Fibroblast-derived conditioned media promotes lung cancer progression.

Lung cancer is the leading cause of cancer death in men and women in the United States. Recent studies have implicated the tumor microenvironment as a new chemotherapeutic target by demonstrating the importance of tumor cell-stromal interactions in cancer progression. However, the exact mechanisms by which tumor cell-stromal interactions drive lung cancer progression remain undefined, particularly in the lung. We suspect host fibroblasts represent an important component of the tumor microenvironment that drives tumor progression. We found that human non-small cell lung carcinoma cell lines show alterations in cell morphology, proliferation, migration, and colony formation on soft agar when exposed to fibroblast-conditioned media (FCM). Interestingly, FCM also promoted tumor cell resistance to cisplatin-induced apoptosis. These effects varied depending on the cancer cell line used. Similar observations were made when exposing murine Lewis Lung Carcinoma cells to conditioned media harvested from primary murine lung fibroblasts. Certain effects of FCM, but not all, could be prevented by using a cMET inhibitor. In vivo, we observed enhanced growth of the primary tumors when treated with FCM, but no changes in metastatic behavior. Although the identity of the stimulating agent(s) in the fibroblast-conditioned media was not unveiled, further studies revealed that the activity is more than one factor with a high-molecular weight (over 100 kDa). These studies implicate lung fibroblast-derived factors in lung cancer progression. These data suggest that targeting the lung tumor stroma alone, or in combination with other interventions, is a promising concept that warrants further study in the setting of lung cancer.

Mice lacking α4 nicotinic acetylcholine receptors are protected against alcohol-associated liver injury.

BACKGROUND: Chronic heavy alcohol consumption is a major risk factor for the development of liver steatosis, fibrosis, and cirrhosis, but the mechanisms by which alcohol causes liver damage remain incompletely elucidated. This group has reported that α4 nicotinic acetylcholine receptors (α4 nAChRs) act as sensors for alcohol in lung cells. This study tested the hypothesis that α4 nAChRs mediate the effects of alcohol in the liver. METHODS: Expression of acetylcholine receptor subunits in mouse liver was determined by RNA sequencing (RNA-seq). α4 nAChR knockout (α4 KO) mice were generated in C57BL/6J mice by introducing a mutation encoding an early stop codon in exon 4 of Chrna4, the gene encoding the α4 subunit of the nAChR. The presence of the inactivating mutation was established by polymerase chain reaction and genomic sequencing, and the lack of α4 nAChR function was confirmed in primary fibroblasts isolated from the α4 KO mice. Wild-type (WT) and α4 KO mice were fed the Lieber-DeCarli diet (with 36% of calories from alcohol) or pair fed an isocaloric maltose-dextrin control diet for a 6-week period that included a ramping up phase of increasing dietary alcohol. RESULTS: Chrna4 was the most abundantly expressed nAChR subunit gene in mouse livers. After 6 weeks of alcohol exposure, WT mice had elevated serum transaminases and their livers showed increased fat accumulation, decreased Sirt1 protein levels, and accumulation of markers of oxidative stress and inflammation including Cyp2E1, Nos2, Sod1, Slc7a11, TNFα, and PAI1. All these responses to alcohol were either absent or significantly attenuated in α4 KO animals. CONCLUSION: Together, these observations support the conclusion that activation of α4 nAChRs by alcohol or one of its metabolites is one of the initial events promoting the accumulation of excess fat and expression of inflammatory mediators. Thus, α4 nAChRs may represent viable targets for intervention in chronic alcohol-related liver disease.

The profibrotic and senescence phenotype of old lung fibroblasts is reversed or ameliorated by genetic and pharmacological manipulation of Slc7a11 expression.

Increased senescence and expression of profibrotic genes in old lung fibroblasts contribute to disrepair responses. We reported that primary lung fibroblasts from old mice have lower expression and activity of the cystine transporter Slc7a11/xCT than cells from young mice, resulting in changes in both the intracellular and extracellular redox environments. This study examines the hypothesis that low Slc7a11 expression in old lung fibroblasts promotes senescence and profibrotic gene expression. The levels of mRNA and protein of Slc7a11, senescence markers, and profibrotic genes were measured in primary fibroblasts from the lungs of old (24 mo) and young (3 mo) mice. In addition, the effects of genetic and pharmacological manipulation of Slc7a11 were investigated. We found that decreased expression of Slc7a11 in old cells was associated with elevated markers of senescence (p21, p16, p53, and ß-galactosidase) and increased expression of profibrotic genes (Tgfb1, Smad3, Acta2, Fn1, Col1a1, and Col5a1). Silencing of Slc7a11 in young cells replicated the aging phenotype, whereas overexpression of Slc7a11 in old cells decreased expression of senescence and profibrotic genes. Young cells were induced to express the senescence and profibrotic phenotype by sulfasalazine, a Slc7a11 inhibitor, whereas treatment of old cells with sulforaphane, a Slc7a11 inducer, decreased senescence without affecting profibrotic genes. Like aging cells, idiopathic pulmonary fibrosis fibroblasts show decreased Slc7a11 expression and increased profibrotic markers. In short, old lung fibroblasts manifest a profibrotic and senescence phenotype that is modulated by genetic or pharmacological manipulation of Slc7a11.

Interplay between aging, lung inflammation/remodeling, and fibronectin EDA in lung cancer progression.

Lung cancer remains the leading cause of cancer death in the United States. Since most lung cancers occur in aged individuals with chronic lung disorders characterized by inflammation and/or fibrosis, we hypothesized that aging and tissue inflammation/remodeling act in concert to promote lung cancer progression. To test this, we engaged in studies using young and aged C57BL/6 mice in conjunction with bleomycin treatment in a syngeneic model of lung cancer. Wildtype young (3 months) and aged (9 months) C57BL/6 mice were injected with Lewis Lung Carcinoma (LLC) cells at day 14 after injection with phosphate-buffered saline or bleomycin. Untreated aged mice were found to develop more lung metastases than young mice. Bleomycin induced weight loss and lung inflammation/remodeling in both young and aged mice, and it increased the number of lung metastases in aged lungs, but not in young lungs. Since aged lungs show alterations in the expression of fibronectin EDA, we repeated studies in aged WT and aged FN EDA KO mice. In the absence of tissue remodeling/inflammation, WT and FN EDA KO mice developed the same number of metastases when injected with LLC cells. However, the increase in lung metastasis due to bleomycin treatment was abolished in FN EDA KO mice, but only in aged and injured lungs. Together, these studies show increased lung cancer metastasis in aging animals and point to the influence of FN EDA and injury in this process.

The Integrin Inhibitor Cilengitide and Bleomycin-Induced Pulmonary Fibrosis : Cilengitide and Bleomycin-Induced Pulmonary Fibrosis.

PURPOSE: Fibroproliferation and excess deposition of extracellular matrix (ECM) are the pathologic hallmarks of idiopathic pulmonary fibrosis (IPF), a chronic progressive disorder with high mortality and suboptimal treatment options. Although the etiologic mechanisms responsible for the development and progression of IPF remain unclear, cell-ECM interactions and growth factors are considered important. Cilengitide is a cyclic RGD pentapeptide with anti-angiogenic activity that targets αvß3, αvß5 and α5ß1, integrins known to mediate cell-ECM interactions and activate the pro-fibrotic growth factor Transforming Growth Factor beta (TGF-ß). METHODS: Cilengitide was studied in vitro with the use of NIH/3T3 cells and primary lung fibroblasts, and in vivo in the well-characterized bleomycin-induced lung injury model. The extent of ECM deposition was determined by RT-PCR, Western blot, histologic analysis and hydroxyproline assay of lung tissue. Bronchoalveolar lavage analysis was used to determine cell counts. RESULTS: Cilengitide treatment of cultured fibroblasts showed decreased adhesion to vitronectin and fibronectin, both integrin-dependent events. Cilengitide also inhibited TGF-ß-induced fibronectin gene expression and reduced the accumulation of mRNAs and protein for fibronectin and collagen type I. Both preventive and treatment effects of daily injections of cilengitide (20 mg/kg) failed to inhibit the development of pulmonary fibrosis as determined by histological analysis (Ashcroft scoring), bronchoalveolar lavage (BAL) fluid cell counts, and hydroxyproline content. CONCLUSIONS: Overall, our data suggest that, despite its in vitro activity in fibroblasts, daily injections of cilengitide (20 mg/kg) did not inhibit the development of or ameliorate bleomycin-induced pulmonary fibrosis in mice.

Impact of sex, age and diet on the cysteine/cystine and glutathione/glutathione disulfide plasma redox couples in mice.

Age, sex and diet are well-established risk factors for several diseases. In humans, each of these variables has been linked to differences in plasma redox potentials (Eh) of the glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox couples. Mice have been very useful for modeling human disease processes, but it is unknown if age, sex and diet affect redox couples in mice as they do in humans. The purpose of the present study was to examine the effects of these factors on plasma redox potentials in C57BL/6J mice. We found that age had no effect on either redox couple in either sex. Plasma Eh Cys/CySS and Eh GSH/GSSG were both more oxidized (more positive) in females than in males. A 24-hour fast negated the sex differences in both redox potentials by oxidizing both redox couples in male mice, while having no effect on Eh Cys/CySS and a smaller effect on Eh GSH/GSSG in female mice. A diet with excess sulfur amino acids reduced the plasma Eh Cys/CySS in females to a level comparable to that seen in male mice. Thus, sex-specific differences in plasma Eh Cys/CySS could be normalized by two different dietary interventions. Some of these findings are consistent with reported human studies, while others are not. Most strikingly, mice do not exhibit age-dependent oxidation of plasma redox potentials. Care must be taken when designing and interpreting mouse studies to investigate redox regulation in humans.

Nicotine stimulates collagen type I expression in lung via α7 nicotinic acetylcholine receptors.

BACKGROUND: Tobacco-related chronic lung diseases are characterized by alterations in lung architecture leading to decreased lung function. Knowledge of the exact mechanisms involved in tobacco-induced tissue remodeling and inflammation remains incomplete. We hypothesize that nicotine stimulates the expression of extracellular matrix proteins, leading to relative changes in lung matrix composition, which may affect immune cells entering the lung after injury. METHODS: Pulmonary fibroblasts from wildtype and α7 nicotinic acetylcholine receptor knockout (α7KO) mice were exposed to nicotine and examined for collagen type 1 mRNA and protein expression. Testing the potential role on immune cell function, pulmonary fibroblasts were retained in culture for 120 h. The fibroblasts were eliminated by osmotic lysis and the remaining matrix-coated dishes were washed thoroughly. U937 cells were incubated on the matrix-coated dishes for 24 h followed by evaluation of IL-1ß gene expression. Wildtype or α7KO C57BL/6 mice (female, 8-12 weeks) were fed normal diet and exposed to nicotine in their drinking water (100 µg/ml) for 8-12weeks. Lungs were processed for mRNA, protein, and histology. Statistical significance was determined at p ≤ .05 by two-tailed test or 2-way ANOVA with Bonferroni posttest. RESULTS: We found that nicotine stimulated collagen type I mRNA and protein expression in a dose-dependent manner and up to 72 h in primary lung fibroblasts. The stimulatory effect of nicotine was inhibited in α7KO primary lung fibroblasts. Testing the potential role of these events on immune cell function, U937 monocytic cells were cultured atop matrices derived from nicotine-treated lung fibroblasts. These cells expressed more IL-1ß than those cultured atop matrices derived from untreated fibroblasts, and antibodies against the α2ß1 collagen integrin receptor inhibited the effect. Nicotine also stimulated fibroblast proliferation via MEK-1/ERK, unveiling a potentially amplifying pathway. In vivo, nicotine increased collagen type I expression was detected in wildtype, but not in α7KO mice. Wildtype mice showed increased collagen staining in lung, primarily around the airways. CONCLUSIONS: These observations suggest that nicotine stimulates fibroblast proliferation and their expression of collagen type I through α7 nAChRs, thereby altering the relative composition of the lung matrix without impacting the overall lung architecture; this may influence inflammatory responses after injury.

Differential Regulation of the Extracellular Cysteine/Cystine Redox State (EhCySS) by Lung Fibroblasts from Young and Old Mice.

Aging is associated with progressive oxidation of plasma cysteine (Cys)/cystine (CySS) redox state, expressed as EhCySS. Cultured cells condition their media to reproduce physiological EhCySS, but it is unknown whether aged cells produce a more oxidized extracellular environment reflective of that seen in vivo. In the current study, we isolated primary lung fibroblasts from young and old female mice and measured the media EhCySS before and after challenge with Cys or CySS. We also measured expression of genes related to redox regulation and fibroblast function. These studies revealed that old fibroblasts produced a more oxidizing extracellular EhCySS than young fibroblasts and that old fibroblasts had a decreased capacity to recover from an oxidative challenge due to a slower rate of reduction of CySS to Cys. These defects were associated with 10-fold lower expression of the Slc7a11 subunit of the xCT cystine-glutamate transporter. Extracellular superoxide dismutase (Sod3) was the only antioxidant or thiol-disulfide regulating enzyme among 36 examined that was downregulated in old fibroblasts by more than 2-fold, but there were numerous changes in extracellular matrix components. Thus, aging fibroblasts not only contribute to remodeling of the extracellular matrix but also have a profound effect on the extracellular redox environment.

Baclofen, a GABABR agonist, ameliorates immune-complex mediated acute lung injury by modulating pro-inflammatory mediators.

Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC) deposition-induced acute lung injury (ALI). Components of gamma amino butyric acid (GABA) signaling, including GABA B receptor 2 (GABABR2), GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF). Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1ßAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1ß-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting a physiological role for GABABR2 in the repair process of lung damage. GABABR2 agonists may play a potential therapeutic role in ALI.

Inhibition of the CXCL12/CXCR4-axis as preventive therapy for radiation-induced pulmonary fibrosis.

BACKGROUND: A devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process. METHODOLOGY/PRINCIPAL FINDINGS: The radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process. CONCLUSIONS/SIGNIFICANCE: CXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation.

Role of endoplasmic reticulum stress in age-related susceptibility to lung fibrosis.

The incidence of idiopathic pulmonary fibrosis (IPF) increases with age. The mechanisms that underlie the age-dependent risk for IPF are unknown. Based on studies that suggest an association of IPF and γherpesvirus infection, we infected young (2-3 mo) and old (≥18 mo) C57BL/6 mice with the murine γherpesvirus 68. Acute murine γherpesvirus 68 infection in aging mice resulted in severe pneumonitis and fibrosis compared with young animals. Progressive clinical deterioration and lung fibrosis in the late chronic phase of infection was observed exclusively in old mice with diminution of tidal volume. Infected aging mice showed higher expression of transforming growth factor-ß during the acute phase of infection. In addition, aging, infected mice showed elevation of proinflammatory cytokines and the fibrocyte recruitment chemokine, CXCL12, in bronchoalveolar lavage. Analyses of lytic virus infection and virus reactivation indicate that old mice were able to control chronic infection and elicit antivirus immune responses. However, old, infected mice showed a significant increase in apoptotic responses determined by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling assay, levels of caspase-3, and expression of the proapoptotitc molecule, Bcl-2 interacting mediator. Apoptosis of type II lung epithelial cells in aging lungs was accompanied by up-regulation of endoplasmic reticulum stress marker, binding immunoglobulin protein, and splicing of X-box-binding protein 1. These results indicate that the aging lung is more susceptible to injury and fibrosis associated with endoplasmic reticulum stress, apoptosis of type II lung epithelial cells, and activation of profibrotic pathways.

Effect of bone marrow-derived mesenchymal stem cells on endotoxin-induced oxidation of plasma cysteine and glutathione in mice.

Bone marrow-derived mesenchymal stem cells (BMDMSC) are emerging as a therapeutic modality in various inflammatory disease states, including acute lung injury (ALI). A hallmark of inflammation, and a consistent observation in patients with ALI, is a perturbation in the systemic redox environment. However, little is known about the effects of BMDMSC on the systemic redox status. The objective of the present study was to determine whether exogenously infused BMDMSC protect against endotoxin-induced oxidation of plasma cysteine (Cys) and glutathione (GSH) redox states. To determine the effect on the redox state if BMDMSC, mice received endotoxin intraperitoneally (1 mg/kg), followed by intravenous infusion of either 5 × 10(5) BMDMSC or an equal volume of saline solution. Control mice received intraperitoneal endotoxin followed by 5 × 10(5) lung fibroblasts given intravenously. Cys, cystine (CySS), GSH, and glutathione disulfide (GSSG) concentrations were determined by HPLC. Results showed sequential preservation of plasma Cys and GSH levels in response to BMDMSC infusion. The data show that BMDMSC infusion leads to a more reducing Cys and GSH redox state. The findings are the first to demonstrate that BMDMSC have antioxidant effects in vivo, and add to our understanding of the systemic effects of BMDMSC in lung injury.

Direct tracheal instillation of solutes into mouse lung.

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 µL instillate volume can be introduced into mouse lung.

Inhibition of NF-kappaB signaling reduces virus load and gammaherpesvirus-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disorder of unknown etiology. Several studies have demonstrated an association between pulmonary infection with a herpesvirus and IPF. Based on those observations, we have developed a mouse model in which interferon (IFN)gammaR(-/-) mice infected intranasally with murine gammaherpesvirus 68 (MHV68) develop lung fibrosis. We hypothesize that viral load was a critical factor for the development of fibrosis. Because nuclear factor (NF)-kappaB signaling is required to efficiently establish gammaherpesvirus, latency we infected IFNgammaR(-/-) mice with a MHV68 virus that expresses a mutant dominant inhibitor of the NF-kappaB signaling pathway, called IkappaBalphaM. Striking differences were observed at the onset of the chronic infection, which correlated with a decreased virus load in mice infected with MHV68-IkappaBalphaM compared with mice infected with control MHV68 (MHV68-MR). IFNgammaR(-/-) mice infected with MHV68-IkappaBalphaM lacked vasculitis and fibrosis 15 to 120 days post infection. Inhibition of NF-kappaB in MHV68-infected cells of the lungs diminished the expression of the fibrocyte recruiting chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL12, ameliorated macrophage expression of markers of alternative activation, and failed to increase expression of the integrin alphavbeta6, which is implicated in the activation of the profibrotic factor TGF-beta. Thus, the inhibition of NF-kappaB signaling in the infected lung cells of IFNgammaR(-/-) mice reduces virus persistence and ameliorates profibrotic events. Host determinants of latency might therefore represent new therapeutic targets for gammaherpesvirus-associated pulmonary fibrosis.

A senescence accelerated mouse model to study aging in the larynx.

OBJECTIVE: Age-related changes in the larynx lead to significant voice impairment and reduced quality of life. There is a need for aged animal models that have practical generation times to study the fundamental changes and new therapeutics for the aging voice. The senescence accelerated prone mouse strain (SAMP) animals experience rapid aging without any experimental manipulation. The main objective of this study was to demonstrate the use of senescence accelerated mice to study aging in the larynx. STUDY DESIGN: Murine model. SETTING: Department of Animal Resources, Emory University. SUBJECTS AND METHODS: Larynges from five senescence accelerated prone mice, five normal aging senescence resistant mice, and five C57BL/6 mice were harvested and processed for paraffin sections. Histomorphometry was performed for assessment of collagen and hyaluronic acid distribution. In addition, frozen laryngeal tissue was harvested for transcriptional and translational assessment of collagen-1, using real-time polymerase chain reaction with specific primers and Western blots. Myofibroblast assessment was performed by immunostaining for the presence of alpha-smooth muscle actin. RESULTS: The deposition of collagen increased at six months of age in the SAMP vocal fold, and the level of collagen-1 mRNA increased with age. The myofibroblast protein alpha-smooth muscle actin was also found at a higher concentration in the SAMP vocal tissue. In contrast, the levels of hyaluronic acid in the vocal folds of SAMP mice decreased with age when compared to age-matched C57BL/6 mice. CONCLUSION: SAMP mice show accelerated, age-related changes in the vocal fold that were evident at as early as six months of age. The use of senescence accelerated mice offers promise as a model to study age-related laryngeal changes.

Slow-release nanoparticle-encapsulated delivery system for laryngeal injection.

OBJECTIVES/HYPOTHESIS: There is a need for a slow-release system for local delivery of therapeutics to the larynx. Most therapeutic substances, such as steroids or chemotherapeutic agents that are injected into the larynx are cleared rapidly. Repeated laryngeal injection of these substances at short intervals is impractical. Injectable encapsulated poly(lactide-co-glycolide) (PLGA) nanoparticles offer a potential slow-release delivery system for biologically active substances in the larynx. STUDY DESIGN: Controlled animal study. METHODS: PLGA nanoparticles were fabricated using a double emulsion method and were loaded with Texas Red-dextran (NPTR), hepatocyte growth factor (NPHGF), and bovine serum albumin (NPBSA). In vitro release of NPTR, NPBSA, and NPHGF was determined over approximately 2 weeks to assess potential duration of PLGA nanoparticle delivery. In vivo release of NPTR was assessed in a murine vocal fold injection model. The transcriptional effect of NPHGF on procollagen was measured in vitro to assess whether released growth factor retained functionality. RESULTS: In vitro release kinetics demonstrated slow release of NPTR, NPBSA, and NPHGF over 12 to 14 days. In vitro NPTR release correlated with in vivo results. In vivo presence of NPTR occurred up to 7 days compared to 1 day for Texas Red control. In addition, NPHGF ameliorated transforming growth factor-beta induced procollagen in vitro in 3T3 fibroblast cells. CONCLUSIONS: The results demonstrate the potential utility of nanoparticle encapsulation as an effective method for long-term delivery of specific drugs and biologically active substances to the larynx.

Twist: a regulator of epithelial-mesenchymal transition in lung fibrosis.

BACKGROUND: Several studies have implicated viral infection as an important factor in the pathogenesis of IPF and related fibrotic lung disorders. Viruses are thought to cause epithelial cell injury and promote epithelial-mesenchymal transition (EMT), a process whereby differentiated epithelial cells undergo transition to a mesenchymal phenotype, and considered a source of fibroblasts in the setting of lung injury. We have demonstrated an association between the epithelial injury caused by chronic herpes virus infection with the murine gamma-herpes virus, MHV68, and lung fibrosis. We hypothesize that EMT in this model of virus-induced pulmonary fibrosis is driven by the expression of the transcription factor Twist. METHODS/FINDINGS: In vitro MHV68 infection of murine lung epithelial cells induced expression of Twist, and mesenchymal markers. Stable overexpression of Twist promoted EMT in MLE15 lung epithelial cells. Transient knockdown expression of Twist resulted in preservation of epithelial phenotype after in vitro MHV68 infection. In concordance, high expression of Twist was found in lung epithelial cells of MHV68 infected mice, but not in mock infected mice. Alveolar epithelial cells from lung tissue of idiopathic pulmonary fibrosis (IPF) patients were strongly positive for Twist. These cells demonstrated features of EMT with low expression of E-cadherin and upregulation of the mesenchymal marker N-cadherin. Finally, IPF tissue with high Twist protein levels was also positive for the herpesvirus, EBV. CONCLUSIONS/SIGNIFICANCE: We conclude that Twist contributes to EMT in the model of virus-induced pulmonary fibrosis. We speculate that in some IPF cases, gamma-herpes virus infection with EBV might be a source of injury precipitating EMT through the expression of Twist.

Oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis.

Several lines of evidence indicate that depletion of glutathione (GSH), a critical thiol antioxidant, is associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, GSH synthesis depends on the amino acid cysteine (Cys), and relatively little is known about the regulation of Cys in fibrosis. Cys and its disulfide, cystine (CySS), constitute the most abundant low-molecular weight thiol/disulfide redox couple in the plasma, and the Cys/CySS redox state (E(h) Cys/CySS) is oxidized in association with age and smoking, known risk factors for IPF. Furthermore, oxidized E(h) Cys/CySS in the culture media of lung fibroblasts stimulates proliferation and expression of transitional matrix components. The present study was undertaken to determine whether bleomycin-induced lung fibrosis is associated with a decrease in Cys and/or an oxidation of the Cys/CySS redox state and to determine whether these changes were associated with changes in E(h) GSH/glutathione disulfide (GSSG). We observed distinct effects on plasma GSH and Cys redox systems during the progression of bleomycin-induced lung injury. Plasma E(h) GSH/GSSG was selectively oxidized during the proinflammatory phase, whereas oxidation of E(h) Cys/CySS occurred at the fibrotic phase. In the epithelial lining fluid, oxidation of E(h) Cys/CySS was due to decreased food intake. Thus the data show that decreased precursor availability and enhanced oxidation of Cys each contribute to the oxidation of extracellular Cys/CySS redox state in bleomycin-induced lung fibrosis.

Control of virus reactivation arrests pulmonary herpesvirus-induced fibrosis in IFN-gamma receptor-deficient mice.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disorder of unknown cause. Several studies suggest an association between Epstein-Barr virus pulmonary infection and the development of IPF. OBJECTIVES: To determine whether reduction of gamma-herpesvirus reactivation from latency would alter progressive lung fibrogenesis in an animal model of virus-induced pulmonary fibrosis. METHODS: IFN-gamma receptor-deficient (IFN-gammaR(-/-)) mice infected intranasally with murine gamma-herpesvirus 68 (MHV68) develop lung fibrosis that progresses for up to at least 180 days after initial infection. Viral replication during the chronic phase of infection was controlled by two methods: the administration of cidofovir, an antiviral drug effective at clearing lytic but not latent virus, and by using a mutant gamma-herpesvirus defective in virus reactivation from latency. MEASUREMENTS AND MAIN RESULTS: Ten percent of the asymptomatic MHV68-infected animals that received antiviral treatment beginning on Day 45 postinfection had severe pulmonary fibrosis compared with 40% of the control saline-treated animals. Absence of severe fibrosis was also observed in IFN-gammaR(-/-) mice infected with the defective reactivation mutant MHV68 v-cyclin stop. Decreased fibrosis was associated with lower levels of transforming growth factor-beta, vascular endothelial growth factor, and markers of macrophage alternative activation. When antiviral treatment was administered on Day 60 in symptomatic animals, survival improved from 20 to 80% compared with untreated symptomatic animals, but lung fibrosis persisted in 60% of the mice. CONCLUSIONS: MHV68-induced fibrosis is a result of viral lytic replication during chronic lung herpesvirus infection in mice. We speculate that antiviral therapy might help to control lung fibrosis in humans with IPF and associated herpesvirus infection.

Activation of alveolar macrophages via the alternative pathway in herpesvirus-induced lung fibrosis.

The etiology of idiopathic pulmonary fibrosis (IPF) is unknown. Because viral pathogenesis of IPF has been suggested, we have established a murine model of progressive pulmonary fibrosis by infecting IFN-gammaR-deficient mice (IFN-gammaR(-/-)) with the murine gamma-herpesvirus 68. Because alveolar macrophages in humans with IPF have been implicated in driving the profibrotic response, we studied their role in our model. Chronic herpesvirus infection of the lung was associated with recruitment of alveolar macrophages to areas with epithelial hyperplasia and fibrosis in infected lungs. Using immunohistochemistry, Western blot, and RT-PCR techniques, we demonstrated that recruited alveolar macrophages showed high levels of expression of the proteins Ym1/2, FIZZ1 (found in inflammatory zone 1), insulin-like growth factor-1, and arginase I, and also active transcription of fibronectin, indicative of activation of macrophages by an alternative pathway. Arginase I expression was also evident in interstitial fibroblasts, and increased arginase activity was found in lungs of infected animals. Lung tissue from patients with IPF showed increased expression of arginase I in epithelial cells, fibroblast foci, and alveolar macrophages compared with normal lung. These results suggest that virus-induced upregulation of arginase I could be a mechanism driving lung fibrogenesis.