NEDD9 is Upregulated by Hypoxia in Human Pulmonary Artery Endothelial Cells Selectively and Modulates Platelet-Endothelial Adhesion
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2 George Alba, MD Pulmonary & Critical Care Medicine Massachusetts General Hospital November 10 th, 2017 NEDD9 is Upregulated by Hypoxia in Human Pulmonary Artery Endothelial Cells Selectively and Modulates Platelet-Endothelial Adhesion George A. Alba, Andriy O. Samokhin, Bradley M. Wertheim, Elena Arons, Ying-Yi Zhang, Julia R. Ceglowski, Sachiko Seo, Richard N. Channick, Elisabeth M. Battinelli, Bradley A. Maron
3 Disclosures I have no relevant financial disclosures.
4 CTEPH is Characterized by Distinct Pulmonary Vascular Remodeling Acute PE CTEPH?
5 Systems Biology Predicts NEDD9 is Relevant to Pulmonary Vascular Disease Gene Collection Fibrosome Subnetwork Curated Literature GeneCards Fibrosis Genes Consolidated Human Interactome Adaptive (Dermal) Fibrosis Pathogenic (Vascular) Fibrosis Lung Fibrosis ,174 proteins 170,303 interactions Menche J, et al. Science 2015;347: NEDD9 111 Adaptive Fibrosis Pathogenic Fibrosis Adaptive Fibrosis BC( a ) i ( s, t a ) i s F1, t F ( s, t) 2 Pathogenic Fibrosis
6 NEDD9, a CAS-L Protein, Regulates Cell Migration and Proliferation HYPOXIA Nature Reviews Cancer. 2010;10:
7 Hypothesis: NEDD9 Promotes Endothelial Dysfunction in CTEPH NEDD9? Hypoxia 1 Platelet-PAEC interaction 4 Pro-inflammatory/thrombotic factors (ICAM-1) 2 Anti-fibrinolytic factors (PAI-1) 3 1 Cancer Res. 2010; 70(10): Circulation. 1994;89: J Heart Lung Transplant. 2017;36(3): Arterioscler Thromb Vasc Biol. 2014;34:
8 a.u. a.u. a.u. Hypoxia Upregulates NEDD9 Expression in HPAECs Selectively NEDD9 ß-actin O 2 21% 0.2% HPAECs * P< % O 2 0.2% O 2 NEDD9 ß-actin O 2 21% 0.2% HPASMCs 30 NS % O 2 0.2% O 2 NEDD9 ß-actin O 2 21% 0.2% NS 21% O 2 0.2% O 2 N=3-4 HCAECs
9 NEDD9 Inhibition Regulates PAI-1 and ICAM-1 in Hypoxia-Treated HPAECs si-scr si-n9 (24 hrs) si-n9 (48 hrs) UN = Untreated L = Lipofectamine Scr = Scrambled sirna PAI-1 inhibits clot breakdown ICAM-1 promotes inflammation & clot formation
10 Density (a.u.) Density (a.u.) NEDD9 Inhibition Prevents Upregulation of PAI-1 and ICAM-1 in Hypoxia-Treated HPAECs NS * 0.6 # P<0.01 PAI-1 inhibits clot breakdown si- N N= UN L UN si-n % O 2 0.2% O 2 * ICAM-1 promotes inflammation & clot formation si -N # P<0.02 UN = Untreated L = Lipofectamine 0 UN L UN si-n9 21% O 2 0.2% O 2 N=3
11 Molecular Inhibition of NEDD9 Decreases Platelet Adhesion to HPAECs HPAEC 100% confluent EC monolayer Control resting platelets Normal controls TRAP (25 μm) CMFDA Labeled Platelets Control activated platelets 492/517 nm + serial washing remaining fluorescence = adhesion
12 side scatter side scatter Platelet Adhesion (%) Molecular Inhibition of NEDD9 Decreases Platelet Adhesion to HPAECs Resting Platelets 1.4% * ** P<0.01 forward scatter p-selectin 10 Activated Platelets 78% 5 forward scatter p-selectin 0 Rest TRAP (25 µm) Rest TRAP (25 µm) N=4 Untreated si-rna-nedd9
13 Adhesion (%) Time (s) NEDD9 Regulates Platelet Function in vivo 433 bp 310 bp NEDD9 -/- +/- +/+ Courtesy of S. Seo (Tokyo, Japan) Platelet-PAEC Adhesion P < 0.01 P < Bleeding Time P = P = NS WT NEDD9 +/- NEDD9 -/- N = 4 0 WT NEDD9 +/- NEDD9 -/- N = 3-5
14 NEDD9 is Expressed on the Surface of Human Platelets Immunofluorescence 100x NEDD9 VEGF Merged Transmission Electron Microscopy Anti-NEDD9, Protein A Gold
15 NEDD9 is Expressed in Human Pulmonary Thromboemboli Ex Vivo H&E 4x Masson s Trichrome IF: NEDD9 10x IF: P-Selectin 10x IF: Merged
16 Conclusions NEDD9 is upregulated by hypoxia in HPAECs in vitro NEDD9 modulates upregulation of ICAM-1 and PAI-1 by hypoxia in HPAECs in vitro Inhibition of NEDD9 impairs platelet-paec adhesion in vitro which is associated with changes in bleeding time in vivo NEDD9 is present on the surface of platelets NEDD9 is expressed in fibrotic pulmonary thromboemboli from patients with CTEPH and co-localizes with P-selectin
17 Take Away Identifying NEDD9 as a novel molecular target to inhibit pulmonary arterial thrombosis may have therapeutic implications for patients with thrombotic pulmonary vascular diseases including CTEPH
18 Acknowledgements BWH Cardiovascular Medicine Bradley Maron, M.D. Andriy Samohkin, Ph.D. Elena Arons, M.D. Bradley Wertheim, M.D. Wusheng Xiao, Ph.D. Ruisheng Wang, Ph.D. William Oldham, M.D., Ph.D. Ying-Yi Zhang, Ph.D. Joseph Loscalzo, M.D., Ph.D. BWH Hematology Elisabeth Battinelli, M.D. Julia Ceglowski, B.A. MGH Pulmonary Medicine Richard Channick, M.D. Josalyn Cho, M.D. Benjamin Medoff, M.D. MGH Pathology George Eng, M.D., Ph.D. Carolyn McDonagh, B.A. Richard Kradin, M.D. Research Support GAA: NIH F32 (NHLBI), Harvard Catalyst BAM: NIH, AHA, CMREF, Scleroderma Foundation, BWH BWH EM Core Maria Ericsson, Ph.D.
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