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）是血管损伤引起的肺动脉高压（PAH）的一个亚组，是一种与严重发病率和高死亡率相关的罕见疾病。 对于 IPAH 或更广泛的 PAH 群体，尚无常规筛查试验或经过验证的疾病活动标记。 因此，患者通常已处于疾病晚期。 IPAH 和其他形式的 PAH 的发病机制仍不清楚。目前的想法集中在双重打击假设上：1）遗传易感性，2）引发肺血管损伤的触发刺激，导致内皮细胞功能障碍。 内皮细胞通常脱落到循环系统中，是研究以内皮细胞功能障碍为特征的疾病的临床材料的宝贵来源。 不幸的是，没有明确的方法来分离临床相关数量的循环内皮细胞（CEC）。在该项目的试验阶段，我们正在使用流式细胞术开发一种方法，从健康志愿者和 PAH 患者中分离出临床相关数量的活 CEC。 我们假设 CEC 和/或外周血单核细胞 (PBMC) 可用于定义 IPAH 和其他形式的 PAH 中差异调节的生物标志物子集，这可能导致早期诊断和测量治疗反应的更好方法。 我们还希望为未来的治疗干预确定新的靶点。 在该项目的临床阶段，我们招募了健康志愿者以及患有IPAH和其他形式的PAH（血管损伤引起的肺动脉高压）的患者。获取外周血样本的 CEC 和 PBMC 用于微阵列；剩余的血浆被保存以供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压并获取肺血标本。 我们于 2006 年 6 月开始积极加入该方案。在 2009 年关闭该方案之前，我们招募了 31 名个体。初步数据表明，与外周血 (PB) 相比，肺静脉血中的 CEC 没有富集的趋势。健康志愿者（4.4 CEC/ml vs. 4.8 CEC/ml）和 PAH 患者（2.4 CEC/ml vs. 3.0 CEC/ml）。与 PB 相比，健康志愿者（13.8 CEC/ml 与 4.8 CEC/ml）和 PAH 患者（3.3 CEC/ml 与 3.0 CEC/ml）的肺动脉血液中存在 CEC 富集的趋势。 2010年，从PBMCs制备了用于微阵列的总RNA。为了更全面地表征其转录组，2011 年对其中 16 个样本进行了进一步处理，用于高密度寡核苷酸微阵列分析。 除了进一步研究基因表达之外，2011 年，还对来自健康志愿者和 PAH 患者的 24 份血浆样本进行了处理，用于培养微血管内皮细胞。 2011 年美国胸科学会年度会议上发表了一份基于 PAH 中 PBMC 差异基因表达模式的摘要。这些模式反映了治疗相关特征和潜在疾病病理生理学。 2011-12 年，我们将 10 名 PAH 患者的外周血单核细胞 (PBMC) 与匹配的对照进行了比较。研究发现，PAH 中的循环 PBMC 存在基因表达变化，这可能反映了对与受损血管床持续接触的反应。主题分析将这种转录组特征与炎症、细胞间信号传导和相互作用、细胞骨架重排、细胞运动、止血和细胞死亡联系起来。螺内酯是一种盐皮质激素 (MR) 和雄激素受体 (AR) 拮抗剂，在体外被发现可以抑制 PAH 患者上调的选定基因。 此外，2011年-12年，我们继续开发一种生物测定法，使用Affymetrix寡核苷酸微阵列评估PAH受试者血浆与健康对照相比引起的整体转录组变化。 将人类 PAEC 暴露于 5 名 PAH 受试者的血浆中，与 5 名年龄、性别和种族匹配的对照进行比较，以 10% 的错误发现率鉴定出 300 多个差异表达的转录本。未来的实验将利用目前从 PAH 患者和健康对照中获得的储存血浆来检查循环介质对 BMPR2 缺陷的 PAEC 中基因表达的影响。 该协议仍然开放用于进一步的数据处理、分析和手稿准备。