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BACKGROUND: Asthma affects 300 million people worldwide. In asthma, the major cause of morbidity and mortality is acute airway narrowing, due to airway smooth muscle (ASM) hypercontraction, associated with airway remodelling. However, little is known about the transcriptional differences between healthy and asthmatic ASM cells. OBJECTIVES: To investigate the transcriptional differences between asthmatic and healthy airway smooth muscle cells (ASMC) in culture and investigate the identified targets using in vitro and ex vivo techniques. METHODS: Human asthmatic and healthy ASMC grown in culture were run on Affymetrix_Hugene_1.0_ST microarrays. Identified candidates were confirmed by PCR, and immunohistochemistry. Functional analysis was conducted using in vitro ASMC proliferation, attachment and contraction assays and ex vivo contraction of mouse airways. RESULTS: We suggest a novel role for latrophilin (LPHN) receptors, finding increased expression on ASMC from asthmatics, compared with non-asthmatics in vivo and in vitro, suggesting a role in mediating airway function. A single nucleotide polymorphism in LPHN1 was associated with asthma and with increased LPHN1 expression in lung tissue. When activated, LPHNs regulated ASMC adhesion and proliferation in vitro, and promoted contraction of mouse airways and ASMC. CONCLUSIONS: Given the need for novel inhibitors of airway remodelling and bronchodilators in asthma, the LPHN family may represent promising novel targets for future dual therapeutic intervention.

Fibulin-1 is an extracellular matrix (ECM) protein, levels of which are elevated in serum and lung tissue from patients with idiopathic pulmonary fibrosis compared to healthy volunteers. Inhibition of fibulin-1C, one of four fibulin-1 isoforms, reduced proliferation and wound healing in human airway smooth muscle (ASM) cells. This study identified the bioactive region/s of fibulin-1C which promotes fibrosis. Seven fibulin-1C peptides were synthesized and used to pre-coat tissue culture plates before lung derived ASM cells and fibroblasts from patients with pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD) or neither disease (Control) were plated. Peptide effects on in vitro measures of fibrosis: cell attachment, proliferation and viability, and ECM deposition, were examined. Among these peptides, peptide 1C1 (FBLN1C1) enhanced ASM cell and fibroblast attachment. FBLN1C1 increased mitochondrial activity and proliferation in fibroblasts. In addition, FBLN1C1 stimulated fibulin1 deposition in PF and COPD fibroblasts, and augmented fibronectin and perlecan deposition in all three groups. Peptides FBLN1C2 to FBLN1C7 had no activity. The active fibulin-1C peptide identified in this study describes a useful tool for future studies. Ongoing investigation of the role of fibulin-1 may reveal the mechanisms underlying the pathphysiology of chronic lung diseases.

Lymphangioleiomyomatosis (LAM) is a rare and progressive cystic lung condition affecting approximately 3.4-7.5/million women, with an average lag time between symptom onset and diagnosis of upwards of 4 years. The aim of this work was to identify altered proteins in LAM serum which may be potential biomarkers of disease. Serum from LAM patient volunteers and healthy control volunteers were pooled and analysis carried out using quantitative 4-plex iTRAQ technology. Differentially expressed proteins were validated using ELISAs and pathway analysis was carried out using Ingenuity Pathway Analysis. Fourteen proteins were differentially expressed in LAM serum compared to control serum (p<0.05). Further screening validated the observed differences in extracellular matrix remodelling proteins including fibronectin (30% decrease in LAM, p = 0.03), von Willebrand Factor (40% reduction in LAM, p = 0.03) and Kallikrein III (25% increase in LAM, p = 0.03). Pathway networks elucidated the relationships between the ECM and cell trafficking in LAM. This study was the first to highlight an imbalance in networks important for remodelling in LAM, providing a set of novel potential biomarkers. These understandings may lead to a new effective treatment for LAM in the future.

Background The underlying mechanisms of idiopathic pulmonary fibrosis (IPF) are unknown. This progressive disease has high mortality rates and current models for prediction of mortality have limited value in identifying which patients will progress. We previously showed that the glycoprotein fibulin-1 is involved in enhanced proliferation and wound repair by mesenchymal cells, thus may contribute to lung fibrosis in IPF. Methods Serum, lung tissue and lung function values were obtained from four independent locations (Sydney and Perth, Australia, San Francisco, USA and Modena, Italy). Patients with IPF were followed for a minimum of one year and progression was defined as a significant decline in lung function or death. Primary parenchymal lung fibroblasts of 15 patients with and without IPF were cultured under non-stimulatory conditions. Fibulin-1 levels in serum and secreted or deposited by fibroblasts were measured by western blot and in lung tissue by immunohistochemistry. Results Serum fibulin-1 levels were increased in patients with IPF compared to subjects without lung disease (p=0.006). Furthermore, tissue fibulin-1 levels were increased in patients with IPF (p=0.02) and correlated negatively with lung function (r=-0.9, p<0.05). Primary parenchymal fibroblasts from patients with IPF produced more fibulin-1 than those from subjects without IPF (p<0.05). Finally, serum fibulin-1 levels at first blood draw predicted disease progression in IPF within 1 year (AUC 0.71, 95%CI 0.57 0.86, p=0.012). Conclusions Fibulin-1 is a novel potential biomarker for disease progression IPF and raise the possibility that it could be used as a target for the development of new treatments.

Background: Tumstatin is a segment of the collagen-IV protein that is markedly reduced in the airways of asthmatics. Tumstatin can play an important role in the development of airway remodelling associated with asthma due to its antiangiogenic properties. This study assessed the anti-angiogenic properties of smaller peptides derived from tumstatin, which contain the interface tumstatin uses to interact with the aVb3 integrin. Methods: Primary human lung endothelial cells were exposed to the LF-15, T3 and T7 tumstatin-derived peptides and assessed for cell viability and tube formation in vitro. The impact of the anti-angiogenic properties on airways hyperresponsiveness (AHR) was then examined using a murine model of chronic OVA-induced allergic airways disease. Results: The LF-15 and T7 peptides significantly reduced endothelial cell viability and attenuated tube formation in vitro. Mice exposed to OVA+ LF-15 or OVA+T7 also had reduced total lung vascularity and AHR was attenuated compared to mice exposed to OVA alone. T3 peptides reduced cell viability but had no effect on any other parameters. Conclusion: The LF-15 and T7 peptides may be appropriate candidates for use as novel pharmacotherapies due to their small size and anti-angiogenic properties observed in vitro and in vivo. © 2014 Grafton et al.

Lymphangioleiomyomatosis (LAM) is a rare and progressive cystic lung condition affecting approximately 3.4-7.5/million women, with an average lag time between symptom onset and diagnosis of upwards of 4 years. The aim of this work was to identify altered proteins in LAM serum which may be potential biomarkers of disease. Serum from LAM patient volunteers and healthy control volunteers were pooled and analysis carried out using quantitative 4-plex iTRAQ technology. Differentially expressed proteins were validated using ELISAs and pathway analysis was carried out using Ingenuity Pathway Analysis. Fourteen proteins were differentially expressed in LAM serum compared to control serum (p<0.05). Further screening validated the observed differences in extracellular matrix remodelling proteins including fibronectin (30% decrease in LAM, p = 0.03), von Willebrand Factor (40% reduction in LAM, p = 0.03) and Kallikrein III (25% increase in LAM, p = 0.03). Pathway networks elucidated the relationships between the ECM and cell trafficking in LAM. This study was the first to highlight an imbalance in networks important for remodelling in LAM, providing a set of novel potential biomarkers. These understandings may lead to a new effective treatment for LAM in the future. © 2014 Banville et al.

Background and Objective: Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired b-agonist induced ASM relaxation in asthmatics, but the mechanism is not known. Objective: To characterize the potential defect in b-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism. Methods: We examined b2-adrenergic (b2AR) receptor expression and basal b-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined. Results: In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ,50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ,2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM. Conclusion: Decreased b-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal b-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be ``hard-wired into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed.

Airway remodeling, which includes increases in the extracellular matrix (ECM), is a characteristic feature of asthma and is correlated to disease severity. Rhinovirus (RV) infections are associated with increased risk of asthma development in young children and are the most common cause of asthma exacerbations. We examined whether viral infections can increase ECM deposition and whether this increased ECM modulates cell proliferation and migration. RV infection of nonasthmatic airway smooth muscle (ASM) cells significantly increased the deposition of fibronectin (40% increase, n = 12) and perlecan (80% increase, n = 14), while infection of asthmatic ASM cells significantly increased fibronectin (75% increase, n = 9) and collagen IV (15% increase, n = 9). We then treated the ASM cells with the Toll-like receptor (TLR) agonists polyinosinic:polycytidylic acid, imiquimod, and pure RV RNA and were able to show that the mechanism through which RV induced ECM deposition was via the activation of TLR3 and TLR7/8. Finally, we assessed whether the virus-induced ECM was bioactive by measuring the amount of migration and proliferation of virus-naive cells that seeded onto the ECM. Basically, ECM from asthmatic ASM cells induced twofold greater migration of virus-naive ASM cells than ECM from nonasthmatic ASM cells, and these rates of migration were further increased on RV-modulated ECM. Increased migration on the RV-modulated ECM was not due to increased cell proliferation, as RV-modulated ECM decreased the proliferation of virus-naive cells. Our results suggest that viruses may contribute to airway remodeling through increased ECM deposition, which in turn may contribute to increased ASM mass via increased cell migration. © 2011 the American Physiological Society.

Background and objective: A hallmark of asthma is airway remodelling, which includes increased deposition of extracellular matrix (ECM) protein. Viral infections may promote the development of asthma and are the most common causes of asthma exacerbations. We evaluated whether rhinovirus (RV) infection induces airway remodelling, as assessed by ECM deposition. Methods: Primary human bronchial epithelial cells and lung parenchymal fibroblasts were infected with RV-2 or RV-16, or treated with RV-16 RNA, imiquimod (Toll-like receptor (TLR) 7/8 agonist) or polyinosinic: polycytidylic acid (poly I: C) (activator of TLR 3, retinoic-acid-inducible protein I and melanoma-differentiated-associated gene 5). Changes in ECM proteins and their transcription were measured by ELISA and quantitative real-time PCR. In addition, gene expression for ECM proteins was assessed in a mouse model of RV infection. Results: RV infection increased deposition of the ECM protein, perlecan, by human bronchial epithelial cells, and collagen V and matrix-bound vascular endothelial growth factor were increased in both human bronchial epithelial cell and fibroblast cultures. Purified RV-16 RNA, poly I: C and imiquimod induced similar increases in ECM deposition to those observed with RV-infected fibroblasts. However, only poly I: C induced ECM deposition by bronchial epithelial cells, suggesting that RV-induced ECM deposition is mediated through TLR. Furthermore, gene expression for fibronectin and collagen I was increased in lung homogenates of mice infected with RV-1b. Conclusions: RV infection and TLR ligands promote ECM deposition in isolated cell systems and RV induces ECM gene expression in vivo, thus demonstrating that RV has the potential to contribute to remodelling of the airways through induction of ECM deposition. Airway remodelling, as indicated by increased extracellular matrix production, was induced by rhinovirus in both in vitro and in vivo models. This study provides important infor

Background: The extracellular matrix is a dynamic and complex network of macromolecules responsible for maintaining and influencing cellular functions of the airway. The role of fibronectin, an extracellular matrix protein, is well documented in asthma. However, the expression and function of fibulin-1, a secreted glycoprotein which interacts with fibronectin, has not been reported. Fibulin-1 is widely expressed in basement membranes in many organs including the lung. There are four isoforms in humans (A-D) of which fibulin-1C and 1D predominate. The objective of this study was to study the expression of fibulin-1 in volunteers with and without asthma, and to examine its function in vitro. Methodology/Principal Findings: We used immunohistochemistry and dot-blots to examine fibulin-1 levels in bronchial biopsies, bronchoalveolar lavage fluid and serum. Real-time PCR for fibulin-1C and 1D, and ELISA and western blotting for fibulin-1 were used to study the levels in airway smooth muscle cells. The function of fibulin-1C was determined by assessing its role, using an antisense oligonucleotide, in cell proliferation, migration and wound healing. A murine model of airway hyperresponsiveness (AHR) was used to explore the biological significance of fibulin-1. Levels of fibulin-1 were significantly increased in the serum and bronchoalveolar lavage fluid of 21 asthmatics compared with 11 healthy volunteers. In addition fibulin-1 was increased in asthma derived airway smooth muscle cells and fibulin-1C contributed to the enhanced proliferation and wound repair in these cells. These features were reversed when fibulin-1C was suppressed using an antisense oligomer. In a mouse model of AHR, treatment with an AO inhibited the development of AHR to methacholine. Conclusions: Our data collectively suggest fibulin-1C may be worthy of further investigation as a target for airway remodeling in asthma. © 2010 Lau et al.

Human bronchial epithelial (HBE) cells contribute to asthmatic airway inflammation by secreting cytokines, chemokines, and growth factors, including interleukin (IL)-6, IL-8 and transforming growth factor (TGF) ß1, all of which are elevated in asthmatic airways. This study examines the signaling pathways leading to TGFß1 induced IL-6 and IL-8 in primary HBE cells from asthmatic and non-asthmatic volunteers. HBE cells were stimulated with TGFß1 in the presence or absence of signaling inhibitors. IL-6 and IL-8 protein and mRNA were measured by ELISA and real-time PCR respectively, and cell signaling kinases by Western blot. TGFß1 increased IL-6, but inhibited IL-8 production in both asthmatic and non-asthmatic cells; however, TGF induced significantly more IL-6 in asthmatic cells. Inhibition of JNK MAP kinase partially reduced TGFß1 induced IL-6 in both cell groups. TGFß1 induced Smad2 phosphorylation, and blockade of Smad2/3 prevented both the TGFß1 modulated IL-6 increase and the decrease in IL-8 production in asthmatic and non-asthmatic cells. Inhibition of Smad2/3 also increased basal IL-8 release in asthmatic cells but not in non-asthmatic cells. Using CHIP assays we demonstrated that activated Smad2 bound to the IL-6, but not the IL-8 promoter region. We conclude that the Smad2/3 pathway is the predominant TGFß1 signaling pathway in HBE cells, and this is altered in asthmatic bronchial epithelial cells. Understanding the mechanism of aberrant pro-inflammatory cytokine production in asthmatic airways will allow the development of alternative ways to control airway inflammation