Rozprawy doktorskie na temat „Lymphangioleiomyomatosis”

Matrix metalloproteinases (MMPs) have been implicated in the lung destruction seen in lymphangioleiomyomatosis (LAM) as elevated MMP levels have been detected in tissue, serum and urine from LAM patients but a causal link has not been established. It has also been hypothesised that inhibition of MMPs with the tetracycline antibiotic doxycycline may provide a potential treatment for LAM. Previously proposed mechanisms of actions of doxycycline were examined. In Eker rat derived ELT3 cells doxycycline at doses ~25 micrograms/ml inhibited cell proliferation and adhesion, possibly by a toxic effect. This was also seen in human angiomyolipoma-derived cells. There was no effect of doxycycline on MMP-2 expression or activity in vitro. In a xenograft model, doxycycline had no effect on tumour growth, final tumour weight, or tumour lysate MMP levels. The role of MMPs as potential biomarkers was also compared with vascular endothelial growth factor D (VEGF-D). Serum VEGF-D, Angiotensin coverting enzyme (ACE) and total matrix metalloproteinase 2 (MMP-2) levels were elevated in patients. VEGF-D was the strongest discriminator between patients and controls and the addition VEGF-D measurement to ERS criteria reduced the need for biopsy to confirm diagnosis. Finally, we performed a 2 year, randomised, double blind placebo controlled study of doxycycline in 23 patients with LAM and found no evidence that it prevented decline in lung function, or improved exercise tolerance of quality of life.

Lymphangioleiomyomatosis (LAM) is a rare lung disease in young women. Dysfunction of the tuberous sclerosis gene complex (TSC)-1 and TSC2 contributes to the manifestations of enhanced proliferation and migration of smooth muscle-like cells (LAM cells), elevated matrix metalloproteinase (MMP) activity and increased lymphangiogenesis. Currently there is no effective treatment for this fatal disease. This study aimed to assess the effectiveness of potential pharmacotherapies (doxycycline, statins and lamstatin) in targeting the different pathological aspects of LAM. Studies in this thesis examined the effects of doxycycline in human LAM cells and in a cellular model for LAM. Doxycycline had no effect on cell proliferation, but reduced elevated MMP2 activity, migratory capability and wound closure. In addition, doxycycline reduced cell migration through the inhibition of RhoA-GTPase and focal adhesion kinase (FAK), proteins that are involved in the regulation of cell motility. These findings were extended to show that the combination of doxycycline and rapamycin (inhibitor of the mammalian target of rapamycin compelx 1 [mTORC1] which is a potent inhibitor of cell proliferation) exhibited an additive effect on the inhibition of wound closure compared to the individual drugs alone. MMP profiles in people with LAM were also examined and confirmed previous findings of elevated serum MMP2 and MMP9. In addition elevated serum levels of MMP1 and MMP3 were identified. The effectiveness of statins as a treatment for LAM was examined in studies which investigated their effects on the enhanced proliferation, migration, wound closure and MMP2 activity in TSC2-deficient cells. All parameters were reduced by simvastatin and fluvastatin. The anti-lymphangiogenic protein lamstatin (NC1 domain of collagen IV α5) is absent in the lungs of people with LAM. Results showed that lamstatin and its consensus peptide, CP17 inhibited the proliferation, migration and wound closure in a cellular model of LAM. These studies have enhanced our understanding of the disease mechanisms underlying LAM and have revealed potential molecular targets for future therapy in this disease.

LAM is a rare metastasizing and destructive disorder of the lung. The disease is caused by cells carrying mutations in the TSC1/2 genes, but the tissue of origin of diseased cells remains unknown. The standard of care for LAM is the inhibition of mTOR with sirolimus. The standard of care for LAM is the inhibition of mTOR with sirolimus. However, the therapy has variable tolerability. LAM diagnosis and monitoring can be challenging due to the heterogeneity of symptoms. In this thesis, we aimed to provide evidence for improving LAM care through the identification of useful biomarkers and exploration of novel therapeutic approaches. In parallel, we also studied disease origin. At the level of biomarkers, the major histamine-derived metabolite MIAA has been found to be more abundant in LAM plasmas. Combined studies of LAM lung tissues and cell models showed enhanced monoamine metabolism and histamine-mediated signaling. In addition, the inhibition of these two pathways decreased LAM tumorigenesis in mouse models. The results of this thesis may help improve the diagnostic process, clinical monitoring and therapeutic management of LAM patients.
LAM es un trastorno pulmonar raro, destructivo y metastásico. Esta enfermedad está causada por células que tienen mutaciones en los genes TSC1/2 cuyo origen sigue siendo desconocido. El tratamiento de LAM consiste en la inhibición de mTOR con sirolimus. Sin embargo, esta terapia presenta una tolerabilidad variable. El diagnóstico de LAM y su monitorización puede ser un desafío ya que los síntomas son muy heterogéneos. En esta tesis, nuestro objetivo era mejorar la atención de LAM a través de la identificación de biomarcadores y la exploración de nuevos enfoques terapéuticos. Paralelamente, también estudiamos el origen de la enfermedad. En cuanto a los biomarcadores, el principal metabolito derivado de la histamina MIAA estaba aumentado en el plasma de pacientes de LAM. Estudios combinados de tejidos pulmonares y modelos celulares mostraron un aumento en el metabolismo de las monoaminas y en la vía de señalización de la histamina. Además, la inhibición de estas dos rutas redujo la tumorogénesis de modelos LAM de ratón. Los resultados de esta tesis podrían ayudar a mejorar el diagnóstico, la monitorización y el tratamiento de las pacientes de LAM.

Background: Lymphangioleiomyomatosis (LAM), a rare progressive disease, is characterized by the proliferation of abnormal smooth muscle cells (LAM cells) in the lung, which leads to cystic parenchymal destruction and progressive respiratory failure. Estrogen receptors are present in LAM cells. LAM affects almost exclusively women of childbearing age. These findings, along with reports of disease progression during pregnancy or treatment with exogenous estrogens, have led to the assumption that hormonal factors play an important role in the pathogenesis of LAM. So, various therapies aim at preventing estrogen receptors (ER) by lowering circulating estrogen levels, by trying to block ER activity, or by attempting to lower ER expression in LAM. Prior experience have yielded conflicting results. Objective: The goal of this study was to evaluate, retrospectively, the effect of estrogen suppression in 21 patients with LAM. Design: We evaluated hormonal assays, pulmonary function tests and gas-exchange at baseline and after 12, 24 and 36 months after initiating hormonal manipulation. Results: The mean yearly rates of decline in FEV1 and DLCO are lower than those observed in prior studies and just DLCO decline was statistically significant. We also found an improvement of mean value of FVC and PaO2. Conclusions: Estrogen suppression appears to prevent decline in lung function in LAM.

Background: Lymphangioleiomyomatosis (LAM), a rare progressive disease, is characterized by the proliferation of abnormal smooth muscle cells (LAM cells) in the lung, which leads to cystic parenchymal destruction and progressive respiratory failure. Estrogen receptors are present in LAM cells. LAM affects almost exclusively women of childbearing age. These findings, along with reports of disease progression during pregnancy or treatment with exogenous estrogens, have led to the assumption that hormonal factors play an important role in the pathogenesis of LAM. So, various therapies aim at preventing estrogen receptors (ER) by lowering circulating estrogen levels, by trying to block ER activity, or by attempting to lower ER expression in LAM. Prior experience have yielded conflicting results. Objective: The goal of this study was to evaluate, retrospectively, the effect of estrogen suppression in 21 patients with LAM. Design: We evaluated hormonal assays, pulmonary function tests and gas-exchange at baseline and after 12, 24 and 36 months after initiating hormonal manipulation. Results: The mean yearly rates of decline in FEV1 and DLCO are lower than those observed in prior studies and just DLCO decline was statistically significant. We also found an improvement of mean value of FVC and PaO2. Conclusions: Estrogen suppression appears to prevent decline in lung function in LAM.

The multisystemic tumors characteristic of the monogenic neoplastic diseases, tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), share common signaling aberrations upon the loss of heterozygosity in either the TSC1 or TSC2 genes. However, their physical manifestations are vastly different and can generally be classified as being either neurological (TSC) or mesenchymal (TSC & LAM; referred to herein as LAM for simplicity) in origin. In this study, I present a comprehensive stem cell model of LAM utilizing multiple TSC2 knockout (TSC2-/-) pluripotent stem cell lines differentiated to the putative cell of origin for mesenchymal tumors, neural crest cells (NCCs). TSC2-/- NCCs faithfully recapitulate LAM phenotypes and temporal RNA-seq analysis of neural and neural crest differentiation was performed to model disease pathogenesis. Analysis revealed immediate activation of stress response signaling resulting in protein aggregation and lysosome and autophagosome accumulation upon neuralization in TSC2-/- cells. This resulted in acute and lasting effects specific to neural progenitor cells (NPCs), that are transient and ameliorated in NCCs. These lineage-specific effects resulted in selective sensitization of NPCs to cell death via proteasome inhibition, suggesting a potential therapeutic avenue for neurological TSC, but not LAM. Thus, a genome-wide CRISPR knockout screen was performed in TSC2-/- NCCs. Analysis of synthetic lethal genes reveals pathways previously targeted for LAM, but provides gene-level resolution to the vulnerable nodes within these pathways. Importantly, 18 novel gene targets were identified that display synthetic lethality to TSC2-/- cells with high specificity. 3 genes within this list were targetable using commercially available small molecule inhibitors, one of which, FGFR1, shows highly selective lethal targeting of TSC2-/- NCCs. Importantly, this model system, paired with the expansive resource of transcriptomic and synthetic lethal data, serves as a foundation for the development of next generation treatment strategies for LAM, and potentially the entire spectrum of TSC manifestations.

Lymphangioleiomyomatosis (LAM) is a rare lung disease recognized by abnormal growth of smooth muscle cells proliferating in lungs parenchyma, developing benign tumors, migrating to the other organs, and ultimately leading to respiratory failure and death. Despite existing literature mainly on clinical aspects of LAM, there is a gap of literature in regards to the knowledge, attitude, and lifestyle practices (KAPs) of LAM patients and their effects on their quality of life. The purpose of this quantitative study was to investigate the KAPs of the sporadic LAM patients as measured by the Bristol Chronic Obstructive Pulmonary Disease Knowledge Questionnaire, Beliefs and Behavior Questionnaire, Determinants of Lifestyle Behavior Questionnaire; these KAPs were then analyzed for their relationship to quality of life reports as measured by the St George’s Quality of Life Questionnaire. Transtheoretical model (TTM) was used to describe the relationship among the variables. The data were collected through online survey questionnaires from 143 sporadic LAM patients registered at the LAM Foundation. Pearson’s correlations and linear regression were used to analyze the data. The results of the analysis showed that there was a significant positive relationship between attitude, lifestyle practices, and quality of life and a negative relationship between knowledge and quality of life. The outcome achieved by this study and its implication on social change identifies the need to initiate more study-specific KAPs within LAM populations, including individuals with tuberous sclerosis complex LAM. The results could also encourage the LAM community as well as other stakeholders to implement programs, workshops, and interventions that could promote and enhance quality of life.

Tuberous sclerosis is an autosomal dominant multisystem disorder characterised by the development of benign tumours in many organs, including the brain, skin, kidneys and heart, seizures and intellectual disability. The condition results from mutations in either of two genes, TSC1 (encoding TSC1) or TSC2 (encoding TSC2). Loss of functional TSC1 or TSC2 leads to activation of mTORCl (mammalian target of rapamycin complex 1), a key regulator of multiple cellular processes including cell growth and division. Lymphangioleiomyomatosis (LAM) is a lung disorder which can lead to respiratory failure and is characterised by the proliferation of abnormal 'LAM' cells LAM occurs in patients with tuberous sclerosis and can also occur as a sporadic disorder due to acquired mutations in TSC2. Renal angiomyolipomas are benign tumours which affects80% of patients with tuberous sclerosis and 40% of patients with sporadic LAM. Sirolimus is an inhibitor of mTORCl and is used in clinical practice as an immunosuppressant. Preclinical studies suggest that TSC1 or TSC2 deficiency renders cells sensitive to mTOR inhibition. This thesis describes a phase 2 trial to assess the safety and efficacy of 2 years of treatment with sirolimus for renal angiomyolipomas in patients with tuberous sclerosis or LAM. Response of angiomyolipoma was the primary efficacy end point. Effects of sirolimus on lung function and neurocognitive function are also reported. Our data show an angiomyolipoma response rate, by RECIST criteria, of 50% in the intention to treat population. There was little change in lung function. Recall but not recognition memory tended to improve. Adverse events were common and consistent with the known toxicities of sirolimus. Our findings suggest that mTOR inhibition is a potential therapeutic strategy in the treatment of tuberous sclerosis and lymphangioleiomyomatosis.

Asthma is an inflammatory disease characterised by tissue remodelling. A prominent feature of this remodelling is an increase in the number and size of the blood vessels- formed from pre-existing capillaries – angiogenesis (Siddiqui et al., 2007; Wilson, 2003). This is triggered by many different endogenous angiogenic stimulators such as vascular endothelial growth factor (VEGF), and inhibited by endogenous angiogenic inhibitors such as tumstatin. Tumstatin is the non-collagenous domain (NC1) of the collagen IV α3 chain which, when cleaved, inhibits endothelial cell proliferation and induces apoptosis. Experiments described in this thesis have for the first time demonstrated the absence of tumstatin in the airways of individuals with asthma and lymphangioleiomyomatosis (LAM) as well as the functional responses to tumstatin as an angiogenic inhibitor, both in vitro and in vivo, in the airway. Although tumstatin was absent from the airways of asthmatic and LAM individuals it was present in the airways of individuals with no airways disease, chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis. No significant difference was seen in the levels of the Goodpasture Binding Protein (GPBP), a phosphorylating protein responsible for the alternate folding of tumstatin, between asthmatic, LAM and individuals with no airways disease. The αvβ3 integrin, reported to be necessary for the activity of tumstatin, as well as the individual αv and β3 sub-units were shown to be equally expressed in the airways of all patient groups. Co-localisation of tumstatin, VEGF and the αvβ3 integrin was seen in the disease free airways, however, a different pattern of VEGF and the αvβ3 integrin expression was observed in asthmatic and LAM airways with minimal co-localisation. Tumstatin was detected in serum and bronchoalveolar lavage fluid (BAL-f) samples from asthmatics and individuals with no airway disease, however there was no significant difference in the level of expression between the two groups. It was demonstrated that the tumstatin detected in the serum and BAL-f samples from asthmatics and individuals with no airway disease was part of the whole collagen IV α3 chain and not in its free and potentially active form. The ability of recombinant tumstatin to inhibit tube formation and proliferation of primary pulmonary endothelial cells was demonstrated for the first time. Further, the functional response of tumstatin was demonstrated in vivo in a mouse model of allergic airway disease. Tumstatin inhibited angiogenesis in the airway and decreased airway hyperresponsiveness. Whether there is potential for tumstatin, or a derivative thereof, to be of therapeutic value in airways diseases in which angiogenesis is a component should be the subject of future studies.

Doctor of Philosophy (PhD)
Asthma is an inflammatory disease characterised by tissue remodelling. A prominent feature of this remodelling is an increase in the number and size of the blood vessels- formed from pre-existing capillaries – angiogenesis (Siddiqui et al., 2007; Wilson, 2003). This is triggered by many different endogenous angiogenic stimulators such as vascular endothelial growth factor (VEGF), and inhibited by endogenous angiogenic inhibitors such as tumstatin. Tumstatin is the non-collagenous domain (NC1) of the collagen IV α3 chain which, when cleaved, inhibits endothelial cell proliferation and induces apoptosis. Experiments described in this thesis have for the first time demonstrated the absence of tumstatin in the airways of individuals with asthma and lymphangioleiomyomatosis (LAM) as well as the functional responses to tumstatin as an angiogenic inhibitor, both in vitro and in vivo, in the airway. Although tumstatin was absent from the airways of asthmatic and LAM individuals it was present in the airways of individuals with no airways disease, chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis. No significant difference was seen in the levels of the Goodpasture Binding Protein (GPBP), a phosphorylating protein responsible for the alternate folding of tumstatin, between asthmatic, LAM and individuals with no airways disease. The αvβ3 integrin, reported to be necessary for the activity of tumstatin, as well as the individual αv and β3 sub-units were shown to be equally expressed in the airways of all patient groups. Co-localisation of tumstatin, VEGF and the αvβ3 integrin was seen in the disease free airways, however, a different pattern of VEGF and the αvβ3 integrin expression was observed in asthmatic and LAM airways with minimal co-localisation. Tumstatin was detected in serum and bronchoalveolar lavage fluid (BAL-f) samples from asthmatics and individuals with no airway disease, however there was no significant difference in the level of expression between the two groups. It was demonstrated that the tumstatin detected in the serum and BAL-f samples from asthmatics and individuals with no airway disease was part of the whole collagen IV α3 chain and not in its free and potentially active form. The ability of recombinant tumstatin to inhibit tube formation and proliferation of primary pulmonary endothelial cells was demonstrated for the first time. Further, the functional response of tumstatin was demonstrated in vivo in a mouse model of allergic airway disease. Tumstatin inhibited angiogenesis in the airway and decreased airway hyperresponsiveness. Whether there is potential for tumstatin, or a derivative thereof, to be of therapeutic value in airways diseases in which angiogenesis is a component should be the subject of future studies.

Women’s experiences of living with a rare disease, lymphangioleiomyomatosis (LAM): A life history study LAM is a rare, potentially life limiting, multisystem condition, affecting almost exclusively women and characterised by progressive cystic lung disease. This study aimed to understand the meaning of women’s experiences of living with LAM over time and how they were affected by the rarity of LAM. Life history methodology was used to fulfill the aim of the study. Interviews were conducted with 19 women living with LAM. Each participant's life story was analysed for turning points and themes using Rosenthal's (1993) biographic interpretive method. Following cross case analysis, a collective life history focused on the participants’ illness experiences was constructed. The participants revealed diagnosis, commencing oxygen therapy, respiratory failure, and receiving a transplant as turning points of significant life disruption. The rarity of LAM created feelings of isolation and uncertainty and a need for self-reliance and self-advocacy to access appropriate information, care and treatment. The participants developed resilience as they adapted to their illness in a period of transition. Resilience was a dynamic learning process of finding meaning and gaining knowledge and competence in illness self-management. It enabled them to experience wellness and constructively manage health-related and social changes over the course of living with LAM.

Background: Lymphangioleiomyomatosis (LAM) is a rare progressive neoplastic cystic lung disease that primarily affects women of child bearing age leading to lung destruction, respiratory failure and death. Thought to be a consequence of dysregulated protease expression, cells of unknown origin accumulate in the lung, often forming clusters or nodules of cells with both melanocytic and smooth muscle properties. Some of these cells, known as LAM cells, have bi-allelic mutations in TSC2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures and genotyping analyses suggest that cells without LOH for TSC2 including wild-type fibroblasts are also common within LAM nodules. Hypotheses and aims: We hypothesise that LAM cells recruit wild-type fibroblasts and modify their properties to generate a permissive microenvironment, akin to a tumour stroma including the production and activation of matrix-degrading proteases which contribute to the destruction of the lung parenchyma. This study has therefore deigned in vitro co-culture models with an aim to study the expression patterns and activation of proteases in a LAM lung leading to matrix destruction. Another aim was also to characterise transcriptional differences normal human lung fibroblasts (NHLFs) and LAM-associated fibroblasts (LAFs) and to investigate changes in their gene expression when cultured together with a model LAM cell line, 621-101 angiomyolipoma cells which were derived from a LAM patient and have bi-allelic loss of TSC2. Methods: In vitro 2-dimensional (2D) and 3-dimensionalD (3D) co-culture models were designed and validated using fibroblasts characterised and isolated from 4 LAM lung donors, now termed LAFs, and 621-101 cells. The 3D extracellular matrix (ECM) incorporated the two cell types in a 10:1 ratio embedded in a basement membrane extract (BME) mimicking the lung matrix. An organotypic spheroid model was also developed incorporating both cell types thereby mimicking a LAM nodule. 6 LAM lung and 3 normal lung tissue donors were screened for candidate proteases in LAM pathology using qRT-PCR and identified upregulated proteases which may contribute to a role in LAM pathology. These findings were verified in the 2D and 3D in vitro models as well as ex vivo tissue using a variety of immunostaining techniques, activity assays and ELISA. Lastly, commercially bought NHLF (n=3) and LAF (n=3) were cultured in the presence or absence of 621-101 cells in the 2D Boyden chamber co-culture model. LAF and NHLF RNA was analysed using Affymetrix Human Genome U133 Plus 2.0 Arrays and Genomics Suite and Pathway (Partek). Findings were validated by multiplex assay and immunohistochemistry in 2D and 3D in vitro models and tissue respectively. Inflammatory cell migration and function was examined in co-culture model and LAM tissue. Results: The 3D BME model showed that TSC2-/- 621-101 cells and fibroblasts spontaneously form aggregates and clump together akin to a LAM nodule. The two cell types exhibited strong heterotypic cell-cell adhesive forces and resulted in strictly spherical spheroids. The 3D models designed all showed expression of markers of LAM nodules thereby representing LAM nodules in a dish. Of 30 proteases screened, cathepsin K gene expression was increased almost 15-fold in LAM lung compared to normal tissue and was also found to be elevated in 3D BME model. Cathepsin K in LAM tissue was expressed in the LAM nodules associated with cysts and was expressed exclusively by fibroblasts in the 3D spheroid model. As cathepsin K requires low pH for activity it was determined if LAFs and TSC2-/- cells can acidify the extracellular space. TSC2-/- cells but not LAFs decreased extracellular pH, over 24 hours and pH values < 7 were associated with increased cathepsin K activity in co-cultures. TSC2-/- cells expressed membrane transporters associated with extra-cellular acidification and inhibitors of the sodium bicarbonate co-transporters, carbonic anhydrases and mTOR reduced the pH gradient and decreased CTSK activity in co-cultures. Transcriptomic analysis using the 2D co-culture model showed 148 genes were significantly altered in both NHLF and LAF by 621-101 cells. Soluble factors from 621-101 cells induce pro-inflammatory transcriptional changes in both NHLFs and LAFs and pathway analysis showed enhanced chemokine signalling which highlighted stimulation of mainly the C-X-C motif chemokines and chemokine receptor signalling. The analysis identified 6 C-X-C motif chemokines all possessing a cognate receptor. The gene and protein expression of these chemokines was validated in the in vitro models and in ex vivo LAM lung tissue. Conclusions: The in vitro models are versatile and mimic the LAM lung nodule and environment. A potent matrix degrading protease possibly playing a role in LAM has been identified and using the in vitro models a possible mechanism of activation of CTSK resulting from a synergistic relationship between TSC2-/- cells and LAFs has been demonstrated. Also, soluble factors from the TSC2-/- LAM cell line elicit changes in gene expression in co-cultured fibroblasts. Chemokine signalling is associated with cell migration; elevated chemokine expression may be associated with the recruitment of inflammatory cells to the LAM nodule. The identification of these mechanisms and pathways opens up new avenues for therapeutic interventions in LAM.

Lymphangioleiomyiomatosis (LAM) is a rare progressive cystic lung disease that affects almost exclusively women. LAM can occur sporadically, or can be associated with tuberous sclerosis complex (TSC); a rare disorder with multiorgan involvement effecting the brain, kidneys, heart, liver, skin and eyes and is associated with intellectual disability, epilepsy and autism spectrum disorders. Dr.Terraneo PhD project was developed with the aim to expand clinical knowledge about diagnosis and follow up as well as to analyze pathogenic aspect of the development of the disease. As a first step of the PhD project, the association between LAM and other features of TSC (e.g. demography, extrapulmonary manifestations, genetic mutations..) was investigated as well as the role of pulmonary function tests (PFTs) for LAM diagnosis. Our results demonstrate that age, but not PFTs, is independently associated with LAM development in patients with TSC. PFTs, even if indicated to assess impairment in lung function, result feasible in a limited number of patients due to cognitive impairment, and are not significantly useful for LAM diagnosis in women with TSC. Successively, the case of a patients with coexistence of three rare diseases (autoimmune hepatitis/primary biliary cirrhosis overlap syndrome, lymphangioleiomyomatosis/tuberous sclerosis complex (LAM-TSC), and sarcoidosis) was described. We speculated that the dysregulation of the pathway involving mTOR and MAPK and their interaction might play a role in the pathogenesis of diseases other than TSC, including sarcoidosis. In the last part of PhD project, the serum levels of VEGF-D, VEGF-C, MMP-2 and MMP-7 were assessed in a cohort of patients affected with S-LAM and TSC with and without LAM. Our results showed that VEGF-D, MMP-2 and MMP7 were higher in patients with LAM than in patients without. VEGF-D was confirmed as the biomarkers with the highest accuracy for LAM diagnosis. MMP-2 and MMP-7 could be a promising biomarker of LAM.

Introdução: A linfangioleiomiomatose (LAM) está associada a HP e está incluída no grupo 5 da classificação atual (mecanismos multifatoriais desconhecidos). No entanto, os dados referentes à ocorrência de HP na LAM são escassos. Os objetivos do estudo foram avaliar a prevalência e as características da HP em pacientes com LAM em diferentes estágios de evolução, além de comparar as características clínicas, funcionais, do teste de caminhada de 6 minutos (TC6M) e da qualidade de vida das pacientes com e sem HP. Metodologia: Cento e cinco pacientes com LAM foram submetidos a ecocardiograma, prova de função pulmonar (PFP) e TC6M. Pacientes com suspeita de HP no ecocardiograma, definida pela presença de pressão arterial pulmonar sistólica estimada (PsAP) acima de 35 mmHg, ou PFP mostrando DLco abaixo de 40% do valor previsto, foram submetidos a cateterismo cardíaco direito para confirmar o diagnóstico de HP. Resultados: Oito pacientes (7,6%) tinham HP confirmada no cateterismo cardíaco direito, seis pacientes (5,7%) tinham padrão pré-capilar e dois pacientes (1,9%) tinham padrão pós-capilar. Apenas um paciente (1%) apresentou pressão média de artéria pulmonar (PAPm) acima de 35 mmHg. Os pacientes com HP apresentaram menor VEF1 e DLco em PFP e maior dessaturação de oxigênio e intensidade de dispneia durante o TC6M comparado com aqueles sem PH. Em 63% dos pacientes com HP confirmada, o cateterismo cardíaco direito foi realizado devido ao resultado do DLco. Conclusões: A prevalência de HP é baixa em pacientes com LAM. A hipertensão pulmonar é de pouca gravidade e significativamente associada ao envolvimento parenquimatoso pulmonar. A capacidade de difusão de monóxido de carbono foi bastante útil na identificação de HP em pacientes com LAM
Introduction: Lymphangioleiomyomatosis (LAM) is associated with pulmonary hypertension (PH) and is included in group 5 of the current classification (unknown multifactorial mechanisms). However, data regarding the occurrence of PH in LAM are scarce. The objectives of the study were to evaluate the prevalence and characteristics of PH in patients with LAM at different stages of evolution, as well as to compare the clinical and functional characteristics of the 6-minute walk test (6MWT) and the quality of life of patients with and without PH. Methodology: One hundred and five patients with LAM underwent echocardiogram, pulmonary function test (PFT) and 6MWT. Patients with suspected PH on the echocardiogram, defined as the presence of estimated systolic pulmonary arterial pressure (PsAP) above 35 mmHg, or PFT showing carbon monoxide diffusion (DLco) below 40% of the predicted value, were submitted to right cardiac catheterization to confirm the diagnosis of PH. Results: Eight patients (7.6%) had PH confirmed in right cardiac catheterization, six patients (5.7%) had a pre-capillary pattern and two patients (1.9%) had a post capillary pattern. Only one patient (1%) presented mean pulmonary artery pressure (PAPm) above 35 mmHg. Patients with PH had lower FEV1 and DLco in PFP and greater oxygen desaturation and dyspnea intensity during the 6MWT compared to those without PH. In 63% of patients with confirmed PH, right heart catheterization was performed because of the DLco result. Conclusions: The prevalence of PH is low in patients with LAM. Pulmonary hypertension is commonly mild and is significantly associated with pulmonary parenchymal involvement. The measure DLco has improved the identification of PH in patients with LAM

Tuberous sclerosis complex (TSC), an autosomal dominant disease, is characterized by the formation of hamartomas in various organs such as brain, kidney, skin, retina and heart, and is caused by mutations in TSC1 e TSC2 tumor suppressor genes, encoding hamartin and tuberin, respectively. Lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by cystic lung destruction leading to progressive respiratory failure and formation of abdominal tumors. LAM can be sporadic or associated to TSC and LAM lung alterations resulting from proliferation of neoplastic cells bearing mutations in either TSC1 or TSC2 genes. A metastatic process has been proposed in dissemination of LAM and TSC cells to explain the identical TSC2 mutations and loss of heterozigosity (LOH) patterns in LAM cells of lung nodules, angiomyolipomas and lymph nodes of the same sporadic LAM patients, that is consistent with metastatic spread among organs. The aim of this project is to study the role of cytokines and matrix metalloproteinases in LAM and TSC. LAM/TSC cells, isolated from chylous of a patient affected by LAM associated to TSC, bear a TSC2 germline mutation and do not express tuberin for an epigenetic silencing of the TSC2 second allele that confirms our previously published evidence that an epigenetic alterations of TSC2 can cause the loss of tuberin in TSC cells. Treatment of LAM/TSC cells with 5-azacytidine that inhibits CpG DNA methylation led to the expression of tuberin as consequence of the chromatin remodeling. Proliferation of LAM/TSC cells is epidermal growth factor (EGF)-dependent and blockade of EGF receptor causes cell death, as we previously showed in cells lacking tuberin, while rapamycin, an mTOR inhibitor, significantly reduced proliferation. LAM/TSC cells have the ability to grow independently from adhesion and survive in adherent and nonadherent condition. For these features these cells are a good model to study the mechanisms of motility in LAM and TSC and the relation to tuberin expression. We studied the effect of anti-EGFR Ab and rapamycin on motility, cytokines and MMPs (Matrix metalloproteinases) expression. LAM/TSC cells spontaneously detach and undergo spontaneous cycles of adhesion and nonadhesion conditions likely for the inactivation of the focal adhesion kinase (FAK)/Akt/mTOR pathway. In LAM/TSC cells FAK inhibition caused the reduction of AKT phosphorylation which was followed by inhibition of mTOR phosphorylation and mTOR autophosphorylation and, consequently, by a strong reduction of S6 phosphorilation as it occurs in nonadherent cells. Anti-EGFR antibody and rapamycin reduced the viability of adherent and nonadherent cells while 5-azacytidine slightly increased the percentage of detaching cells. Nonadherent LAM/TSC cells underwent an extremely low proliferation rate consistent with tumour stem cell characteristics. One of the step driving EMT is the repression of E-cadherin, resulting in the loss of cell-cell adhesion. LAM/TSC cells did not express E-cadherin, but they express vimentin, marker of mesenchymal cells. EMT process controls the migration of cancer cells from primary tumors depending on an inflammatory microenvironment. LAM/TSC cells secreted high amount of IL-6, IL-8 and IL-1α, cytokines crucial for a variety of cancer cells. The levels of IL-6, IL-8 and IL-1α were not affected by rapamycin or anti-EGFR antibody but were regulated by tuberin expression since their levels were reduced by 5-azacytidine incubation. MMPs degrade and modify the extracellular matrix (ECM), facilitating detachment of cells from the tissue. Levels of MMP-2 and MMP-9 in urine are predictive of disease status in a variety of cancers and also in LAM. MMP-2 and MMP-9 levels are high in urine of LAM patients and MMP-2 is substantially up-regulated in their lung tissue. Matrilisin (MMP-7) contributes to tumor progression, invasion and is overexpressed in several types of invasive cancers. In adherent status LAM/TSC cells expressed higher levels of MMP-2 mRna and lower of MMP-7 than in nonadherent condition. Consistent with ECM degration and invasive features respectively,. MMP-2 and MMP-7 mRna expression appeared to be related to tuberin since they were significantly reduced by 5-azacytidine incubation. Anti-EGFR Ab and rapamycin significantly decreased MMP-2 mRna expression while their effect was the opposite on MMP-7. Extracellular matrix metalloproteinase inducer (EMMPRIN), is thought to affect tumor progression through its ability to stimulate MMPs expression. EMMPRIN expression, quantified by cytometric analysis, was reduced by 5-azacytidine and was much higher in LAM/TSC cells than in MCF7, breast cancer cells. Anti-EGFR antibody and rapamycin did not change EMMPRIN levels. For the mesenchymal feature of LAM/TSC cells and their ability to survive in adherent and nonadherent conditions, we evaluated LAM/TSC motility in wound healing assay providing an indication of cell migration rate. LAM/TSC cells closed wounds in about 11h, much faster than tumoral cells such as MCF7. Cell motility of LAM/TSC cells is related to tuberin expression since 5-azacytidine treatment, and the consequent induced-tuberin expression, increased the time of wound closure to 19h. However, 5-azacytidine treatment did not have any effect on MMP-2 and MMP-7 mRna expression in wound healing assay. Rapamycin and anti-EGFR Ab decreased the migration rate of LAM/TSC cells leading to the closure of wound in about 25h and causing an induced an increase of MMP-2 and MMP-7 levels. However during wound closure (50%) MMP-2 secretion was increased by anti-EGFR and rapamycin treatments and normalized at complete wound closure. In conclusion, LAM/TSC cells express invasive and migratory features which appear to be related to tuberin expression . Phosphorylation of FAK and MMP-2 expression was reduced in nonadherent LAM/TSC cells compared to adherent cells suggesting that FAK signaling was required for the activation of MMP-2 expression. MMP-2 and MMP-7 are related to the adherent and nonadherent conditions and are involved in the metastatic process proposed in dissemination of LAM cells. LAM/TSC cells have the ability to migrate and their motility is related to tuberin expression and reduced by anti-EGFR antibody and rapamycin. Rapamycin and anti-EGFR Ab control MMP-2 and MMP-7 expression and secretion during cell migration. These data suggest a specific role for MMP-2 and MMP-7 in LAM/TSC cell motility and thereby in the pathogenesis of LAM and TSC indicating their involvement in the metastatic process proposed in dissemination of LAM cells. The understanding of LAM/TSC cell features in motility is important for the assessment of cell invasiveness in LAM and TSC and provide highlights to sustain MMPs pathways as potential target for LAM. TSC and TSC related disorders.

A linfangioleiomiomatose pulmonar é uma enfermidade cística progressiva que afeta mulheres jovens e cuja sobrevida média estimada em algumas casuísticas é de cerca de dez anos após o diagnóstico. A deterioração da função pulmonar é uma de suas principais características e resulta em dispnéia progressiva e hipoxemia grave nos casos avançados. O esforço físico é um desencadeador de dessaturação e correlaciona-se com a progressão funcional da doença. Assim como o esforço, o sono é um importante momento fisiológico durante o qual o sistema cardiorrespiratório dessas pacientes pode ser estressado. A partir da ocorrência de hipoxemia durante o sono ao longo dos anos pode surgir elevação progressiva da pressão na circulação pulmonar, resultando em cor pulmonale. Com o intuito de estudar o comportamento da saturação de oxigênio durante o sono e sua correlação com variáveis funcionais respiratórias estáticas e dinâmicas e de qualidade de vida, foram avaliadas consecutivamente 21 pacientes com diagnóstico de linfangioleiomiomatose pulmonar (LAM) atendidas no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo no período de março de 2005 a dezembro de 2007. Todas as pacientes realizaram polissonografia completa (PSG), prova de função pulmonar (PFP), teste de caminhada de 6 minutos (TC6M) e responderam aos questionários de Berlim, Epworth e SF-36, após assinatura de termo de consentimento livre e esclarecido. Verificou-se que a idade média das pacientes foi de 38 8 anos ao início dos sintomas, 41 10 ao diagnóstico e 45 11 anos à realização dos testes. O sintoma clínico mais freqüente foi dispnéia aos esforços, presente em 20 das 21 pacientes. Funcionalmente as pacientes apresentavam um padrão obstrutivo leve (%VEF1 72% e VEF1/CVF 0,68) com redução moderada da DLCO (48% predito). Das 21 pacientes, 14 apresentaram queda da saturação de hemoglobina abaixo de 90% durante a PSG. O tempo médio de queda da saturação foi de 2 horas e 25 minutos, com os principais períodos de dessaturação ocorrendo durante o sono REM e de ondas lentas. Estes caracterizaram-se por períodos de hipoventilação, sem aumento do índice de apnéia-hipopnéia (mediana IAH = 1,7). As principais variáveis que se correlacionaram através do teste de Pearson com a dessaturação abaixo de 90% durante o sono foram o delta de dessaturação durante o TC6M (r = 0,57), o %VEF1 (r = -0,80), %CVF (r = -0,80) e %DLCO (r = - 0,64) em relação ao predito, todas com p < 0,01. A partir da estratificação pela ocorrência de SpO2 < 90% durante a PSG e análise pelo teste de Wilcoxon, evidenciou-se que além das variáveis acima a relação VR/CPT mostrou-se significativamente diferente do ponto de vista estatístico (p = 0,04) nos grupos com e sem hipoxemia noturna. Em relação à qualidade de vida observou-se redução em todos os domínios particularmente quanto aos aspectos emocionais nas pacientes com hipoxemia noturna. O ecocardiograma evidenciou ocorrência de hipertensão pulmonar em cinco pacientes. Verificou-se com esse estudo pela primeira vez a ocorrência de hipoxemia durante o sono em pacientes com LAM. A hipoxemia noturna, apesar de não rotineiramente avaliada nessa população de pacientes, correlacionou-se com aspectos corriqueiramente avaliados no seguimento ambulatorial dessas pacientes como parâmetros da PFP e do TC6M, além de indicar uma tendência a prejuízo da qualidade de vida avaliada pelo SF-36.
Pulmonary lymphangioleiomyomatosis (LAM) is a progressive cistic lung disease of unknown etiology and no established medical treatment. It basically occurs in child bearing age women and its mean survival is about 10 years in some case series. LAM is caractherized by pulmonary function deterioration: obstructive pattern on espirometry and progressive impairment on DLCO capacity which results in rest dyspnea and hypoxemia in advanced cases. Exercise is an important factor that causes hypoxemia in this set of patients and correlates with progressive functional impaiment. Sleep is also another important physiologic moment when cardiorespiratory system may be stressed in these patients, leading to chronic hypoxemia though the years. This phenomenum may be responsible by cor pulmonale observed in advanced stages of LAM. In order to study hemoglobin saturation by oxygen during sleep and its correlation with lung function test (LFT), six minute walk test (6MWT), ecocardiography and SF-36 quality of life variables, 21 consecutive patients with LAM from Hospital das Clínicas University of São Paulo Medical School were evaluated from march 2005 to december 2007. All patients performed full polysomnography (PSG), lung function test, 6MWT and answered Berlim, Epworth and SF-36 quality of life questionnaire. Mean age at beginning of symptoms was 38 8 years, 41 10 years at diagnosis and 45 11 years by the time of the protocol. Dyspnea during routine daily activities was the most frequent symptom (20 / 21). LFT yielded mild obstruction (% FEV1 = 72%; FEV1/FVC = 0,68) and moderate impairment in DLCO (48%). Fourteen patients presented desaturation with SpO2 lower than 90% on PSG. Mean time with SpO2 lower than 90% was 2 hours and 25 minutes (mainly during REM and S3 and S4). Median apnea-hipopnea index (AHI) was 1,7 (normal). Pearson and Spearman correlations yielded that the main variables related to desaturation below 90% on PSG with p < 0.01 were: Desaturation on TC6M rs = 0,57 Spearman %FEV1 r = -0,80 Pearson %FVC r = -0,80 Pearson %DLCO rs = -0,64 Spearman After that, functional variables were stratified by SpO2 < 90% during PSG. Wilcoxon test showed that besides all 4 previous variables RV/TLC (p=0.04) was also statistically different between patients who did and did not desaturate. SF-36 yielded impairments in all domains, particularly in emotional aspects among patients who desaturated on PSG. Heart ecocardiography detected pulmonary hypertension in 5 patients. Our study found out for the first time that patients with LAM desaturate during sleep. Although pulmonary function variables are related to sleep desaturation, desaturation occured even in patients with mild or no impairments in lung function. 6MWT desaturation ,but not distance walked, correlated to sleep desaturation. SF-36 analysis yielded impairments in all domains of quality of life, particularly in emotional aspects in patients who desaturated.

Introdução: A linfangioleiomiomatose (LAM) é uma neoplasia de baixo grau, frequentemente associada à redução na capacidade de exercício, secundária a múltiplos fatores incluindo alteração de troca gasosa, limitação ventilatória e hiperinsuflação dinâmica (HD). A reabilitação pulmonar (RP) tem benefícios bem estabelecidos em diversas doenças pulmonares crônicas, porém não foi estudada na LAM. Objetivos: Avaliar o impacto de um programa de RP, comparativamente a um grupo controle, em portadoras de LAM, nos seguintes parâmetros: capacidade de exercício (objetivo primário), HD, dispneia, nível de atividade física diária, qualidade de vida, ansiedade e depressão, função pulmonar e força muscular. Metodologia: Ensaio clínico, controlado, não-randomizado, incluindo 21 pacientes com LAM no grupo RP e 19 no grupo controle. A RP teve duração de 3 meses, compreendendo 24 sessões de uma hora de duração (30 minutos de exercício aeróbico e 30 minutos de treinamento de força muscular). A avaliação inicial incluiu um teste de exercício cardiopulmonar (TECP) máximo incremental. As seguintes variáveis foram avaliadas antes e após a RP ou observação (grupo controle): capacidade de exercício, através do tempo até o limite da tolerância (Tlim) no teste de exercício cardiopulmonar (TECP) com carga constante; distância percorrida e dessaturação de oxigênio no teste de caminhada de 6 minutos (TC6M); dispneia (escala de dispneia do Medical Research Council modificada - mMRC, Índice de Dispneia Basal - BDI, e Índice Transicional de Dispneia - TDI); nível de atividade física diária (pedômetro); qualidade de vida relacionada à saúde (Questionário Respiratório de St George\'s, SGRQ); ansiedade e depressão (Escala Hospitalar de Ansiedade e Depressão, HADS); provas de função pulmonar (PFP) e força muscular (uma repetição máxima, 1 RM). Resultados: Não houve diferença nas características basais entre os grupos RP e controle em relação à: idade (45 ± 11 vs. 40 ± 9 anos, p = 0,21), VEF1 (74 ± 30 vs. 70 ± 27% pred, p = 0,67), DLCO (67 ± 33 vs. 64 ± 30% pred, p = 0,79), carga máxima (77 ± 33 vs. 76 ± 35 W, p = 0,93) e O2 pico (17 ± 5 vs. 16 ± 4 ml/ kg/ min; p = 0,52) no TECP incremental. O grupo RP apresentou melhora comparativamente ao grupo controle em (expressos em mediana [intervalo interquartil]): Tlim (169 s [2 - 303 s] vs. -33 s [-129 - 39 s], p = 0,001) e O2 (11% [2 - 26%] vs. -2% [-7 - 5% pred], p = 0,001) no TECP com carga constante, mMRC (0 [-1 - 0] vs. 0 [0 - 1], p < 0,001), TDI (3 [2 - 3] vs. 0 [-2 - 0], p < 0,001), números de passos diários (752 [-694 - 1814] vs. -138 [-830 - 208], p= 0,02), SGRQ (-8 [-16 - 2] vs. 2 [-4 - 5], p = 0,002, distância caminhada no TC6M (59 m [13 - 81] vs. 20 [-12 - 30], p = 0,002) e 1 RM para todos os grupamentos musculares treinados (ex. quadríceps 39% [20 - 70%] vs. 4% [0 - 17%], p <0,001). Houve uma tendência de melhora nos sintomas de depressão e HADS total. HD, dessaturação ao exercício, sintomas de ansiedade e PFP não melhoraram após RP. Houve correlação moderada entre o aumento do Tlim e as variações da mMRC, do O2 de pico no TECP com carga constante, do Borg dispneia isotime e do Borg de pernas isotime. Conclusões: A RP está associada à melhora na capacidade de exercício, dispneia, nível de atividade física diária, qualidade de vida relacionada à saúde e força muscular em pacientes com LAM. O principal mecanismo sugerido é adaptação da musculatura periférica
Introduction: Lymphangioleiomyomatosis (LAM) is a low-grade neoplasm, which is frequently associated with reduced exercise capacity, secondary to multiple factors including gas exchange impairment, ventilatory limitation and dynamic hyperinflation (DH). Pulmonary rehabilitation (PR) has proven benefits in many chronic pulmonary diseases but it was not evaluated in LAM. Objectives: To evaluate the impact of a PR program in women with LAM, when compared to a control group, in the following parameters: exercise capacity (primary outcome), DH, dyspnea, daily physical activity, quality of life, anxiety and depression, lung function and muscle strength. Methods: A non-randomized controlled clinical trial that included 21 LAM patients in the PR group and 19 in the control group. The PR program lasted 3 months, comprising 24 sessions of 1 hour (30 minutes of aerobic exercise and 30 minutes of muscle strength training). The initial evaluation included a maximum incremental cardiopulmonary exercise test (CPET). The following variables were assessed before and after PR or observation (control group): exercise capacity using the tolerable limit duration (Tlim) in constant work rate (CWR) exercise testing; walking distance and oxygen desaturation (six-minute walk test, 6MWT), dyspnea (Modified Medical Research Council Dyspnea Scale -mMRC; Basal Dyspnea Index - BDI, and Transitional Dyspnea Index -TDI); daily physical activity (pedometer); health-related quality of life (St George\'s Respiratory Questionnaire, SGRQ); anxiety and depression (Hospital Anxiety and Depression Scale, HADS); pulmonary function tests (PFT) and muscle strength (one-repetition maximum, 1RM). Results: There was no difference in baseline characteristics between the PR and control groups related to age (45 ± 11 vs. 40 ± 9 years, p = 0.12), FEV1 (74 ± 30 vs. 70 ± 27% pred, p = 0.67), DLCO (67 ± 33 vs. 64 ± 30% pred, p = 0.79), maximum work rate (77 ± 33 vs. 76 ± 35 W, p = 0.93) and peak O2 (17 ± 5 vs. 16 ± 4 ml/ kg/ min; p = 0.52) in incremental CPET. The PR group had a significant improvement when compared to the control group in (expressed in median [interquartile range]): Tlim (169 s [2 - 303 s] vs. -33 s [-129 - 39 s], p = 0.001) and O2 (11% [2 - 26%] vs. -2% [-7 - 5% pred], p = 0.001) in CWR exercice testing, mMRC (0 [-1 - 0] vs. 0 [0 - 1], p< 0.001), ( TDI (3 [2 - 3] vs.0 [-2 - 0], p< 0.001), daily steps (752 [-694 - 1814] vs. -138 [-830 - 208], p= 0.02), SGRQ (-8 [-16 - 2] vs. 2 [-4 - 5], p = 0.002), walking distance in 6MWT (median 59 m [13 - 81] vs. 20 [-12 - 30], p = 0.002) and 1 RM for all muscle groups trained (ex. quadriceps 39% [20 - 70%] vs. 4% [0 - 17%], p <0.001). There was a trend towards improvement in depression symptoms and total HADS. DH, desaturation during exercise, anxiety symptoms and PFT did not improve after PR. There was a moderate correlation between increased Tlim and variations of mMRC, peak O2 in CWR exercise testing, Borg dyspnea isotime and Borg leg discomfort isotime. Conclusions: PR is associated with improvements in exercise capacity, dyspnea, daily physical activity, health-related quality of life and muscle strength in patients with LAM. The main mechanism suggested is peripheral muscles adaptation

LAM is a rare and progressive disease characterized by widespread proliferation of abnormal smooth muscle-like cells (LAM cells). LAM cells cause cystic destruction of lung parenchyma, abdominal tumours (angiomyolipoma, AML) and infiltration of axial lymphatics in torax and abdomen (adenopathy and lymphangioleiomyoma). LAM occurs sporadically or in association with tuberous sclerosis complex (TSC), an inherited disorder with variable penetrance, which results from mutations in TSC1 or TSC2 genes. TSC1 or TSC2 genes, encoding hamartin and tuberin respectively, regulate mammalian target of rapamycin (mTOR). LAM affects primarly women of child-bearing age and the mechanisms causing the disease are not yet clarified. To explain the multisystemic clinical manifestations of LAM an experimental model is needed to study the pathological mechanism causing LAM. It may help to explain how LAM cells migrate from tissue to tissue and to develop a pharmacological approach. We recently isolated and characterized α-actin positive smooth muscle cells from chylous of a patient affected by LAM/TSC (LAM/TSC cells). These circulating cells showed reactivity to HMB45 and CD44v6 antibodies, markers of TSC and LAM, and bear a germline TSC2 mutation in exon 21. Like TSC2 smooth muscle cells previously isolated (TSC2-/- and TSC2-/meth ASM cells), LAM/TSC cells from chylous required epidermal growth factor (EGF) to proliferate and the blockade of EGF receptor (EGFR) caused progressive cell death. To better study LAM pathogenesis we developed a procedure for a quick invasion of the respiratory system by endonasally administrating LAM/TSC cells. LAM/TSC cells were administrated in immunodeficient female nude mice (nu/nu Hsd: athymic nude mice, 3 weeks old) and after 26 weeks anti-EGFR antibody and rapamycin were intraperitoneally injected 2 times a week for 4 weeks. 30 weeks after endonasal administration LAM/TSC cells were detected in lungs, lymph nodes and uterus. In lung parenchyma, LAM/TSC cells proliferated and caused cystic destruction with emphysematous-like picture such as in LAM patient lungs. This lesion and the proliferating rate were reverted by anti-EGFR antibody, while rapamycin was less effective and caused hemoptysis. In lungs blood vessel number was increased and, using LYVE-1 antibody, a significant increase of lymphatic vessel density (LVD) was observed in animals that received LAM/TSC cells suggesting a possible correlation between LAM/TSC cells and lymphangiogenesis. LVD decreased following anti-EGFR antibody and rapamycin treatments. When treatments were stopped, hemoptysis caused by rapamycin was reverted. Anti-EGFR antibody was more effective than rapamycin in reducing lung injury caused by LAM/TSC cells administration and in decreasing lymphangiogenesis. In some lung parenchyma noduli were detected. Such in lungs of LAM patients they were positive to human COX IV, ER, PR and phosphoS6. In lymph nodes, LAM/TSC cells promoted a lymphatic vessel invasion, as showed by PROX-1-reactivity. Anti-EGFR antibody and rapamycin treatments decreased lymphatic vessels. Differently from lungs, blood vessels in lymph nodes were not altered after LAM/TSC cells administration, as shown by CD31 immunoreactivity. LAM/TSC cells were also detected in uteri where they promoted a significant increase of cells with high levels of estrogen (ER) and progesterone receptors (PR). In uteri afterLAM/TSC cells administration the morphological structure was unchanged. Pharmacological treatments decreased ER and PR expressions. Our data show that endonasal administration of cells isolated from chylous of LAM/TSC patient developed a mouse LAM model. LAM/TSC cells invaded lungs, lymph nodes and uteri causing LAM-like lesions. Anti-EGFR antibody is more effective than rapamycin in promoting lung restauration and reducing lymphangiogenesis; its efficacy persists also when treatment is stopped. These data suggest that anti-EGFR antibody treatment may represent a useful pharmacological approach for LAM therapy.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a rare disease characterized by cystic destruction of the lung. It occurs in 80% of people with Tuberous Sclerosis Complex disorder (TSC), a multisystem, autosomal dominant disorder caused by mutations in tumor suppressor genes TSC1 and TSC2. Spontaneous biallelic mutations in these genes can give rise to sporadic LAM. Mammalian target of rapamycin complex I (mTORC1), a master regulator of cellular anabolic metabolism is hyperactivated in LAM cells. Upregulation of protein synthesis and downregulation of autophagy creates a state of starvation stress that upregulates pathways of extracellular nutrient acquisition. Macropinocytosis, a form of clathrin-independent endocytosis, is upregulated in TSC2-deficient cells. We performed a high-throughput compound screen utilizing a repurposing drug library. We identified that ritanserin, a diacylglycerol kinase alpha (DGKA) inhibitor, synergizes with Chloroquine (CQ) to selectively inhibit proliferation of TSC2-deficient mouse embryonic fibroblasts (MEFs) compared to TSC2+/+ MEFs. OBJECTIVE: We hypothesized that TSC2-deficient cells rely on macropinocytosis to support their growth during the periods of stress and starvation and that ritanserin synergizes with CQ to inhibit proliferation in TSC2-deficient cells by inhibiting macropinocytosis. METHODS: Crystal violet-based proliferation assays were used to monitor the effect of pharmacological and genetic inhibition of DGKA on cell proliferation. Immunoblotting was used to measure the expression levels of TSC2, tS6R, pS6R, Cleaved PARP, Cleaved Caspase 3 and Actin. siRNA induced Htr2a knockdown and shRNA induced DGKA knockdown cell culture models were used to define the dual functions of ritanserin and observe their effects on macropinocytosis and cell proliferation. LC/MS was used to measure cell lipid content and how it changes in response to ritanserin. Fluorophore-labeled BSA and 70-kDa Dextran were used to measure macropinocytosis. Lysotracker was used to measure the number of lysosomes, while DQ-BSA was used to measure lysosomal functionality. RESULTS: TSC2-deficient cells express higher levels and show upregulated activity of DGKA. Genetic and pharmacologic inhibition of DGKA prevents TSC2-deficient cells from acquiring nutrients via macropinocytosis. Phospholipid metabolism is altered in TSC2-deficient cells, marked by the accumulation of phosphatidic acid and ceramides. Treatment with ritanserin leads to the accumulation of diacylglycerol and phospholipids, as well as a reduction in phosphatidic acid. CONCLUSIONS: TSC2-deficient cells rely on macropinocytosis to meet their metabolic needs. Diacylglycerol kinase alpha (DGKA) is required for macropinocytic nutrient uptake. Pharmacologic or genetic inhibition of DGKA creates metabolic stress in TSC2-deficient cells, which ultimately leads to increased apoptotic response to treatment with CQ. This project identifies a novel connection between mTOR signaling, lysosome metabolism and macropinocytosis, and a vulnerability that allows the selective targeting of LAM cells.
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