Effect of beta-agonists on LAM progression and treatment.

Lymphangioleiomyomatosis (LAM), a rare disease of women, is associated with cystic lung destruction resulting from the proliferation of abnormal smooth muscle-like LAM cells with mutations in the tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2 The mutant genes and encoded proteins are responsible for activation of the mechanistic target of rapamycin (mTOR), which is inhibited by sirolimus (rapamycin), a drug used to treat LAM. Patients who have LAM may also be treated with bronchodilators for asthma-like symptoms due to LAM. We observed stabilization of forced expiratory volume in 1 s over time in patients receiving sirolimus and long-acting beta-agonists with short-acting rescue inhalers compared with patients receiving only sirolimus. Because beta-agonists increase cAMP and PKA activity, we investigated effects of PKA activation on the mTOR pathway. Human skin TSC2+/- fibroblasts or LAM lung cells incubated short-term with isoproterenol (beta-agonist) showed a sirolimus-independent increase in phosphorylation of S6, a downstream effector of the mTOR pathway, and increased cell growth. Cells incubated long-term with isoproterenol, which may lead to beta-adrenergic receptor desensitization, did not show increased S6 phosphorylation. Inhibition of PKA blocked the isoproterenol effect on S6 phosphorylation. Thus, activation of PKA by beta-agonists increased phospho-S6 independent of mTOR, an effect abrogated by beta-agonist-driven receptor desensitization. In agreement, retrospective clinical data from patients with LAM suggested that a combination of bronchodilators in conjunction with sirolimus may be preferable to sirolimus alone for stabilization of pulmonary function.

The Lymphangioleiomyomatosis Lung Cell and Its Human Cell Models.

Lymphangioleiomyomatosis (LAM) is a multisystem disease of women, affecting lungs, kidneys, and lymphatics. It is caused by the proliferation of abnormal smooth muscle-like LAM cells, with mutations and loss of heterozygosity in the TSC1 or, more frequently, TSC2 genes. Isolated pulmonary LAM cells have been difficult to maintain in culture, and most studies of LAM lung cells involve mixtures of TSC2 wild-type and TSC2-null cells. A clonal population of LAM lung cells has not been established, making analysis of the cells challenging. Cell lines have been established from angiomyolipomas, a common manifestation of LAM, and from tumors from patients with TSC. Circulating LAM cells have also been isolated from blood and other body fluids. LAM cells may also be identified in clusters apparently derived from lymphatic vessels. Genetics, patterns of antigen expression, and signaling pathways have been studied in LAM lung tissue and in LAM cell models, although rarely all in the same study. We show here that LAM cells manifest differences in these characteristics, depending on the source investigated, suggesting further studies.

Official American Thoracic Society/Japanese Respiratory Society Clinical Practice Guidelines: Lymphangioleiomyomatosis Diagnosis and Management.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease that primarily affects women. The purpose of these guidelines is to provide recommendations for the diagnosis and treatment of LAM. METHODS: Systematic reviews were performed to summarize evidence pertinent to our questions. The evidence was summarized and discussed by a multidisciplinary panel. Evidence-based recommendations were then formulated, written, and graded using the Grading of Recommendations, Assessment, Development, and Evaluation approach. RESULTS: After considering the panel's confidence in the estimated effects, the balance of desirable (i.e., benefits) and undesirable (i.e., harms and burdens) consequences of treatment, patient values and preferences, cost, and feasibility, recommendations were formulated for or against specific interventions. These included recommendations for sirolimus treatment and vascular endothelial growth factor D testing and recommendations against doxycycline and hormonal therapy. CONCLUSIONS: Evidence-based recommendations for the diagnosis and treatment of patients with LAM are provided. Frequent reassessment and updating will be needed.

Osteoprotegerin contributes to the metastatic potential of cells with a dysfunctional TSC2 tumor-suppressor gene.

In addition to its effects on bone metabolism, osteoprotegerin (OPG), a soluble member of the tumor necrosis factor family of receptors, promotes smooth muscle cell proliferation and migration and may act as a survival factor for tumor cells. We hypothesized that these cellular mechanisms of OPG may be involved in the growth and proliferation of lymphangioleiomyomatosis (LAM) cells, abnormal smooth muscle-like cells with mutations in one of the tuberous sclerosis complex tumor-suppressor genes (TSC1/TSC2) that cause LAM, a multisystem disease characterized by cystic lung destruction, lymphatic infiltration, and abdominal tumors. Herein, we show that OPG stimulated proliferation of cells cultured from explanted LAM lungs, and selectively induced migration of LAM cells identified by the loss of heterozygosity for TSC2. Consistent with these observations, cells with TSC2 loss of heterozygosity expressed the OPG receptors, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules showed reactivities to antibodies to tumor necrosis factor-related apoptosis-inducing ligand, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules also produced OPG, as shown by expression of OPG mRNA and colocalization of reactivities to anti-OPG and anti-gp100 (HMB45) antibodies in LAM lung nodules. Serum OPG was significantly higher in LAM patients than in normal volunteers. Based on these data, it appears that OPG may have tumor-promoting roles in the pathogenesis of lymphangioleiomyomatosis, perhaps acting as both autocrine and paracrine factors.

Unexpected sirolimus-stimulated airway hyperreactivity in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a multisystem disease affecting primarily women, characterised in the lung by proliferation of LAM cells, abnormal smooth muscle-like cells with dysfunctional tuberous sclerosis complex genes. This dysfunction results in activation of mechanistic target of rapamycin (mTOR), leading to LAM cell proliferation. Sirolimus (rapamycin) is the only United States Food and Drug Administration-approved treatment for pulmonary LAM, resulting in decreased LAM cell growth/size and stabilised lung function [1].

Pulmonary Langerhans Cell Histiocytosis and Lymphangioleiomyomatosis Have Circulating Cells With Loss of Heterozygosity of the TSC2 Gene.

BACKGROUND: Lymphangioleiomyomatosis (LAM) and pulmonary Langerhans cell histiocytosis (PLCH) are cystic lung diseases in which a neoplastic cell is thought to be responsible for disease pathogenesis. The neoplastic LAM cell has mutations in the TSC genes, TSC1 or TSC2, whereas the neoplastic PLCH cell may have mutations in several genes (eg, BRAF, NRAS, MAP2K1). These mutations are not specific for PLCH and have been described in multiple cancers. TSC1 or TSC2 mutations and loss of heterozygosity (LOH) have also been described in cancers. RESEARCH QUESTION: Is TSC2 LOH specific to LAM or is it also found in PLCH? STUDY DESIGN AND METHODS: We recruited patients with LAM (n = 53) and healthy volunteers (n = 22) and compared the presence of cells with TSC2 LOH with patients with PLCH (n = 12). Blood and urine samples were collected for analysis. Fluorescence-activated cell sorting (FACS) was used to identify subpopulations of cells from blood and urine samples. We isolated CD45-CD235a-, CD45-CD235a+, and CD45+CD235a- cells from blood after density gradient separation. Cells were screened for TSC2 LOH at five microsatellites markers (ie, kg8, D16S3395, D16S3024, D16S521, D16S291). We obtained four cell subpopulations from urine (ie, CD44v6+CD9+, CD44v6+CD9-, CD44v6-CD9+, CD44v6-CD9-). RESULTS: Using FACS, cells were isolated from blood and urine from patients with PLCH that showed TSC2 LOH. Healthy volunteers did not have cells with TSC2 LOH. As a control, cells isolated from blood and urine from patients with LAM gave results similar to those reported previously. These data show that TSC2 LOH is found in patients with cystic lung diseases with potential neoplastic characteristics, and in patients with cancer. INTERPRETATION: The presence of TSC2 LOH in circulating cells is not specific for LAM. The data suggest that chromosomal abnormalities affecting the TSC2 gene are found in other diseases associated with cells having cancer-like neoplastic cells.

ETV2 regulates PARP-1 binding protein to induce ER stress-mediated death in tuberin-deficient cells.

Lymphangioleiomyomatosis (LAM) is a rare progressive disease, characterized by mutations in the tuberous sclerosis complex genes (TSC1 or TSC2) and hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1). Here, we report that E26 transformation-specific (ETS) variant transcription factor 2 (ETV2) is a critical regulator of Tsc2-deficient cell survival. ETV2 nuclear localization in Tsc2-deficient cells is mTORC1-independent and is enhanced by spleen tyrosine kinase (Syk) inhibition. In the nucleus, ETV2 transcriptionally regulates poly(ADP-ribose) polymerase 1 binding protein (PARPBP) mRNA and protein expression, partially reversing the observed down-regulation of PARPBP expression induced by mTORC1 blockade during treatment with both Syk and mTORC1 inhibitors. In addition, silencing Etv2 or Parpbp in Tsc2-deficient cells induced ER stress and increased cell death in vitro and in vivo. We also found ETV2 expression in human cells with loss of heterozygosity for TSC2, lending support to the translational relevance of our findings. In conclusion, we report a novel ETV2 signaling axis unique to Syk inhibition that promotes a cytocidal response in Tsc2-deficient cells and therefore maybe a potential alternative therapeutic target in LAM.

Erythropoietin-driven proliferation of cells with mutations in the tumor suppressor gene TSC2.

Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction, resulting from proliferation of smooth-muscle-like cells, which have mutations in the tumor suppressor genes TSC1 or TSC2. Among 277 LAM patients, severe disease was associated with hypoxia and elevated red blood cell indexes that accompanied reduced pulmonary function. Because high red cell indexes could result from hypoxemia-induced erythropoietin (EPO) production, and EPO is a smooth muscle cell mitogen, we investigated effects of EPO in human cells with genetic loss of tuberin function, and we found that EPO increased proliferation of human TSC2-/-, but not of TSC2+/-, cells. A discrete population of cells grown from explanted lungs was characterized by the presence of EPO receptor and loss of heterozygosity for TSC2, consistent with EPO involvement. In LAM cells from lung nodules, EPO was localized to the extracellular matrix, supporting evidence for activation of an EPO-driven signaling pathway. Although the high red cell mass of LAM patients could be related to advanced disease, we propose that EPO, synthesized in response to episodic hypoxia, may increase disease progression by enhancing the proliferation of LAM cells.

Chemokine-enhanced chemotaxis of lymphangioleiomyomatosis cells with mutations in the tumor suppressor TSC2 gene.

Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction caused by LAM cells (smooth-muscle-like cells) that have mutations in the tumor suppressor genes tuberous sclerosis complex (TSC) 1 or 2 and have the capacity to metastasize. Since chemokines and their receptors function in chemotaxis of metastatic cells, we hypothesized that LAM cells may be recruited by chemokine(s) in the lung. Quantification of 25 chemokines in bronchoalveolar lavage fluid from LAM patients and healthy volunteers revealed that concentrations of CCL2, CXCL1, and CXCL5 were significantly higher in samples from LAM patients than those from healthy volunteers. In vitro, CCL2 or MCP-1 induced selective migration of cells, showing loss of heterozygosity of TSC2 from a heterogeneous population of cells grown from explanted LAM lungs. Additionally, the frequencies of single-nucleotide polymorphisms in the CCL2 gene promoter region differed significantly in LAM patients and healthy volunteers (p = 0.018), and one polymorphism was associated significantly more frequently with the decline of lung function. The presence (i.e., potential functionality) of chemokine receptors was evaluated using immunohistochemistry in lung sections from 30 LAM patients. Expression of chemokines and these receptors varied among LAM patients and differed from that seen in some cancers (e.g., breast cancer and melanoma cells). These observations are consistent with the notion that chemokines such as CCL2 may serve to determine mobility and specify the site of metastasis of the LAM cell.

Effects of prolactin on TSC2-null Eker rat cells and in pulmonary lymphangioleiomyomatosis.

RATIONALE: Lymphangioleiomyomatosis, a cystic lung disease of women, is characterized by proliferation of smooth muscle-like lymphangioleiomyomatosis cells, which possess mutations in the tuberous sclerosis complex genes, TSC1/TSC2. Growth factors involved in lymphangioleiomyomatosis cell proliferation are unknown. Prolactin, an important reproductive hormone in women, is known to promote cell proliferation and survival in other tissues. OBJECTIVES: To determine the role of prolactin in signaling and proliferation in lymphangioleiomyomatosis. METHODS: Prolactin levels in the sera of patients with lymphangioleiomyomatosis were correlated with clinical status. Components of prolactin signal transduction pathways were assessed in lymphangioleiomyomatosis lesions from human lung explants by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Prolactin effects on proliferation and signaling were quantified in tuberin-deficient and tuberin-expressing rat cells in vitro. MEASUREMENTS AND MAIN RESULTS: Higher prolactin levels in the sera of patients with lymphangioleiomyomatosis were associated with a faster rate of decline in FEV(1) and an increased history of pneumothorax (P < 0.01). Higher levels of prolactin and prolactin receptor mRNA and immunoreactivity were found in lymphangioleiomyomatosis lesions when compared with vascular smooth muscle cells in the same region of tissue. This was accompanied by evidence of activation of signal transducer and activator of transcription-1 (STAT1), STAT3, p44/42, and p38 mitogen-activated protein kinase. Tsc2(-/-) Eker rat embryonic fibroblasts expressed more prolactin receptor than did Tsc2(+/+) cells, and responded to prolactin with increased proliferation and activation of the same signaling pathways seen in vivo. CONCLUSIONS: Prolactin may be an important growth factor in the pathogenesis of lymphangioleiomyomatosis.

Phenotypic characterization of disseminated cells with TSC2 loss of heterozygosity in patients with lymphangioleiomyomatosis.

RATIONALE: Lymphangioleiomyomatosis (LAM), occurring sporadically (S-LAM) or in patients with tuberous sclerosis complex (TSC), results from abnormal proliferation of LAM cells exhibiting mutations or loss of heterozygosity (LOH) of the TSC genes, TSC1 or TSC2. OBJECTIVES: To identify molecular markers useful for isolating LAM cells from body fluids and determine the frequency of TSC1 or TSC2 LOH. METHODS: Candidate cell surface markers were identified using gene microarray analysis of human TSC2⁻(/)⁻ cells. Cells from bronchoalveolar lavage fluid (BALF), urine, chylous effusions, and blood were sorted based on reactivity with antibodies against these proteins (e.g., CD9, CD44v6) and analyzed for LOH using TSC1- and TSC2-related microsatellite markers and single nucleotide polymorphisms in the TSC2 gene. MEASUREMENTS AND MAIN RESULTS: CD44v6(+)CD9(+) cells from BALF, urine, and chyle showed TSC2 LOH in 80%, 69%, and 50% of patient samples, respectively. LAM cells with TSC2 LOH were detected in more than 90% of blood samples. LAM cells from different body fluids of the same patients showed, in most cases, identical LOH patterns, that is, loss of alleles at the same microsatellite loci. In a few patients with S-LAM, LAM cells from different body fluids differed in LOH patterns. No patients with S-LAM with TSC1 LOH were identified, suggesting that TSC2 abnormalities are responsible for the vast majority of S-LAM cases and that TSC1-disease may be subclinical. CONCLUSIONS: Our data support a common genetic origin of LAM cells in most patients with S-LAM, consistent with a metastatic model. In some cases, however, there was evidence for genetic heterogeneity between LAM cells in different sites or within a site.

Pulmonary nontuberculous mycobacterial disease: prospective study of a distinct preexisting syndrome.

RATIONALE: Pulmonary nontuberculous mycobacterial (PNTM) disease is increasing, but predisposing features have been elusive. OBJECTIVES: To prospectively determine the morphotype, immunophenotype, and cystic fibrosis transmembrane conductance regulator genotype in a large cohort with PNTM. METHODS: We prospectively enrolled 63 patients with PNTM infection, each of whom had computerized tomography, echocardiogram, pulmonary function, and flow cytometry of peripheral blood. In vitro cytokine production in response to mitogen, LPS, and cytokines was performed. Anthropometric measurements were compared with National Health and Nutrition Examination Survey (NHANES) age- and ethnicity-matched female control subjects extracted from the NHANES 2001-2002 dataset. MEASUREMENTS AND MAIN RESULTS: Patients were 59.9 (+/-9.8 yr [SD]) old, and 5.4 (+/-7.9 yr) from diagnosis to enrollment. Patients were 95% female, 91% white, and 68% lifetime nonsmokers. A total of 46 were infected with Mycobacterium avium complex, M. xenopi, or M. kansasii; 17 were infected with rapidly growing mycobacteria. Female patients were significantly taller (164.7 vs. 161.0 cm; P < 0.001) and thinner (body mass index, 21.1 vs. 28.2; P < 0.001) than matched NHANES control subjects, and thinner (body mass index, 21.1 vs. 26.8; P = 0.002) than patients with disseminated nontuberculous mycobacterial infection. A total of 51% of patients had scoliosis, 11% pectus excavatum, and 9% mitral valve prolapse, all significantly more than reference populations. Stimulated cytokine production was similar to that of healthy control subjects, including the IFN-gamma/IL-12 pathway. CD4(+), CD8(+), B, and natural killer cell numbers were normal. A total of 36% of patients had mutations in the cystic fibrosis transmembrane conductance regulator gene. CONCLUSIONS: Patients with PNTM infection are taller and leaner than control subjects, with high rates of scoliosis, pectus excavatum, mitral valve prolapse, and cystic fibrosis transmembrane conductance regulator mutations, but without recognized immune defects.

Angiotensin-converting enzyme inhibitors may affect pulmonary function in lymphangioleiomyomatosis.

INTRODUCTION: A local renin-angiotensin system exists in the pulmonary nodules of lymphangioleiomyomatosis patients. Sirolimus, the standard treatment for lymphangioleiomyomatosis, stabilizes lung function, but all patients do not respond to or tolerate sirolimus. As renin-angiotensin systems may affect tumor growth and metastasis, we questioned if angiotensin-converting enzyme inhibitors affected lymphangioleiomyomatosis disease progression. METHODS: Retrospective study of 426 patients was performed, examining angiotensin-converting enzyme levels, pulmonary function data, and angiotensin-converting enzyme inhibitor treatment. RESULTS: Serum angiotensin-converting enzyme levels were elevated in approximately 33% of patients, increased with duration of disease, and were inversely correlated with pulmonary function. Levels decreased significantly over time with sirolimus treatment. Treatment with angiotensin-converting enzyme inhibitors was reported by approximately 15% of patients and was significantly associated with a slower rate of decline in percentage predicted forced expiratory volume (FEV1) and diffusing capacity of the lungs for carbon monoxide (DLCO) in patients not treated with sirolimus. No significant differences in rates of decline of FEV1 or DLCO were seen in patients treated with both inhibitors and sirolimus versus sirolimus alone. CONCLUSIONS: Angiotensin-converting enzyme inhibitors may slow decline of pulmonary function in patients with lymphangioleiomyomatosis not treated with sirolimus. These inhibitors may be an option or adjunct in the treatment of lymphangioleiomyomatosis. A clinical trial may be warranted to examine this possibility. FUNDING: NIH.

Use of CT Imaging to Quantify Progression and Response to Treatment in Lymphangioleiomyomatosis.

BACKGROUND: In lymphangioleiomyomatosis (LAM), infiltration of the lungs with smooth muscle-like LAM cells results in cystic destruction and decline in lung function, effects stabilized by sirolimus therapy. LAM lung disease is followed, in part, by high-resolution CT scans. To obtain further information from these scans, we quantified changes in lung parenchyma by analyzing image "texture." METHODS: Twenty-six texture properties were quantified by analyzing the distribution and intensity of pixels with a computer-aided system. Both cross-sectional and longitudinal studies were performed to examine the relationships between texture properties, cyst score (percentage of lung occupied by cysts), FEV1, and diffusion capacity for carbon monoxide (Dlco), and to determine the effect of sirolimus treatment. RESULTS: In the cross-sectional study, 18 texture properties showed significant positive correlations with cyst score. Cyst score and 13 of the 18 texture properties showed significant differences in rates of change after sirolimus treatment; 11 also significantly predicted FEV1 and Dlco. CONCLUSIONS: Increased cyst score was associated with increased texture degradation near cysts. Sirolimus treatment improved lung texture surrounding cysts and stabilized cyst score. Eleven texture properties were associated with FEV1, Dlco, cyst score, and response to sirolimus. Texture analysis may be valuable in evaluating LAM severity and treatment response.