George A Alba1, Andriy O Samokhin2, Rui-Sheng Wang2, Ying-Yi Zhang2, Bradley M Wertheim3, Elena Arons2, Edward A Greenfield4, Martina H Lundberg Slingsby5, Julia R Ceglowski5, Kathleen J Haley3, Frederick P Bowman2, Yen-Rei Yu6, John C Haney7, George Eng8, Richard N Mitchell9, Anthony Sheets9, Sara O Vargas10, Sachiko Seo11, Richard N Channick12, Peter J Leary13, Sudarshan Rajagopal14, Joseph Loscalzo2, Elisabeth M Battinelli5, Bradley A Maron2. 1. Division of Pulmonary and Critical Care Medicine, Department of Medicine, and. 2. Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts. 3. Division of Cardiovascular Medicine. 4. Division of Pulmonary and Critical Care Medicine. 5. Division of Hematology and Oncology, Department of Medicine, and. 6. Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts. 7. Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts. 8. Division of Pulmonary and Critical Care Medicine, Department of Medicine. 9. Division of Cardiovascular and Thoracic Surgery, Department of Surgery, and. 10. Division of Cardiology, Department of Medicine, Duke University Medical Center, Duke University, Durham, North Carolina. 11. Department of Pathology, Boston Children's Hospital, Boston, Massachusetts. 12. Department of Hematology and Oncology, Dokkyo Medical University, Tochigi, Japan. 13. Division of Pulmonary and Critical Care Medicine, Department of Medicine, Ronald Reagan UCLA Medical Center, University of California, Los Angeles, Los Angeles, California; and. 14. Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Washington, Seattle, Washington.
Abstract
Rationale: Data on the molecular mechanisms that regulate platelet-pulmonary endothelial adhesion under conditions of hypoxia are lacking, but may have important therapeutic implications. Objectives: To identify a hypoxia-sensitive, modifiable mediator of platelet-pulmonary artery endothelial cell adhesion and thrombotic remodeling. Methods: Network medicine was used to profile protein-protein interactions in hypoxia-treated human pulmonary artery endothelial cells. Data from liquid chromatography-mass spectrometry and microscale thermophoresis informed the development of a novel antibody (Ab) to inhibit platelet-endothelial adhesion, which was tested in cells from patients with chronic thromboembolic pulmonary hypertension (CTEPH) and three animal models in vivo. Measurements and Main Results: The protein NEDD9 was identified in the hypoxia thrombosome network in silico. Compared with normoxia, hypoxia (0.2% O2) for 24 hours increased HIF-1α (hypoxia-inducible factor-1α)-dependent NEDD9 upregulation in vitro. Increased NEDD9 was localized to the plasma-membrane surface of cells from control donors and patients with CTEPH. In endarterectomy specimens, NEDD9 colocalized with the platelet surface adhesion molecule P-selectin. Our custom-made anti-NEDD9 Ab targeted the NEDD9-P-selectin interaction and inhibited the adhesion of activated platelets to pulmonary artery endothelial cells from control donors in vitro and from patients with CTEPH ex vivo. Compared with control mice, platelet-pulmonary endothelial aggregates and pulmonary hypertension induced by ADP were decreased in NEDD9-/- mice or wild-type mice treated with the anti-NEDD9 Ab, which also decreased chronic pulmonary thromboembolic remodeling in vivo. Conclusions: The NEDD9-P-selectin protein-protein interaction is a modifiable target with which to inhibit platelet-pulmonary endothelial adhesion and thromboembolic vascular remodeling, with potential therapeutic implications for patients with disorders of increased hypoxia signaling pathways, including CTEPH.
Rationale: Data on the molecular mechanisms that regulate platelet-pulmonary endothelial adhesion under conditions of hypoxia are lacking, but may have important therapeutic implications. Objectives: To identify a hypoxia-sensitive, modifiable mediator of platelet-pulmonary artery endothelial cell adhesion and thrombotic remodeling. Methods: Network medicine was used to profile protein-protein interactions in hypoxia-treated human pulmonary artery endothelial cells. Data from liquid chromatography-mass spectrometry and microscale thermophoresis informed the development of a novel antibody (Ab) to inhibit platelet-endothelial adhesion, which was tested in cells from patients with chronic thromboembolic pulmonary hypertension (CTEPH) and three animal models in vivo. Measurements and Main Results: The protein NEDD9 was identified in the hypoxia thrombosome network in silico. Compared with normoxia, hypoxia (0.2% O2) for 24 hours increased HIF-1α (hypoxia-inducible factor-1α)-dependent NEDD9 upregulation in vitro. Increased NEDD9 was localized to the plasma-membrane surface of cells from control donors and patients with CTEPH. In endarterectomy specimens, NEDD9 colocalized with the platelet surface adhesion molecule P-selectin. Our custom-made anti-NEDD9 Ab targeted the NEDD9-P-selectin interaction and inhibited the adhesion of activated platelets to pulmonary artery endothelial cells from control donors in vitro and from patients with CTEPH ex vivo. Compared with control mice, platelet-pulmonary endothelial aggregates and pulmonary hypertension induced by ADP were decreased in NEDD9-/- mice or wild-type mice treated with the anti-NEDD9 Ab, which also decreased chronic pulmonary thromboembolic remodeling in vivo. Conclusions: The NEDD9-P-selectin protein-protein interaction is a modifiable target with which to inhibit platelet-pulmonary endothelial adhesion and thromboembolic vascular remodeling, with potential therapeutic implications for patients with disorders of increased hypoxia signaling pathways, including CTEPH.
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