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: 8952821
• 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/8952821
• 关键词: Acetates; Age; American; Anti-Inflammatory Agents; Anti-inflammatory; Bioinformatics; Biological Assay; Biological Markers; Blood; Blood Circulation; Blood Tests; Blood Vessels; Blood specimen; Cardiac Catheterization Procedures; Cell Communication; Cell Death; Cells; Chest; Clinical; Computer software; DNA Sequence Rearrangement; Data; Disease; Disease Marker; Dose; Early Diagnosis; Endothelial Cells; Enrollment; Flow Cytometry; Functional disorder; Future; Gender; Gene Expression; Gene Expression Profile; Gene Silencing; Genes; Genetic Predisposition to Disease; Genetic Transcription; Hemostatic function; Human; In Vitro; Individual; Inflammation; Inflammatory; Injury; Laboratories; Lead; Lung; Measures; Mediating; Mediator of activation protein; Meta-Analysis; Methodology; Methods; Molecular; Molecular Profiling; Mononuclear; Morbidity - disease rate; Movement; Oligonucleotide Microarrays; Pathogenesis; Pathway Analysis; Patients; Pattern; Peripheral Blood Mononuclear Cell; Phase; Plasma; Process; Protocols documentation; Publishing; Pulmonary Hypertension; Pulmonary artery structure; Pulmonary veins; RNA; Race; Rare Diseases; Recruitment Activity; Reporter; Sampling; Societies; Source; Spironolactone; Staging; Stimulus; Subgroup; Techniques; Testing; Therapeutic Intervention; Thinking; Tissue-Specific Gene Expression; Transcript; Transcription Factor AP-1; Work; abstracting; base; bone morphogenic protein; clinical material; clinically relevant; exposed human population; genome-wide; healthy volunteer; intercellular communication; meetings; mortality; novel; overexpression; peripheral blood; phorbol-12-myristate; pressure; promoter; pulmonary arterial hypertension; receptor; research study; response; screening; transcriptomics; trend; vascular inflammation; vector

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 were 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 and 2011, total RNA was processed from PBMCs for genome-wide expression analysis. 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 addition to completing expression profiling of PBMCs, plasma samples from healthy volunteers and patients with PAH were processed for application to cultured microvascular endothelial cells. In 2011 to 2013, using PBMC expression profiles from 10 PAH subjects with 10 age, gender and race matched healthy control subjects we identified over 230 differentially regulated genes at a 20% false discovery rate. Ingenuity Pathway Analysis identified gene signatures for inflammation, cell-to-cell signaling and interaction, cytoskeletal rearrangement, cellular movement, hemostasis and cell death. In vitro data from our collaborating laboratory demonstrates that spironolactone suppresses phorbol 12-myristate 13-acetate-induced (PMA; an AP-1 activator) inflammatory gene transcription in primary human PAECs. In order to explore the effect of spironolactone on PAH-associated vascular inflammation we conducted a promoter level analysis of the up-regulated genes we identified in subjects with PAH. Biobase ExPlain and Transfac bioinformatics software identified activator protein-1 (AP-1) as a key transcriptional regulator. Experiments using PBMCs isolated from healthy subjects and stimulated with PMA demonstrate that spironolactone suppresses these AP-1 inducible, PAH-associated genes in a dose-dependent manner. Similarly in PBMCs from healthy subjects and PAH subjects, spironolactone strongly suppressed the basal expression of genes that had been up regulated in the PBMCs of 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. In additional work done in 2012-13, we explored the gene expression changes in cultured human PAECs induced by plasma from PAH subjects and found that 20% of this signature overlapped with the gene expression changes induced following bone morphogeneic protein receptor-2 (BMPR2) gene silencing in PAECs. Importantly, more than 90% of this overlap was directionally discordant, suggesting circulating factors may work to counter-regulate genotypic and phenotypic abnormalities that drive PAH. 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. In 2013-14, we have continued to actively investigate the mechanisms that mediate the anti-inflammatory effects of spironolactone using molecular techniques such as overexpression vectors and AP-1 promoter based reporter assays. Furthermore, in order to expand upon our expression findings in circulating mononuclear cells, we have collected data from all of the other published human PAH PBMC genome-wide expression profiling studies for subsequent meta-analysis. Once data annotation and aggregation is completed, we plan to compare PBMC gene expression of IPAH and disease-associated PAH to healthy controls as well as to each other. This protocol remains open to continue bioinformatic analyses of the gene expression data, completing downstream in vitro work as well as finishing a meta-analysis of the published PAH PBMC expression profiling studies.

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 年和 2011 年，对 PBMC 进行了总 RNA 处理，用于全基因组表达分析。 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。除了完成 PBMC 的表达谱外，还对健康志愿者和 PAH 患者的血浆样本进行了处理，以应用于培养的微血管内皮细胞。 2011 年至 2013 年，利用 10 名 PAH 受试者（其中 10 名年龄、性别和种族匹配的健康对照受试者）的 PBMC 表达谱，我们以 20% 的错误发现率鉴定了超过 230 个差异调节基因。 Ingenuity 通路分析确定了炎症、细胞间信号传导和相互作用、细胞骨架重排、细胞运动、止血和细胞死亡的基因特征。我们合作实验室的体外数据表明，螺内酯可抑制原代人 PAEC 中佛波醇 12-肉豆蔻酸酯 13-乙酸酯诱导的（PMA；一种 AP-1 激活剂）炎症基因转录。为了探索螺内酯对 PAH 相关血管炎症的影响，我们对在 PAH 受试者中鉴定的上调基因进行了启动子水平分析。 Biobase ExPlain 和 Transfac 生物信息学软件将激活蛋白-1 (AP-1) 确定为关键的转录调节因子。使用从健康受试者中分离并用 PMA 刺激的 PBMC 进行的实验表明，螺内酯以剂量依赖性方式抑制这些 AP-1 诱导型 PAH 相关基因。同样，在健康受试者和 PAH 受试者的 PBMC 中，螺内酯强烈抑制 PAH 患者 PBMC 中上调基因的基础表达。 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。在 2012-13 年完成的其他工作中，我们探索了由 PAH 受试者血浆诱导的培养的人类 PAEC 中的基因表达变化，发现该特征的 20% 与骨形态发生蛋白受体 2 (BMPR2) 诱导的基因表达变化重叠PAEC 中的基因沉默。重要的是，超过 90% 的重叠在方向上不一致，这表明循环因素可能会起到反调节驱动 PAH 的基因型和表型异常的作用。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。 2013-14年，我们继续积极研究介导螺内酯抗炎作用的机制，使用分子技术，例如过表达载体和基于AP-1启动子的报告基因检测。此外，为了扩展我们在循环单核细胞中的表达发现，我们收集了所有其他已发表的人类 PAH PBMC 全基因组表达谱研究的数据，用于后续荟萃分析。一旦数据注释和聚合完成，我们计划将 IPAH 和疾病相关 PAH 的 PBMC 基因表达与健康对照以及彼此之间进行比较。 该协议仍然开放，以继续对基因表达数据进行生物信息学分析，完成下游体外工作以及完成已发表的 PAH PBMC 表达谱研究的荟萃分析。