Debbie Clements

Effect of doxycycline on proliferation, MMP production, and adhesion in LAM-related cells.

Matrix metalloproteinases (MMPs) have been implicated in lung cyst formation in lymphangioleiomyomatosis (LAM). As doxycycline inhibits MMP activity in vivo, some patients take doxycycline, as one report has suggested a possible benefit in LAM. However, there have been no randomized controlled clinical trials of doxycycline for LAM, and any mechanism of action is unclear. Here, we examine previously proposed mechanisms of actions. Cell proliferation and adhesion were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and Cytomatrix cell adhesion kits. Apoptosis was examined by TdT-mediated dUTP nick end labeling (TUNEL) assay. MMP-2 expression was examined by quantitative real-time PCR and zymography in doxycycline-treated ELT3 cells and tumor growth using angiomyolipoma-derived tumor xenografts in nude mice. In ELT3 cells, >or=25 microg/ml doxycycline decreased proliferation, increased apoptosis, and caused a change in cell morphology associated with redistribution of actin stress filaments. Reduction in proliferation was also seen in human angiomyolipoma-derived cells. Cell adhesion to ECM proteins was decreased by doxycycline at 50 microg/ml and prevented detachment of already adherent cells. There was no effect of doxycycline on MMP-2 expression or activity in vitro. In the xenograft model, doxycycline (30 mg*kg(-1)*day(-1)) had no effect on tumor growth, final tumor weight, or tumor lysate MMP levels. Doxycycline at doses >or= 25 microg/ml inhibited cell proliferation and adhesion, possibly by a toxic effect. Doxycycline had no effect on MMP-2 expression or activity or tumor growth in the xenograft model. Any possible in vivo effect is unlikely to be mediated by MMP-2 or reduced cell proliferation.

Analysis of the oestrogen response in an angiomyolipoma derived xenograft model

Angiomyolipomas are benign mesenchymal tumours of smooth muscle, blood vessels and fat which occur sporadically or associated with tuberous sclerosis and lymphangioleiomyomatosis (LAM), a rare cystic lung disease. Angiomyolipoma and LAM are caused by loss of function of either the tuberous sclerosis-1 or -2 genes resulting in activation of p70S6kinase (S6K1) and uncontrolled cellular proliferation. LAM and angiomyolipoma can be exacerbated by oestrogens but how this occurs is not understood. To address this question, we created a xenograft tumour system in nude mice using immortalised angiomyolipoma cells. Angiomyolipoma xenografts had active S6K1, p38, p42/44 MAPK and Akt; they grew more rapidly and had greater Akt phosphorylation after oestrogen treatment of tumour-bearing mice. Transcriptional profiling showed oestrogen induced 300 genes including extracellular matrix proteins, proteases, cell cycle regulatory proteins and growth factors including platelet derived growth factor-C (PDGF-C). Biologically active PDGF-C was produced by primary angiomyolipoma cells in culture and PDGF-C protein was present in the neoplastic smooth muscle cells of 5/5 human angiomyolipoma and 4/5 LAM tissues examined by immunohistochemistry. These findings suggest that the response to oestrogen in this model is mediated by activation of Akt and transcriptional events. This model may prove useful for studying the biology and effect of drugs on angiomyolipoma and diseases related to TSC.

Role of the CXCR4/CXCL12 Axis in Lymphangioleiomyomatosis and Angiomyolipoma

Lymphangioleiomyomatosis (LAM) is a progressive disease caused by accumulation of metastatic (LAM) cells in the lungs, lymphatics, and the tumor angiomyolipoma (AML). LAM cells have biallelic loss of either tuberous sclerosis complex gene (but predominantly TSC-2) and resultant dysregulation of the mammalian target of rapamycin (mTOR) pathway. Chemokines are associated with neoplastic cell growth, survival, and homing to specific organs and may play similar roles in LAM. Our objective was to study comprehensively the expression and function of chemokine receptors and how their function interacts with dysregulation of the mTOR pathway in LAM and AML. We used RT-PCR and FACS to study receptor expression in primary AML cells and immunohistochemistry to investigate expression in tissues. Chemokine receptor function was analyzed in AML cells by Western blotting of signaling proteins and cell proliferation and apoptosis assays. Primary AML cells, LAM, and AML tissues expressed CCR3, CXCR4, CXCR6, and CXC3CR1. In AML cells, their ligands CXCL12 CX3CL1, CCL11, CCL24, and CCL28 caused robust phosphorylation of p42/44 MAPK and Akt. CXCL12 was expressed in type II pneumocytes covering LAM nodules and caused AML cell growth and protection from apoptosis, which was blocked by AMD3100, a CXCR4 inhibitor. The mTOR inhibitor rapamycin, but not AMD3100, inhibited growth of AML tumor xenografts. We conclude that the CXCL12/CXCR4 axis promotes, but is not absolutely required for, AML/LAM cell growth and survival.

Extra-cellular matrix proteins induce matrix metalloproteinase-1 (MMP-1) activity and increase airway smooth muscle contraction in asthma.

Airway remodelling describes the histopathological changes leading to fixed airway obstruction in patients with asthma and includes extra-cellular matrix (ECM) deposition. Matrix metalloproteinase-1 (MMP-1) is present in remodelled airways but its relationship with ECM proteins and the resulting functional consequences are unknown. We used airway smooth muscle cells (ASM) and bronchial biopsies from control donors and patients with asthma to examine the regulation of MMP-1 by ECM in ASM cells and the effect of MMP-1 on ASM contraction. Collagen-I and tenascin-C induced MMP-1 protein expression, which for tenascin-C, was greater in asthma derived ASM cells. Tenascin-C induced MMP-1 expression was dependent on ERK1/2, JNK and p38 MAPK activation and attenuated by function blocking antibodies against the β1 and β3 integrin subunits. Tenascin-C and MMP-1 were not expressed in normal airways but co-localised in the ASM bundles and reticular basement membrane of patients with asthma. Further, ECM from asthma derived ASM cells stimulated MMP-1 expression to a greater degree than ECM from normal ASM. Bradykinin induced contraction of ASM cells seeded in 3D collagen gels was reduced by the MMP inhibitor ilomastat and by siRNA knockdown of MMP-1. In summary, the induction of MMP-1 in ASM cells by tenascin-C occurs in part via integrin mediated MAPK signalling. MMP-1 and tenascin-C are co-localised in the smooth muscle bundles of patients with asthma where this interaction may contribute to enhanced airway contraction. Our findings suggest that ECM changes in airway remodelling via MMP-1 could contribute to an environment promoting greater airway narrowing in response to broncho-constrictor stimuli and worsening asthma symptoms.

Wild Type Mesenchymal Cells Contribute to the Lung Pathology of Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare disease leading to lungs cysts and progressive respiratory failure. Cells of unknown origin accumulate in the lungs forming nodules and eventually resulting in lung cysts. These LAM cells are described as clonal with bi-allelic mutations in TSC-2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures containing cells of different phenotypes; we investigated whether recruited wild type cells were also present alongside mutation bearing cells. Cells were isolated from LAM lung tissue, cultured and characterised using microscopy, immunocytochemistry and western blotting. Fibroblast-like cells were identified in lung tissue using immunohistochemical markers. Fibroblast chemotaxis toward LAM cells was examined using migration assays and 3D cell culture. Fibroblast-like cells were obtained from LAM lungs: these cells had fibroblast-like morphology, actin stress fibres, full length tuberin protein and suppressible ribosomal protein S6 activity suggesting functional TSC-1/2 protein. Fibroblast Activation Protein, Fibroblast Specific Protein/S100A4 and Fibroblast Surface Protein all stained subsets of cells within LAM nodules from multiple donors. In a mouse model of LAM, tuberin positive host derived cells were also present within lung nodules of xenografted TSC-2 null cells. In vitro, LAM 621-101 cells and fibroblasts formed spontaneous aggregates over three days in 3D co-cultures. Fibroblast chemotaxis was enhanced two fold by LAM 621-101 conditioned medium (p=0.05), which was partially dependent upon LAM cell derived CXCL12. Further, LAM cell conditioned medium also halved fibroblast apoptosis under serum free conditions (p=0.03). Our findings suggest that LAM nodules contain a significant population of fibroblast-like cells. Analogous to cancer associated fibroblasts, these cells may provide a permissive environment for LAM cell growth and contribute to the lung pathology of LAM lung disease.

CCR3 induced‐p42/44 MAPK activation protects against staurosporine induced‐DNA fragmentation but not apoptosis in airway smooth muscle cells

Chemokine receptors (CCRs) are expressed on airway smooth muscle (ASM) cells. As their ligands are present in the airways in asthma, we hypothesized that ASM CCR activation could promote the increase in ASM mass seen in patients with chronic asthma.

ECM crosslinking enhances fibroblast growth and protects against matrix proteolysis in lung fibrosis

&NA; Idiopathic pulmonary fibrosis (IPF) is characterized by accumulation of extracellular matrix (ECM) proteins and fibroblast proliferation. ECM cross‐linking enzymes have been implicated in fibrotic diseases, and we hypothesized that the ECM in IPF is abnormally cross‐linked, which enhances fibroblast growth and resistance to normal ECM turnover. We used a combination of in vitro ECM preparations and in vivo assays to examine the expression of cross‐linking enzymes and the effect of their inhibitors on fibroblast growth and ECM turnover. Lysyl oxidase‐like 1 (LOXL1), LOXL2, LOXL3, and LOXL4 were expressed equally in control and IPF‐derived fibroblasts. Transglutaminase 2 was more strongly expressed in IPF fibroblasts. LOXL2‐, transglutaminase 2‐, and transglutaminase‐generated cross‐links were strongly expressed in IPF lung tissue. Fibroblasts grown on IPF ECM had higher LOXL3 protein expression and transglutaminase activity than those grown on control ECM. IPF‐derived ECM also enhanced fibroblast adhesion and proliferation compared with control ECM. Inhibition of lysyl oxidase and transglutaminase activity during ECM formation affected ECM structure as visualized by electron microscopy, and it reduced the enhanced fibroblast adhesion and proliferation of IPF ECM to control levels. Inhibition of transglutaminase, but not of lysyl oxidase, activity enhanced the turnover of ECM in vitro. In bleomycin‐treated mice, during the postinflammatory fibrotic phase, inhibition of transglutaminases was associated with a reduction in whole‐lung collagen. Our findings suggest that the ECM in IPF may enhance pathological cross‐linking, which contributes to increased fibroblast growth and resistance to normal ECM turnover to drive lung fibrosis.

S92 Matrix metalloproteinase-1 activation by mast cell tryptase causes airway remodelling and is associated with bronchial hyper-responsiveness in patients with asthma

Introduction Matrix Metalloproteinase-1 (MMP-1) is a collagenase, which is present, in its inactive form, in the airways, lung parenchyma and in broncho-alveolar lavage (BAL) fluid of patients with asthma. We hypothesised that MMP-1 could be activated during asthma exacerbations leading to extra-cellular matrix (ECM) processing which contributes to airway remodelling. Methods Patients with stable, BTS stage 2/3 asthma, and healthy controls underwent Juniper asthma questionnaire, spirometry, methacholine challenge and bronchoscopy. Bronchial washings were processed for MMP-1 protein and activation. A second cohort of 14 patients with mild and 16 with moderate asthma and 10 controls underwent rhinovirus inoculation and had BAL fluid collected 14 days before and 4 days after inoculation. MMP-1 activity was assessed by fluorescent activity assay. ECM was prepared from decellularised airway smooth muscle (ASM) cultures. Cell proliferation was measured by MTT reduction assay and cell counts. Mast cell supernatants were obtained from cultures of HMC-1 cells activated using Phorbol 12-myristate 13-acetate/Calcium ionophore. Results Pro-MMP-1 was expressed more strongly in bronchial washings in asthma than controls (P = 0.0003). After rhinovirus inoculation, asthma symptoms increased and lung function fell. BAL MMP-1 activity increased in asthma patients compared with controls (P = 0.047). MMP-1 protein and activity was positively associated with fall in FEV1 (R square = 0.3618) (P = 0.0039) post viral inoculation. Activated, but not control, mast cell supernatants increased both expression of pro- and active MMP-1 by ASM cell cultures. This was blocked almost completely by inhibitors of tryptase but not chymase or MMPs. Recombinant tryptase activated MMP-1 in vitro. ECM obtained from both control and asthma derived ASM cells treated with activated mast cell supernatants during matrix synthesis and ECM treated directly after decellularization with active MMP-1 (10 ng/ml) enhanced subsequent ASM growth by 1.5 fold (P < 0.05). Conclusions MMP-1 expression and activity in bronchial fluid is enhanced during asthma exacerbations and is associated with increased BHR. MMP-1 activation by mast cell tryptase processes ASM derived ECM to enhance ASM growth in-vitro. Our findings suggest that ASM/mast cell interactions during exacerbations may contribute to airway remodelling by generating a pro-proliferative matrix.

Formation of the Endoderm in Xenopus

Throughout most of the last century the study of the amphibian embryo was a dominant theme in embryology, the balance scarcely shifting until the fusion of molecular biology and Drosophila genetics and the introduction of targeted gene inactivation in mice. Within amphibian embryology the concept of the Organiser has been predominant. This was initially important in emphasising the importance of cell interactions in patterning the embryo, but it has somewhat skewed experiments and concepts towards a primacy of the dorsal tissues and particularly the head and brain. Part of the reason for this was heuristic; these structures are the most easily recognised, particularly without molecular markers. However, a second reason has perhaps been an unconscious focus on the region of the animal we find most interesting, i.e. the head and brain. This way of thinking about the embryo naturally led to the naming of the Nieuwkoop Centre, the part of the vegetal hemisphere which generates the Organiser, and hence the head, through sperm-directed redistribution of elements of the Wnt pathway (Gerhart et al. 1989). Of course, Nieuwkoop actually discovered that the vegetal pole generates a polarised signal that induces both the posterior and ventral mesoderm (Nieuwkoop 1969a,b, 1973). However, we have associated his name with what we find more interesting.

P254 Identifying MMP-12 Substrates as Therapeutic Targets in COPD

Background Matrix metalloprotease (MMP)-12 is a key protease in COPD which cleaves pulmonary extracellular matrix and non-matrix substrates. Variation in MMP-12 activity affects severity of COPD, yet the mechanism of this, including MMP-12’s non-matrix substrates in COPD lungs are unknown. Targeting MMP-12 substrates may lead to the development of drugs for COPD with reduced side effects compared to the broader spectrum MMP inhibitors. Aims To identify MMP-12 substrates of relevance to COPD and determine how their activity affects disease progression in vitro and in vivo. Methods In vitro cleavage assays: After literature review the pro-inflammatory mediators osteopontin and tumour necrosis factor (TNF)-α were selected as potential MMP-12 substrates in COPD. Both were incubated with MMP-12 and reaction products analysed by silver stain and western blot. EDTA was used as a metalloprotease inhibitor and thrombin as positive control. COPD cohort: Patients with COPD were recruited during exacerbations at the Nottingham University Hospitals NHS Trust. Sputum, lung function and other data were collected on Day 0 and 1 and 4 weeks later. Sputum was analysed by western blot for proteins of interest. The study was approved by the local research ethics committee and all patients gave informed consent. Results MMP-12 cleaved osteopontin and pro-TNF-α in a dose and time-dependent manner when visualised by silver staining. Cleavage was dependent on MMP-12 activity as it was inhibited by EDTA. Western blot of cleaved protein fragments gave a characteristic band signature. MMP-12 was present in sputum of patients with COPD as demonstrated by western blotting, ELISA and casein zymography. Western blot analysis of sputum with anti-osteopontin antibodies showed a similar band signature to the in vitro cleavage suggesting osteopontin is cleaved in the airways of patients with COPD. Discussion MMP-12 possesses proteolytic activity against osteopontin and pro-TNF-α in vitro. MMP-12, osteopontin and TNFα are present in COPD sputum and our data suggest that MMP-12 may target osteopontin in COPD. Further work is needed to determine the precise mechanisms of such MMP-12 substrate activity in COPD.