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Afshin Ehsan, MD, Adam P. McGraw, PhD, Mark J

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1 Mineralocorticoid receptor antagonism inhibits vein graft remodeling in mice 
Afshin Ehsan, MD, Adam P. McGraw, PhD, Mark J. Aronovitz, MS, Carol Galayda, BS, Michael S. Conte, MD, Richard H. Karas, MD, PhD, Iris Z. Jaffe, MD, PhD  The Journal of Thoracic and Cardiovascular Surgery  Volume 145, Issue 6, Pages e1 (June 2013) DOI: /j.jtcvs Copyright © 2013 The American Association for Thoracic Surgery Terms and Conditions

2 Figure 1 Expression of renin–angiotensin–aldosterone system (RAAS) components in human saphenous vein tissue and smooth muscle cells (HSVSMC). A, Quantitative reverse transcription–polymerase chain reaction was conducted to identify RAAS gene messenger RNA (mRNA) isolated from human saphenous vein (white bars) and HSVSMC (black bars). n = 4. B, Immunoblots on HSVSMC lysates with antibody specific for mineralocorticoid receptor (MR; top) and 11-β-hydroxysteroid dehydrogenase-2 (11βHSD2; bottom) protein. Lysate from HEK293 cells transfected with empty plasmid (−) or with plasmid that expresses MR or 11βHSD2 (+) serve as negative and positive controls, respectively. ND, Not detected; GR, glucocorticoid receptor; ACE, angiotensin-converting enzyme; AT1R, angiotensin type 1 receptor; AS, aldosterone synthase; MR, mineralocorticoid receptor. The Journal of Thoracic and Cardiovascular Surgery  , e1DOI: ( /j.jtcvs ) Copyright © 2013 The American Association for Thoracic Surgery Terms and Conditions

3 Figure 2 Functional mineralocorticoid receptor (MR) in human saphenous vein smooth muscle cells (HSVSMC). Primary HSVSMC were infected with an adenovirus containing a mineralocorticoid-inducible response element–luciferase transcriptional reporter. A, Infected cells were treated with increasing doses of aldosterone (Aldo), and Aldo-induced changes in luciferase activity were measured. B, Cells were treated with 10 nM Aldo and/or the MR antagonist spironolactone (Spiro). Dash indicates reagents not added. *P < .05 versus vehicle. †P < .05 versus 10 nM Aldo. The Journal of Thoracic and Cardiovascular Surgery  , e1DOI: ( /j.jtcvs ) Copyright © 2013 The American Association for Thoracic Surgery Terms and Conditions

4 Figure 3 Mineralocorticoid receptor antagonism reduces vein graft thickening in mice. A, Representative hematoxylin & eosin–stained sections from the native inferior vena cava (IVC) and throughout the grafted IVC from placebo- and spironolactone (Spiro)-treated mice. Images are arranged from proximal (left) to distal (right) orientation. Scale bar = 0.1 mm. B, Quantification of the intima–media area of the section with maximal vessel thickness (black boxes) in A from placebo- (white bar) and Spiro-treated (black bar) mice. *P < .05 versus placebo. The Journal of Thoracic and Cardiovascular Surgery  , e1DOI: ( /j.jtcvs ) Copyright © 2013 The American Association for Thoracic Surgery Terms and Conditions

5 Figure 4 Vein graft smooth muscle cell (SMC) hyperplasia with mineralocorticoid receptor antagonism. Immunohistochemical staining of native and grafted inferior vena cava (IVC) sections using SMC-specific α-actin antibody. Actin-positive area and nuclei count in SMC actin-positive regions are quantified in vein sections from spironolactone-treated animals (black bars) and placebo-treated animals (white bars). Scale bar = 0.1 mm. *P < .05 versus placebo. The Journal of Thoracic and Cardiovascular Surgery  , e1DOI: ( /j.jtcvs ) Copyright © 2013 The American Association for Thoracic Surgery Terms and Conditions

6 Figure 5 Inhibition of the mineralocorticoid receptor reduces vein graft fibrosis and inflammation. A, Quantification of vein graft trichrome-stained collagen area on the thickest sections from placebo- (white bars) and spironolactone (Spiro)-treated (black bars) mice. Scale bar = 0.1 mm. B, Quantification of inflammatory cells in high-power (40×) images of hematoxylin & eosin–stained sections of placebo- and spironolactone-treated animals. Representative mononuclear (MN) cells are indicated by white arrows; polymorphonuclear (PMN) cells are indicated by white asterisks. Scale bar = 0.02 mm. *P < .05 versus placebo. The Journal of Thoracic and Cardiovascular Surgery  , e1DOI: ( /j.jtcvs ) Copyright © 2013 The American Association for Thoracic Surgery Terms and Conditions

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