Comparative Histopathological Analysis of Mitral Valves in Barlow Disease and Fibroelastic Deficiency Jesper Hjortnaes, MD, PhD, Josh Keegan, BS, Patrick.

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Comparative Histopathological Analysis of Mitral Valves in Barlow Disease and Fibroelastic Deficiency Jesper Hjortnaes, MD, PhD, Josh Keegan, BS, Patrick Bruneval, MD, Eugenia Schwartz, BS, Frederick J. Schoen, MD, PhD, Alain Carpentier, MD, Robert A. Levine, MD, Albert Hagège, MD, Elena Aikawa, MD, PhD Seminars in Thoracic and Cardiovascular Surgery Volume 28, Issue 4, Pages 757-767 (December 2016) DOI: 10.1053/j.semtcvs.2016.08.015 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Parasternal long-axis 2-dimensional echocardiographic images of DMVD. BD (left): a 45-year-old man demonstrating prolapse of the anterior and posterior leaflets with increased leaflet thickness and markedly enlarged (48mm) mitral annulus (dotted line); note the leaflet elongation and posterior annular calcifications (asterisk). FED (right): a 78-year-old woman with a prolapse limited to the middle segment of the posterior leaflet, with thin leaflets and absence (33mm) of mitral annulus dilatation (dotted line); note the flail posterior leaflet due to chordal rupture (asterisk). AO, aorta; AML, anterior mitral leaflet; LA, left atrium; LV, left ventricle; PML, posterior mitral leaflet; RA, right atrium; RV, right ventricle. (Color version of figure is available online at http://www.semthorcardiovascsurg.com.) Seminars in Thoracic and Cardiovascular Surgery 2016 28, 757-767DOI: (10.1053/j.semtcvs.2016.08.015) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Morphologic features of normal, Barlow and FED mitral valves. A. Movat pentachrome stain of (A) normal, (B) BD, and (C) FED valves (collagen yellow; proteoglycans blue-green; elastin, black). Picosirius red staining of (D) normal, (E) BD, and (F) FED valves. White arrows indicate change of the spongiosa layer. Orange arrows point toward intimal thickening in both BD and FED and tissue overgrowth around. The 3-layered structure, characteristic of nondiseased mitral valves, was observed in both BD and FED, including the zona fibrosa (f), primarily composed of densely packed and uniformly arranged collagen; zona spongiosa (s), which lacks the uniformity of the fibrosa and predominantly contains glycosaminoglycans (GAGs); and atrialis (a), mainly composed of elastic fibers. Bar = 1μm. (G and H) BD and FED exhibit both shared and distinct morphologic features. Both pathologies show elastin fragmentation. BD often shows formation of spherical plaque-like structures composed of elastin (arrows). BD exhibits diminished elastin staining while FED has an excessive deposition of elastin, particularly around chordea. Movat stain. Bar = 50μm. (I) Mitral leaflets were analyzed for leaflet (left panel) and chordae (right panel) thickness. BD valves demonstrated significantly increased overall leaflet thickness and decreased chordae thickness compared to FED valves. *P < 0.05. (Color version of figure is available online at http://www.semthorcardiovascsurg.com.) Seminars in Thoracic and Cardiovascular Surgery 2016 28, 757-767DOI: (10.1053/j.semtcvs.2016.08.015) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Differentfiation and proliferation of VICs in leaflets vs chordae BD and FED mitral valves. Valves were stained for (A and B) α-SMA, (C and D) CD34, (E and F) CD45, (G and H) Ki67, and (I and J) MMP-2. Quantification was performed by counting positively stained cells per high power field (n = 15); P < 0.05. Bar = 50μm. (Color version of figure is available online at http://www.semthorcardiovascsurg.com.) Seminars in Thoracic and Cardiovascular Surgery 2016 28, 757-767DOI: (10.1053/j.semtcvs.2016.08.015) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Signaling markers in BD and FED mitral valves. Valves were stained for (A and B) Fliman A, (C and D) TGF-β, and (E and F) pERK. Quantification was performed by counting positively stained cells per high power field (n = 5) (HPF); P < 0.05. Bar = 50μm. (Color version of figure is available online at http://www.semthorcardiovascsurg.com.) Seminars in Thoracic and Cardiovascular Surgery 2016 28, 757-767DOI: (10.1053/j.semtcvs.2016.08.015) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Schematic depiction of pathologic mechanism of BD and FED. DMVD leading to MR can be considered either BD or FED. (A) BD is characterized by diffuse increased thickness, elastin fragmentation, increased cell proliferation (Ki67), and high proteolytic enzyme activity (MMP-2). This contributes to overall increase of the leaflet thickness and eventual billowing or floppy leaflets. (B) FED is characterized by increased focal leaflet and chordal thickness, hallmarked by focal deposition of elastin and collagen in the chordae, increased expression of myofibroblast-like cells and fibrocytes in the chordae, and increased expression of profibrotic TGF-β and pERK, probably contributing to the typical chordae rupture or flail leaflet of FED. (Color version of figure is available online at http://www.semthorcardiovascsurg.com.) Seminars in Thoracic and Cardiovascular Surgery 2016 28, 757-767DOI: (10.1053/j.semtcvs.2016.08.015) Copyright © 2016 The Authors Terms and Conditions

Morphological features of Barlow and FED mitral valves show distinct pathologies. Video. Seminars in Thoracic and Cardiovascular Surgery 2016 28, 757-767DOI: (10.1053/j.semtcvs.2016.08.015) Copyright © 2016 The Authors Terms and Conditions