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: 8440548
• 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/8440548
• 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; Functional disorder; 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; 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; 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; Functional disorder; 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; 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，具有明显的发病率和死亡率，经常使影响退伍军人人群的疾病复杂化。现有的pH疗法很昂贵且不最佳，这表明迫切需要新的pH治疗方法。该建议旨在定义pH发病机理中的新途径，并制定新的pH疗法策略。拟议的研究集中于核能受体，过氧化物体增生剂激活的受体伽马（PPARG），该受体伽玛（PPARG）已在pH发病机理中实施。 pPARG表达降低引起pH，而现有药物减弱PPARG的激活。不幸的是，这些相同的PPARG配体也会引起显着的副作用。为了避免这些不良反应，这种建议检查在pH值下调的提案检查机制以及防止其作为pH疗法的新替代策略的方法。研究人员将检查由暴露于慢性缺氧治疗的小鼠中的pH值 - 血管内皮生长因子受体（VEGF-R）拮抗剂治疗。互补的研究将在体外暴露于缺氧或从对照或pH患者中分离出的HPAEC或HPASMC中的人类肺动脉内皮或平滑肌细胞（HPAEC或HPASMC）中进行体外。研究者假设缺氧诱导的肺血管壁中PPARG的降低促进了引起血管细胞增殖，重塑和pH的下游介质的表达。此外，可以预测，预防或减少PPARG表达和功能的减少将减少增殖介质表达和pH。该提案将解决2个具体目标。第一个目的侧重于调节PPARG表达的转录机制。 AIM 1将确定NF-KB在抑制PPARG启动子活性中的作用。已发布和初步数据表明，NF-KB在pH中被激活，并暴露于慢性缺氧。 PPARG和下游PPARG调节的增生剂（包括NADPH氧化酶4，内皮素-1和血小板素-1）的表达将在建议的模型中使用定量的实时聚合酶链反应（QRT-PCR）和Western Blotting进行测量。血管细胞增殖，血管重塑和pH将由调查小组发布的技术确定。 NF-KB的激活将由电泳迁移率转移评估确定。 siRNA将被施用以抑制NF-KB，以确定其在降低PPARG表达以及增加引起pH的增殖介质表达中的作用。 AIM 2将重点介绍MicroRNA-27（miR-27）对PPARG的转录后调节。使用QRT-PCR在AIM 1中采用的模型中将测量miR-27的水平，并将使用miR-27拮抗剂或过表达来确定其在pH病理生物学中的作用。 MiR-27与PPARG 3'-UTR的结合将使用荧光素酶报告基因构建体确认。鼻内抗MIR-27向下呼吸道的递送将在体内使用 如果防止miR-27的增加可以避免降低PPARG并减少下游的增殖介体的增加。拟议的研究将阐明pH值的致病机制，并定义转录和转录后途径对PPARG减少的相对贡献。结果将确定防止减少肺血管PPARG表达的策略是否可以提供一种新型的pH疗法方法。提出的策略还可能增强现有PPARG配体的治疗作用，从而降低剂量和相关的副作用。该提案的结果可以定义新的有效的治疗策略来调节与pH发病机理有关的基因表达程序。