权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

项目总结/文摘