Mechanisms and Consequences of Reduced PPAR gamma in Pulmonary Hypertension

描述(由申请人提供)： 肺动脉高压(PH)是一种破坏性的心肺疾病，具有显著的发病率和死亡率，经常使影响退伍军人患者群体的疾病复杂化。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。这一建议旨在定义PH发病机制的新途径，并为PH治疗开发新的策略。建议的研究集中在核激素受体，过氧化物酶体增殖物激活受体γ(PPARg)，它与PH的发病机制有关。PPARg表达减少导致PH，而用现有药物激活PPARg则减弱PH。不幸的是，这些相同的PPARg配体也会引起显著的副作用。因此，为了避免这些不利影响，本提案研究了PPARg在PH中下调的机制以及如何防止它作为PH治疗的一种新的替代策略。研究人员将使用血管内皮生长因子受体(VEGF-R)拮抗剂检测暴露于慢性低氧治疗下的小鼠的PH。补充研究将在体外对暴露于低氧中的人肺动脉内皮细胞或平滑肌细胞(HPAEC或HPASMC)或从对照组或PH患者分离的HPAEC或HPASMC进行。研究人员假设，低氧导致肺血管壁中PPARg的减少，促进下游介质的表达，从而导致血管细胞增殖、重塑和肺动脉高压。此外，据预测，阻止或减轻PPARg表达和功能的减少将减少增殖介质的表达和PH。该提案将涉及两个具体目标。第一个目标集中在调节PPARg表达的转录机制上。目的1确定核因子-kB在抑制PPARg启动子活性中的作用。已发表的和初步的数据表明，在PH和慢性低氧暴露下，核因子-kB被激活。在所提出的模型中，将使用定量实时聚合酶链式反应(qRT-PCR)和Western blotting来检测PPARg和PPARg调节的下游增殖介质(包括NADPH氧化酶4、内皮素-1和血栓反应蛋白-1)的表达。血管细胞增殖、血管重塑和PH将由研究小组发表的技术来确定。核因子-kB的激活将通过电泳迁移率改变分析来确定。SiRNA将被用来抑制核因子-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。目的2将重点研究microRNA-27(miR-27)对PPARg的转录后调控。MiR-27的水平将在AIM 1中使用的模型中使用qRT-PCR进行测量，其在PH病理生物学中的作用将使用miR-27拮抗剂或过度表达来确定。将使用荧光素酶报告构建来确认miR-27与PPARg 3‘-UTR的结合。抗miR-27抗体鼻腔内下呼吸道给药将在体内用于确定 如果阻止miR-27的增加，可以避免PPARg的降低，并减弱下游增殖性介质的增加。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。结果将决定防止肺血管PPARg表达减少的策略是否可以为PH治疗提供一种新的方法。拟议的策略还可能增强现有PPARg配体的治疗效果，从而减少剂量和相关的副作用。这一建议的结果可以因此确定新的和有效的治疗策略来调节参与PH发病机制的基因表达程序。

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.