Characterization of the Pathogenesis of Lymphangioleiomyomatosis (LAM)

淋巴管平滑肌瘤病 (LAM) 发病机制的特征

基本信息

批准号：
7734978
负责人：
Joel Moss
金额：
$ 210.08万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7734978
关键词：
A549 ARNT protein Abdomen Activities of Daily Living Adenocarcinoma Cell Adverse effects Affect Air Alleles Alveolar Cell Alveolar Macrophages Antibodies Apoptosis Binding Biological Markers Breathing CD44 gene Cardiopulmonary Cell Count Cell Proliferation Cell Size Cells Chronic Collagen Collecting Cell Condition Cultured Cells Cyst Data Deposition Disease Disease Progression Disease susceptibility Effectiveness Endothelial Cells Epithelial Erythrocyte Indices Erythrocytes Erythrocytoses Erythroid Cells Erythropoiesis Erythropoietin Erythropoietin Receptor Exercise Exercise stress test Exhibits Extracellular Matrix Fc Receptor Fibroblasts Fluorescence-Activated Cell Sorting Functional disorder Gases Gene Expression Genes Genetic Genetic Transcription Glycolysis Goals Growth HIF1A gene Hamartoma Hamman-Rich syndrome Head and neck structure Hematocrit procedure Hemoglobin Hemoglobin concentration result Histology Human Hypoxemia Hypoxia Immunohistochemistry Impairment Kidney Lasers Lead Lesion Link Loss of Heterozygosity Lung Lung Lymphangioleiomyomatosis Lung diseases Lung nodule Lymphangioleiomyomatosis Lymphangiomyoma Lymphatic Lymphatic Abnormalities Malignant neoplasm of lung Measures Membrane Messenger RNA Molecular Target Multivariate Analysis Mutation Neuroblastoma Neurocutaneous Syndromes Nodule Obstruction Oxygen Pathogenesis Pathway interactions Patients Pharmaceutical Preparations Phenotype Phosphorylation Play Population Production Proliferating Proteins Pulmonary artery structure RNA Rate Receptor Activation Recurrence Red Blood Cell Count Renal Angiomyolipoma Reporting Respiratory physiology Rest Role Score Severities Severity of illness Shapes Signal Pathway Signal Transduction Signal Transduction Pathway Sirolimus Site Skin Skin Neoplasms Smooth Muscle Smooth Muscle Myocytes Squamous Cell Neoplasms Staining method Stains Structure of parenchyma of lung Sturge-Weber Syndrome TSC2 gene Testing Therapy Clinical Trials Time Transplantation Tuberous sclerosis protein complex Tumor Suppressor Genes Vascular Smooth Muscle Woman alveolar type II cell base body system cancer cell cell growth cell motility experience fibroma human FRAP1 protein human TSC2 protein immunoreactivity indexing kidney cortex laser capture microdissection matrigel neoplastic cell pulmonary function receptor recombinant human erythropoietin response transcription factor tumor

项目摘要

Since hypoxia may lead to changes in red cell indices, we asked whether there was a relationship between these indices and severity of lung disease. Pulmonary function, cardiopulmonary exercise data, and red blood cell indices from 277 LAM patients, grouped according to use of oxygen, were analyzed. Patients who used supplemental oxygen intermittently or continuously had higher hematocrit and hemoglobin levels than those who did not. Those using supplemental oxygen continuously also had higher red blood cell counts than patients who did not use oxygen. Red blood cell count was significantly correlated with DLCO for patients not using supplemental oxygen, those on supplemental oxygen, and for both groups combined. Lower resting PaO2 while breathing room air was also associated with higher red blood cell count, hematocrit, and hemoglobin. Resting PaO2 was a marker of lung disease severity, and was significantly correlated negatively with the LAM histology scores, a measure of severity of lung disease that is a predictor of survival or time to transplantation. Based on a multivariate analysis, DLCO was a significant predictor of the hematocrit, hemoglobin, and red blood cell count. SaO2 at peak exercise was also significantly correlated with red blood cell count. To assess the relationship between red blood cell count and rate of progression of lung disease, we correlated red blood cell count with the yearly rate of DLCO decline. Although this relationship did not reach statistical significance for patients not receiving supplemental oxygen, the correlation between red blood cell count and the yearly rate of decline in DLCO was significant for patients who used supplemental oxygen and for all patients combined. Thus, higher red blood cell indices were associated with greater severity of lung disease and greater rate of decline in lung function. Since hypoxia increases red blood cell indices, through increased synthesis of EPO, the accelerated loss of lung function in those with erythrocytosis could be due to a growth-enhancing effect of EPO upon LAM cells. The next question was, therefore, whether EPO effects could be found on LAM lung nodules or on LAM cells in culture. Both epithelioid and spindle-shaped LAM cells in LAM lung nodules exhibited immunoreactivity for the EPO receptor (EPOR) as, to a lesser extent, did normal vascular smooth muscle and endothelial cells. Ca. 90% of cultured cells grown from explanted LAM lungs reacted with the anti-EPOR antibody as did pulmonary artery smooth muscle cells (PASM). Microscopically sections of explanted LAM lungs showed reactivity with antibodies against EPO in LAM lung nodules. Anti-EPO antibodies did not react with proliferating fibroblasts sections of lung tissue from patients with idiopathic pulmonary fibrosis, which is characterized by fibroblast proliferation and large depositions of collagen. However, we observed immunoreactivity of anti-EPO anitbodies with alveolar macrophages and type II pneumocytes in these sections. We then investigated the activation state of EPOR in LAM cells. EPOR is phosphorylated at multiple sites after binding of EPO. Phosphorylation of Tyr479 is responsible for EPOR activation and plays a role in cell proliferation. Although smooth muscle cells reacted weakly with anti-phospho-EPOR(Tyr479) antibodies, staining of alveolar cells was stronger. Greatest immunoreactivity with antibodies against activated EPOR(Tyr479) was observed, however, in cells within the LAM lung nodules and in adjacent type II pneumocytes. These data suggest that the activated EPOR and pathway necessary for EPO-induced cell proliferation are activated in LAM cells. EPOR mRNA was detected in total RNA from LAM cells collected by laser-capture microdissection from LAM nodules, and in total RNA extracted from lung, kidney, PASM, and A549 cells (human lung epithelial adenocarcinoma cells), but EPO was not produced by microdiseccted LAM cells. Because EPO might be associated with the collagen that is abundant in LAM lesions, we next tested the ability of recombinant human EPO to bind collagen or Matrigel. EPO binding to collagen was higher than the binding to Matrigel, suggesting that EPO was associated with the extracellular matrix. We had shown that cells from LAM lung explants that react with antibodies against the membrane-associated CD44v6 molecule have dysfunctional TSC2. Therefore, we assessed the presence of EPO and EPOR in cells separated by fluorescence-activated cell sorting using CD44 and CD44v6 antibodies to collect four cell populations (CD44+/CD44v6+, CD44-/CD44v6-, CD44-/CD44v6+, and CD44+/CD44v6-) from the heterogeneous cultured LAM cells. RNA from each population contained EPOR mRNA, but cultured LAM cells (CD44+/CD44v6+, and CD44-/CD44v6+), like the laser-captured microdissected LAM cells, did not produce EPO, which was produced by CD44+/CD44v6- cells. Thus, although LAM cells contained EPOR and could be sensitive to EPO from non-LAM cells in the lung, they did not produce EPO. Because EPO appears to increase the proliferation of cells lacking the TSC2 gene, its effects on cells with loss of heterozygosity for TSC2 grown from LAM lung explants, were tested. Increases of 50-100% in rates of proliferation were seen with the addition of EPO. Because these cultures were not homogeneous and had non-LAM cells as well a substantial loss of TSC2 heterozygosity, EPO may have affected the proliferation of non-LAM as well as LAM cells. To verify an effect of EPO on rate of proliferation of TSC2-/- cells, we used homogeneous TSC2-/- cells grown from a TSC skin tumor (periungual fibroma). These cells lack tuberin due to inactivating mutations in both TSC2 alleles, as has been reported in lung LAM cells. We found that TSC2+/- and TSC2-/- cells produced EPOR but not EPO. Comparison of gene expression by TSC2+/- and TSC2-/- cells showed an amount of EPOR in TSC2-/- cells three-fold that in TSC2+/-. In the presence of EPO, the TSC2-/- skin tumor cells proliferated at two- to three-times the rate of fibroblasts grown from normal-appearing skin of the same patient, upon which EPO had little effect.This study demonstrated the presence of activated EPOR in LAM lung lesions and in LAM cells in culture, along with the existence, by immunohistochemistry, of an activated EPO signaling pathway, which could lead to LAM cell proliferation and the continued growth of LAM lesions. Further, we showed that EPO promoted the growth of lung LAM cells and TSC2 -/- skin tumor cells. Although we found no evidence for the production of EPO by LAM cells, our data suggest that EPO, produced by either the renal cortex or non-LAM cells in the lung, could enhance LAM cell proliferation and thereby accelerate disease progression. Elevated red cell count, probably in response to hypoxia, was associated with more rapid decline in lung function, establishing a link between the pathophysiology of LAM and activation of signaling pathways that promote LAM cell growth. This finding is unique because it demonstrates a correlation between hypoxia-enhanced erythrocytosis and accelerated loss of lung function.

由于缺氧可能导致红细胞指数的变化，因此我们询问这些指数与肺部疾病严重程度之间是否存在关系。分析了根据使用氧气分组的277名LAM患者的肺功能，心肺运动数据和红细胞指数。间歇性或连续使用补充氧的患者的血细胞比容和血红蛋白水平高于那些没有的患者。与不使用氧气的患者相比，使用补充氧气的人的红细胞计数也更高。对于不使用补充氧气的患者，补充氧气的患者以及两组合并的患者，红细胞计数与DLCO显着相关。较低的静息PAO2同时呼吸空气还与较高的红细胞计数，血细胞比容和血红蛋白有关。静止的PAO2是肺部疾病严重程度的标志，并且与LAM组织学评分显着相关，LAM组织学评分是肺部疾病严重程度的量度，这是生存或移植时间的预测指标。基于多元分析，DLCO是血细胞比容，血红蛋白和红细胞计数的重要预测指标。峰值运动中的SAO2也与红细胞计数显着相关。为了评估红细胞计数与肺部疾病进展率之间的关系，我们将红细胞计数与DLCO下降的年龄相关。尽管这种关系对于未接受补充氧气的患者没有达到统计学意义，但是红细胞计数与DLCO年度下降速率之间的相关性对于使用补充氧气以及所有患者合并的患者而言是显着的。因此，较高的红细胞指数与肺部疾病的严重程度更高，肺功能下降率更高。由于缺氧增加了红细胞指数，通过增加了EPO的合成，因此在红细胞增多症患者中，肺功能的加速丧失可能是由于EPO对LAM细胞的生长增强作用。因此，下一个问题是，是否可以在LAN肺结节或培养物中的LAM细胞上发现EPO效应。 LAM肺结节中的上皮细胞和纺锤形的LAM细胞均表现出对EPO受体（EPOR）的免疫反应性，因为正常的血管平滑肌和内皮细胞在较小程度上表现出。大约90％的培养细胞与肺动脉平滑肌细胞（PASM）一样，与抗ePOR抗体反应的外植型肿瘤生长。在肿瘤肺结节中，植物的LAM肺的显微切片与针对EPO的抗体的反应性。抗EPO抗体与来自特发性肺纤维化患者的肺组织的增殖成纤维细胞的反应，其特征是成纤维细胞增殖和胶原蛋白的大沉积。但是，我们观察到这些切片中具有肺泡巨噬细胞和II型肺细胞的抗epo抗疾病的免疫反应性。然后，我们研究了LAM细胞中EPOR的激活状态。 EPO结合后在多个位点磷酸化。 Tyr479的磷酸化负责EPOR活化，并在细胞增殖中起作用。尽管平滑肌细胞与抗磷酸 - ePor（Tyr479）抗体反应较弱，但牙槽细胞的染色较强。然而，在lam肺结节内和相邻的II型肺炎细胞中，观察到具有针对活化EPOR的抗体（Tyr479）抗体的最大免疫反应性。这些数据表明，在LAM细胞中激活了EPO诱导的细胞增殖所必需的激活的EPOR和途径。通过从LAM结节中从LASER捕获显微解剖收集的LAM细胞中检测到Epor mRNA，在肺，肾脏，PASM和A549细胞中提取的总RNA（人肺上皮腺癌细胞），但没有由微膜片摄取的叶片细胞产生EPO。因为EPO可能与LAM病变中丰富的胶原蛋白有关，因此我们接下来测试了重组人EPO结合胶原蛋白或基质凝胶的能力。 EPO与胶原蛋白的结合高于与Matrigel的结合，这表明EPO与细胞外基质有关。我们已经表明，来自Lam Lung Epplants的细胞与抗体相关的CD44V6分子反应的细胞具有功能失调的TSC2。因此，我们评估了使用CD44和CD44V6抗体分离的细胞中EPO和EPOR的存在，用于收集四个细胞群（CD44+/CD44V6+，CD44-/CD44-/CD44V6-，CD444V6-，CD444-/CD44-/CD44V6+CD44V6+，以及CD44444+/cd44444444444444444v6-）。来自每个群体的RNA包含Epor mRNA，但是像激光捕获的微解析的LAM细胞一样，培养的LAM细胞（CD44+/CD44V6+和CD44-/CD44V6+）并未产生EPO，该EPO是由CD44+/CD444V6-细胞产生的。因此，尽管LAM细胞含有EPOR，并且可能对肺中非LAM细胞的EPO敏感，但它们没有产生EPO。由于EPO似乎增加了缺乏TSC2基因的细胞的增殖，因此测试了其对细胞的影响，其对细胞的作用因LAM肺外植体而生长的TSC2的杂合性损失。通过添加EPO，可以看到50-100％的增殖率增加。由于这些培养物不是均匀的，并且具有非LAM细胞以及TSC2杂合性的大量丧失，因此EPO可能影响了非LAM和LAM细胞的增殖。为了验证EPO对TSC2 - / - 细胞增殖速率的影响，我们使用了从TSC皮肤肿瘤（周期纤维瘤）生长的均质TSC2 - / - 细胞。这些细胞由于在两个TSC2等位基因中灭活突变而缺乏结核素，正如肺lam细胞中报道的那样。我们发现TSC2 +/-和TSC2 - / - 细胞产生了EPOR，但未产生EPO。 TSC2 +/-和TSC2 - / - 细胞对基因表达的比较显示了TSC2 - / - 细胞中的EPOR量三倍，在TSC2 +/-中。 In the presence of EPO, the TSC2-/- skin tumor cells proliferated at two- to three-times the rate of fibroblasts grown from normal-appearing skin of the same patient, upon which EPO had little effect.This study demonstrated the presence of activated EPOR in LAM lung lesions and in LAM cells in culture, along with the existence, by immunohistochemistry, of an activated EPO signaling pathway, which could lead to LAM细胞增殖和LAM病变的持续生长。此外，我们表明EPO促进了肺LAM细胞和TSC2 - / - 皮肤肿瘤细胞的生长。尽管我们没有发现通过LAM细胞产生EPO的证据，但我们的数据表明，肺部肾皮质或非LAM细胞产生的EPO可以增强LAM细胞增殖，从而加速疾病的进展。升高的红细胞计数可能是针对缺氧的，与肺功能的快速下降有关，建立了LAM的病理生理学与促进LAM细胞生长的信号传导途径的激活之间的联系。这一发现是独一无二的，因为它表明了缺氧增强的红细胞增多和加速肺功能丧失之间的相关性。

项目成果

期刊论文数量（36）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Computer-Aided Grading of Lymphangioleiomyomatosis (LAM) using HRCT.

使用 HRCT 的计算机辅助淋巴管平滑肌瘤病 (LAM) 分级。

DOI：
10.1109/icpr.2008.4760991
发表时间：
2008
期刊：
Proceedings of the ... IAPR International Conference on Pattern Recognition. International Conference on Pattern Recognition
影响因子：
0
作者：
Yao,Jianhua;Avila,Nilo;Dwyer,Andrew;Taveira-Dasilva,AngeloM;Hathaway,OlandaM;Moss,Joel
通讯作者：
Moss,Joel

Association of lymphangioleiomyomatosis (LAM) with endosalpingiosis in the retroperitoneal lymph nodes: report of two cases.

淋巴管平滑肌瘤病（LAM）与腹膜后淋巴结输卵管内膜异位症的关联：两例报告。