Subcellular Mechanisms in Pathogenesis of Pulmonary Arterial Hypertension

肺动脉高压发病机制的亚细胞机制

基本信息

批准号：
8230450
负责人：
Jason Edward Lee
金额：
$ 1.1万
依托单位：
NEW YORK MEDICAL COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-02-07 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8230450
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 public health relevance pulmonary arterial hypertension receptor response sildenafil simian human immunodeficiency virus trafficking two-photon vector

项目摘要

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（由申请人提供）：特发性肺动脉高压（PAH）是一种不懈的进行性疾病，其特征是增生，肿大和空泡的内皮和平滑肌细胞具有抗细胞凋亡的抗性。关于PAH的发病机理的讨论奇异的是考虑到质膜和细胞内部之间的亚细胞膜和蛋白/受体贩运机制。我们将检验以下假设：该疾病中血管脂蛋白蛋白质的整个变化（包括各种受体水平的增加/降低，信号通路分子和eNOS）在很大程度上是由于这些蛋白质错误地定位于错误的亚细胞障碍物，因为它们是错误的亚细胞障碍物，因此在细胞内的缺陷范围内，在golgi级别的golgi级别（golgi golgi part of golgi partect）（假设”）。我们暗示了新的玩家（各自的高尔基tethers，网罗和1-SNAP和NSF），以及“引起” PAH的亚细胞机制中的老参与者（错误定位的eNOS和减少了突变的BMPRII物种）。目的，我将处理高分辨率3-D成像研究研究，以在各种模仿条件下[无清除，缺氧，缺氧和单脂素pyrolle（MCTP）在主要HPAECS和HPASMCS中培养的培养物和HPASMCS培养物中，对GM1和1-SN的培养中的培养物的变化[无清除，缺氧和单细胞素pylolle（MCTP）。 AIM II将根据eNOS的亚细胞错误定位来处理这些变化的后果。这些研究将在暴露于无扫描，缺氧或MCTP的HPAEC培养物中进行，以及（2）在福尔马林固定的肺部病变的细胞中，源自MCT治疗的大鼠，人类PAH，人类PAH和SHIV-NEF-NEF-NEF-NEF-NEF-NEF-NEF-NEF-NEF-NEF-NEF-NEF-NEF-NEF-NEF-NEF-NEFECTER-NEFECTER-NEFECTENF IMACAQUE。 AIM III将检验以下假设：与PAH酶相关的BMPRII突变物种类可能会错误地定位于HPAEC和HPAEC中的ENOS和运输中介质蛋白，从而导致对细胞内运输的显性阴性作用。综上所述，提出的研究代表了测试PAH中高尔基封锁假设的下一步。该项目完成后，我们将阐明这种疾病中细胞培养和组织切片中细胞培养和细胞中高分辨率3-D中高尔基体形态的变化，获得了eNOS和1-SNAP的亚细胞不定位的证据，并为BMpriiral inclyarbornular clanciration callminartim clarmintim clarmintial climintial climination climination提供了新的机制。 PAH病变中的细胞。公共卫生相关性：每年在美国每年死于肺动脉高压（PAH）的15,000例患者，尚不清楚导致PAH的原因尚不清楚。尽管已经涉及BMPR2基因中的突变，但尽管进行了10年的研究，但仍未阐明该机制。我们提出了一种新颖的方式来研究这种疾病的发病机理，这是在肺部动脉内皮和平滑肌细胞中细胞内运输的全球破坏，从而导致血管纤维含量不正确在细胞表面和细胞内部的位置。