Background Lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm that causes progressive cystic lung destruction and is often associated with renal angiomyolipomas (AMLs). Given evidence of pleiotropy linking LAM risk to pulmonary traits, we investigated whether glucocorticoid receptor (GR) signalling might influence LAM biology and clinical features. Methods We combined cell-based studies, GR inhibition/activation assays, gene expression and single-cell RNA sequencing analyses, and hormone profiling in retrospective and prospective LAM cohorts. Cellular experiments employed murine Tsc2 −/− embryonic fibroblasts and human TSC2 −/− AML cells. Circulating steroid levels were measured in women with LAM and healthy controls, and associations with clinical variables were evaluated. Results In LAM/AML models, GR activation by glucocorticoids elicited transcriptional responses, whereas GR inhibition reduced clonogenic potential. GR stimulation was associated with CDKN1C upregulation through enhancer binding, and single-cell profiling suggested a shift towards slower proliferation and differentiation-prone states enriched for a LAM cell signature. Clinically, our analyses suggest that women with LAM may show altered circulating hormone profiles, including elevated adrenocorticotropic hormone (ACTH) and cortisol levels, together with reduced 17-hydroxyprogesterone, compared with controls. In a prospective cohort, ACTH levels were suggestively associated with advanced radiological disease stage. AML cells showed elevated expression of POMC , which encodes the precursor of ACTH, and POMC peptide was detected in LAM lung tissue. Conclusions Our findings suggest that GR signalling may contribute to aspects of LAM cell behaviour and disease status. Further investigation of this pathway could clarify its role as a disease modifier and potential therapeutic target.

RATIONALE:Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease driven by nodules containing TSC2 -/- "LAM cells" and recruited LAM-associated fibroblasts. Although rapamycin reduces lung function loss, some patients continue to decline, meaning additional therapies are needed. OBJECTIVES:To investigate how the LAM nodule environment affects LAM cell proliferation and the response to rapamycin. METHODS:Proteins altered in advanced LAM were identified using shotgun proteomics and immunohistochemistry in tissue from closely phenotyped patients. Genes associated with rapamycin insensitivity on LAM derived extracellular matrix were identified by RNA sequencing and validated using pharmacological inhibitors. RESULTS:More advanced disease was associated a greater decline in forced expiratory volume in 1 s when treated with rapamycin (p=0.005). In advanced LAM, using proteomics analysis, an upregulation of protein clusters comprising extracellular matrix, glucose metabolism and the actin cytoskeleton was identified. RNA sequencing and immunohistochemistry confirmed expression of collagens I and VI in LAM-associated fibroblasts and LAM nodules, and increased markers of collagen turnover in patient serum (p=0.0048). Growth of LAM patient-derived cells in vitro was faster on LAM associated fibroblast-derived extracellular matrix (p<0.0001) and incompletely suppressed by rapamycin. RNA sequencing identified upregulation of pathways driving cell cycle control, transcription and metabolism by extracellular matrix. Tractable, pro-proliferative, upregulated genes included CDK7, GAS6, PLAUR and PLAU. Inhibitors of these pathways reduced LAM cell proliferation and enhanced the antiproliferative effect of rapamycin. CONCLUSIONS:Extracellular matrix deposition upregulates the expression of genes which may blunt the response to rapamycin, but offer additional therapeutic opportunities for patients with established LAM.

Lymphangioleiomyomatosis (LAM) is a rare disease of women in which TSC2 deficient 'LAM cells' with dysregulated mTOR signalling and recruited fibroblasts form nodules causing lung cysts and respiratory failure. We examine if mTOR dysregulation can induce senescence and impair the response to lung injury in LAM. The senescence markers p21, p16 and the SenMayo gene set are increased in LAM lungs and colocalise with alveolar type 2 cells. LAM models induce mTOR dependent senescence in alveolar type 2 cell organoids in vitro and in vivo. IL-6 produced by LAM cells, induces p16 and p21 in alveolar type 2 cells, inhibits epithelial wound resolution and is related to lung function in LAM patients. Rapamycin and the IL-6 receptor antagonist Tocilizumab reduce alveolar type 2 cell organoid p21 accumulation and Tocilizumab enhances epithelial wound repair. Targeting IL-6 signalling in parallel with mTOR inhibition, may reduce lung damage in LAM.

Rationale Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease driven by nodules containing TSC2-/- ‘LAM cells’ and recruited LAM associated fibroblasts (LAFs). Although rapamycin reduces lung function loss, some patients continue to decline meaning additional therapies are needed.Objectives To investigate how the LAM nodule environment affects LAM cell proliferation and the response to rapamycin.Methods Changes in advanced LAM were identified using shotgun proteomics and immunohistochemistry in tissue from carefully phenotyped patients. Genes potentially associated with rapamycin insensitivity of cells grown on LAF-derived extracellular matrix were identified by RNA sequencing and validated using repurposed pharmacologic inhibitors.Main Results More advanced disease was associated with increasing nodules adjacent to lung cysts and greater decline in forced expiratory volume in 1 sec (FEV1) when treated with rapamycin (p=0.005). In late-stage LAM, proteomics identified upregulation of pathways associated with accumulation of activated fibroblasts, including extracellular matrix deposition, glucose metabolism and the actin cytoskeleton. Picrosirius red staining and immunohistochemistry confirmed deposition of extracellular matrix within LAM nodules. The growth of TSC2-/- model LAM cells was increased on LAF-derived extracellular matrix (LAF ECM), and incompletely supressed by rapamycin (p<0.0001). RNA sequencing of cells grown on LAF ECM identified upregulation of pathways driving cell cycle control, transcription and metabolism in cells. Tractable, pro-proliferative, rapamycin insensitive genes included CDK7 , GAS6 and PLAU. Repurposed inhibitors of these pathways inhibited LAM cell proliferation and enhanced the anti-proliferative effect of rapamycin.Conclusions Extracellular matrix deposited by LAM associated fibroblasts upregulates expression of genes which potentially blunt the response to rapamycin, but offer additional therapeutic opportunities for patients with established LAM.### Competing Interest StatementThe authors have declared no competing interest.

Introduction and Objectives Lymphangioleiomyomatosis (LAM) is a rare, female-specific cystic lung disease in which destruction of the lung parenchyma is driven by lesions containing TSC2-/- LAM cells and recruited stromal LAM associated fibroblasts (LAFs). LAM patients can be treated with rapamycin to stabilise lung function, but some patients continue to decline, and additional therapies are needed for these patients. We hypothesised that extracellular matrix (ECM) deposited by LAFs within lesions could affect LAM cell behaviour and promote disease progression; previously we showed that decellularised LAF-deposited ECM could stimulate TSC2-/- LAM-derived cell proliferation in vitro. We aimed to investigate the transcriptional response of LAM cells to ECM, to identify druggable, ECM-driven, pro-proliferative pathways. Methods RNA sequencing was used to identify genes that were differentially expressed in TSC2-/- LAM-derived cells grown on decellularised LAF-deposited matrix relative to cells grown on standard tissue culture plastic. We detected 458 genes with elevated expression on matrix, 155 genes with decreased expression. We mined these data for components of pro-proliferative pathways which were elevated on matrix, insensitive to rapamycin inhibition and targeted by drugs currently under clinical development, for rapid translation to the clinic. Results We identified several promising candidates including: All three components (CDK7, CCNH, MNAT1) of the CDK activating kinase (CAK) complex, responsible for the activating phosphorylation of CDK1, CDK2, CDK4 and CDK6 and promoting cell cycle progression. GAS6, the ligand for the AXL receptor tyrosine kinase, which has demonstrated pro-proliferative and anti-apoptotic activity in multiple cancer cell models. Urokinase (PLAU) and its receptor uPAR (PLAUR): UPAR interacts directly with integrins and vitronectin, to initiate signalling cascades involved in cell proliferation and survival. Inhibitors of these molecules (Samuraciclib/CDK7, Dubermatinib/AXL, IPR-803/UPAR) proved effective against TSC2-/- LAM-derived cells, significantly reducing proliferation in vitro. Conclusion LAF-derived ECM enhances TSC2-/- cell proliferation in vitro and may contribute to disease progression by providing a pro-proliferative microenvironment for LAM cells in vivo. A number of pro-proliferative molecules are upregulated when TSC2-/- LAM-derived cells are grow on decellularised ECM in vitro, and targeting these pathways may provide novel therapies for LAM patients with reduced response to rapamycin. Please refer to page A285 for declarations of interest related to this abstract.

Background Lymphangioleiomyomatosis (LAM) is a destructive monogenic disease in which clonal mTOR dysregulated mesenchymal 'LAM cells' recruit fibroblasts and immune cells forming discreet lung parenchymal nodules resulting in protease activation, lung cysts and respiratory failure. We hypothesised that mTOR driven senescence in LAM cells, induces senescence in adjacent LAM associated fibroblasts (LAF) in turn impairing ATII cell mediated repair of protease induced lung injury. Methods We examined LAM cell interactions using scRNAseq, laser microdissection, dual label immunohistochemistry, primary cell co-cultures and a TSC null murine homograft model. Results p21 and to a lesser extent, p16 proteins were increased in human LAM lung. Within LAM nodules p21 co-localised with both PNL2 (LAM cells) and PNL2 negative cells. Outside nodules, p21 co-localised with SPC (ATII cells). In immunocompetent LAM homograft models, senescence associated beta-galactosidase activity increased with time and was greater than control animals. scRNAseq of human LAM lungs showed alterations in ATII cell regulation of cell death, apoptotic pathways, senescence and Wnt signalling. A Stat 3/p53 dependent pathway governing apoptosis and alterations in lipolysis and ATI/II differentiation were present. In vitro LAM cell/LAF/epithelial co-cultures show that SA-beta-galactosidase activity and associated genes are upregulated in an mTOR dependent manner. Interrogation of scRNA seq data from nodules and epithelial areas validates these findings and is associated with lung function and disease duration in humans. Conclusions mTOR dysregulated LAM cells induce fibroblast and epithelial senescence and reduce ATII cell viability to impair the repair response to lung injury. Please refer to page A210 for declarations of interest related to this abstract.

Lymphangioleiomyomatosis (LAM) is a rare, female-specific cystic lung disease in which destruction of the lung parenchyma is driven by lesions containing TSC2-/- LAM cells and recruited stromal LAM associated fibroblasts (LAFs). LAM patients can be treated with rapamycin to stabilise lung function, but some patients continue to decline, and additional therapies are needed for these patients. We hypothesised that extracellular matrix (ECM) deposited by LAFs within lesions could affect LAM cell behaviour and promote disease progression. We aimed to quantify ECM deposition in LAM, investigate its association with disease severity and study the effect of LAF deposited matrix on LAM cell behaviour in vitro.MethodsCollagen neoepitopes were measured in sera from 96 LAM patients and 22 controls using Nordic Bioscience assays. Collagen deposition in paraffin sections of LAM lung tissue was assayed by picrosirius red (PSR) staining and immunohistochemistry from 19 lung samples with linked clinical data. In vitro assays were performed on TSC2-/- cells grown on cell-free LAF-derived ECM.ResultsSerum markers of collagen, but not elastin turnover tended to be greater in women with LAM than controls (C6M, p=0.057). Quantification of PSR staining revealed trends toward ECM accumulation with increasing disease duration (r=0.62, p=0.060) and reducing DLCO (r=-0.55, p=0.057). LAF-derived ECM increased proliferation (p<0.0001) and reduced the anti-proliferative effect of rapamycin in TSC2-/- cells (p=0.0004) in vitro.ConclusionCollagen deposition can be observed in LAM lesions. LAF-derived ECM enhances TSC2-/- cell proliferation in vitro and may contribute to disease progression by providing a pro-proliferative microenvironment for LAM cells in vivo. This may reduce response to rapamycin in some patients. These data support the investigation of anti-fibrotic therapies for LAM patients who respond poorly to rapamycin.

Introduction and Objectives Lymphangioleiomyomatosis (LAM) is a rare multisystem disease of women characterised by lung cysts, lymphatic abnormalities and angiomyolipomas. 'LAM' cells present in the lungs harbour mutations in either TSC1 or TSC2 genes giving rise to constituitive mTOR pathway activation. Lung damage in LAM is thought to result from aberrant protease activation. We previously showed the cysteine protease Cathepsin K (CTSK) was the most strongly expressed protease in LAM compared to control lung, increased with worsening disease and could be activated in LAM lung tissue. Here, we evaluate the inhibition of Cathepsin K in cell and murine models of LAM, and assess levels of CTSK in the sera of patients from a UK cohort of LAM. Methods TSC2-null cells were treated with the Cathepsin K inhibitor Odanacatib, viability and proliferation assessed. An immunocompetent TSC2-null murine model of LAM was treated with Odanacatib, rapamycin or both, tumour burden and cell proliferation were assessed. Cathepsin K activity was quantified in mouse lung tissue and BAL by Cathepsin K activity assay and ELISA. Serum Cathepsin K was measured in 53 women with LAM with linked phenotype and lung function data. Results Odanacatib inhibited the proliferation of murine TSC2-null TTJ cells in vitro (IC50=0.68 nMol, p=0.01). In a murine model of LAM over 6 weeks, Odanacatib reduced Cathepsin K activity in both BAL and lung (p=0.005), decreased the size of lung nodules (p<0.0001), reduced Ki67 immunopositivity in TSC2 null nodules (p=0.01) and tended to be synergistic with rapamycin. Serum Cathepsin K levels in 53 women with LAM were 7.8–84.7 pg/ml (35.3±17.5). Cathepsin K was higher in 27 patients with active LAM (12.4–84.7pg/ml, 40.8±19.0) compared to 26 with stable disease (7.8–62.1, 29.5±13.5), p=0.034 (figure 1). A trend was observed for higher levels of Cathepsin K in patients with lower FEV1 (p=0.067) but Cathepsin K was not associated with serum VEGFD (p=0.13). Conclusions Our findings suggest that Cathepsin K may be involved in lung damage in LAM and inhibition of Cathepsin K should be investigated further as a treatment for LAM. Please refer to page A208 for declarations of interest related to this abstract.

Lymphangioleiomyomatosis (LAM) is a female-specific cystic lung disease in which tuberous sclerosis complex 2 (TSC2)-deficient LAM cells, LAM-associated fibroblasts (LAFs), and other cell types infiltrate the lungs. LAM lesions can be associated with type II alveolar epithelial (AT2) cells. We hypothesized that the behavior of AT2 cells in LAM is influenced locally by LAFs. We tested this hypothesis in the patient samples and in vitro. In human LAM lung, nodular AT2 cells show enhanced proliferation when compared with parenchymal AT2 cells, demonstrated by increased Ki67 expression. Furthermore, nodular AT2 cells express proteins associated with epithelial activation in other disease states including matrix metalloproteinase 7, and fibroblast growth factor 7 (FGF7). In vitro, LAF-conditioned medium is mitogenic and positively chemotactic for epithelial cells, increases the rate of epithelial repair, and protects against apoptosis. In vitro, LAM patient-derived TSC2 null cells cocultured with LAFs upregulate LAF expression of the epithelial chemokine and mitogen FGF7, a potential mediator of fibroblast-epithelial cross talk, in a mechanistic target of rapamycin (mTOR)-dependent manner. In a novel in vitro model of LAM, ex vivo cultured LAM lung-derived microtissues promote both epithelial migration and adhesion. Our findings suggest that AT2 cells in LAM display a proliferative, activated phenotype and fibroblast accumulation following LAM cell infiltration into the parenchyma contributes to this change in AT2 cell behavior. Fibroblast-derived FGF7 may contribute to the cross talk between LAFs and hyperplastic epithelium in vivo, but does not appear to be the main driver of the effects of LAFs on epithelial cells in vitro.

Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene ftmction results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 +/- 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% +/- 23.6%; SCG: 5.5% +/- 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.

Cystic lung diseases are a group of rare diseases characterised by discrete areas of parenchymal destruction. This chapter will contrast the diverse mechanisms of parenchymal loss that occur in common cystic lung diseases such as chronic obstructive pulmonary disease (COPD) with those of rare disorders associated with dysregulation of specific molecular pathways. Four representative cystic lung diseases are described with reference to underlying molecular pathogenesis, mechanisms of cyst formation and pathological and radiological manifestations. We discuss how normal repair mechanisms may be deranged by processes such as senescence in COPD and how in lymphangioleiomyomatosis mechanistic target of rapamycin (mTOR) dysregulation leads to protease activation and a tumor-like microenvironment resulting in parenchymal destruction. In pulmonary Langerhans cell histiocytosis (PLCH), activation of the BRAF/MEK/ERK pathway in dendritic cells interacts with cigarette smoke to cause a destructive bronchiolitis. In Birt-Hogg-Dubé syndrome (BHD), loss of the tumour suppressor folliculin leads to defects in cell-cell interactions and shear-mediated lung damage. Finally, we discuss the poorly understood group of cystic lung diseases caused by abnormal immunoglobulin deposition in the lung. The end result in each case is thin-walled cystic change in the pulmonary parenchyma, which can have consequences that range from no symptoms, to recurrent pneumothorax, to chronic respiratory failure.

AbstractLymphangioleiomyomatosis (LAM) is a rare multisystem disease with a variable clinical course. The lungs are infiltrated by nodules of LAM cells, stromal cells and inflammatory cells, causing lung cysts and respiratory failure. We used immunohistochemical markers in lung biopsy and transplant samples from a national cohort of women with LAM with linked clinical data to understand how LAM nodule cell populations changed with disease progression. Marker distribution was examined qualitatively by dual immunohistochemistry, and markers for LAM cells, fibroblasts, lymphatics, mast cells, proliferation, cathepsin K and mTOR pathway activity were quantitated in LAM nodules and compared with clinical features and prospective lung function loss. The LAM cell marker PNL2 was more extensively expressed in those with higher forced expiratory volume in one second (FEV1), higher diffusion in the lung for carbon monoxide (DLCO) and less extensive disease involvement whilst the converse was true for the protease cathepsin K. Each percentage increase in cathepsin K reactivity was associated with a 0.65% decrease in FEV1 (95% CI −1.11 to −0.18) and a 0.50% decrease in DLCO (95% CI −0.96 to −0.05). Higher reactivity to the mTOR complex 1 activation marker, phospho‐ribosomal protein S6, was associated with a better lung function response to rapamycin (p = 0.0001). We conclude that LAM nodules evolve with disease progression, with LAM cells becoming outnumbered by fibroblasts. Increasing cathepsin K expression is associated with more severe disease and lung function loss. Markers of mTOR activation predict the response to rapamycin, suggesting that more advanced LAM may be less mTOR responsive and treatments specifically targeted towards LAM associated fibroblasts may have roles as adjuncts to mTOR inhibition.

Lymphangioleiomyomatosis (LAM) is a cystic lung disease mainly affecting women, in which degradation of the lung parenchyma is associated with a cell of unknown provenance, known as a LAM cell. LAM cells carry TSC2 mutations and can be identified in the lung parenchyma by their expression of both smooth muscle actin and antigens characteristic of melanocytes and melanocytic tumors. The nature of the cell-of-origin of LAM is controversial, and despite continued research effort remains elusive. Further, it has not been possible to culture pulmonary LAM cells in vitro, and current research relies on cells and animal models which may not recapitulate all features of the disease. We noted aberrant expression of melanoma antigens in pleural mesothelial cells in lung tissue from LAM patients, indicating that these cells could be the precursors of parenchymal LAM cells. We hypothesise that loss of tuberin function following TSC2 mutation in the mesothelial cell lineage gives rise to the cell-of-origin of pulmonary LAM (P-LAM), and of other associated conditions commonly noted in LAM patients. The unique properties of mesothelial cells provide a straightforward explanation of the diverse presentation of LAM.

Introduction: LAM is a rare lung disease characterised by progressive cystic destruction of the lung. There is no effective treatment. The hallmark lesion of LAM is the LAM nodule, a complex structure consisting of proliferating smooth muscle-like ‘LAM cells’, fibroblasts, lymphatic endothelial and inflammatory cells. KCa3.1 ion channels are implicated in several cell processes including proliferation, migration, differentiation and collagen secretion. We hypothesise that KCa3.1-dependent cell processes in LAM cells and LAM associated fibroblasts (LAFs) contribute to the development of LAM nodules and the accompanying lung damage. Methods: TSC null LAM cells and primary LAFs were obtained from the National Centre for LAM, Nottingham. We examined these cells for the presence of KCa3.1 channels using qRT-PCR and whole-cell patch clamp electrophysiology, and used immunohistochemistry to detect KCa3.1 in LAM tissue. We also examined the effects of KCa3.1 blockers on the formation of 3D LAM spheroids resulting from LAM cell and LAF co-culture. Results: Both LAM cells and LAFs (N=3 donors) expressed KCa3.1 mRNA. At rest, both LAM cells and LAFs displayed similar membrane currents with slight outward rectification at positive potentials. Both cell types responded to the KCa3.1 channel opener 1-EBIO with the development of characteristic KCa3.1 whole cell currents, which were blocked by the selective KCa3.1 blocker senicapoc. Immunoreactive KCa3.1 was present in LAM tissue, and inhibition of KCa3.1 channels inhibited the formation of LAM spheroids dose-dependently. Conclusion: LAM cells and primary LAFs express functional KCa3.1 channels which may contribute to the development of LAM nodules.

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.

Introduction and objectives LAM is a rare, progressive interstitial lung disease characterised by progressive cystic destruction of the lung. There is currently no effective treatment for LAM. The hallmark lesion of LAM lung disease is the LAM nodule, a complex structure consisting of proliferating smooth muscle-like ‘LAM cells’, fibroblasts, lymphatic endothelial cells and inflammatory cells. KCa3.1 ion channels are expressed by a number of structural airway and lung parenchymal cells including airway smooth muscle cells (ASM) and parenchymal fibroblasts, and are implicated in several cell processes including proliferation, migration, differentiation and collagen secretion. We hypothesise that KCa3.1-dependent cell processes in LAM cells and LAM associated fibroblasts (LAFs) are a common denominator in the development of LAM nodules and the accompanying lung destruction. Methods TSC null 621–101 LAM cells (human angiomyolipoma cell line) and primary LAFs were obtained from National Centre for LAM, Nottingham. We examined both cell types for the presence and activity of KCa3.1 channels using q-PCR and whole-cell variant patch clamp electrophysiology. Results Both LAM cells and LAFs (n=3) expressed KCa3.1 mRNA. At baseline, both LAM 621–101 cells and LAFs displayed similar membrane currents with slight outward rectification at positive potentials. Both cell types responded to the KCa3.1 channel opener 1-ethyl-2-benzimidazolinone (1-EBIO;100 µM) with the development of characteristic KCa3.1 whole cell currents. Furthermore, the currents were completely blocked by the selective KCa3.1 blocker TRAM 34 (200 nM). Conclusion LAM cells and primary LAFs express functional KCa3.1 channels. The role of these in the development and progression of LAM nodules requires further investigation.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 ajp.amjpathol.org 63 64 65 66 67 68 69 70 71 72 73 74 Cathepsin K in Lymphangioleiomyomatosis 75 76 77 78 79 80 81 LAM CelleFibroblast Interactions Enhance Protease Activity by Extracellular Acidification Arundhati Dongre,* Debbie Clements,* Andrew J. Fisher,y and Simon R. Johnson* 82 83 84 85 From the Division of Respiratory Medicine and Respiratory Research Unit,* University of Nottingham, Nottingham; and the Institute of Transplantation,y Newcastle upon Tyne Hospitals NHS FT and Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom