using human subjects have also reported inflammation as a critical element in IPF. Inflammatory cytokinesserve amajor rolein commencing theinflammatoryresponse in the lungs. Several cytokines are reported to be involved in different molecular mechanisms underlying IPF, someof which alsocontribute additionally by acting as growth factors. The present review addressed to explore the contribution of various inflammatory cytokines, growth factors, and various other inflammatory molecules activating the major molecular pathways involved during the development of IPF.

Curcumin Targets p53-Fibrinolytic System in TGF-ß1 Mediated Alveolar Epithelial Mesenchymal Transition in Alveolar Epithelial Cells.

AIMS: We aim to investigate curcumin interaction with p53-fibrinolytic system, smad dependent and independent pathways underlying their prime role during lung injury and fibrosis. BACKGROUND: Curcumin, an active component of Curcuma longa plant, substantially modulates respiratory conditions. TGF-ß1 plays a central role in lung remodeling by balancing extracellular matrix (ECM) production and degradation, which is a hallmark for alveolar EMT. However, the crosstalk of curcumin is not known yet with TGF- ß1 mediated p53-Fibrinolytic system regulating alveolar EMT leading to IPF. In the present study, the potential molecular mechanism of curcumin in TGF-ß1 mediated p53-fibrinolytic system in basal alveolar epithelial cells was explored. OBJECTIVES: To understand the potential molecular mechanism of curcumin in TGF-ß1 mediated p53-fibrinolytic system in basal alveolar epithelial cells. METHODS: Basal alveolar epithelial cells were treated with TGF- ß1 to induce alveolar EMT and after 24 hrs curcumin was administered to study its anti-fibrotic effects. Molecular techniques like immunoblot, RT-PCR and immunofluorescence were performed to assess the anti-fibrotic role of curcumin on EMT markers, IL-17A, p53-smad interaction to investigate the anti-fibrotic role of curcumin. RESULTS: The results indicated that TGF-ß1-induced EMT in A549 cells exhibited altered expression of the IL-17A, p53-fibrinolytic markers and EMT markers at the mRNA and protein level. Intervention with curcumin attenuated alveolar EMT and inactivated TGF-ß1 induced Smad/non Smad signaling pathways via blocking p53-fibrinolytic system. CONCLUSION: This study provides the first evidence of the dynamic response of curcumin on TGF- ß1 mediated p53-fibrinolytic system during alveolar injury in vitro.

Bioresponsive supramolecular hydrogels for hemostasis, infection control and accelerated dermal wound healing.

Injectable, drug-releasing hydrogel scaffolds with multifunctional properties including hemostasis and anti-bacterial activity are essential for successful wound healing; however, designing ideal materials is still challenging. Herein, we demonstrate the fabrication of a biodegradable, temperature-pH dual responsive supramolecular hydrogel (SHG) scaffold based on sodium alginate/poly(N-vinyl caprolactam) (AG/PVCL) through free radical polymerization and the subsequent chemical and ionic cross-linking. A natural therapeutic molecule, tannic acid (TA)-incorporated SHG (AG/PVCL-TA), was also fabricated and its hemostatic and wound healing efficiency were studied. In the AG/PVCL-TA system, TA acts as a therapeutic molecule and also substitutes as an effective gelation binder. Notably, the polyphenol-arm structure and diverse bonding abilities of TA can hold polymer chains through multiple bonding and co-ordinate cross-linking, which were vital in the formation of the mechanically robust AG/PVCL-TA. The SHG formation was successfully balanced by varying the composition of SA, VCL, TA and cross-linkers. The AG/PVCL-TA scaffold was capable of releasing a therapeutic dose of TA in a sustained manner under physiological temperature-pH conditions. AG/PVCL-TA displayed excellent free radical scavenging, anti-inflammatory, anti-bacterial, and cell proliferation activity towards the 3T3 fibroblast cell line. The wound healing performance of AG/PVCL-TA was further confirmed in skin excision wound models, which demonstrated the potential application of AG/PVCL-TA for skin regeneration and rapid wound healing.

Radiation induced apoptosis and pulmonary fibrosis: curcumin an effective intervention?

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by interstitial remodeling, leading to compromised lung function. Extra vascular fibrin deposition and abnormalities in the fibrinolysis are the major clinical manifestations of lung diseases such as acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS). ALI progresses to pulmonary fibrosis (PF) and makes patient's life miserable. Anti-fibrinolysis and apoptosis are involved in the progression of PF. Apoptotic markers are detectable within IPF lung tissue and senescent cell deletion can rejuvenate pulmonary health. Enhanced expression of p53 due to DNA damage is seen in irradiated lung tissue. The role of fibrinolytic components such as Urokinase Plasminogen activator (uPA), uPA receptor (uPAR) and Plasminogen activator inhibitor-1 (PAI-1) has been detailed in I. Curcumin is known to possess anti-inflammatory and anti-fibrotic effects. Radioprotective effect of curcumin enables it to attenuate radiation-induced inflammation and fibrosis. Understanding the mechanism of radioprotective effect of curcumin in radiation-induced PF and apoptosis can lead to the development of an effective therapeutic to combat acute lung injury and fibrosis.

Effect of calcium glucoheptonate on proliferation and osteogenesis of osteoblast-like cells in vitro.

Calcium is the key macromineral having a role in skeletal structure and function, muscle contraction, and neurotransmission. Bone remodeling is maintained through a constant balance between calcium resorption and deposition. Calcium deficiency is resolved through calcium supplementation, and among the supplements, water-soluble organic molecules attracted great pharmaceutical interest. Calcium glucoheptonate is a highly water-soluble organic calcium salt having clinical use; however, detailed investigations on its biological effects are limited. We assessed the effects of calcium glucoheptonate on cell viability and proliferation of osteoblast-like MG-63 cells. Calcium uptake and mineralization were evaluated using Alizarin red staining of osteoblast-like MG-63 cells treated with calcium glucoheptonate. Expression of osteogenic markers were monitored by western blotting, immunofluorescence, and qRT-PCR assays. Increased proliferation and calcium uptake were observed in the MG-63 cells treated with calcium glucoheptonate. The treatment also increased the expression of osteopontin and osteogenic genes such as collagen-1, secreted protein acidic and cysteine rich (SPARC), and osteocalcin. Calcium glucoheptonate treatment did not exert any cytotoxicity on colorectal and renal epithelial cells, indicating the safety of the treatment. This is the first report with evidence for its beneficial effect for pharmaceutical use in addressing calcium deficiency conditions.

Curcumin down-regulates IL-17A mediated p53-fibrinolytic system in bleomycin induced acute lung injury in vivo.

Bleomycin (BLM) induced cellular damage causes inflammation in the alveolar compartment and impairment of fibrinolytic system leads to alveolar epithelial cell apoptosis. Here, we describe novel inflammatory pathway associated with p53-fibrinolytic system and apoptosis of alveolar epithelial cells and pharmacological efficiency of curcumin against this action. In the present study we used C57BL/6 mice. The specific dose and time interval of curcumin were analyzed to assess the intervention. Experiments were designed to investigate the IL-17A mediated modulation in the alveolar epithelial cell apoptosis and injury. Various techniques such as Western blot, RT-PCR, Immunohistochemistry were used for this study. We observed that the BLM-induced lung injury and its progression were successfully regulated by the effective dose and time intervention of curcumin. There was also decreased expression of chemokines, p53, and fibrinolytic components such as PAI-1 and increased uPA, uPAR expression, and decreased alveolar epithelial cell apoptosis, which indicates the IL-17A mediated novel inflammatory pathway. It is confirmed that the IL-17A involved in the modulation of p53-fibrinolytic system and epithelial cell apoptosis in BLM induced mice. The cross-talk between the inflammatory, fibrinolytic, and apoptotic pathways were resolved by curcumin intervention. This pathway and intervention could serve as a modern therapy to resolve the complications to cure the lung injury and its progression.

Curcumin alleviates IL-17A-mediated p53-PAI-1 expression in bleomycin-induced alveolar basal epithelial cells.

Bleomycin-mediated inflammatory pathway is known to play an important role in the up regulation of oxidative stress. IL-17A is a pro-inflammatory cytokine involved in the modulation of fibrosis. The complex underlying mechanism for the said phenomenon remains unclear. This newly defined investigation was designed to understand the changes associated with 1L-17A mediated up-regulation of p53 and PAI-1 expression and the role of curcumin in attenuating this process. A549 cells were treated with bleomycin (BLM) and IL-17A to induce the inflammatory response in vitro. Curcumin, a known anti-inflammatory bioactive compound was administered as an intervention. Cytotoxicity in the treatment groups was assessed using Methyl thiazolyl tetrazolium (MTT) assay. Cell migration was evaluated using scratch assay. Protein expressions were studied using Western blot analysis for the downstream effector molecules of IL-17A mediated inflammatory pathways. In MTT assay, BLM treatment showed cytotoxicty upto 88% at a concentration of 1000 µM after 48 h of treatment. Cell migration assay results revealed that curcumin blocked the migration of cells to the area of the scratch. BLM treatment to the cells significantly induced the expression of pro-inflammatory cytokine IL-17A, which in turn modulated p53-PAI-1 expression. Bioactive compound curcumin showed anti-inflammatory and anti-apoptotic activity. Curcumin also regulated the BLM and IL-17A mediated changes in p53-PAI-1 expression. Curcumin has the ability to regulate inflammatory cytokines during BLM-induced injury and their effect on p53-PAI-1 expression. It can be used as a potential anti-inflammatory and anti-fibrinolytic component for intervening the epithelial cell damage.Very little information is provided till date on the inflammatory mechanism controlling the fibrinolytic system in acute lung injury (ALI). Damage to alveolar epithelial cells during ALI is important in the development of pulmonary fibrosis (PF). Most forms of ALI are characterized by defective alveolar fibrinolysis, inflammation, and fibrotic lesions. Recent reports show that alveolar epithelial cells express uPA, uPAR, and p53-mediated changes inhibit epithelial cell viability contributing to ALI. Thus, the roles of pulmonary epithelial cells in the inflammatory cascades activated after noninfectious injury, and the key signaling mediators of this process were actively investigated in this study. This investigation revealed that curcumin is an effective inhibitor of BLM-induced inflammation, apoptosis, and migration of basal alveolar epithelial cells. These results throw an insight into the possibility of developing curcumin as a novel therapeutic for ALI.

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.

Plasminogen activator inhibitor-1 in cigarette smoke exposure and influenza A virus infection-induced lung injury.

Parenchymal lung inflammation and airway and alveolar epithelial cell apoptosis are associated with cigarette smoke exposure (CSE), which contributes to chronic obstructive pulmonary disease (COPD). Epidemiological studies indicate that people exposed to chronic cigarette smoke with or without COPD are more susceptible to influenza A virus (IAV) infection. We found increased p53, PAI-1 and apoptosis in AECs, with accumulation of macrophages and neutrophils in the lungs of patients with COPD. In Wild-type (WT) mice with passive CSE (PCSE), p53 and PAI-1 expression and apoptosis were increased in AECs as was lung inflammation, while those lacking p53 or PAI-1 resisted AEC apoptosis and lung inflammation. Further, inhibition of p53-mediated induction of PAI-1 by treatment of WT mice with caveolin-1 scaffolding domain peptide (CSP) reduced PCSE-induced lung inflammation and reversed PCSE-induced suppression of eosinophil-associated RNase1 (EAR1). Competitive inhibition of the p53-PAI-1 mRNA interaction by expressing p53-binding 3'UTR sequences of PAI-1 mRNA likewise suppressed CS-induced PAI-1 and AEC apoptosis and restored EAR1 expression. Consistent with PCSE-induced lung injury, IAV infection increased p53, PAI-1 and apoptosis in AECs in association with pulmonary inflammation. Lung inflammation induced by PCSE was worsened by subsequent exposure to IAV. Mice lacking PAI-1 that were exposed to IAV showed minimal viral burden based on M2 antigen and hemagglutination analyses, whereas transgenic mice that overexpress PAI-1 without PCSE showed increased M2 antigen and inflammation after IAV infection. These observations indicate that increased PAI-1 expression promotes AEC apoptosis and exacerbates lung inflammation induced by IAV following PCSE.

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.

Role of p53-fibrinolytic system cross-talk in the regulation of quartz-induced lung injury.

Silica is the major component of airborne dust generated by wind, manufacturing and/or demolition. Chronic occupational inhalation of silica dust containing crystalline quartz is by far the predominant form of silicosis in humans. Silicosis is a progressive lung disease that typically arises after a very long latency and is a major occupational concern with no known effective treatment. The mechanism of silicosis is not clearly understood. However, silicosis is associated with increased cell death, expression of redox enzymes and pro-fibrotic cytokines and chemokines. Since alveolar epithelial cell (AEC) death and disruption of alveolar fibrinolysis is often associated with both acute and chronic lung injuries, we explored whether p53-mediated changes in the urokinase-type plasminogen activator (uPA) system contributes to silica-induced lung injury. We further sought to determine whether caveolin-1 scaffolding domain peptide (CSP), which inhibits p53 expression, mitigates lung injury associated with exposure to silica. Lung tissues and AECs isolated from wild-type (WT) mice exposed to silica exhibit increased apoptosis, p53 and PAI-1, and suppression of uPA expression. Treatment of WT mice with CSP inhibits PAI-1, restores uPA expression and prevents AEC apoptosis by suppressing p53, which is otherwise induced in mice exposed to silica. The process involves CSP-mediated inhibition of serine-15 phosphorylation of p53 by inhibition of protein phosphatase 2A-C (PP2A-C) interaction with silica-induced caveolin-1 in AECs. These observations suggest that changes in the p53-uPA fibrinolytic system cross-talk contribute to lung injury caused by inhalation of silica dust containing crystalline quartz and is protected by CSP by targeting this pathway.

Regulation of lung injury and fibrosis by p53-mediated changes in urokinase and plasminogen activator inhibitor-1.

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.

Regulation of airway and alveolar epithelial cell apoptosis by p53-Induced plasminogen activator inhibitor-1 during cigarette smoke exposure injury.

Increased expression of tumor suppressor protein p53 and of plasminogen activator inhibitor (PAI)-1 is associated with cigarette smoke (CS) exposure-induced lung epithelial injury. p53 induces PAI-1 through mRNA stabilization in lung epithelial cells. However, it is unclear how this process affects lung epithelial damage. Here, we show that CS induces p53 and PAI-1 expression and apoptosis in cultured Beas2B and primary alveolar type (AT)II cells. CS exposure augmented binding of p53 protein with PAI-1 mRNA. Inhibition of p53 from binding to PAI-1 mRNA through expression of p53-binding 70 nt PAI-1 mRNA 3'UTR sequences suppressed CS-induced PAI-1 expression. Treatment of Beas2B cells with caveolin-1 scaffolding domain peptide (CSP) suppressed p53 expression and p53-PAI-1 mRNA interaction. These changes were associated with parallel inhibition of CS-induced PAI-1 expression and apoptosis in Beas2B cells. Wild-type mice exposed to passive CS likewise show augmented p53 and PAI-1 with parallel induction of ATII cell apoptosis, whereas mice deficient for p53 or PAI-1 expression resisted apoptosis of ATII cells. CSP suppressed CS-induced ATII cell apoptosis in wild-type mice and abrogated p53-PAI-1 mRNA interaction with parallel inhibition of p53 and PAI-1 expression. The protection against ATII cell apoptosis by CSP involves inhibition of passive CS-induced proapoptotic Bax and Bak expression and restoration of the prosurvival proteins Bcl-X(L). These observations demonstrate that inhibition of p53 binding to PAI-1 mRNA 3'UTR attenuates CS-induced ATII cell apoptosis. This presents a novel link between p53-mediated PAI-1 expression and CS-induced ATII cell apoptosis.