Mary P. Kotlarczyk, PhD, Marie Billaud, PhD, Benjamin R

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Regional Disruptions in Endothelial Nitric Oxide Pathway Associated With Bicuspid Aortic Valve Mary P. Kotlarczyk, PhD, Marie Billaud, PhD, Benjamin R. Green, MS, Jennifer C. Hill, MFS, Sruti Shiva, PhD, Eric E. Kelley, PhD, Julie A. Phillippi, PhD, Thomas G. Gleason, MD The Annals of Thoracic Surgery Volume 102, Issue 4, Pages 1274-1281 (October 2016) DOI: 10.1016/j.athoracsur.2016.04.001 Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Nitric oxide synthase 3 (NOS3) gene expression (n = 10–11 per valve type, per right [R], left [L], and noncoronary (N) sinus regions). Values are normalized to platelet endothelial cell adhesion molecule-1 expression. The error bars indicate SD. *p < 0.05 (BAV = bicuspid aortic valve; TAV = tricuspid aortic valve.) The Annals of Thoracic Surgery 2016 102, 1274-1281DOI: (10.1016/j.athoracsur.2016.04.001) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Expression of endothelial nitric oxide synthase (eNOS). (A) Analysis of eNOS immunoblots normalized to CD31; n = 12 tricuspid aortic valve (TAV) specimens or n = 20 to 21 bicuspid aortic valve (BAV) specimens per right (R), left (L), and noncoronary (N) sinus regions. *p < 0.05. The error bars indicate SD. (B) Representative immunoblots. The Annals of Thoracic Surgery 2016 102, 1274-1281DOI: (10.1016/j.athoracsur.2016.04.001) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Phosphorylated (p)Ser1177-endothelial nitric oxide synthase (eNOS) quantification. (A) Analysis of pSer1177-eNOS immunoblots normalized to CD31; n = 12 tricuspid aortic valve (TAV) specimens or 20 to 21 bicuspid aortic valve (BAV) specimens per right (R), left (L), and noncoronary (N) sinus regions. *p < 0.05 (B) Representative immunoblots. The Annals of Thoracic Surgery 2016 102, 1274-1281DOI: (10.1016/j.athoracsur.2016.04.001) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 pSer239 vasodilator-stimulated phosphoprotein (VASP) as an indicator of nitric oxide bioavailability. Analysis of (A) pSer239-VASP and (B) VASP immunoblots normalized to β-actin; n = 12 tricuspid aortic valve (TAV) specimens or 20 to 21 bicuspid aortic valve (BAV) specimens per right (R), left (L), and noncoronary (N) sinus regions. (C) Representative immunoblots. (p = phosphorylated.) The Annals of Thoracic Surgery 2016 102, 1274-1281DOI: (10.1016/j.athoracsur.2016.04.001) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Proposed disruption of endothelial nitric oxide (NO) synthase (eNOS) pathway in bicuspid aortic valve (BAV) aortopathy. (A) Typically, eNOS produces NO in endothelial cells. NO increases activation of guanylyl cyclase (GC) in smooth muscle cells, leading to phosphorylation of vasodilator-stimulated phosphoprotein (VASP). (B) Under pathologic conditions such as bicuspid aortic valve, eNOS function may become uncoupled from NO synthesis. Production of superoxide anion predominates and NO levels decline. Uncoupled eNOS function can lead to oxidative stress and altered vascular wall remodeling. (BH4 = tetrahydrobiopterin; cGMP = cyclic guanosine monophosphate; GTP = guanosine 5′-triphosphate.) The Annals of Thoracic Surgery 2016 102, 1274-1281DOI: (10.1016/j.athoracsur.2016.04.001) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions