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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：
10649448
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/10649448
关键词：
3-Dimensional Adhesions Affect Alteplase Antibodies Aorta Arteries Autologous Binding Biological Availability Blood Platelets Blood Vessels Blood coagulation C-terminal COS-7 Cell CRISPR/Cas technology Cardiopulmonary Cell Nucleus Cell Separation Cell membrane Chronic Disease Cicatrix Clinical Coagulation Process Complementary DNA Complex Coupling Custom Data Development Disease Endarterectomy Endothelium Event Failure Fibrin Fibrinolysis Fibrosis Genes Genetic Transcription Human Hypoxia Impairment In Situ In Vitro Ligands Ligase Ligation Lung MADH3 gene Mediating Mediator Medical Microfluidics Microscopy Modeling Modification Molecular Mus Nebulizer Neoplasm Metastasis Operative Surgical Procedures Oxidation-Reduction P-Selectin Pathogenesis Patients Peptides Pharmaceutical Preparations Phosphorylation 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 Transfection 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 drug repurposing experience hemodynamics improved in vivo mortality mutant novel overexpression oxidant stress oxidation patient subsets prevent protein complex protein expression protein protein interaction pulmonary arterial pressure pulmonary artery endothelial cell right ventricular failure therapeutic target thrombotic 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/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，纤维蛋白溶解受损、血小板-内皮粘附增加和血管纤维化。肺动脉内膜切除术是 CTEPH的主要治疗方法，但在大量患者亚群中不合适或不成功。目前尚不存在针对CTEPH的疾病特异性药物治疗，目前批准的单一药物不靶向纤维蛋白溶解因此，鉴定CTEPH特异性纤溶治疗靶点是CTEPH治疗中未满足的主要需求。领域在这个NIH研究项目资助计划提案中，我们关注缺氧信号的后果，和人肺动脉内皮细胞（HPAECs）中氧化应激增加，了解CTEPH的发病机制。具体来说，我们建议研究HIF-1α依赖性上调转移蛋白NEDD 9，以及NEDD 9蛋白-蛋白与SMAD 3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。在所附的建议中，我们提出了新的初步数据，表明缺氧增加了 HPAEC质膜表面富含酪氨酸的NEDD 9肽。我们开发了一种定制的反- 靶向该序列的NEDD 9单特异性抗体（msAb-N9），其抑制血小板粘附于HPAEC，体外和离体从CTEPH患者分离的HPAEC，以及体内小鼠中的血小板-内皮聚集体。与疾病相比，在动脉内膜切除术样品中观察到msAb-N9靶点的表达增加。对照此外，NEDD 9在Cys 18处的氧化防止NEDD 9-SMAD 3复合物形成以稳定NEDD 9 SMAD3这导致NEDD 9依赖性血管纤维化增加。重要的是，SMAD 3已经在其他细胞类型上调SERPINE-1，其编码抗纤维蛋白溶解蛋白纤溶酶原激活物抑制剂- 1（派-1）。在这里，我们观察到CTEPH-HPAEC表达增加的SMAD 3，并且siRNA-SMAD 3抑制CTEPH-HPAEC表达增加的SMAD 3。派-1这些集体数据支持当前提案的中心假设：SMAD稳定3 由于NEDD 9的氧化还原调节增加了HPAEC中派-1的SMAD 3依赖性上调，其抑制纤维蛋白溶解以促进CTEPH中的血栓重塑。我们进一步假设SMAD 3 与msAb-N9组合的抑制分别恢复纤维蛋白溶解和拮抗血小板-PAEC粘附。目的是：（1）使用CRISP-Cas9、突变SMAD 3 cDNA和微流控显微镜来显示NEDD 9-Cys 18 氧化促进体外HPAECS和离体CTEPH-HPAEC中SMAD 3依赖性血栓形成，和 (2)研究NEDD 9-/-、SMAD 3-/-和msAb-N9对血栓形成和心肺血流动力学的影响，在两个CTEPH模型中的体内。我们建议探索NEDD 9-SMAD 3和SMAD 3-派的翻译相关性， 1动脉内膜切除术样品和原位疾病对照的相互作用。总的来说，这个项目旨在表明， msAb-N9加SMAD 3抑制是一种新的潜在CTEPH特异性治疗策略。

项目成果

期刊论文数量（27）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Harnessing Big Data to Advance Treatment and Understanding of Pulmonary Hypertension.

DOI：
10.1161/circresaha.121.319969
发表时间：
2022-04-29
期刊：
CIRCULATION RESEARCH
影响因子：
20.1
作者：
Rhodes, Christopher J.;Sweatt, Andrew J.;Maron, Bradley A.
通讯作者：
Maron, Bradley A.

Evidence of Advanced Pulmonary Vascular Remodeling in Obstructive Hypertrophic Cardiomyopathy With Pulmonary Hypertension.

阻塞性肥厚型心肌病合并肺动脉高压的晚期肺血管重塑的证据。

DOI：
10.1016/j.chest.2022.09.040
发表时间：
2023
期刊：
Chest
影响因子：
9.6
作者：
Maron,BradleyA;Kleiner,DavidE;Arons,Elena;Wertheim,BradleyM;Sharma,NirmalS;Haley,KathleenJ;Samokhin,AndriyO;Rowin,EthanJ;Maron,MartinS;Rosing,DouglasR;Maron,BarryJ
通讯作者：
Maron,BarryJ

Pulmonary endothelial NEDD9 and the prothrombotic pathophenotype of acute respiratory distress syndrome due to SARS-CoV-2 infection.

DOI：
10.1002/pul2.12071
发表时间：
2022-04
期刊：
PULMONARY CIRCULATION
影响因子：
2.6
作者：
Alba, George A.;Samokhin, Andriy O.;Wang, Rui-Sheng;Wertheim, Bradley M.;Haley, Kathleen J.;Padera, Robert F.;Vargas, Sara O.;Rosas, Ivan O.;Hariri, Lida P.;Shih, Angela;Thompson, Boyd Taylor;Mitchell, Richard N.;Maron, Bradley A.
通讯作者：
Maron, Bradley A.

Individualized interactomes for network-based precision medicine in hypertrophic cardiomyopathy with implications for other clinical pathophenotypes.

DOI：
10.1038/s41467-021-21146-y
发表时间：
2021-02-08
期刊：
Nature communications
影响因子：
16.6
作者：
Maron BA;Wang RS;Shevtsov S;Drakos SG;Arons E;Wever-Pinzon O;Huggins GS;Samokhin AO;Oldham WM;Aguib Y;Yacoub MH;Rowin EJ;Maron BJ;Maron MS;Loscalzo J
通讯作者：
Loscalzo J

Network medicine in Cardiovascular Research.