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，我们询问这些指数与肺部疾病的严重程度之间是否存在关系。对 277 名 LAM 患者（根据氧气使用情况分组）的肺功能、心肺运动数据和红细胞指数进行了分析。间歇或连续使用吸氧的患者比不使用吸氧的患者具有更高的血细胞比容和血红蛋白水平。持续吸氧的患者红细胞计数也高于不吸氧的患者。对于不使用补充氧气的患者、使用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。呼吸室内空气时静息 PaO2 较低也与红细胞计数、血细胞比容和血红蛋白较高相关。静息 PaO2 是肺部疾病严重程度的标志，并且与 LAM 组织学评分显着负相关，LAM 组织学评分是衡量肺部疾病严重程度的指标，是生存或移植时间的预测因子。根据多变量分析，DLCO 是血细胞比容、血红蛋白和红细胞计数的重要预测因子。运动高峰时的 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每年 DLCO 下降率相关联。尽管这种关系对于未接受吸氧的患者并未达到统计学显着性，但红细胞计数与 DLCO 年下降率之间的相关性对于使用吸氧的患者以及所有合并的患者而言均显着。因此，较高的红细胞指数与较严重的肺部疾病和较大的肺功能下降速度相关。由于缺氧会通过增加 EPO 的合成来增加红细胞指数，红细胞增多症患者肺功能的加速丧失可能是由于 EPO 对 LAM 细胞的生长促进作用。因此，下一个问题是，是否可以在 LAM 肺结节或培养的 LAM 细胞上发现 EPO 效应。 LAM 肺结节中的上皮样和纺锤形 LAM 细胞均表现出对 EPO 受体 (EPOR) 的免疫反应性，正常血管平滑肌和内皮细胞也表现出较小程度的免疫反应性。约。与肺动脉平滑肌细胞 (PASM) 一样，从移植的 LAM 肺中生长的培养细胞中 90% 与抗 EPOR 抗体发生反应。移植的 LAM 肺的显微镜切片显示与 LAM 肺结节中的 EPO 抗体有反应。抗EPO抗体与特发性肺纤维化患者肺组织中增殖的成纤维细胞切片不发生反应，特发性肺纤维化的特征是成纤维细胞增殖和大量胶原蛋白沉积。然而，我们在这些切片中观察到抗EPO抗体与肺泡巨噬细胞和II型肺细胞的免疫反应性。然后我们研究了 LAM 细胞中 EPOR 的激活状态。 EPOR 与 EPO 结合后在多个位点被磷酸化。 Tyr479 的磷酸化负责 EPOR 激活并在细胞增殖中发挥作用。尽管平滑肌细胞与抗磷酸 EPOR(Tyr479) 抗体反应较弱，但肺泡细胞的染色较强。然而，在 LAM 肺结节内的细胞和邻近的 II 型肺细胞中观察到针对激活的 EPOR（Tyr479）的抗体具有最大的免疫反应性。这些数据表明，LAM 细胞中激活的 EPOR 和 EPO 诱导细胞增殖所需的途径被激活。通过激光捕获显微切割从 LAM 结节收集的 LAM 细胞的总 RNA 中，以及从肺、肾、PASM 和 A549 细胞（人肺上皮腺癌细胞）提取的总 RNA 中检测到 EPOR mRNA，但显微切割不产生 EPO。 LAM 细胞。由于 EPO 可能与 LAM 病变中丰富的胶原蛋白相关，因此我们接下来测试了重组人 EPO 结合胶原蛋白或基质胶的能力。 EPO 与胶原蛋白的结合高于与 Matrigel 的结合，表明 EPO 与细胞外基质相关。我们已经证明，来自 LAM 肺外植体的细胞与膜相关 CD44v6 分子的抗体发生反应，其 TSC2 功能失调。因此，我们使用 CD44 和 CD44v6 抗体评估了通过荧光激活细胞分选分离的细胞中 EPO 和 EPOR 的存在，以收集四种细胞群（CD44+/CD44v6+、CD44-/CD44v6-、CD44-/CD44v6+ 和 CD44+/CD44v6- ）来自异质培养的 LAM 细胞。每个群体的 RNA 均含有 EPOR mRNA，但培养的 LAM 细胞（CD44+/CD44v6+ 和 CD44-/CD44v6+）与激光捕获的显微解剖 LAM 细胞一样，不产生 EPO，而 EPO 是由 CD44+/CD44v6- 细胞产生的。因此，虽然LAM细胞含有EPOR并且可能对肺中非LAM细胞的EPO敏感，但它们不产生EPO。由于 EPO 似乎会增加缺乏 TSC2 基因的细胞的增殖，因此测试了它对从 LAM 肺外植体生长的 TSC2 杂合性丧失的细胞的影响。添加 EPO 后，增殖率增加了 50-100%。由于这些培养物不是同质的，并且含有非 LAM 细胞以及 TSC2 杂合性的大量损失，因此 EPO 可能影响非 LAM 和 LAM 细胞的增殖。为了验证 EPO 对 TSC2-/- 细胞增殖率的影响，我们使用从 TSC 皮肤肿瘤（甲周纤维瘤）生长的同质 TSC2-/- 细胞。正如在肺 LAM 细胞中所报道的那样，由于 TSC2 等位基因的失活突变，这些细胞缺乏马铃薯蛋白。我们发现 TSC2+/- 和 TSC2-/- 细胞产生 EPOR，但不产生 EPO。 TSC2+/-和TSC2-/-细胞的基因表达比较显示，TSC2-/-细胞中的EPOR量是TSC2+/-细胞中的三倍。在 EPO 存在的情况下，TSC2-/- 皮肤肿瘤细胞的增殖速度是同一患者正常皮肤成纤维细胞增殖速度的两到三倍，而 EPO 对此几乎没有影响。这项研究证明了 TSC2-/- 皮肤肿瘤细胞的存在在 LAM 肺部病变和培养的 LAM 细胞中，EPOR 被激活，并且通过免疫组织化学显示，存在激活的 EPO 信号通路，这可能导致 LAM 细胞增殖和 LAM 病变的持续生长。此外，我们还发现 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LAM细胞产生EPO的证据，但我们的数据表明，肾皮质或肺部非LAM细胞产生的EPO可以增强LAM细胞增殖，从而加速疾病进展。红细胞计数升高（可能是缺氧的反应）与肺功能更快下降有关，从而在 LAM 的病理生理学与促进 LAM 细胞生长的信号通路激活之间建立了联系。这一发现是独一无二的，因为它证明了缺氧增强的红细胞增多与肺功能加速丧失之间的相关性。