Mechanisms and Consequences of Reduced PPAR gamma in Pulmonary Hypertension

肺动脉高压中 PPAR γ 减少的机制和后果

• 批准号: 8598800
• 负责人: C MICHAEL HART
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-10-01 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598800
• 关键词: Address; Adverse effects; Affect; Attenuated; Behavior; Binding; Blood Vessels; Cell Proliferation; Cell Wall; Chronic; Chronic Obstructive Airway Disease; Collagen; Complex; Complication; Congestive Heart Failure; Data; Development; Disease; Down-Regulation; Electrophoretic Mobility Shift Assay; Endothelin-1; Event; Exposure to; Gene Expression; HIV Infections; Healthcare; Human; Hypertension; Hypoxia; In Vitro; Interstitial Lung Diseases; Lead; Ligands; Lower respiratory tract structure; Luciferases; Lung; Measures; Mediator of activation protein; MicroRNAs; Modeling; Morbidity - disease rate; Mus; NADPH Oxidase; NF-kappa B; Names; Nuclear Hormone Receptors; Obstructive Sleep Apnea; Outcome; PPAR gamma; Pathogenesis; Pathway interactions; Patients; Peroxisome Proliferator-Activated Receptors; Pharmaceutical Preparations; Polymerase Chain Reaction; Post-Transcriptional Regulation; Prevention; Publishing; Pulmonary Heart Disease; Pulmonary Hypertension; Pulmonary artery structure; Pulmonary vessels; Receptor Activation; Relative (related person); Reporter; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Small Interfering RNA; Smooth Muscle Myocytes; Stimulus; Techniques; Therapeutic; Therapeutic Effect; Thrombospondin 1; Time; Untranslated Regions; Vascular Diseases; Vascular Endothelial Growth Factor Receptor; Vascular remodeling; Veterans; Western Blotting; dosage; effective therapy; endothelial dysfunction; hypertension treatment; improved; in vivo; mortality; novel; novel strategies; novel therapeutics; overexpression; patient population; peroxisome; prevent; programs; promoter; public health relevance; pulmonary arterial hypertension; receptor; vasoconstriction

DESCRIPTION (provided by applicant): Pulmonary hypertension (PH) is a devastating cardiopulmonary disorder with significant morbidity and mortality that frequently complicates disorders that affect the veteran patient population. Existing PH therapies are expensive and not optimally effective, indicating that novel approaches to PH treatment are urgently needed. This proposal seeks to define new pathways in PH pathogenesis and develop novel strategies for PH therapy. The proposed studies focus on the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARg) which has been implicated in PH pathogenesis. Reduced PPARg expression caused PH whereas activation of PPARg with existing medications attenuated PH. Unfortunately, these same PPARg ligands also cause significant side effects. Thus to avoid these adverse effects, this proposal examines mechanisms of PPARg downregulation in PH and ways to prevent it as a new and alternative strategy in PH therapy. The investigators will examine PH in mice caused by exposure to chronic hypoxia ¿ treatment with a vascular endothelial growth factor receptor (VEGF-R) antagonist. Complementary studies will be performed in vitro in human pulmonary artery endothelial or smooth muscle cells (HPAEC or HPASMC) exposed to hypoxia or in HPAEC or HPASMC isolated from control or PH patients. The investigators hypothesize that hypoxia- induced reductions in PPARg in the pulmonary vascular wall promote the expression of downstream mediators that cause vascular cell proliferation, remodeling and PH. Further, it is predicted that preventing or attenuating reductions in PPARg expression and function will reduce proliferative mediator expression and PH. The proposal will address 2 specific aims. The first aim focuses on transcriptional mechanisms that regulate PPARg expression. Aim 1 will determine the role of NF-kB in suppressing PPARg promoter activity. Published and preliminary data demonstrate that NF-kB is activated in PH and by exposure to chronic hypoxia. The expression of PPARg and downstream PPARg-regulated proliferative mediators (including NADPH oxidase 4, endothelin-1, and thrombospondin-1) will be measured in the proposed models using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Vascular cell proliferation, vascular remodeling, and PH will be determined with techniques published by the investigative team. Activation of NF-kB will be determined by electrophoretic mobility shift assays. siRNA will be administered to inhibit NF-kB to determine its role in reduced PPARg expression and in the increased expression of proliferative mediators that cause PH. Aim 2 will focus on the post-transcriptional regulation of PPARg by microRNA-27 (miR-27). Levels of miR-27 will be measured in the models employed in Aim 1 using qRT-PCR, and its role in PH pathobiology will be established using miR-27 antagonists or overexpression. Binding of miR-27 to the PPARg 3'-UTR will be confirmed using luciferase reporter constructs. Intranasal delivery of anti-miR-27 to the lower respiratory tract will be used in vivo to determine if preventing increases in miR-27 can avert reductions in PPARg and attenuate downstream increases in proliferative mediators. The proposed studies will clarify pathogenic mechanisms in PH and define the relative contributions of transcriptional and post-transcriptional pathways to reductions in PPARg. The outcomes will determine if strategies to prevent reductions in pulmonary vascular PPARg expression can provide a novel approach to PH therapy. The proposed strategies might also enhance the therapeutic effects of existing PPARg ligands thereby permitting reductions in dosage and associated side effects. The results of this proposal can thereby define novel and effective therapeutic strategies to regulate programs of gene expression involved in PH pathogenesis.

描述（由申请人提供）： 肺动脉高压 (PH) 是一种毁灭性的心肺疾病，具有显着的发病率和死亡率，经常使影响退伍军人群体的疾病复杂化。现有的 PH 治疗方法昂贵且效果不佳，这表明迫切需要新的 PH 治疗方法。确定 PH 发病机制的新途径并制定 PH 治疗的新策略。拟议的研究重点是与 PH 相关的核激素受体、过氧化物酶体增殖物激活受体 γ (PPARg)。 PPARg 表达减少导致 PH，而现有药物激活 PPARg 会减弱 PH，不幸的是，这些相同的 PPARg 配体也会引起显着的副作用，因此，为了避免这些副作用，本提案研究了 PH 中 PPARg 下调的机制和预防方法。它作为 PH 治疗的一种新的替代策略，研究人员将检查因暴露于慢性缺氧而引起的小鼠的 PH。使用血管内皮生长因子受体 (VEGF-R) 拮抗剂治疗 将在暴露于缺氧的人肺动脉内皮细胞或平滑肌细胞 (HPAEC 或 HPASMC) 或从对照或 PH 患者中分离的 HPAEC 或 HPASMC 中进行体外补充研究。研究人员发现，缺氧引起的肺血管壁 PPARg 减少会促进下游介质的表达，从而导致血管细胞增殖、重塑和 PH。预计防止或减弱 PPARg 表达和功能的减少将减少增殖介质表达和 PH。第一个目标是调节 PPARg 表达的转录机制，目标 1 将确定 NF-kB 的作用。已发表的数据和初步数据表明，NF-kB 在 PH 中被激活，并通过暴露于慢性缺氧而激活 PPARg 和下游 PPARg 的表达。将使用实时聚合酶链式反应 (qRT-PCR) 和蛋白质印迹在所提出的模型中定量测量增殖介质（包括 NADPH 氧化酶 4、内皮素-1 和血小板反应蛋白-1）。将通过研究小组公布的技术来确定 NF-kB 的激活将通过电泳迁移率变化测定来确定，并且将施用 siRNA 来抑制。 NF-kB 以确定其在 PPARg 表达减少和导致 PH 的增殖介质表达增加中的作用。目标 2 将重点关注 microRNA-27 (miR-27) 对 PPARg 的转录后调节。将使用 qRT-PCR 在目标 1 中使用的模型中进行测量，并将使用 miR-27 拮抗剂或过表达来确定其在 PH 病理学中的作用。将使用荧光素酶报告构建体确认 miR-27 与 PPARg 3'-UTR 的抗 miR-27 鼻内递送至下呼吸道将用于体内测定。 如果阻止 miR-27 的增加可以避免 PPARg 的减少并减弱下游增殖介质的增加，拟议的研究将阐明 PH 的致病机制，并确定转录和转录后途径对 PPARg 减少的相对贡献。如果防止肺血管 PPARg 表达减少的策略可以提供一种新的 PH 治疗方法，那么所提出的策略也可能增强现有 PPARg 配体的治疗效果，从而减少剂量。因此，该提案的结果可以定义新颖且有效的治疗策略来调节与 PH 发病机制相关的基因表达程序。