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

• 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
• 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

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.

项目摘要/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，由 纤溶功能受损，血小板-内皮细胞黏附增加，血管纤维化。肺内膜切除术是 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。 目前尚不存在针对CTEPH的针对疾病的治疗方法，目前批准的单一药物也没有靶向 纤溶作用。因此，确定CTEPH特异性纤溶治疗靶点是CTEPH尚未满足的主要需求。 菲尔德。在这份美国国立卫生研究院研究项目拨款计划提案中，我们重点研究了缺氧信号的后果 并增加人肺动脉内皮细胞(HPAECs)的氧化应激 栓塞术有助于了解CTEPH的发病机制。具体地说，我们建议研究HIF-1α依赖的hIF-1基因上调 转移蛋白NEDD9及其与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，我们提出了新的初步数据，表明低氧增加了 HPAEC质膜表面有富含酪氨酸的NEDD9多肽。我们开发了一种定制的反- 针对该序列的NEDD9单特异性抗体(msAb-N9)，可抑制血小板与HPAECs的黏附 体外和体外分离的CTEPH患者的HPAECs，以及体内小鼠的血小板-内皮聚集体。 与疾病相比，动脉内膜切除术样本中msAb-N9靶标的表达增加 控制。此外，NEDD9在Cys18处的氧化阻止NEDD9-Smad3复合体的形成以稳定NEDD9 和SMAD3。这会导致依赖NEDD9的血管纤维化增加。重要的是，SMAD3已经在 其他类型的细胞上调SERPINE-1，它编码抗纤溶蛋白纤溶酶原激活物抑制物- 1(PAI-1)。在这里，我们观察到CTEPH-HPAECs表达Smad3增加，而siRNA-Smad3抑制 PAI-1。这些集体数据支持当前提议的中心假设：稳定SMAD3 由于NEDD9的氧化还原调节增加了HPAECs中Smad3依赖的PAI-1的上调， 其抑制纤溶，促进CTEPH的血栓重塑。我们进一步假设SMAD3 联合应用msAb-N9可恢复纤溶功能，拮抗血小板-PAEC黏附。 本研究的目的是：(1)利用CRISP-Cas9、突变型Smad3基因和微流控显微镜显示NEDD9-Cys18 氧化促进体外HPAECs和CTEPH-HPAECs中SMAD3依赖性血栓的形成 (2)研究NEDD9-/-、Smad3-/-和msAb-N9对大鼠血栓形成和心肺血流动力学的影响。 两种CTEPH模型中的活体模型。我们建议探索NEDD9-SMAD3和SMAD3-PAI-的翻译相关性。 1动脉内膜剥脱术标本与原位疾病对照的相互作用。总体而言，该项目旨在表明 MsAb-N9联合Smad3抑制是一种新的潜在的CTEPH特异性治疗策略。