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Phosphodiesterase-1 in Pulmonary Hypertension: Expression, Regulation, and Target

肺动脉高压中的磷酸二酯酶 1：表达、调节和靶点

基本信息

批准号：
8309855
负责人：
Fiona Murray
金额：
$ 24.48万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-10 至 2013-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Pulmonary hypertension (PHT) is characterized by active vasoconstriction and structural changes in the small pulmonary arteries (PAs); proliferation of pulmonary artery smooth muscle cells (PASMC) contribute to the remodeling. The abnormal pathophysiology in the pulmonary vasculature may relate to decreased cyclic nucleotide levels. Phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP. In PASMC isolated from patients with PHT an increase in the expression of PDE1A and PDE1C, at least in part, accounts for lower agonist-induced cAMP levels and increased proliferation. The goal of this proposal is to study the expression, function, regulation, and therapeutic potential of PDE1 isoforms using PASMC isolated from PHT patients, and from an animal model for PHT [monocrotaline (MCT)-treated rats]. The objective of the proposal is to show that PDE1 contributes to the remodeling of the PA and that PDE1 isoforms represent novel targets for the treatment of PHT. The Specific Aims are: 1) Define the expression, localization and activity of PDE1 isoforms in human and rat PAs, focusing on PASMC, and determine if this is altered in PHT, 2) Determine the functional impact of increased PDE1 isoforms in human and rat PHT-PASMC, 3) Identify mechanisms that regulate PDE1 expression in human- and rat-PASMC, 4) Assess, using an animal model of PHT, if administration of PDE1 inhibitors can provide a therapeutic approach for PHT. Immunohistochemistry, real-time PCR, Western blotting, and PDE assays will be used to define the expression of PDE1 isoforms in human and rat lung, in particular in PASMC, and changes with PHT. The functional response of inhibition or over expression of PDE1 isoforms will be assessed by measuring cAMP, cGMP, proliferation, and apoptosis in the presence and absence of PDE1A- or PDE1 C-targeted siRNA or PDE1C adenovirus. Gel shift assay will be used to determine the transcriptional regulation of PDE1C by Nuclear factor of activated T-cells (NFAT). Finally, PDE1 inhibitors for PHT will be tested in vivo in MCT-treated rats. This proposal has the potential to reveal new aspects regarding the cellular signaling that contribute to PHT and provide novel information to support the therapeutic potential of PDE1 inhibitors in the disease, which currently has no cure.

描述（由申请人提供）：肺动脉高压（pulmonary hypertension，PHT）是以肺小动脉（small pulmonary arteries，PA）主动收缩和结构改变为特征，肺动脉平滑肌细胞（pulmonary artery smooth muscle cells，PASMC）增殖参与肺动脉重构。肺血管系统的异常病理生理学可能与环核苷酸水平下降有关。磷酸二酯酶（PDE）催化cAMP和cGMP的水解。在从PHT患者分离的PASMC中，PDE 1A和PDE 1C表达的增加至少部分地解释了较低的激动剂诱导的cAMP水平和增加的增殖。本提案的目的是使用从PHT患者和PHT动物模型[野百合碱（MCT）治疗大鼠]分离的PASMC研究PDE 1亚型的表达、功能、调节和治疗潜力。该提案的目的是表明PDE 1有助于PA的重塑，并且PDE 1亚型代表了治疗PHT的新靶点。具体目标是：1）确定PDE 1亚型在人和大鼠PA中的表达、定位和活性，重点是PASMC，并确定这在PHT中是否改变，2）确定PDE 1亚型在人和大鼠PHT-PASMC中增加的功能影响，3）确定调节人和大鼠PASMC中PDE 1表达的机制，4）使用PHT动物模型评估，如果PDE 1抑制剂给药可以为PHT提供治疗方法。将使用免疫组织化学、实时PCR、蛋白质印迹和PDE测定来确定PDE 1亚型在人和大鼠肺中的表达，特别是在PASMC中，以及PHT的变化。在存在和不存在PDE 1A-或PDE 1C-靶向siRNA或PDE 1C腺病毒的情况下，通过测量cAMP、cGMP、增殖和凋亡来评估抑制或过度表达PDE 1同种型的功能反应。凝胶迁移试验将用于确定活化T细胞核因子（NFAT）对PDE 1C的转录调节。最后，将在MCT给药大鼠中对PHT的PDE 1抑制剂进行体内试验。这一提议有可能揭示有关促进PHT的细胞信号传导的新方面，并提供新的信息来支持PDE 1抑制剂在目前无法治愈的疾病中的治疗潜力。