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

描述(由申请人提供)： 肺动脉高压(PHT)是以肺小动脉(PAS)血管收缩活跃和结构改变为特征的疾病，肺动脉平滑肌细胞(PASMC)的增殖参与了血管重构。肺血管的异常病理生理可能与环核苷酸水平降低有关。磷酸二酯酶(PDE)催化cAMP和cGMP的水解。在从PHT患者分离的PASMC中，PDE1A和PDE1C表达的增加至少在一定程度上是激动剂诱导的cAMP水平降低和增殖增加的原因。这项建议的目的是利用从PHT患者和野百合碱(MCT)治疗的大鼠的动物模型分离的PASMC，研究PDE1亚型的表达、功能、调节和治疗潜力。该提案的目的是表明PDE1有助于PA的重塑，并且PDE1亚型是治疗PHT的新靶点。 其具体目标是：1)明确PDE1亚型在人和大鼠PAS中的表达、定位和活性，重点是PASMC，并确定这种变化在PHT中是否发生改变；2)确定PDE1异构体在人和大鼠PHT-PASMC中增加的功能影响；3)识别调节人和大鼠PASMC中PDE1表达的机制；4)使用PHT的动物模型，评估PDE1抑制剂的应用是否能为PHT提供治疗方法。 免疫组织化学、实时定量聚合酶链式反应、Western blotting和PDE分析将被用来确定PDE1亚型在人和大鼠肺中的表达，特别是在PASMC中的表达，以及随着PHT的变化。在存在或不存在PDE1A或PDE1C靶向siRNA或PDE1C腺病毒的情况下，通过检测cAMP、cGMP、增殖和凋亡来评估抑制或过度表达PDE1亚型的功能反应。凝胶漂移实验将用于检测活化T细胞核因子(NFAT)对PDE1C的转录调控。最后，治疗PHT的PDE1抑制剂将在接受MCT治疗的大鼠体内进行测试。这一提议有可能揭示导致PHT的细胞信号的新方面，并提供新的信息来支持PDE1抑制剂对目前尚不能治愈的疾病的治疗潜力。

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.