Pulmonary Fibrotic Tissue Research Articles

Lactate accumulation induced by Akt2-PDK1 signaling promotes pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with an abnormal accumulation of fibrotic tissue in the lung parenchyma and elevated glycolysis level in associated cells without effective therapy options. Lactate accumulation in pulmonary fibrotic tissue is a significant factor aggravating IPF development, but the main mechanism regulating glycolysis needs further investigation. In this study, lung fibrosis model was induced by bleomycin (BLM) intratracheally in female C57BL/6 mice. The changes of lactate level and fibrotic markers were detected. For invitro studies, cell lines of alveolar epithelial cell and lung fibroblast cell were stimulated with TGF-β1 and BLM respectively, to detect changes in their fibrotic properties. The function of lactate accumulation on facilitating fibrosis was verified. We demonstrated that BLM-induced pulmonary fibrosis is accompanied by lactate accumulation owing to glycolysis upregulation. Significantly high PDK1 expression in lung fibrotic tissue promotes glycolysis. Moreover, PDK1 stimulated trans-differentiation of lung fibroblasts and epithelial-mesenchymal transition (EMT) of alveolar epithelial cells. Furthermore, phosphorylated Akt2 activated PDK1 to cause pulmonary fibrosis and inhibitors of Akt2 and PDK1 could suppress fibrotic process. This study is the first to consider PDK1 facilitated lactate accumulation through glycolysis as a vital factor in pulmonary fibrosis and could be initiated by Akt2. We concluded that the pro-fibrotic properties of PDK1 are associated with Akt2 phosphorylation and thus provide new potential therapeutic targets for pulmonary fibrosis.

Vincamine as an agonist of G protein-coupled receptor 40 effectively ameliorates pulmonary fibrosis in mice

BackgroundPulmonary fibrosis (PF) is an irreversible and fatal lung disease with limited therapeutic options. G protein-coupled receptor 40 (GPR40) has been developed as a promising therapeutic target for metabolic disorders and functions potently in varied pathological and physiological processes. Vincamine (Vin) is a monoterpenoid indole alkaloid originated from Madagascar periwinkle and was reported as a GPR40 agonist in our previous work. PurposeHere, we aimed to clarify the role of GPR40 in PF pathogenesis by using the determined GPR40 agonist Vin as a probe and explore the potential of Vin in ameliorating PF in mice. MethodsPulmonary GPR40 expression alterations were assessed in both PF patients and bleomycin-induced PF mice (PF mice). Vin was used to evaluate the therapeutic potential of GPR40 activation for PF and the underlying mechanism was intensively investigated by assays against GPR40 knockout (Ffar1−/−) mice and the cells transfected with si-GPR40 in vitro. ResultsPulmonary GPR40 expression level was highly downregulated in PF patients and PF mice. Pulmonary GPR40 deletion (Ffar1−/−) exacerbated pulmonary fibrosis as evidenced by the increases in mortality, dysfunctional lung index, activated myofibroblasts and extracellular matrix (ECM) deposition in PF mice. Vin-mediated pulmonary GPR40 activation ameliorated PF-like pathology in mice. Mechanistically, Vin suppressed ECM deposition by GPR40/β-arrestin2/SMAD3 pathway, repressed inflammatory response by GPR40/NF-κB/NLRP3 pathway and inhibited angiogenesis by decreasing GPR40-mediated vascular endothelial growth factor (VEGF) expression in the region of interface to normal parenchyma in pulmonary fibrotic tissues of mice. ConclusionPulmonary GPR40 activation shows promise as a therapeutic strategy for PF and Vin exhibits high potential in treating this disease.

Exploration and validation of the hub genes involved in hypoxia-induced endothelial-mesenchymal transition of systemic sclerosis.

During the development of systemic sclerosis (SSc), endothelial-mesenchymal transition (EndoMT) has been shown to be one of the mechanisms leading to pulmonary fibrosis. However, the correlation between hypoxia and EndoMT was mostly unknown. R software was used to analyse differentially expressed genes (DEGs) in vascular endothelial cells under hypoxic conditions, and fibroblasts derived from SSc-related pulmonary fibrotic tissues, respectively. Using a web-based online Venn diagram tool, we analysed overlapping genes of DEGs between endothelial cells and fibroblasts. Finally, the protein-protein interaction network of EndoMT hub genes were constructed using the STRING database. The hub genes were knockdown by transfection of siRNAs in the hypoxia model of HULEC-5a cells constructed by liquid paraffin closure and then used to detect the effect on EndoMT-related biomarkers by western blot. In this study, we found that INHBA, DUSP1, NOX4, PLOD2, BHLHE40 were upregulated in SSc fibroblasts and hypoxic-treated endothelial cells, while VCAM1, RND3, CCL2, and TXNIP were downregulated. In the hypoxia model of HULEC-5a cells, the expression of these 9 hub genes was confirmed by western blot. In addition, through Spearman's correlation analysis and Western blot, we confirmed that these hub genes were closely related to the EndoMT-related markers. The mechanisms of these hypoxia-induced EndoMT hub genes may be related to TGF-β, Notch, Wnt, NF-κ B, TNF and mTOR signalling pathways. Our study provides new insights into the occurrence and development of SSc-related pulmonary fibrosis resulting from hypoxia-induced EndoMT.

Identification of hub genes and key pathways of paraquat-induced human embryonic pulmonary fibrosis by bioinformatics analysis and in vitro studies.

Objective: Paraquat (N,N0-dimethyl-4,40-bipyridinium dichloride;PQ) is a highly toxic pesticide, which usually leads to acute lung injury and subsequent development of pulmonary fibrosis. The exact mechanism underlying PQ-induced lung fibrosis remain largely unclear and as yet, no specific treatment drugs have been approved. Our study aimed to identify its potential mechanisms of PQ-induced fibrosis through a modeling study in vitro studies and bioinformatics analysis.Methods: Gene expression datasets associated with PQ-induced lung fibrosis were obtained from the Gene Expression Omnibus, wherefrom differentially expressed genes (DEGs) were identified using GEO2R. Functional enrichment analyses were performed using the Database for Annotation Visualization and Integrated Discovery. The DEGs analyzed by a protein–protein interaction network was constructed with the Search Tool for the Retrieval of Interacting Genes database. MCODE, a Cytoscape plugin, was subsequently used to identify the most significant modules. The expression of the key genes in PQ-induced pulmonary fibrotic tissues was verified by reverse transcription-quantitative PCR (RT-qPCR).Results: Two datasets were analyzed and revealed 92 overlapping DEGs. Functional analysis demonstrated that these 92 DEGs were enriched in the ‘TNF signaling pathway’, ‘CXCR chemokine receptor binding’, and ‘core promoter binding’. Moreover, nine hub genes were identified from the protein–protein interaction network formed from the DEGs. These results suggested that the TNF signaling pathway and nine hub genes are possibly involved in PQ-induced lung fibrosis progression.Conclusions: This integrative analysis identified candidate genes and pathways potentially involved in PQ-induced lung fibrosis, and could benefit future development of novel approaches for controlling and treating this disease.

Clodronate-loaded liposomal and fibroblast-derived exosomal hybrid system for enhanced drug delivery to pulmonary fibrosis

Pulmonary fibrosis is a rapidly progressive and fatal fibrotic lung disease with high mortality and morbidity. However, pulmonary fibrosis therapy in the clinic has been limited by poor selectivity and inefficiency of drug delivery to fibroblasts. Herein, a clodronate (CLD)-loaded liposome and fibroblast-derived exosome (EL-CLD) hybrid drug delivery system with non-specific phagocytosis inhibition and fibroblast homing properties, was designed for the treatment of pulmonary fibrosis. EL-CLD effectively depleted Kupffer cells via apoptosis by passive targeting after intravenous injection, and thus significantly reduced accumulation in the liver. Notably, the EL-CLD hybrid system preferentially accumulated in the fibrotic lung, and significantly increased penetration inside pulmonary fibrotic tissue by targeted delivery due to the specific affinity for fibroblasts of the homologous exosome. Nintedanib (NIN), an anti-fibrotic agent used to treat pulmonary fibrosis, was loaded in the EL-CLD system, and achieved a remarkable improvement in curative effects. The enhanced therapeutic efficacy of NIN was a result of enhanced pulmonary fibrotic tissue accumulation and delivery, combined with a diminished macrophage-induced inflammatory response. Hence, the EL-CLD hybrid system acts as an efficient carrier for pulmonary anti-fibrotic drug delivery and should be developed as an efficient fibroblast specific therapy.

Upregulation of ACE2 and TMPRSS2 by particulate matter and idiopathic pulmonary fibrosis: a potential role in severe COVID-19

BackgroundAir pollution exposure and idiopathic pulmonary fibrosis (IPF) cause a poor prognosis after SARS-CoV-2 infection, but the underlying mechanisms are not well explored. Angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are the keys to the entry of SARS-CoV-2. We therefore hypothesized that air pollution exposure and IPF may increase the expression of ACE2 and TMPRSS2 in the lung alveolar region. We measured their expression levels in lung tissues of control non-IPF and IPF patients, and used murine animal models to study the deterioration of IPF caused by particulate matter (PM) and the molecular pathways involved in the expression of ACE2 and TMPRSS2.ResultsIn non-IPF patients, cells expressing ACE2 and TMPRSS2 were limited to human alveolar cells. ACE2 and TMPRSS2 were largely upregulated in IPF patients, and were co-expressed by fibroblast specific protein 1 (FSP-1) + lung fibroblasts in human pulmonary fibrotic tissue. In animal models, PM exposure increased the severity of bleomycin-induced pulmonary fibrosis. ACE2 and TMPRSS2 were also expressed in FSP-1+ lung fibroblasts in bleomycin-induced pulmonary fibrosis, and when combined with PM exposure, they were further upregulated. The severity of pulmonary fibrosis and the expression of ACE2 and TMPRSS2 caused by PM exposure were blocked by deletion of KC, a murine homologue of IL-8, or treatment with reparixin, an inhibitor of IL-8 receptors CXCR1/2.ConclusionsThese data suggested that risk of SARS-CoV-2 infection and COVID-19 disease severity increased by air pollution exposure and underlying IPF. It can be mediated through upregulating ACE2 and TMPRSS2 in pulmonary fibroblasts, and prevented by blocking the IL-8/CXCR1/2 pathway.

Physiopathology and prospectives for therapeutic treatment of pulmonary fibrotic state in COVID-19 patients

The COVID-19 global pandemic has caused about 4,30 Mln deaths. Recently the first vaccines have been licensed, representing the most powerful weapon available to stop the pandemic. The COVID-19 viral infection in the most severe cases can cause severe lung lesions with the presence of fibrotic tissue. Even among cured individuals, the presence of pulmonary fibrotic tissue may be the major cause of long-term complications of COVID-19 requiring antifibrotic therapeutic treatment even in the post-COVID-19 infection phase to accelerate the healing process and fully recover lung function. This article reviews the fibrogenic mechanism of SARS-CoV-2-induced viral damage and the antifibrotic treatments indicated to treat sequelae post COVID-19 infection.

<p>Tranilast Inhibits Pulmonary Fibrosis by Suppressing TGFβ/SMAD2 Pathway</p>

PurposeIdiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) β-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFβ expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified.MethodsWe investigated the in vitro effect of tranilast on ECM production and TGFβ/SMAD2 pathway in TGFβ2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin.ResultsTreatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFβ2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFβ, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice.ConclusionThese findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFβ/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

COVID-19 Patients with Pulmonary Fibrotic Tissue: Clinical Pharmacological Rational of Antifibrotic Therapy.

In December 2019, the first data emerged from Wuhan, China, of a serious acute respiratory disease caused by a new coronavirus, SARS-CoV-2 (COVID-19). In a short time, the health emergency became a global pandemic. To date, there are about 18.8 million infected people and about 700,000 deaths. There are currently no effective vaccines, and treatments are mostly experimental. The symptoms associated with COVID-19 are different, ranging from mild upper respiratory tract symptoms to severe acute respiratory distress syndrome (SARS). Data from previous coronavirus outbreaks such as SARS-CoV (2003 outbreak) and emerging epidemiological data from the current global COVID-19 pandemic suggest that there could be substantial tissue fibrotic consequences following SARS-CoV-2 infection, responsible for severe and in some cases fatal lung lesions. Some data show that even patients cured of viral infection have lung fibrotic tissue residues responsible for incorrect respiratory function even after healing. The role of antifibrotic drug therapy in patients with ongoing SARS-CoV-2 infection or in patients cured of residual pulmonary fibrosis is still to be defined and unclear; the scientific rationale for initiating, continuing, or discontinuing therapy is poorly defined. In this article, we describe the advantages of antifibrotic therapy in patients with ongoing SARS-CoV-2 viral infection to prevent the worsening and aggravation of the clinical situation, and the advantages it could have in the role of preventing pulmonary fibrosis after SARS-CoV-2 infection, and in accelerating the complete healing process.

The circadian clock protein REVERBα inhibits pulmonary fibrosis development

Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinβ1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.

Bioengineered in Vitro Tissue Model of Fibroblast Activation for Modeling Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a complex disease of unknown etiology with no current curative treatment. Modeling pulmonary fibrotic (PF) tissue has the potential to improve our understanding of IPF disease progression and treatment. Rodent animal models do not replicate human fibroblastic foci (Hum-FF) pathology, and current iterations of in vitro model systems (e.g., collagen hydrogels, polyacrylamide hydrogels, and fibrosis-on-chip systems) are unable to replicate the three-dimensional (3D) complexity and biochemical composition of human PF tissue. Herein, we fabricated a 3D bioengineered pulmonary fibrotic (Eng-PF) tissue utilizing cell laden silk collagen type I dityrosine cross-linked hydrogels and Flexcell bioreactors. We show that silk collagen type I hydrogels have superior stability and mechanical tunability compared to other hydrogel systems. Using customized Flexcell bioreactors, we reproduced Hum-FF-like pathology with airway epithelial and microvascular endothelial cells. Eng-PF tissues can model myofibroblast differentiation and permit evaluation of antifibrotic drug treatments. Further, Eng-PF tissues could be used to model different facets of IPF disease, including epithelial injury with the addition of bleomycin and cellular recruitment by perfusion of cells through the hydrogel microchannel.

Notch1 promotes the pericyte-myofibroblast transition in idiopathic pulmonary fibrosis through the PDGFR/ROCK1 signal pathway

The goals of this study were to investigate the role of the Notch1/PDGFRβ/ROCK1 signaling pathway in the pathogenesis of pulmonary fibrosis and to explore the possibility of treating fibrosis by targeting Notch1. Lung tissues from patients with pulmonary fibrosis were examined for the expression of Notch1/PDGFRβ/ROCK1 using RT-qPCR, western blotting, and immunostaining. Cultured mouse lung pericytes were transfected with Notch1-overexpressed vectors or shRNA targeting PDGFRβ/ROCK1 to examine cell behaviors, including proliferation, cell cycle arrest, and differentiation toward myofibroblasts. Finally, a mouse pulmonary fibrosis model was prepared, and a Notch1 inhibitor was administered to observe tissue morphology and pericyte cell behaviors. Human pulmonary fibrotic tissues presented with overexpression of Notch1, PDGFRβ, and ROCK1, in addition to a prominent transition of pericytes into myofibroblasts. In cultured mouse lung pericytes, overexpression of Notch1 led to the accelerated proliferation and differentiation of cells, and it also increased the expression of the PDGFRβ and ROCK1 proteins. The knockdown of PDGFRβ/ROCK1 in pericytes remarkably suppressed pericyte proliferation and differentiation. As further substantiation, the administration of a Notch1 inhibitor in a mouse model of lung fibrosis inhibited the PDGFRβ/ROCK1 pathway, suppressed pericyte proliferation and differentiation, and alleviated the severity of fibrosis. Our results showed that the Notch1 signaling pathway was aberrantly activated in pulmonary fibrosis, and this pathway may facilitate disease progression via mediating pericyte proliferation and differentiation. The inhibition of the Notch1 pathway may provide one promising treatment strategy for pulmonary fibrosis.

Reduced expression of BMP3 contributes to the development of pulmonary fibrosis and predicts the unfavorable prognosis in IIP patients.

Idiopathic pulmonary fibrosis (IPF) and idiopathic nonspecific interstitial pneumonia (INSIP) are two related diseases involving varying degrees of pulmonary fibrosis with no effective cure. Bone morphogenetic protein 3 (BMP3) is a member of the transforming growth factor-β (TGF-β) super-family, which has not been implicated in pulmonary fibrosis previously. In this study, we aimed to investigate the potential role of BMP3 playing in pulmonary fibrosis from clinical diagnosis to molecular signaling regulation. RNA sequencing was performed to explore the potential biomarker of IIP patients. The expression of BMP3 was evaluated in 83 cases of IPF and INSIP by immunohistochemistry. The function of BMP3 was investigated in both fibroblast cells and a bleomycin-induced murine pulmonary fibrosis model. The clinical relevance of BMP3 expression were analyzed in 47 IIP patients, which were included in 83 cases and possess more than five-year follow-up data. Both RNA-sequencing and immunohistochemistry staining revealed that BMP3 was significantly down-regulated in lung tissues of patients with IPF and INSIP. Consistently, lower expression of BMP3 also was found in pulmonary fibrotic tissues of bleomycin-induced mice model. Up-regulation of BMP3 prevented pulmonary fibrosis processing through inhibiting cellular proliferation of fibroblasts as well as TGF-β1 signal transduction. Finally, the relatively higher expression of BMP3 in IPF patients was associated with less/worse mortality. Intravenous injection of recombinant BMP3. Taken together, our results suggested that the low expression level of BMP3 may indicate the unfavorable prognosis of IPF patients, targeting BMP3 may represent a novel potential therapeutic method for pulmonary fibrosis management.

Increased AGE-RAGE ratio in idiopathic pulmonary fibrosis.

BackgroundThe abnormal epithelial-mesenchymal restorative capacity in idiopathic pulmonary fibrosis (IPF) has been recently associated with an accelerated aging process as a key point for the altered wound healing. The advanced glycation end-products (AGEs) are the consequence of non-enzymatic reactions between lipid and protein with several oxidants in the aging process. The receptor for AGEs (RAGEs) has been implicated in the lung fibrotic process and the alveolar homeostasis. However, this AGE-RAGE aging pathway has been under-explored in IPF.MethodsLung samples from 16 IPF and 9 control patients were obtained through surgical lung biopsy. Differences in AGEs and RAGE expression between both groups were evaluated by RT-PCR, Western blot and immunohistochemistry. The effect of AGEs on cell viability of primary lung fibrotic fibroblasts and alveolar epithelial cells was assessed. Cell transformation of fibrotic fibroblasts cultured into glycated matrices was evaluated in different experimental conditions.ResultsOur study demonstrates an increase of AGEs together with a decrease of RAGEs in IPF lungs, compared with control samples. Two specific AGEs involved in aging, pentosidine and Nε-Carboxymethyl lysine, were significantly increased in IPF samples. The immunohistochemistry identified higher staining of AGEs related to extracellular matrix (ECM) proteins and the apical surface of the alveolar epithelial cells (AECs) surrounding fibroblast foci in fibrotic lungs. On the other hand, RAGE location was present at the cell membrane of AECs in control lungs, while it was almost missing in pulmonary fibrotic tissue. In addition, in vitro cultures showed that the effect of AGEs on cell viability was different for AECs and fibrotic fibroblasts. AGEs decreased cell viability in AECs, even at low concentration, while fibroblast viability was less affected. Furthermore, fibroblast to myofibroblast transformation could be enhanced by ECM glycation.ConclusionsAll of these findings suggest a possible role of the increased ratio AGEs-RAGEs in IPF, which could be a relevant accelerating aging tissue reaction in the abnormal wound healing of the lung fibrotic process.

Abstract 3050: Proteotoxic isolevuglandins are a central feature of radiation-induced pulmonary injury

Abstract The development of radiation induced lung injury (RiPI) is a major barrier that limits the dose that can be administered for controlling locally advanced lung cancer. Although progress has been made toward identifying the pathophysiological events responsible for RiPI, there is still a substantial gap in knowledge. It is well established that oxidative stress is central to the progression of RiPR. In addition, reoccurring cellular injury appears to be a critical event for promotion of radiation-induced fibrosis. Yet, the exact mechanism by which reactive oxygen species (ROS) promotes injury and the nature of the reoccurring injury remain to be fully elucidated. Isolevuglandins are generated by free radical peroxidation of phospholipid-esterified arachidonic acid. Isoketalation, defined as covalent adduction of isolevuglandins to the ϵ-amino group of protein lysine residues, is emerging as a novel mechanism by which ROS contributes to the genesis of some diseases. Although isoketalation promotes proteotoxicity and cytotoxicity that can contribute to disease progression, there is currently a lack of knowledge concerning its role in RiPI, the identity of susceptible proteins or whether the process can be genetically regulated. We used a multifaceted approach that included wild type, Nrf2 and p47 null mice, confocal microscopy, and LC-MS/MS analysis of affinity purified proteins to address these questions. Mass spectrometry and gene ontology analysis identified several potential protein targets involved in cytoskeletal regulation, Wnt signaling, integrin signaling, chemokine and cytokine signaling and histone biology. Genetic evidence linking oxidant challenge to protein adduction was provided from the mouse knockout studies where it was shown that Nrf2 expression suppressed, while NADPH oxidase activity promoted isoketalation. We found that when isoketalation exceeded a critical level, cells underwent apoptosis. We identified the cell types in mouse lung that are susceptible to adduction. In a C57BL/6j murine model of radiation induced pulmonary injury we found that ionizing radiation increased the level of adduction concomitant with the development of a chronic apoptotic phenotype. We used human idiopathic pulmonary fibrotic (IPF) tissue as a surrogate for radiation-induced pulmonary fibrotic tissue, which is very hard to obtain. Human IPF and radiation-induced lung injury share a common phenotype that includes slow/chronic development and a prominent ROS component. We found an abundance of adducted protein in human IPF compared to control lung tissue, identified collagen 1α1 as one of the highly adducted proteins and show that adduction impairs collagen degradation by MMP1. In summary, these data support the hypothesis that radiation-induced oxidant stress promotes pulmonary injury, in part, by a hitherto, unrecognized mechanism: isoketalation. Supported in part by NIH/NHLBI Grant RO1HL112286. Citation Format: Stacy Mont, Sean S. Davies, L Jackson Roberts, Raymond L. Mernaugh, W Hayes McDonald, Brahm H. Segal, Jonathan A. Kropski, Timothy S. Blackwell, Konjeti R. Sekhar, James J. Galligan, Lawrence J. Marnett, Michael L. Freeman. Proteotoxic isolevuglandins are a central feature of radiation-induced pulmonary injury. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3050.

Proteoglycan and proteome profiling of central human pulmonary fibrotic tissue utilizing miniaturized sample preparation: a feasibility study.

The objective of this study was to isolate fibrotic cells from human lung biopsies taken from different central pulmonary locations. A comparison was made of cell morphology, proteoglycan- and protein-expression in mesenchymal cell cultures obtained from human bronchial biopsies from patients with asthmatic-like disorders. We isolated viable cells from 10 out of the 12 biopsies. The fibroblast-like cells were positive for the biomarker a-smooth muscle actin, indicating that the cells were in an activated state. Two different types of fibroblast-like cells were observed from human pulmonary connective tissue; one of contractile type with lamellipodia that facilitate migration and a second cell type with an increased cell size, which most likely is of a synthetic phenotype. This is the first evidence of alterations in the proteoglycan expression pattern of versican, perlecan, biglycan and decorin which can be linked to the pathophysiological state of asthmatics proven by a combination of solid-phase extraction by reversed phase and by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Protein expression analysis using two-dimensional electrophoresis was interfaced to miniaturized sample preparation techniques using microcapillary extraction. Four protein groups were identified; cytoskeletal, adhesion, scavenger and metabolic proteins. These patient's proteomes showed a high degree of heterogeneity between patients but larger homogeneity within biopsies derived from different locations of the same patient.

Cells containing factor XIIIa and pulmonary fibrosis induced by bleomycin.

To show the clinical importance of cells containing FXIIIa in pulmonary fibrosis induced by bleomycin, the distributions of FXIIIa and collagenous components were investigated immunohistochemically in both normal lung tissues and those affected by bleomycin. In the normal tissues FXIIIa-containing cells were sparse, but they were numerous in the pulmonary fibrotic tissues, especially in the subpleural area and around the blood vessels of alveolar septa, where slight to moderate fibrosis was seen, and in the intra-alveolar fibrinous exudate. In the collagenous scar-like areas, however, these cells were fewer in number and their FXIIIa expression was depleted. These findings suggest that cells containing FXIIIa have an important role in the development of pulmonary fibrosis induced by bleomycin.