NEDD9-SMAD3, fibrinolysis, and chronic thromboembolic pulmonary hypertension

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， 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。具体地说，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。

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.