Pulmonary Artery Endothelial Cell Phenotypic Alterations in a Large Animal Model of Pulmonary Arteriovenous Malformations After the Glenn Shunt Minoo.

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Pulmonary Artery Endothelial Cell Phenotypic Alterations in a Large Animal Model of Pulmonary Arteriovenous Malformations After the Glenn Shunt Minoo N. Kavarana, MD, Rupak Mukherjee, PhD, Shaina R. Eckhouse, MD, William F. Rawls, BS, Christina Logdon, BS, Robert E. Stroud, MS, Risha K. Patel, BS, Elizabeth K. Nadeau, BS, Francis G. Spinale, MD, PhD, Eric M. Graham, MD, Geoffrey A. Forbus, MD, Scott M. Bradley, MD, John S. Ikonomidis, MD, PhD, Jeffrey A. Jones, PhD The Annals of Thoracic Surgery Volume 96, Issue 4, Pages 1442-1449 (October 2013) DOI: 10.1016/j.athoracsur.2013.05.075 Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 (A) Two-dimensional contrast echocardiogram showing pulmonary arteriovenous malformations. (B) Protein abundance of angiopoietin-1, angiostatin, TEK (Tie-2), endoglin, Kruppel-like factor (KLF-2), and vascular endothelial growth factor (VEGF) in the right lung of superior cavopulmonary connection (SCPC) pigs expressed as a fold change from values obtained in the left lung of the SCPC pigs and controls (set to 100% [dashed vertical line]). (∗p < 0.05 versus right lung; #versus left lung SCPC; SVC = superior vena cava.) The Annals of Thoracic Surgery 2013 96, 1442-1449DOI: (10.1016/j.athoracsur.2013.05.075) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 (A) Proliferation of smooth muscle cells from the left pulmonary artery (LPA), right pulmonary artery (RPA), and controls expressed as a change from the 24-hour time point (set to zero). There were no differences between the LPA and the RPA. (B) Proliferation of RPA endothelial cells was higher than the LPA and controls (∗p < 0.05 versus LPA; #p < 0.05 versus time 0). (C) Photomicrographs of endothelial cells from the LPA and RPA pig at 5 days after plating. (D) Photomicrographs of endothelial cells from the LPA and RPA at similar confluence. (Photomicrographs were recorded using a 20× Nomarski differential contrast objective.) The Annals of Thoracic Surgery 2013 96, 1442-1449DOI: (10.1016/j.athoracsur.2013.05.075) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 (A) Immunoblots for endoglin, Kruppel-like factor (KLF-2), TEK (Tie-2), and β-actin in lysates of endothelial cells from the left pulmonary artery (LPA), right pulmonary artery (RPA), and control. (C) The KLF-2 abundance was statistically similar between LPA, RPA and controls; the (B) endoglin and (D) Tie-2 abundance was higher in the RPA than in the LPA (∗p < 0.05 versus LPA). The Annals of Thoracic Surgery 2013 96, 1442-1449DOI: (10.1016/j.athoracsur.2013.05.075) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Radar plot showing relative gene expression profiles for genes related to extracellular matrix (ECM) degradation, ECM deposition, and cellular signaling. The plot for the left pulmonary artery (LPA [plane defined by the red triangle]) and control (plane defined by the green triangle) was clearly different than the plane defined for the right pulmonary artery (RPA [blue triangle]). Fold expression levels for the genes that resulted in this plot are summarized in Appendix Table 1. The Annals of Thoracic Surgery 2013 96, 1442-1449DOI: (10.1016/j.athoracsur.2013.05.075) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 (A) Photomicrographs of endothelial cells from the left pulmonary artery (LPA), right pulmonary artery (RPA), and controls 3 hours after plating. Cells from the LPA (left) and controls assembled into a number of nodes and tubule extension had commenced as evidenced by thin structures between nodes. In contrast, cells from the RPA—at the same timepoint—had assembled into a well-defined network structure between nodes, consistent with a more proangiogenic phenotype. (Photomicrographs were recorded using a 2.5× objective with phase contrast. Scale bars: 100 μm.) (B) Number of tubes and (C) total length of tubes were higher in the RPA than in the LPA (∗p < 0.05 versus LPA). The Annals of Thoracic Surgery 2013 96, 1442-1449DOI: (10.1016/j.athoracsur.2013.05.075) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions