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-1?-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.
Among patients experiencing a blood clot in the lungs, some will develop a chronic disease called ?CTEPH? that is due to impaired breakdown of the clot, progressive lung artery scar, and increased lung artery blood pressure. We propose that failure for the two proteins NEDD9 and SMAD3 to interact normally prevents clot breakdown resulting in the development of CTEPH, and that inhibiting the function of NEDD9 and SMAD3 is a new potential treatment strategy for affected patients.
