NEDD9-SMAD3, fibrinolysis, and chronic thromboembolic pulmonary hypertension

NEDD9-SMAD3、纤溶和慢性血栓栓塞性肺动脉高压

基本信息

批准号：
10031602
负责人：
Bradley Maron
金额：
$ 43.23万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10031602
关键词：
3-Dimensional Adhesions Affect Alteplase Antibodies Antifibrinolytic Agents Arteries Autologous Binding Bioavailable Blood Platelets Blood Pressure Blood Vessels Blood coagulation C-terminal COS-7 Cell CRISPR/Cas technology Cardiopulmonary Cell Nucleus Cell membrane Chronic Disease Cicatrix Clinical Coagulation Process Complementary DNA Complex Coupling Custom Data Development Disease Endarterectomy Endothelium Event Failure Fibrin Fibrinolysis Fibrosis Genetic Transcription Heart failure Human Hypoxia Impairment In Situ In Vitro Ligands Ligase Ligation Lung MADH3 gene Mediating Mediator of activation protein Medical Microfluidics Microscopy Modeling Modification Molecular Mus Nebulizer Neoplasm Metastasis Operative Surgical Procedures Oxidation-Reduction P-Selectin Pathogenesis Patients Peptides Pharmaceutical Preparations Plasminogen Activator Inhibitor 1 Platelet aggregation Program Research Project Grants Proteins Pulmonary Embolism Pulmonary Heart Disease Rattus Regulation Resistance SERPINE1 gene Sampling Signal Transduction Slide Small Interfering RNA Specimen Surface Technology Testing Therapeutic Thrombosis Thrombus Tyrosine United States National Institutes of Health Up-Regulation Ventricular arterial remodeling cell type chromatin immunoprecipitation chronic thromboembolic pulmonary hypertension clinical efficacy density disorder control experience hemodynamics improved in vivo mortality mutant novel overexpression oxidant stress oxidation patient subsets prevent protein complex protein expression protein protein interaction pulmonary artery endothelial cell therapeutic target treatment strategy

项目摘要

Project Summary/Abstract Chronic thromboembolic pulmonary hypertension (CTEPH) is a severe cardiopulmonary disease defined by impaired fibrinolysis, increased platelet-endothelial adhesion, and vascular fibrosis. Pulmonary endarterectomy is the mainstay treatment for CTEPH, but is inappropriate or unsuccessful in a large subpopulation of patients. Disease-specific medical therapies for CTEPH do not exist, and the single currently approved drug does not target fibrinolysis. Thus, identifying CTEPH-specific fibrinolytic therapeutic targets is a principal unmet need in the CTEPH field. In this NIH Research Project Grant Program proposal, we focus on the consequences of hypoxia signaling and increased oxidant stress in human pulmonary artery endothelial cells (HPAECs) that follows luminal pulmonary embolism to understand CTEPH pathogenesis. Specifically, we propose to study HIF-1α-dependent upregulation of the metastasis protein NEDD9, and redox regulation of the NEDD9 protein-protein interaction with SMAD3 to explain CTEPH thrombosis and dysregulated fibrinolysis, respectively. In the accompanying proposal, we present novel preliminary data showing that hypoxia increases expression of a tyrosine-rich NEDD9 peptide on the HPAEC plasma membrane surface. We developed a custom-made anti- NEDD9 monospecific antibody (msAb-N9) targeting that sequence, which inhibits platelet adhesion to HPAECs in vitro and HPAECs isolated from CTEPH patients ex vivo, as well as platelet-endothelial aggregates in mice in vivo. Increased expression of the msAb-N9 target was observed in endarterectomy samples compared to disease controls. Additionally, oxidation of NEDD9 at Cys18 prevents NEDD9-SMAD3 complex formation to stabilize NEDD9 and SMAD3. This leads to increased NEDD9-dependent vascular fibrosis. Importantly, SMAD3 has been shown in other cell types to upregulate SERPINE-1, which encodes the antifibrinolytic protein plasminogen activator inhibitor- 1 (PAI-1). Here, we observed that CTEPH-HPAECs express increased SMAD3, and that siRNA-SMAD3 inhibits PAI-1. These collective data support the central hypothesis of the current proposal: Stabilization of SMAD3 due to redox regulation of NEDD9 increases SMAD3-dependent upregulation of PAI-1 in HPAECs, which inhibits fibrinolysis to promote thrombotic remodeling in CTEPH. We postulate further that SMAD3 inhibition combined with msAb-N9 restores fibrinolysis and antagonizes platelet-PAEC adhesion, respectively. The Aims are: (1) use CRISP-Cas9, mutant SMAD3 cDNA, and microfluidic microscopy to show that NEDD9-Cys18 oxidation promotes SMAD3-dependent thrombus formation in HPAECS in vitro and CTEPH-HPAECs ex vivo, and (2) study the effect of NEDD9-/-, SMAD3-/-, and msAb-N9 on thrombosis and cardiopulmonary hemodynamics in vivo in two CTEPH models. We propose to explore the translational relevance of NEDD9-SMAD3 and SMAD3-PAI- 1 interactions in endarterectomy samples and disease controls in situ. Overall, this project aims to show that msAb-N9 plus SMAD3 inhibition is a novel potential CTEPH-specific therapeutic strategy.

项目概要/摘要慢性血栓栓塞性肺动脉高压（CTEPH）是一种严重的心肺疾病，定义为纤维蛋白溶解受损、血小板内皮粘附增加和肺动脉内膜切除术。 CTEPH 的主要治疗方法，但在很大一部分患者中不合适或不成功。针对 CTEPH 的疾病特异性药物疗法尚不存在，且目前批准的单一药物不针对因此，确定 CTEPH 特异性纤溶治疗靶点是 CTEPH 中未满足的主要需求。在这个 NIH 研究项目拨款计划提案中，我们重点关注缺氧信号传导的后果。以及管腔肺动脉内皮细胞（HPAEC）氧化应激增加具体而言，我们建议研究 HIF-1α 依赖性上调。转移蛋白 NEDD9，以及 NEDD9 蛋白-蛋白与 SMAD3 相互作用的氧化还原调节分别解释 CTEPH 血栓形成和纤溶失调。在随附的提案中，我们提出了新颖的初步数据，表明缺氧会增加我们开发了一种定制的抗-HPAEC 质膜表面富含酪氨酸的 NEDD9 肽。 NEDD9 单特异性抗体 (msAb-N9) 靶向该序列，可抑制血小板与 HPAEC 的粘附体外和从 CTEPH 患者离体分离的 HPAEC，以及小鼠体内的血小板内皮聚集物。与疾病相比，在动脉内膜切除术样本中观察到 msAb-N9 靶标的表达增加此外，NEDD9 Cys18 处的氧化可防止 NEDD9-SMAD3 复合物形成，从而稳定 NEDD9。 SMAD3 会导致 NEDD9 依赖性血管纤维化增加。其他细胞类型上调 SERPINE-1，它编码抗纤维蛋白溶解蛋白纤溶酶原激活剂抑制剂 - 1 (PAI-1) 在这里，我们观察到 CTEPH-HPAEC 表达增加的 SMAD3，并且 siRNA-SMAD3 抑制。 PAI-1。这些集体数据支持当前提案的中心假设：SMAD3 的稳定性由于 NEDD9 的氧化还原调节增加了 HPAEC 中 PAI-1 的 SMAD3 依赖性上调，它抑制纤维蛋白溶解，促进 CTEPH 中的血栓重塑。我们进一步假设 SMAD3。抑制与 msAb-N9 相结合分别恢复纤维蛋白溶解并拮抗血小板-PAEC 粘附。目标是：(1) 使用 CRISP-Cas9、突变 SMAD3 cDNA 和微流控显微镜来显示 NEDD9-Cys18 氧化促进体外 HPAECS 和离体 CTEPH-HPAEC 中 SMAD3 依赖性血栓形成，以及 (2)研究NEDD9-/-、SMAD3-/-、msAb-N9对血栓形成及心肺血流动力学的影响我们建议探索 NEDD9-SMAD3 和 SMAD3-PAI 的翻译相关性。 1 动脉内膜切除术样本和原位疾病控制的相互作用总体而言，该项目旨在表明这一点。 msAb-N9 联合 SMAD3 抑制是一种新型的潜在 CTEPH 特异性治疗策略。