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，我们使用流式细胞术来开发一种方法，用于从健康的志愿者和PAH患者中隔离临床相关数量的可行CEC。 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。 在2010年和2011年，从PBMCS处理总RNA以进行全基因组表达分析。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。除了完成PBMC的表达分析外，还处理了来自健康志愿者和PAH患者的血浆样品，以应用于培养的微血管内皮细胞。 在2011年至2013年，使用来自10个年龄的10个PAH受试者的PBMC表达曲线，性别和种族与健康对照组相匹配，我们以20％的错误发现率确定了超过230个差异调节的基因。 Ingenition途径分析确定了炎症，细胞到细胞信号和相互作用，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，螺内酯抑制了原代人PAEC中的12-乙酸13-乙酸酯诱导的炎症基因转录。为了探索螺内酯对PAH相关的血管炎症的影响，我们对我们在患有PAH受试者中鉴定的上调基因进行了启动子水平分析。生物酶解释和转换生物信息学软件将激活剂蛋白-1（AP-1）识别为关键转录调节剂。使用从健康受试者中分离并用PMA刺激的PBMC的实验表明，螺内乳肽以剂量依赖性的方式抑制了这些AP-1可诱导的PAH相关基因。同样，在来自健康受试者和PAH受试者的PBMC中，螺内酯强烈抑制了在PAH患者的PBMC中受到调节的基因的基础表达。 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。在2012 - 13年度进行的其他工作中，我们探讨了PAH受试者血浆引起的培养的人类PAEC的基因表达变化，发现该特征中有20％与PAEC中骨形态蛋白受体-2（BMPR2）基因沉默在骨形态蛋白受体-2（BMPR2）基因后诱导的基因表达变化重叠。重要的是，超过90％的重叠在方向上是不一致的，这表明循环因子可能有效地调节驱动PAH的基因型和表型异常。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。 在2013 - 14年度，我们继续使用分子技术（例如过表达矢量和基于AP-1启动子的报告基因测定法）进行积极研究介导螺内酯抗炎作用的机制。此外，为了扩展我们在循环单核细胞中的表达结果，我们从所有其他已发表的人PAH PAH PBMC基因组全基因组表达分析研究中收集了数据，以进行随后的荟萃分析。一旦完成数据注释和聚集完成，我们计划将IPAH和与疾病相关的PAH的PBMC基因表达与健康对照组进行比较。 该方案保持开放，以继续对基因表达数据进行生物信息学分析，完成了下游的体外工作，并完成对已发表的PAH PAH PBMC表达分析研究的荟萃分析。