Subcellular Mechanisms in Pathogenesis of Pulmonary Arterial Hypertension

肺动脉高压发病机制的亚细胞机制

• 批准号: 8057910
• 负责人: Jason Edward Lee
• 金额: $ 2.84万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-07 至 2014-02-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8057910
• 关键词: 3-Dimensional; Apoptosis; BMPR2 gene; Blood Vessels; Cell Culture Techniques; Cell Size; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Cellular biology; Co-Immunoprecipitations; Confocal Microscopy; Data; Defect; Development; Disease; Dominant-Negative Mutation; Doxycycline; Endoplasmic Reticulum; Etiology; Formalin; Functional disorder; Golgi Apparatus; Growth Factor; Human; Hypoxia; Interleukin-6; Investigation; Label; Lead; Lesion; Ligands; Location; Lung; Macaca; Mediator of activation protein; Membrane Protein Traffic; Membrane Proteins; Microscopy; Mitosis; Modality; Modeling; Monocrotaline; Morbidity - disease rate; Morphology; Mutation; Pathogenesis; Pathway interactions; Patients; Phenotype; Play; Progressive Disease; Protein Isoforms; Proteins; Rattus; Receptor Signaling; Recombinants; Research Personnel; Research Proposals; Resistance; Resolution; Rho-associated kinase; Role; S-nitro-N-acetylpenicillamine; Signal Pathway; Small Interfering RNA; Smooth Muscle Myocytes; Structure; System; Testing; Thinking; Three-Dimensional Imaging; Tissues; Transgenic Organisms; Vascular Diseases; Vascular remodeling; Virus; bone morphogenetic protein receptor type II; inhibitor/antagonist; knock-down; macrogolgin; mimetics; monocrotaline pyrrole; mortality; mutant; novel; overexpression; pulmonary arterial hypertension; receptor; response; sildenafil; simian human immunodeficiency virus; trafficking; two-photon; vector

DESCRIPTION (provided by applicant): Idiopathic pulmonary arterial hypertension (PAH) is an unrelentingly progressive disease characterized by proliferative, enlarged, and vacuolated endothelial and smooth muscle cells resistant to apoptosis. Singularly missing from discussions of the pathogenesis of PAH is consideration of subcellular membrane- and protein/receptor-trafficking mechanisms between the plasma membrane and the cell interior. We will test the hypothesis that the entire basket of alterations in vasorelevant proteins (including increases/decreases in levels of various receptors, signaling pathway molecules and eNOS) in this disease is largely due to mislocalization of these proteins to the wrong subcellular compartments due to defects in intracellular trafficking at the level of the Golgi apparatus ("Golgi blockade hypothesis"). We implicate both new players (respective Golgi tethers, SNAREs and 1-SNAP and NSF) and old players (mislocalized eNOS and reduced subcellular NO, mutant BMPRII species) in the subcellular mechanisms that "cause" PAH. Aim I will deal with high-resolution 3-D imaging studies of the alterations of the Golgi apparatus morphology, enlargement and fragmentation under various PAH-mimetic conditions [NO scavenging, hypoxia and monocrotaline pyrolle (MCTP)] in cultures of primary HPAECs and HPASMCs with a focus on giantin, GM130, p115, GS28 and 1-SNAP. Aim II will deal with the consequences of these changes in terms of the subcellular mislocalization of eNOS. The studies will be carried out in (1) cultures of HPAECs exposed to NO-scavenging, hypoxia or MCTP, and (2) in cells in formalin-fixed sections of vascular lesions in lungs derived from the MCT-treated rat, human PAH and the SHIV-nef-infected macaque with pulmonary vasculopathies. Aim III will test the hypothesis that PAH-disease-associated BMPRII mutant species can mislocalize eNOS and trafficking-mediator proteins in HPAECs and HPSMCs causing a dominant-negative effect on intracellular trafficking. Taken together, the proposed studies represent the next step in testing the Golgi blockade hypothesis in PAH. Upon completion of this project we will have elucidated changes in Golgi apparatus morphology in high-resolution 3-D in both cell culture and in cells in tissue sections in this disease, obtained evidence for subcellular mislocalization of eNOS and 1-SNAP, and provide a novel mechanism for how mutations in BMPRII might disrupt intracellular trafficking culminating in the cellular phenotype observed in pulmonary vascular cells in PAH lesions. PUBLIC HEALTH RELEVANCE: Fifteen thousand patients die of pulmonary arterial hypertension (PAH) every year in the U.S. It is not clearly understood what causes PAH. Although mutations in the BMPR2 gene have been implicated, the mechanisms have not been elucidated despite 10 years' of investigations. We propose a novel way of looking at the pathogenesis of this disease in terms of a global disruption of intracellular trafficking in pulmonary arterial endothelial and smooth muscle cells which result in the incorrect placement of vasorelevant proteins on the cell surface and in locations within the cells.

描述（由申请方提供）：特发性肺动脉高压（PAH）是一种以增殖、增大和空泡化内皮细胞和平滑肌细胞耐凋亡为特征的进行性疾病。PAH发病机制的讨论中特别缺少考虑质膜和细胞内部之间的亚细胞膜和蛋白质/受体运输机制。我们将检验这样的假设，即在这种疾病中血管相关蛋白的整个改变篮（包括各种受体、信号传导途径分子和eNOS水平的增加/减少）主要是由于这些蛋白质由于在高尔基体水平的细胞内运输缺陷而错误定位到错误的亚细胞区室（“高尔基体阻断假设”）。我们牵连新的球员（各自的高尔基体系链，SNARE和1-SNAP和NSF）和老球员（错误定位eNOS和减少亚细胞NO，突变BMPRII物种）的亚细胞机制，“导致”PAH。目的利用高分辨率三维成像技术研究不同PAH模拟条件下（NO清除、低氧和野百合碱吡咯（monocrotaline pyrolle，MCTP））培养的原代HPAEC和HPASMCs中高尔基体形态、增大和碎裂的变化，重点研究Giantin、GM 130、p115、GS 28和1-SNAP。目的二将处理这些变化的后果eNOS的亚细胞定位错误。研究将在（1）暴露于NO清除、缺氧或MCTP的HPAEC培养物中进行，以及（2）来自MCT处理的大鼠、人PAH和患有肺血管病的SHIV-nef感染猕猴的肺血管病变的福尔马林固定切片中的细胞中进行。目的III将测试假设PAH疾病相关的BMPRII突变物种可以在HPAEC和HPSMC中错误定位eNOS和转运介导蛋白，导致对细胞内转运的显性负效应。 综上所述，拟议的研究代表了检验PAH中高尔基体阻断假说的下一步。完成本项目后，我们将阐明高尔基体形态的变化，在高分辨率的3-D在细胞培养和在这种疾病的组织切片中的细胞，获得证据的eNOS和1-SNAP的亚细胞错误定位，并提供一种新的机制，如何突变BMPRII可能会破坏细胞内运输最终在PAH病变的肺血管细胞中观察到的细胞表型。 公共卫生相关性：在美国，每年有15，000名患者死于肺动脉高压（PAH）。虽然BMPR 2基因突变已被牵连，但尽管10年的研究，其机制尚未阐明。我们提出了一种新的方式来看待这种疾病的发病机制，在肺动脉内皮细胞和平滑肌细胞的细胞内运输的全球中断，导致血管相关蛋白在细胞表面上的不正确的位置和在细胞内的位置。