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Identification of Novel Biomarkers for Lymphangioleiomyomatosis by Proteomics from Serum Exosomes
Description
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A1069 - Identification of Novel Biomarkers for Lymphangioleiomyomatosis by Proteomics from Serum Exosomes
Author Block: T. Takimoto1, Y. Takeda1, T. Shiromizu2, R. Narumi2, T. Koba1, T. Matsuki1, M. Kuroyama1, H. Kida1, M. Hirose3, Y. Inoue3, T. Tomonaga2, A. Kumanogoh1; 1Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Japan, 2Laboratory of Proteome Research, National Institute of Biomedical Innovation, Ibaragi, Japan, 3Diffuse Lung Diseases and Respiratory Failure, Clinical Research Cente, National Hospital Organization Kinki-Chuo Chest Medical Center, Sakai, Japan.
RATIONALE
Lymphangioleiomyomatosis (LAM) is a rare and progressive cystic lung disease affecting approximately 3.4-7.5/million women, with an average lag time between symptom onset and diagnosis of upwards of 4 years. LAM occurs sporadically (S-LAM) or in about 30% of individuals born with tuberous sclerosis complex (TSC), which is classified as TSC-LAM. The aim of this study was to identify proteomic profiling of serum exosomes in LAM patients, which may be potential biomarkers (BM) of disease. We focused on proteins of serum exosomes to overcome obstacles, including the complexity of samples such as serum as well as immaturity of proteomic technology. Our laboratory has already established a strategy for discovering BM in some lung diseases, including COPD and asthma.
METHODS
Eight LAM patients and four healthy control volunteers (female) were included in this study. Serum exosomes were isolated by ultracentrifugation. A quantitative high throughput proteomics using isobaric tags, tandem mass tag system technique (TMT)-based analysis was performed, and differentially expressed proteins were validated using target proteomics, multiple reaction monitoring (MRM). Pathway analysis was carried out using Ingenuity Pathway Analysis (IPA) and KEGG.
RESULTS
Isolated exosomes were characterized by transmission electron microscope and NanoSight nanoparticle tracking analysis. The size and number of serum exosomes was not different in LAM, compared to healthy controls. We obtained 573 proteins, including abundant membrane proteins as well as extracellular matrix proteins, using TMT based proteomics. Pathway analysis revealed that exosomal proteomic profiles in LAM could reflect the disease characteristics: involvement of coagulation system and inflammation; difference between S-LAM and TSC-LAM. Of note, these BM candidates included membrane proteins that could be derived from LAM lungs. We are verifying these candidate proteins in an independent set of samples by MRM. CONCLUSIONS This study is the first to highlight the LAM signature in serum exosomes, providing a set of novel potential BM. This approach may lead to the further understanding of pathogenesis as well as a drug discovery for LAM.
Author Block: T. Takimoto1, Y. Takeda1, T. Shiromizu2, R. Narumi2, T. Koba1, T. Matsuki1, M. Kuroyama1, H. Kida1, M. Hirose3, Y. Inoue3, T. Tomonaga2, A. Kumanogoh1; 1Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Japan, 2Laboratory of Proteome Research, National Institute of Biomedical Innovation, Ibaragi, Japan, 3Diffuse Lung Diseases and Respiratory Failure, Clinical Research Cente, National Hospital Organization Kinki-Chuo Chest Medical Center, Sakai, Japan.
RATIONALE
Lymphangioleiomyomatosis (LAM) is a rare and progressive cystic lung disease affecting approximately 3.4-7.5/million women, with an average lag time between symptom onset and diagnosis of upwards of 4 years. LAM occurs sporadically (S-LAM) or in about 30% of individuals born with tuberous sclerosis complex (TSC), which is classified as TSC-LAM. The aim of this study was to identify proteomic profiling of serum exosomes in LAM patients, which may be potential biomarkers (BM) of disease. We focused on proteins of serum exosomes to overcome obstacles, including the complexity of samples such as serum as well as immaturity of proteomic technology. Our laboratory has already established a strategy for discovering BM in some lung diseases, including COPD and asthma.
METHODS
Eight LAM patients and four healthy control volunteers (female) were included in this study. Serum exosomes were isolated by ultracentrifugation. A quantitative high throughput proteomics using isobaric tags, tandem mass tag system technique (TMT)-based analysis was performed, and differentially expressed proteins were validated using target proteomics, multiple reaction monitoring (MRM). Pathway analysis was carried out using Ingenuity Pathway Analysis (IPA) and KEGG.
RESULTS
Isolated exosomes were characterized by transmission electron microscope and NanoSight nanoparticle tracking analysis. The size and number of serum exosomes was not different in LAM, compared to healthy controls. We obtained 573 proteins, including abundant membrane proteins as well as extracellular matrix proteins, using TMT based proteomics. Pathway analysis revealed that exosomal proteomic profiles in LAM could reflect the disease characteristics: involvement of coagulation system and inflammation; difference between S-LAM and TSC-LAM. Of note, these BM candidates included membrane proteins that could be derived from LAM lungs. We are verifying these candidate proteins in an independent set of samples by MRM. CONCLUSIONS This study is the first to highlight the LAM signature in serum exosomes, providing a set of novel potential BM. This approach may lead to the further understanding of pathogenesis as well as a drug discovery for LAM.
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