Endothelial Cell Dysfunction in Pulmonary Arterial Hypertension

肺动脉高压中的内皮细胞功能障碍

• 批准号: 8565315
• 负责人: Michael Solomon
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 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/8565315
• 关键词: Age; American; Androgen Receptor; BMPR2 gene; Biological Assay; Biological Markers; Blood; Blood Circulation; Blood Tests; Blood Vessels; Blood specimen; Cardiac Catheterization Procedures; Cell Communication; Cell Death; Cells; Chest; Clinical; DNA Sequence Rearrangement; Data; Disease; Disease Marker; Early Diagnosis; Endothelial Cells; Enrollment; Flow Cytometry; Functional disorder; Future; Gender; Gene Expression; Gene Expression Profile; Genes; Genetic Predisposition to Disease; Hemostatic function; In Vitro; Individual; Inflammation; Injury; Lead; Lung; Manuscripts; Measures; Mediator of activation protein; Methodology; Methods; Microarray Analysis; Mineralocorticoids; Morbidity - disease rate; Movement; Oligonucleotide Microarrays; Pathogenesis; Patients; Pattern; Peripheral Blood Mononuclear Cell; Phase; Plasma; Preparation; Process; Protocols documentation; Pulmonary Hypertension; Pulmonary artery structure; Pulmonary veins; RNA; Race; Rare Diseases; Recruitment Activity; Sampling; Screening procedure; Societies; Source; Spironolactone; Staging; Stimulus; Subgroup; Testing; Therapeutic Intervention; Thinking; Tissue-Specific Gene Expression; Transcript; abstracting; base; clinical material; clinically relevant; computerized data processing; density; exposed human population; healthy volunteer; injured; intercellular communication; meetings; mortality; novel; peripheral blood; pressure; pulmonary arterial hypertension; research study; response; transcriptomics; trend; vascular bed

Idiopathic (primary) pulmonary arterial hypertension (IPAH), a subgroup of the vascular injury-induced forms of pulmonary arterial hypertension (PAH), is a rare disorder associated with severe morbidity and high mortality rates. There are no routine screening tests or validated markers of disease activity in IPAH, or the broader group of PAH. Therefore, patients usually present at advanced stages of disease. The pathogenesis of IPAH and other forms of PAH remain unclear. Current thinking focuses on a two-hit hypothesis: 1) genetic susceptibility, and 2) a triggering stimulus that initiates pulmonary vascular injury, resulting in endothelial cell dysfunction. Endothelial cells are normally shed into the circulation and are a valuable source of clinical material for studying diseases characterized by endothelial cell dysfunction. Unfortunately, no clear methodology exists for isolating clinically relevant numbers of circulating endothelial cells (CECs). In the bench phase of the project we are using flow cytometry to develop a methodology for isolating clinically relevant numbers of viable CECs from healthy volunteers and patients with PAH. We hypothesize that CECs and/or peripheral blood mononuclear cells (PBMC) can be used to define a subset of differentially regulated biomarkers in IPAH and other forms of PAH that may lead to earlier diagnosis and better methods for measuring responses to therapy. We also hope to identify novel targets for future therapeutic interventions. In the clinical phase of the project, we recruited healthy volunteers and patients with IPAH and other forms of PAH (vascular injury induced pulmonary hypertension). Peripheral blood specimens were obtained for CECs and PBMCs for microarrays; the remaining plasma was saved for future application to cultured microvascular cells. A subset of subjects underwent right heart catheterization to assess pulmonary pressures and to obtain pulmonary blood specimens. We started actively enrolling into the protocol in June 2006. We enrolled 31 individuals prior to closing the protocol to enrollment in 2009. Preliminary data suggested that there was no trend towards CEC enrichment in pulmonary vein blood compared to peripheral blood (PB) for both the healthy volunteers (4.4 CEC/ml vs. 4.8 CEC/ml) and the PAH patients (2.4 CEC/ml vs. 3.0 CEC/ml). There was a trend towards CEC enrichment in pulmonary artery blood compared to PB for both the healthy volunteers (13.8 CEC/ml vs. 4.8 CEC/ml) and the PAH patients (3.3 CEC/ml vs. 3.0 CEC/ml). In 2010, total RNA for microarrays was prepared from PBMCs. To more fully characterize their transcriptome, 16 of these samples were further processed in 2011 for high density oligonucleotide microarray analysis. In addition to further study gene expression, in 2011, 24 plasma samples from healthy volunteers and patients with PAH were processed for application to cultured microvascular endothelial cells. An abstract based on the PBMC differential gene expression patterns in PAH was presented at the Annual American Thoracic Society Meeting in 2011. These patterns reflected both treatment related signatures and underlying disease pathophysiology. In 2011-12, peripheral blood mononuclear cells (PBMCs) from 10 patients with PAH were compared to matched controls. Circulating PBMCs in PAH were found to harbor gene expression changes that may reflect a response to persistent contact with an injured vascular bed. Thematic analysis associated this transcriptomic signature with inflammation, cell-to-cell signaling and interaction, cytoskeletal rearrangement, cellular movement, hemostasis and cell death. Spironolactone, a mineralocorticoid (MR) and androgen receptor (AR) antagonist, was found in vitro to suppress selected genes upregulated in patients with PAH. In addition in 2011 -12 we continued to develop a bioassay assessing global transcriptomic changes induced by plasma from PAH subjects compared to healthy controls using Affymetrix oligonucleotide microarrays. Exposure of human PAECs to plasma from 5 PAH subjects compared to 5 age, gender and race matched controls, identified over 300 differentially expressed transcripts at a 10% false discovery rate. Future experiments will utilize stored plasma currently available from PAH patients and healthy controls to examine the effects of circulating mediators on gene expression in BMPR2-deficient PAECs. The protocol remains open for further data processing, analysis, and manuscript preparation.

特发性（原发性）肺动脉高压（IPAH）是血管损伤诱导的肺动脉高压（PAH）的亚组，是一种与严重的发病率和高死亡率相关的罕见疾病。 IPAH或更广泛的PAH没有常规筛查测试或经过验证的疾病活动标记。 因此，患者通常处于疾病晚期阶段。 IPAH和其他形式的PAH的发病机理尚不清楚。当前的思维重点是两次打击的假设：1）遗传敏感性，以及2）引发肺血管损伤的触发刺激，导致内皮细胞功能障碍。 内皮细胞通常被脱落到循环中，是研究以内皮细胞功能障碍为特征的疾病的宝贵临床材料来源。 不幸的是，尚无明确的方法来分离临床相关数量的循环内皮细胞（CEC）。在项目的基准阶段，我们正在使用流式细胞术来开发一种方法，以将临床相关数量的可行CEC与健康的志愿者和PAH患者分离。 我们假设CEC和/或外周血单核细胞（PBMC）可用于定义IPAH中差异调节的生物标志物的子集，以及其他形式的PAH，这可能会导致较早的诊断和更好的方法来测量对治疗的反应。 我们还希望确定未来治疗干预措施的新颖目标。 在该项目的临床阶段，我们招募了健康的志愿者和IPAH和其他形式的PAH（血管损伤引起的肺动脉高压）的患者。获得了CEC和PBMC的外周血标本用于微阵列。将其余的血浆保存为将来应用于培养的微血管细胞。 一部分受试者接受了右心导管插入术，以评估肺部压力并获得肺部血液标本。 我们于2006年6月开始积极入学。在关闭2009年入学方案之前，我们招募了31个人。初步数据表明，与健康志愿者相比，与外周血（PB）相比，肺静脉血液中CEC富集的趋势没有趋势（4.4 CEC/ML vs. 4.8 CEC/ML CEC/ML）和PAH（4.ML CEC/ML）（4. ML）（2.4） CEC/ML）。与健康志愿者（13.8 CEC/ML相比4.8 CEC/mL）和PAH患者（3.3 CEC/ML相比3.0 CEC/ML）相比，与PB的PB相比，肺动脉血液中CEC富集的趋势是一种趋势。 2010年，通过PBMC制备了微阵列的总RNA。为了更充分地表征其转录组，在2011年进一步处理了其中16个样品，以进行高密度寡核苷酸微阵列分析。 除了进一步研究基因表达外，还将处理来自健康志愿者和PAH患者的24个血浆样本，以应用于培养的微血管内皮细胞。 在2011年的年度美国胸腔协会会议上提出了基于PBMC差异基因表达模式的摘要。这些模式反映了相关的特征和潜在的疾病病理生理学。 在2011 - 12年度，将10名PAH患者的外周血单核细胞（PBMC）与匹配的对照组进行了比较。发现PAH中循环的PBMC具有基因表达的变化，可能反映了对与受伤的血管床的持续接触的反应。主题分析将这种转录组特征与炎症，细胞对细胞信号传导和相互作用，细胞骨架重排，细胞运动，止血和细胞死亡相关联。在体外发现螺内乳酮是一种矿物皮质激素（MR）和雄激素受体（AR）拮抗剂，可抑制PAH患者上调的选定基因。 此外，与使用Affymetrix寡核苷酸微阵列相比，在2011年-12 -12-12中，我们继续开发出评估PAH受试者血浆引起的全球转录组变化。 与5个PAH受试者相比，人类PAEC暴露于血浆中的血浆，性别和种族匹配的对照组，以10％的错误发现率确定了300多个差异表达的转录本。未来的实验将利用当前可从PAH患者使用的存储等离​​子体和健康对照组来检查循环介质对BMPR2缺陷型PAEC中基因表达的影响。 该协议保持开放，以进行进一步的数据处理，分析和手稿准备。