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Flow disturbances and the development of endocardial fibroelastosis
Journal Pre-proof Flow Disturbances and the Development of Endocardial Fibroelastosis Viktoria Weixler, MD, Gerald R. Marx, MD, Peter E. Hammer, PhD, Sitaram M. Emani, MD, Pedro J. del Nido, MD, Ingeborg Friehs, MD PII:
S0022-5223(19)31901-4
DOI:
https://doi.org/10.1016/j.jtcvs.2019.08.101
Reference:
YMTC 14982
To appear in:
The Journal of Thoracic and Cardiovascular Surgery
Received Date: 10 May 2019 Revised Date:
21 August 2019
Accepted Date: 24 August 2019
Please cite this article as: Weixler V, Marx GR, Hammer PE, Emani SM, del Nido PJ, Friehs I, Flow Disturbances and the Development of Endocardial Fibroelastosis, The Journal of Thoracic and Cardiovascular Surgery (2019), doi: https://doi.org/10.1016/j.jtcvs.2019.08.101. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Copyright © 2019 by The American Association for Thoracic Surgery
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Flow Disturbances and the Development of Endocardial Fibroelastosis 2 3
Viktoria Weixler1, MD, Gerald R. Marx2, MD, Peter E. Hammer1, PhD, Sitaram M. Emani1,
4
MD, Pedro J. del Nido1, MD, Ingeborg Friehs1, MD
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1
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Longwood Ave, Boston, MA 02115, USA
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2
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Longwood Ave, Boston, MA 02115, USA
Department of Cardiac Surgery, Boston Children’s Hospital, Harvard Medical School, 300
Department of Cardiology, Boston Children’s Hospital, Harvard Medical School, 300
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Running Title: HLHS/EFE and EndMT
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Word Count (body of text): 2810
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Key Words: endocardial fibroelastosis, hypoplastic left heart syndrome, endothelial-to-
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mesenchymal transition
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Send proofs and correspondence to:
Ingeborg Friehs, M.D.
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Department of Cardiac Surgery
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Boston Children’s Hospital
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Harvard Medical School
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300 Longwood Ave.
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Boston, MA 02115
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Telephone:
(617) 919-2311
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Fax:
(617) 730-0235
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E-mail: [email protected]
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24
3
Glossary of Abbreviations
25 26
EFE = endocardial fibroelastosis
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HLHS = hypoplastic left heart syndrome
28
AV = aortic valve
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MV = mitral valve
30
H&E = Hematoxylin&Eosin
31
MT = Masson’s Trichrome
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VG = Van Giessen Elastin
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LA=left atrium
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LV= left ventricle
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LVOT= left ventricle outflow tract
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EndMT= endothelial-to-mesenchymal transition
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MRI=magnetic resonance imaging
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OCT compound=optimal cutting temperature compound
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RV=right ventricle
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ICR=interquartile range
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MMP-2, -9, -12=matrix metalloproteinase-2, -9, -12
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Abstract
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Objective(s): Endothelial-to-mesenchymal transition (EndMT) has been identified as the
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underlying mechanism of endocardial fibroelastosis (EFE) formation. The purpose of this study
45
was to determine whether hemodynamic alterations due to valvar defects promote EndMT, and
46
whether age-specific structural changes affect ventricular diastolic compliance despite extensive
47
surgical resection of EFE tissue.
48
Material and Methods: We analyzed EFE tissue from 24 HLHS patients who underwent LV
49
rehabilitation surgery at Boston Children’s Hospital between 12/2011 to 03/2018. Six patients
50
with flow disturbances across aortic (AV) and/or mitral valve (MV) but no HLHS diagnosis, and
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macroscopic appearance of “EFE-like tissue” in the LV were included for comparison. All
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samples were examined for amount of collagen/elastin production and degradation, and presence
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of active EndMT by histological analysis.
54
Results: EFE tissue from HLHS patients and non-HLHS patients consisted predominantly of
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elastin and collagen fibers. There was no alteration in degradation activity for collagen or elastin
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as shown by in situ zymography. Active EndMT was found in all HLHS patients and all non-
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HLHS patients with flow disturbances (“EFE-like”). In HLHS patients EFE infiltrates into the
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underlying myocardium with increasing age.
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Conclusion: HLHS patients and non-HLHS patients with flow disturbances due to stenotic or
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incompetent valves develop EndMT-derived fibrotic tissue covering the LV. When EFE recurs,
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it is directly associated with flow disturbances and switches to an infiltrative growth pattern with
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increasing age leading to increased diastolic stiffness of the LV.
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Word count: 232
5
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Ultramini Abstract
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Flow disturbances exposing endocardial endothelial cells to altered shear forces are the main
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trigger for induction of EndMT leading to reoccurrence of EFE displaying a more infiltrative
67
growth pattern into the underlying myocardium. These alterations are present in congenital
68
defects with severe valvar defects with jets onto the endocardial surface.
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Word count: 50
6
70
Central Message
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EFE shows infiltrative growth with increasing age, triggered by flow disturbances promoting
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EndMT as underlying mechanism which is independent of HLHS.
73 74
Perspective Statement
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Flow disturbances exposing endocardial endothelial cells to altered shear forces are the main
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trigger for induction of EndMT leading to reoccurrence of EFE displaying a more infiltrative
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growth pattern into the underlying myocardium. These alterations are not limited to HLHS but
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are related to severe valvar defects with jets onto the endocardial surface.
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80
Central Picture
81 82
Legend (Central Picture): Endocardial fibroelastosis (blue) shows infiltrative growth pattern
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into the underlying myocardium (red).
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84
Introduction
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Endocardial fibroelastosis (EFE) was first described by Weinberg and Himmelfarb in
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1943 as thick subendocardial layer of connective tissue and elastin, encapsulating the underlying
87
myocardium of left atrium (LA) and ventricle (LV) (1). Since then, the presence of EFE has been
88
reported in several other cardiac diseases such as cardiomyopathies (2), infectious diseases (3),
89
immunologic diseases (4) and congenital heart diseases such as hypoplastic left heart syndrome
90
(HLHS) (5,6). Macroscopic and microscopic appearance led to this uniform diagnosis but the
91
underlying root cause of EFE development has not been examined until recently.
92
With surgical resection of EFE during routine rehabilitation surgery aimed at preserving
93
the growth potential of the diminutive LV, EFE tissue became available at different time points
94
of occurrence for further analysis. Clinical observation indicates that in younger patients a
95
distinct layer separates EFE tissue from the myocardium which led to our assumption that EFE is
96
of endocardial rather than myocardial origin (7). In pursuit of this hypothesis, we examined EFE
97
tissue and determined that the root cause of this tissue is in fact the endocardial endothelial cells.
98
Following a yet unknown stimulus, endocardial endothelial cells undergo a process called
99
endothelial-to-mesenchymal transition (EndMT), where endothelial cells of the endocardial layer
100
undergo transformation to fibroblasts (6). This process is well established during fetal cardiac
101
development for the formation of the endocardial cushions and valves (8) and it has also been
102
described under pathological conditions such as myocardial fibrosis (9). Stimuli for EndMT
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include hypoxia/ischemia (10), inflammation (11) and mechanical alterations (12,13), whereof
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the latter plays an important role in congenital cardiac defects such as HLHS. Our focus is on
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EFE associated with HLHS which encompasses a spectrum of complex congenital heart lesions
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with various degrees of underdevelopment of the LV and aortic/mitral valve stenosis/atresia (5).
8
107
Our patient cohort included only patients with patent but stenotic mitral valves potentially
108
suitable for left ventricular recruitment surgery.
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In up to 70% of all HLHS patients, EFE restricts the left ventricular outflow tract
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(LVOT) (14) and surgical removal of EFE tissue has been found to result in catch-up growth of
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left ventricular structures (7). At times, several EFE resections during consecutive surgical
112
interventions in the same patients are necessary to potentially alleviate the restrictive forces of
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this stiff tissue. Despite the increase in LV volume indicative of ventricular growth, the
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restrictive component on the LV is not resolved as the patients grow older since they continue to
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present with persistently elevated end-diastolic pressures (15).
116
We recently presented a unique case of EFE reoccurrence in a HLHS patient with severe
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flow disturbances across the mitral valve which was resolved by repetitive EFE resections and
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replacement of the mitral valve to normalize blood flow in the LV (16). For the first time, this
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report associates mechanical forces such as flow disturbances with EFE development and
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identifies EndMT as underlying mechanism.
121
whether hemodynamic alterations due to valvar stenosis and/or insufficiency promote EFE
122
progression independent of the underlying diagnosis of HLHS. Furthermore, we examined age-
123
specific structural changes of EFE tissue potentially affecting diastolic compliance of the
124
ventricle despite extensive resection of EFE tissue in HLHS patients.
The purpose of this study was to determine
9
Materials and Methods
125 126
Ethics Statement:
127
This research project was approved by and conducted in accordance with the standards of
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the institutional review board of Boston Children’s Hospital. Initially only de-identified
129
discarded human tissue was analyzed but following consent prospectively at the time of
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HLHS/EFE diagnosis, patients’ operative details, catheterization details, echocardiographic data,
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magnetic resonance imaging (MRI) data, and clinical information were obtained and correlated
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with histological findings.
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Patients:
134
We analyzed EFE tissue from a total of 24 patients with the diagnosis of HLHS who
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underwent LV rehabilitation surgery with EFE resection as previously described in more detail at
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Boston Children’s Hospital between December 2011 to March 2018 (6). Out of these 24 HLHS
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patients, we received tissue from 17 patients, sent as de-identified discarded tissue, resected
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during open heart surgeries, and tissue from additional seven patients, in which we obtained
139
consent prior to surgery. In the latter group, we followed the patients collecting clinical
140
background and imaging data, and obtained EFE tissue from previous resections if available.
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These patients presented with mitral stenosis and/or aortic stenosis rather than atresia at the time
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of EFE resection as detailed in Table 1.
143
Six patients were included for comparison with diagnoses other than HLHS but with
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known flow disturbances across aortic (AV) and/or mitral valve (MV). In all of these patients,
145
preoperative standard imaging studies (echocardiography and MRI) did not describe EFE.
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Macroscopic appearance of “EFE-like tissue” in the LV outflow tract diagnosed by the cardiac
147
surgeon performing the surgery led to resection of endocardial tissue in the operating room.
10
148
For comparison, we received two de-identified discarded tissue samples from the right
149
ventricle (RV) from patients without flow disturbances, which was resected in both cases
150
because a right ventriculotomy was performed during the surgical procedure. Furthermore, a
151
healthy rat LV was used as negative control.
152
All tissue samples, once received from the operating room were immediately embedded
153
in optimal cutting temperature (OCT) compound, snap frozen and stored at -80°C for future
154
analysis. Frozen sections were produced from OCT blocks and further processed for microscopic
155
analysis as described in more detail below.
156
Composition of EFE Tissue:
157
Age-related changes of EFE tissue, were examined for the amount of collagen and
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elastin, vascularity, and cellularity by staining with Hematoxylin&Eosin (H&E), Masson’s
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Trichrome (MT) and Van Giessen Elastin (VG). All slides were visualized using a Zeiss
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Observer.Z1 fluorescent microscope with a Nikon 20x objective (NA=20x/0.45). Ten randomly
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selected fields from each slide were taken and quantification was performed with ImageJ
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(version 2.0.0-rc-43, obtained from the National Institute of Health, Bethesda, MD). Details
163
about the method of quantifying collagen, elastin and muscle on histological sections have been
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described previously by our group in more detail (17).
165
Analysis of tissue remodeling through degradation of elastin fibers and collagen by
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matrix metalloproteinases was determined by in situ zymography. Unstained frozen slides were
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either incubated with 40ug/ml DQ elastin (EnzChek Elastase Assay Kit) in Dulbecco’s Modified
168
Eagle Medium (DMEM, Gibco, Gaithersburg, MD) and 10% Fetal Bovine Serum (FBS, Gibco,
169
Gaithersburg, MD) for 1 hour at 37°C or in a reaction buffer (0.05M TRIS-HCl, 0.15M NaCl,
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5mM CaCl2 and 0.2mM NaN3 at pH 7.6) containing either 40ug/ml DQ gelatin or collagen type
11
171
I or IV (EnzChek Gelatinase/Collagenase Assay Kit) at 37°C overnight. Slides, incubated with
172
DQ elastin were counterstained tropoelastin (1:50, Abcam, Cambridge, MA) and DAPI (1:1000,
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Dako, Carpinteria, CA), and slides incubated with DQ gelatin were counterstained with collagen
174
I (1:50, Novus Biologicals, Littelton, CO) and DAPI (1:1000, Dako, Carpinteria, CA). All
175
chemicals were obtained from Thermofisher (Thermofisher, Waltham, MA).
176
Diagnosis of EndMT:
177
In support of our hypothesis that EndMT is the underlying mechanism for EFE
178
development, the presence of active EndMT was determined in all tissue samples obtained from
179
the operating room. Immunohistochemical double-staining of endothelial cells with an
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endothelial marker, cluster of differentiation 31 (CD31; 1:100, Dako, Carpinteria, CA), and a
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mesenchymal marker, α-smooth muscle actin (α-SMA; 1:100, Abcam, Cambridge, MA) at the
182
same time, is indicative of active EndMT. Additionally, all samples were stained for the
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transcription factors Twist (1:100, Abcam, Cambridge, MA), which regulate EndMT indicated
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by Twist-positive nuclei. Nuclei were stained for 4',6-Diamidino-2-Phenylindole (DAPI;
185
(1:1000, Dako, Carpinteria, CA). Furthermore, active EndMT was confirmed by staining for the
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transcription factors Slug/Snail (1:100, Abcam Cambridge, MA). Co-localization with nuclei in
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endothelial cells is indicative of active EndMT. As all tissue samples contained some muscle
188
tissue, the myocardium was also analyzed for tissue structure and fibrosis on HE and MT as well
189
as immunochistochemical staining for desmin to assess cardiomyocytes and EndMT (1:50,
190
Abcam, Cambridge, MA).
191
Statistical Analysis:
192
After confirmation of normal distribution of data, ANOVA for multiple group
193
comparisons with Bonferroni post-hoc analysis was performed for calculation of statistically
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194
significant (STATA, StataCorp. 2015. Stata Statistical Software: Release 14. College Station,
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TX: StataCorp LP). Probability values of ≤ 0.05 were regarded statistically significant. Data are
196
expressed as mean ± SEM.
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Results
197 198
Patients:
199
The median age of the HLHS patients at the time of most recent EFE resection was 27
200
months (IQR=0 – 73.5 months) compared to a median age of 36 months (IQR=0.8 – 73.3
201
months) in the non-HLHS patients (P=0.8).
202
In the consented HLHS patients, apart from the main diagnosis of HLHS, aortic
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regurgitation (AR) especially following post-natal balloon valvuloplasty was described in four
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patients; mitral stenosis (MS) due to a dysplastic mitral annulus was found in five of seven
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patients. These valvular diseases caused turbulent flow within the LV in all seven consented
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HLHS patients, which has been described throughout their entire medical history. All except for
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two HLHS patients had previous EFE resections. At the time of the most recent resection, all
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valvar diseases were successfully repaired and no remaining turbulences were described
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postoperatively and at follow-up neither in echocardiography nor in MRI studies. The median
210
follow-up time after the last EFE resection in the consented HLHS patient was 6.8 months
211
(IQR=4.15 - 9.45 months). (Table 1). In the “non-HLHS” patients with valvar disease,
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predominantly a jet across a stenotic MV was present. In two cases a regurgitant AV caused a jet
213
toward the LVOT. (Table 2)
214
Composition of EFE Tissue:
215
The number of nuclei (mean±SEM expressed as nuclei per field of vision) within EFE
216
tissue decreased with increasing age – infants (<1 year of age) 282±63 versus toddlers and
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preschoolers (1-5 years of age) 158±24 versus school-aged children (>5 years of age with the
218
oldest child at 12 years of age) 94±12 - but did not reach significance. All tissue remained
219
avascular in all tissue samples. EFE tissue from HLHS patients consisted predominantly of
14
220
elastin and collagen fibers with some underlying myocardium. The ratio of muscle to elastin and
221
collagen did not show significant age-related differences within the three groups (Figure 1).
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“EFE-like” tissue obtained from non-HLHS patients, showed the same characteristics as EFE
223
tissue from HLHS patients. Collagen and elastin predominated the tissue sections, the number of
224
nuclei decreased with age, and there were no blood vessels found within the tissue.
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In situ zymography determined that impaired elastase/gelatinase activity was not the
226
cause of increased collagen and elastin deposition and no age-related alteration could be
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identified (Figure 2). The same characteristics were found in the non-HLHS patients with flow
228
disturbances.
229
Presence of Active EndMT:
230
Endocardial double-staining with CD31/alpha-SMA as well as Twist and Slug/Snail-
231
positive stained nuclei, respectively, both indicative of active EndMT, were found not only in all
232
of the 24 samples from HLHS patients but also in all six non-HLHS patients. EndMT-positive
233
tissue was not found, however, in right ventricular tissue samples nor in a healthy rat heart
234
(Figure 3).
235
In all patients with EFE and “EFE-like” tissue, some underlying myocardium was
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resected (Figure 4). MT stains showed infiltrative growth of collagen bundles from the
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endocardium into the underlying myocardium of HLHS patients and non-HLHS patients with
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flow disturbances. The degree of infiltrative growth increases with increasing age in HLHS
239
patients. In samples of patients <1 year of age, the fibrotic tissue is separated from the underlying
240
muscle, whereas samples from older children (> 2years of age), no separation between collagen
241
fibers and myocardium is feasible. The endocardial thickness of fibrotic tissue decreases,
242
whereas infiltrative growth of collagen into the underlying myocardium increases (Figure 5). In
15
243
contrast, in a healthy rat heart and RV tissue samples, a thin endocardial layer is present which is
244
separated from the underlying myocardium. Localization of endothelial cells within the collagen-
245
rich tissue which infiltrates the myocardium identified multiple double-stained cell for CD31 and
246
alpha-SMA. The double-staining is indicative that intramyocardial fibrotic tissue is also derived
247
through EndMT (Figure 6).
248
Figure 7 summaries the analytic process from an intraoperative diagnosis of
249
macroscopically appearing EFE tissue, to the unraveling of the mechanistic cause of EFE
250
formation, and the connection to clinical presentation of EFE tissue formation localized to areas
251
of flow disturbances due to valvar defects.
16
Discussion
252 253
In this study, we established that structural changes of EFE tissue toward a more
254
infiltrative growth into the underlying myocardium and less cellularity occurs with increasing
255
age. Furthermore, we describe for the first time that hemodynamic alterations in the way of jets
256
on the level of the mitral valve or regurgitant flow across an incompetent aortic valve promote
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localized aggravated EFE growth and reoccurrence despite excessive surgical resection. “EFE-
258
like” tissue growth independent of an underlying HLHS diagnosis, shows the same pattern.
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Following detailed analysis of this “EFE-like” tissue, we determined that the underlying
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mechanism for development is EndMT which we had already reported in detail in patients with
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EFE associated with HLHS (6). Our data further indicate that HLHS alone does not predispose
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this patient cohort to EFE formation but that the underlying communality is the presence of
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valvar defects represented as either mitral stenosis or aortic insufficiency. Both valvar defects
264
result in flow disturbances, displayed as jets across the valve onto localized areas of the LV
265
cavity which responds by endocardial thickening through subendocardial development of EFE
266
tissue. In HLHS patients, despite several surgical resections of this tissue, progression still occurs
267
unless the valvar defect is repaired.
268
Another novel finding of this study is that unlike previously suggested, EFE develops and
269
progresses as a distinct layer of fibrotic tissue which can be easily dissected from the underlying
270
myocardium (7). Following thorough examination of our resected tissue samples from HLHS
271
and non-HLHS patients, all EFE tissue samples contained some degree of muscle tissue.
272
Macroscopically this finding suggests infiltrative growth of EFE tissue into the underlying
273
myocardium which we confirm microscopically through MT staining. With increasing age,
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subendocardial tissue growth takes on a more infiltrative pattern with collagen and elastin
17
275
bundles protruding into the adjacent myocardium. Thus, our data show that the main trigger for
276
the development and progression of EFE is flow disturbances, which might also occur in other
277
congenital heart diseases leading to macroscopically “EFE-like” tissue unrelated to the primary
278
diagnosis of HLHS.
279
In our EFE tissue samples we could show that localized response of the endocardium to
280
flow disturbances (e.g. shear stress) created EFE. It is well known that endothelial cells lining the
281
vasculature respond to alterations in shear stress by changing their shape and function (18) since
282
they are exposed to a large variety of biochemical and hemodynamic stimuli. Thus, endothelial
283
cells are required to be highly plastic which allows for adaptation to alterations in fluid shear
284
forces. Plasticity is essential to maintain homeostasis but pathological stimuli might alter this
285
process and contribute to disease through adverse plasticity which EndMT is an example of (19).
286
Areas exposed to disturbed blood flow and thus, alterations in shear stress generate a
287
microenvironment conducive for EndMT. As a consequence, activation of EndMT results in
288
intimal hyperplasia with extracellular matrix remodeling (19,20). EFE has the appearance of
289
organized layers resembling degenerative intimal hyperplasia of arterial vessel walls. The details
290
on hemodynamic forces and their role in maintaining vascular integrity have been well studied in
291
the vasculature but little is known whether laminar shear stress is protective also for the
292
endocardial endothelial cells. However, it supports the idea that flow disturbances may play a
293
role in endocardial remodeling through EndMT driven by activation of transcription factors
294
which are actively involved in suppression of VE-cadherins and other endothelial specific
295
proteins. Furthermore, in an animal model of EFE formation which we had previously described
296
in detail, an association with alterations in mechanical forces including flow disturbances across
297
an incompetent aortic valve is associated with formation of EFE (12).
18
298
Functional indicators of EndMT in tissue are increased expression of collagens and
299
elastin, as well as production of enzymes degrading the extracellular matrix, such as matrix
300
metalloproteinase-2 (MMP)-2, MMP-9 or MMP-12 (elastase). The expression of mesenchymal
301
markers corresponds to a newly acquired ability to produce and remodel the extracellular matrix
302
(21). With regard to remodeling, activation of MMPs are important following the interruption of
303
cell-cell or cell-matrix contact. In our patient cohort, no defect in the degradative potential of the
304
extracellular matrix of the endocardium or myocardium was found since all MMPs (MMP12 and
305
MMP2/9) were active in all tissue samples and distribution was focused in areas of active
306
remodeling.
307
In summary, flow disturbances exposing endocardial endothelial cells to altered shear
308
forces are the main trigger for induction of EndMT leading to reoccurrence of EFE displaying a
309
more infiltrative growth pattern into the underlying myocardium. Supported by our data, we can
310
also conclude that these alterations are not limited to congenital cardiac pathologies associated
311
with HLHS but are related to severe valvar defects with jets onto the endocardial surface
312
appearing to respond by EFE thickening. Targeting EFE therapeutically with surgical resection
313
likely is not sufficient to address reoccurrence and prevent infiltrative growth. A more
314
comprehensive approach by addressing the underlying mechanism of EndMT through localized
315
or systemic therapy is warranted but requires more investigation.
19
316
Acknowledgment
317
We would like to thank Breanna Piekarski, BSN, MPH for contribution regarding IRB and
318
patient related organization, and collecting tissue samples.
20
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Circulation After Fetal Aortic Stenosis Intervention. Am J Cardiol. 2018;122(3):511–6.
363 364
16.
Weixler V, Hammer PE, Marx GR, Emani SM, del Nido PJ, Friehs I. Flow disturbances and progression of endocardial fibroelastosis – a case report. Cardiovasc Pathol.
22
2019;42:1-3.
365 366
17.
Illigens BM, Casar Berazaluce A, Poutias D, Gasser R, del Nido PJ, Friehs I. Vascular
367
endothelial growth factor prevents endothelial-to-mesenchymal transition in hypertrophy.
368
Ann Thorac Surg. 2017;104(3):932-9.
369
18.
stress on endothelial cell adherens junctions. Circ Res. 1999;85(6):504–14.
370 371
Noria S, Cowan DB, Gotlieb AI, Langille BL. Transient and steady-state effects of shear
19.
Moonen JRAJ, Lee ES, Schmidt M, Maleszewska M, Koerts JA, Brouwer LA, et al.
372
Endothelial-to-mesenchymal transition contributes to fibro-proliferative vascular disease
373
and is modulated by fluid shear stress. Cardiovasc Res. 2015;108(3):377–86.
374
20.
endothelium in vascular calcification. Circ Res. 2013;113(5):495–504.
375 376 377
Yao Y, Jumabay M, Ly A, Radparvar M, Cubberly MR, Bostrom KI. A role for the
21.
Woessner JFJ. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J Off Publ Fed Am Soc Exp Biol. 1991;5(8):2145–54.
23
Figure Legends
378 379
Table 1: Detailed information of consented patients with hypoplastic left heart syndrome
380
Data presented include age of patients, surgeries in chronological order, number of EFE
381
resections and localization of EFE tissue that was obtained for histological analysis.
382 383
Table 2: Detailed information of patients with diagnosis other than hypoplastic left heart
384
syndrome
385
In these patients, indication for surgical intervention were either mitral valve (MV) stenosis or
386
aortic valve (AV) regurgitation causing flow disturbances in the left ventricle. However, imaging
387
studies did not describe any endocardial abnormalities in the sense of endocardial fibroelastosis
388
(EFE) prior to surgery. EFE resection was performed following the cardiac surgeon’s ad hoc
389
decision intraoperatively. Age of the patients, diagnosis and localization of EFE resection is
390
presented.
391 392
Figure 1:
393
(A) Macroscopic appearance of resected endocardial fibroelastosis (EFE) tissue from the left
394
ventricle of patients with hypoplastic left heart syndrome (HLHS) is shown; left: tissue sample
395
facing the myocardium (red=muscle), right: sample side facing toward the ventricular lumen.
396
(B) A representative picture of an Elastin van Giesson stain shows the distribution of collagen
397
(blue) and elastin (black) and muscle (white-yellow) in EFE tissue samples.
398
(C) A summary of muscle-collagen-elastin distributions are shown where tissue samples were
399
grouped according to age (<1year, 1-5years, >5years). There is no significant difference in the
400
distribution within each specific age groups.
24
401 402
Figure 2:
403
(A) Representative in situ zymography of gelatinase activity and collagen in endocardial
404
fibroelastosis (EFE) tissue obtained from patients diagnosed with hypoplastic left heart
405
syndrome is shown. Gelatinase is shown in green, collagen in red and nuclei in blue.
406
(B) Representative in situ zymography of elastase activity and tropoelastin in in endocardial
407
fibroelastosis (EFE) tissue obtained from patients diagnosed with hypoplastic left heart
408
syndrome. Elastase is shown in green, tropoelastin in red and nuclei in blue.
409
(C and D) Summary of matrix metalloproteinase (MMP) activity (elastase on the left and
410
gelatinase on the right) per field of vision for different age groups (<1year, 1-5years, >5years) is
411
shown. There was no significant age-related difference in the balance of MMP activity to
412
collagen/elastin production.
413 414
Figure 3:
415
Representative sections of endocardial fibroelastosis (EFE) and “EFE-like” tissues are displayed.
416
Double-staining of endothelial cells with an endothelial cell marker (cluster of differentiation 31
417
- CD-31) in red and an fibroblast marker (α-smooth muscle actin – SMA) in green, or endothelial
418
cells with CD-31 (red) and nuclei-positive staining for transcription factors Twist and Slug/Snail
419
(green) is indicative of active endothelial-to-mesenchymal transition (EndMT). Nuclei are
420
stained in blue.
421
(A) EFE tissue from HLHS patients is positive for EndMT (CD-31/αSMA double-staining, white
422
arrows); (B) “EFE-like” tissue from non-HLHS patients is positive for EndMT (CD-31/αSMA
423
double-staining, white arrows); (C) EFE tissue and “EFE-like” tissue stained for the transcription
25
424
factors Twist (white arrows), (D) and Slug/Snail (white arrows), respectively, which are
425
indicative of active EndMT when co-localized with endothelial cell nuclei. (E) Normal rat heart
426
tissue and (F) human right ventricular tissue are negative for EndMT.
427 428
Figure 4:
429
As indicated by these representative left ventricular tissue samples stained with Masson’s
430
Trichrome, infiltrative growth patterns of collagen fibers (blue) into the underlying myocardium
431
(red) is associated with increasing age, (A) infants (<1year) (B) toddler and preschooler (1-
432
5years) (C) school-aged children (5-12years). The summary in (D) shows significantly higher
433
infiltrative growth in the oldest age group (mean±SEM, *p<0.05 versus school-aged children).
434 435
Figure 5:
436
Distribution of collagen and muscle tissue on Masson’s Trichrome stains is shown for a (A)
437
normal rat heart, (B) normal human tissue, (C) left ventricular (LV) tissue from a hypoplastic left
438
heart syndrome (HLHS)-patient, and (D) LV from non-HLHS patient. Muscle is stained in red
439
and collagen in blue where of the latter is not limited to the endocardial surface but grows
440
infiltratively into the underlying myocardium in (C) and (D).
441 442
Figure 6:
443
Endocardial fibroelastosis (EFE) tissue that infiltrates the myocardium stains positive for
444
endothelial-to-mesenchymal transition (EndMT). A representative section of EFE tissue was
445
stained with Masson’s Trichrome (blue: collagen fibers, red: myocardium) and correspondingly
446
on the left, the same areas were stained to identify active EndMT (green: fibroblast marker, α-
26
447
SMA; red: endothelial cell marker, CD31; blue: nuclei marker). Double-staining is indicative for
448
EndMT (white arrows) which is more pronounced in the transition zone between endocardial
449
thickening due to EFE tissue and the underlying myocardium.
450 451
Figure 7:
452
The analytic process from intraoperative diagnosis to mechanistic cause connected with clinical
453
presentation is described here. Macroscopically appearing endocardial fibroelastosis (EFE) tissue
454
is identified intraoperatively and resected for histological analysis. Immunohistochemical
455
staining establishes endothelial-to-mesenchymal transition (EndMT) as the underlying
456
mechanism for endocardial fibroelastosis (EFE) tissue formation. EFE formation is directly
457
localized to areas of flow disturbances due to valvar defects in the left ventricle.
458 459
Video:
460
This video shows a detailed view of the surgical procedure excising endocardial fibroelastosis
461
from the left ventricular cavity and mitral valve surface.
S0022-5223(19)31901-4
DOI:
https://doi.org/10.1016/j.jtcvs.2019.08.101
Reference:
YMTC 14982
To appear in:
The Journal of Thoracic and Cardiovascular Surgery
Received Date: 10 May 2019 Revised Date:
21 August 2019
Accepted Date: 24 August 2019
Please cite this article as: Weixler V, Marx GR, Hammer PE, Emani SM, del Nido PJ, Friehs I, Flow Disturbances and the Development of Endocardial Fibroelastosis, The Journal of Thoracic and Cardiovascular Surgery (2019), doi: https://doi.org/10.1016/j.jtcvs.2019.08.101. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Copyright © 2019 by The American Association for Thoracic Surgery
1
Flow Disturbances and the Development of Endocardial Fibroelastosis 2 3
Viktoria Weixler1, MD, Gerald R. Marx2, MD, Peter E. Hammer1, PhD, Sitaram M. Emani1,
4
MD, Pedro J. del Nido1, MD, Ingeborg Friehs1, MD
5 6
1
7
Longwood Ave, Boston, MA 02115, USA
8
2
9
Longwood Ave, Boston, MA 02115, USA
Department of Cardiac Surgery, Boston Children’s Hospital, Harvard Medical School, 300
Department of Cardiology, Boston Children’s Hospital, Harvard Medical School, 300
10 11
Running Title: HLHS/EFE and EndMT
12
Word Count (body of text): 2810
13
Key Words: endocardial fibroelastosis, hypoplastic left heart syndrome, endothelial-to-
14
mesenchymal transition
15
Send proofs and correspondence to:
Ingeborg Friehs, M.D.
16
Department of Cardiac Surgery
17
Boston Children’s Hospital
18
Harvard Medical School
19
300 Longwood Ave.
20
Boston, MA 02115
21
Telephone:
(617) 919-2311
22
Fax:
(617) 730-0235
23
E-mail: [email protected]
2
24
3
Glossary of Abbreviations
25 26
EFE = endocardial fibroelastosis
27
HLHS = hypoplastic left heart syndrome
28
AV = aortic valve
29
MV = mitral valve
30
H&E = Hematoxylin&Eosin
31
MT = Masson’s Trichrome
32
VG = Van Giessen Elastin
33
LA=left atrium
34
LV= left ventricle
35
LVOT= left ventricle outflow tract
36
EndMT= endothelial-to-mesenchymal transition
37
MRI=magnetic resonance imaging
38
OCT compound=optimal cutting temperature compound
39
RV=right ventricle
40
ICR=interquartile range
41
MMP-2, -9, -12=matrix metalloproteinase-2, -9, -12
4
42
Abstract
43
Objective(s): Endothelial-to-mesenchymal transition (EndMT) has been identified as the
44
underlying mechanism of endocardial fibroelastosis (EFE) formation. The purpose of this study
45
was to determine whether hemodynamic alterations due to valvar defects promote EndMT, and
46
whether age-specific structural changes affect ventricular diastolic compliance despite extensive
47
surgical resection of EFE tissue.
48
Material and Methods: We analyzed EFE tissue from 24 HLHS patients who underwent LV
49
rehabilitation surgery at Boston Children’s Hospital between 12/2011 to 03/2018. Six patients
50
with flow disturbances across aortic (AV) and/or mitral valve (MV) but no HLHS diagnosis, and
51
macroscopic appearance of “EFE-like tissue” in the LV were included for comparison. All
52
samples were examined for amount of collagen/elastin production and degradation, and presence
53
of active EndMT by histological analysis.
54
Results: EFE tissue from HLHS patients and non-HLHS patients consisted predominantly of
55
elastin and collagen fibers. There was no alteration in degradation activity for collagen or elastin
56
as shown by in situ zymography. Active EndMT was found in all HLHS patients and all non-
57
HLHS patients with flow disturbances (“EFE-like”). In HLHS patients EFE infiltrates into the
58
underlying myocardium with increasing age.
59
Conclusion: HLHS patients and non-HLHS patients with flow disturbances due to stenotic or
60
incompetent valves develop EndMT-derived fibrotic tissue covering the LV. When EFE recurs,
61
it is directly associated with flow disturbances and switches to an infiltrative growth pattern with
62
increasing age leading to increased diastolic stiffness of the LV.
63
Word count: 232
5
64
Ultramini Abstract
65
Flow disturbances exposing endocardial endothelial cells to altered shear forces are the main
66
trigger for induction of EndMT leading to reoccurrence of EFE displaying a more infiltrative
67
growth pattern into the underlying myocardium. These alterations are present in congenital
68
defects with severe valvar defects with jets onto the endocardial surface.
69
Word count: 50
6
70
Central Message
71
EFE shows infiltrative growth with increasing age, triggered by flow disturbances promoting
72
EndMT as underlying mechanism which is independent of HLHS.
73 74
Perspective Statement
75
Flow disturbances exposing endocardial endothelial cells to altered shear forces are the main
76
trigger for induction of EndMT leading to reoccurrence of EFE displaying a more infiltrative
77
growth pattern into the underlying myocardium. These alterations are not limited to HLHS but
78
are related to severe valvar defects with jets onto the endocardial surface.
79
80
Central Picture
81 82
Legend (Central Picture): Endocardial fibroelastosis (blue) shows infiltrative growth pattern
83
into the underlying myocardium (red).
7
84
Introduction
85
Endocardial fibroelastosis (EFE) was first described by Weinberg and Himmelfarb in
86
1943 as thick subendocardial layer of connective tissue and elastin, encapsulating the underlying
87
myocardium of left atrium (LA) and ventricle (LV) (1). Since then, the presence of EFE has been
88
reported in several other cardiac diseases such as cardiomyopathies (2), infectious diseases (3),
89
immunologic diseases (4) and congenital heart diseases such as hypoplastic left heart syndrome
90
(HLHS) (5,6). Macroscopic and microscopic appearance led to this uniform diagnosis but the
91
underlying root cause of EFE development has not been examined until recently.
92
With surgical resection of EFE during routine rehabilitation surgery aimed at preserving
93
the growth potential of the diminutive LV, EFE tissue became available at different time points
94
of occurrence for further analysis. Clinical observation indicates that in younger patients a
95
distinct layer separates EFE tissue from the myocardium which led to our assumption that EFE is
96
of endocardial rather than myocardial origin (7). In pursuit of this hypothesis, we examined EFE
97
tissue and determined that the root cause of this tissue is in fact the endocardial endothelial cells.
98
Following a yet unknown stimulus, endocardial endothelial cells undergo a process called
99
endothelial-to-mesenchymal transition (EndMT), where endothelial cells of the endocardial layer
100
undergo transformation to fibroblasts (6). This process is well established during fetal cardiac
101
development for the formation of the endocardial cushions and valves (8) and it has also been
102
described under pathological conditions such as myocardial fibrosis (9). Stimuli for EndMT
103
include hypoxia/ischemia (10), inflammation (11) and mechanical alterations (12,13), whereof
104
the latter plays an important role in congenital cardiac defects such as HLHS. Our focus is on
105
EFE associated with HLHS which encompasses a spectrum of complex congenital heart lesions
106
with various degrees of underdevelopment of the LV and aortic/mitral valve stenosis/atresia (5).
8
107
Our patient cohort included only patients with patent but stenotic mitral valves potentially
108
suitable for left ventricular recruitment surgery.
109
In up to 70% of all HLHS patients, EFE restricts the left ventricular outflow tract
110
(LVOT) (14) and surgical removal of EFE tissue has been found to result in catch-up growth of
111
left ventricular structures (7). At times, several EFE resections during consecutive surgical
112
interventions in the same patients are necessary to potentially alleviate the restrictive forces of
113
this stiff tissue. Despite the increase in LV volume indicative of ventricular growth, the
114
restrictive component on the LV is not resolved as the patients grow older since they continue to
115
present with persistently elevated end-diastolic pressures (15).
116
We recently presented a unique case of EFE reoccurrence in a HLHS patient with severe
117
flow disturbances across the mitral valve which was resolved by repetitive EFE resections and
118
replacement of the mitral valve to normalize blood flow in the LV (16). For the first time, this
119
report associates mechanical forces such as flow disturbances with EFE development and
120
identifies EndMT as underlying mechanism.
121
whether hemodynamic alterations due to valvar stenosis and/or insufficiency promote EFE
122
progression independent of the underlying diagnosis of HLHS. Furthermore, we examined age-
123
specific structural changes of EFE tissue potentially affecting diastolic compliance of the
124
ventricle despite extensive resection of EFE tissue in HLHS patients.
The purpose of this study was to determine
9
Materials and Methods
125 126
Ethics Statement:
127
This research project was approved by and conducted in accordance with the standards of
128
the institutional review board of Boston Children’s Hospital. Initially only de-identified
129
discarded human tissue was analyzed but following consent prospectively at the time of
130
HLHS/EFE diagnosis, patients’ operative details, catheterization details, echocardiographic data,
131
magnetic resonance imaging (MRI) data, and clinical information were obtained and correlated
132
with histological findings.
133
Patients:
134
We analyzed EFE tissue from a total of 24 patients with the diagnosis of HLHS who
135
underwent LV rehabilitation surgery with EFE resection as previously described in more detail at
136
Boston Children’s Hospital between December 2011 to March 2018 (6). Out of these 24 HLHS
137
patients, we received tissue from 17 patients, sent as de-identified discarded tissue, resected
138
during open heart surgeries, and tissue from additional seven patients, in which we obtained
139
consent prior to surgery. In the latter group, we followed the patients collecting clinical
140
background and imaging data, and obtained EFE tissue from previous resections if available.
141
These patients presented with mitral stenosis and/or aortic stenosis rather than atresia at the time
142
of EFE resection as detailed in Table 1.
143
Six patients were included for comparison with diagnoses other than HLHS but with
144
known flow disturbances across aortic (AV) and/or mitral valve (MV). In all of these patients,
145
preoperative standard imaging studies (echocardiography and MRI) did not describe EFE.
146
Macroscopic appearance of “EFE-like tissue” in the LV outflow tract diagnosed by the cardiac
147
surgeon performing the surgery led to resection of endocardial tissue in the operating room.
10
148
For comparison, we received two de-identified discarded tissue samples from the right
149
ventricle (RV) from patients without flow disturbances, which was resected in both cases
150
because a right ventriculotomy was performed during the surgical procedure. Furthermore, a
151
healthy rat LV was used as negative control.
152
All tissue samples, once received from the operating room were immediately embedded
153
in optimal cutting temperature (OCT) compound, snap frozen and stored at -80°C for future
154
analysis. Frozen sections were produced from OCT blocks and further processed for microscopic
155
analysis as described in more detail below.
156
Composition of EFE Tissue:
157
Age-related changes of EFE tissue, were examined for the amount of collagen and
158
elastin, vascularity, and cellularity by staining with Hematoxylin&Eosin (H&E), Masson’s
159
Trichrome (MT) and Van Giessen Elastin (VG). All slides were visualized using a Zeiss
160
Observer.Z1 fluorescent microscope with a Nikon 20x objective (NA=20x/0.45). Ten randomly
161
selected fields from each slide were taken and quantification was performed with ImageJ
162
(version 2.0.0-rc-43, obtained from the National Institute of Health, Bethesda, MD). Details
163
about the method of quantifying collagen, elastin and muscle on histological sections have been
164
described previously by our group in more detail (17).
165
Analysis of tissue remodeling through degradation of elastin fibers and collagen by
166
matrix metalloproteinases was determined by in situ zymography. Unstained frozen slides were
167
either incubated with 40ug/ml DQ elastin (EnzChek Elastase Assay Kit) in Dulbecco’s Modified
168
Eagle Medium (DMEM, Gibco, Gaithersburg, MD) and 10% Fetal Bovine Serum (FBS, Gibco,
169
Gaithersburg, MD) for 1 hour at 37°C or in a reaction buffer (0.05M TRIS-HCl, 0.15M NaCl,
170
5mM CaCl2 and 0.2mM NaN3 at pH 7.6) containing either 40ug/ml DQ gelatin or collagen type
11
171
I or IV (EnzChek Gelatinase/Collagenase Assay Kit) at 37°C overnight. Slides, incubated with
172
DQ elastin were counterstained tropoelastin (1:50, Abcam, Cambridge, MA) and DAPI (1:1000,
173
Dako, Carpinteria, CA), and slides incubated with DQ gelatin were counterstained with collagen
174
I (1:50, Novus Biologicals, Littelton, CO) and DAPI (1:1000, Dako, Carpinteria, CA). All
175
chemicals were obtained from Thermofisher (Thermofisher, Waltham, MA).
176
Diagnosis of EndMT:
177
In support of our hypothesis that EndMT is the underlying mechanism for EFE
178
development, the presence of active EndMT was determined in all tissue samples obtained from
179
the operating room. Immunohistochemical double-staining of endothelial cells with an
180
endothelial marker, cluster of differentiation 31 (CD31; 1:100, Dako, Carpinteria, CA), and a
181
mesenchymal marker, α-smooth muscle actin (α-SMA; 1:100, Abcam, Cambridge, MA) at the
182
same time, is indicative of active EndMT. Additionally, all samples were stained for the
183
transcription factors Twist (1:100, Abcam, Cambridge, MA), which regulate EndMT indicated
184
by Twist-positive nuclei. Nuclei were stained for 4',6-Diamidino-2-Phenylindole (DAPI;
185
(1:1000, Dako, Carpinteria, CA). Furthermore, active EndMT was confirmed by staining for the
186
transcription factors Slug/Snail (1:100, Abcam Cambridge, MA). Co-localization with nuclei in
187
endothelial cells is indicative of active EndMT. As all tissue samples contained some muscle
188
tissue, the myocardium was also analyzed for tissue structure and fibrosis on HE and MT as well
189
as immunochistochemical staining for desmin to assess cardiomyocytes and EndMT (1:50,
190
Abcam, Cambridge, MA).
191
Statistical Analysis:
192
After confirmation of normal distribution of data, ANOVA for multiple group
193
comparisons with Bonferroni post-hoc analysis was performed for calculation of statistically
12
194
significant (STATA, StataCorp. 2015. Stata Statistical Software: Release 14. College Station,
195
TX: StataCorp LP). Probability values of ≤ 0.05 were regarded statistically significant. Data are
196
expressed as mean ± SEM.
13
Results
197 198
Patients:
199
The median age of the HLHS patients at the time of most recent EFE resection was 27
200
months (IQR=0 – 73.5 months) compared to a median age of 36 months (IQR=0.8 – 73.3
201
months) in the non-HLHS patients (P=0.8).
202
In the consented HLHS patients, apart from the main diagnosis of HLHS, aortic
203
regurgitation (AR) especially following post-natal balloon valvuloplasty was described in four
204
patients; mitral stenosis (MS) due to a dysplastic mitral annulus was found in five of seven
205
patients. These valvular diseases caused turbulent flow within the LV in all seven consented
206
HLHS patients, which has been described throughout their entire medical history. All except for
207
two HLHS patients had previous EFE resections. At the time of the most recent resection, all
208
valvar diseases were successfully repaired and no remaining turbulences were described
209
postoperatively and at follow-up neither in echocardiography nor in MRI studies. The median
210
follow-up time after the last EFE resection in the consented HLHS patient was 6.8 months
211
(IQR=4.15 - 9.45 months). (Table 1). In the “non-HLHS” patients with valvar disease,
212
predominantly a jet across a stenotic MV was present. In two cases a regurgitant AV caused a jet
213
toward the LVOT. (Table 2)
214
Composition of EFE Tissue:
215
The number of nuclei (mean±SEM expressed as nuclei per field of vision) within EFE
216
tissue decreased with increasing age – infants (<1 year of age) 282±63 versus toddlers and
217
preschoolers (1-5 years of age) 158±24 versus school-aged children (>5 years of age with the
218
oldest child at 12 years of age) 94±12 - but did not reach significance. All tissue remained
219
avascular in all tissue samples. EFE tissue from HLHS patients consisted predominantly of
14
220
elastin and collagen fibers with some underlying myocardium. The ratio of muscle to elastin and
221
collagen did not show significant age-related differences within the three groups (Figure 1).
222
“EFE-like” tissue obtained from non-HLHS patients, showed the same characteristics as EFE
223
tissue from HLHS patients. Collagen and elastin predominated the tissue sections, the number of
224
nuclei decreased with age, and there were no blood vessels found within the tissue.
225
In situ zymography determined that impaired elastase/gelatinase activity was not the
226
cause of increased collagen and elastin deposition and no age-related alteration could be
227
identified (Figure 2). The same characteristics were found in the non-HLHS patients with flow
228
disturbances.
229
Presence of Active EndMT:
230
Endocardial double-staining with CD31/alpha-SMA as well as Twist and Slug/Snail-
231
positive stained nuclei, respectively, both indicative of active EndMT, were found not only in all
232
of the 24 samples from HLHS patients but also in all six non-HLHS patients. EndMT-positive
233
tissue was not found, however, in right ventricular tissue samples nor in a healthy rat heart
234
(Figure 3).
235
In all patients with EFE and “EFE-like” tissue, some underlying myocardium was
236
resected (Figure 4). MT stains showed infiltrative growth of collagen bundles from the
237
endocardium into the underlying myocardium of HLHS patients and non-HLHS patients with
238
flow disturbances. The degree of infiltrative growth increases with increasing age in HLHS
239
patients. In samples of patients <1 year of age, the fibrotic tissue is separated from the underlying
240
muscle, whereas samples from older children (> 2years of age), no separation between collagen
241
fibers and myocardium is feasible. The endocardial thickness of fibrotic tissue decreases,
242
whereas infiltrative growth of collagen into the underlying myocardium increases (Figure 5). In
15
243
contrast, in a healthy rat heart and RV tissue samples, a thin endocardial layer is present which is
244
separated from the underlying myocardium. Localization of endothelial cells within the collagen-
245
rich tissue which infiltrates the myocardium identified multiple double-stained cell for CD31 and
246
alpha-SMA. The double-staining is indicative that intramyocardial fibrotic tissue is also derived
247
through EndMT (Figure 6).
248
Figure 7 summaries the analytic process from an intraoperative diagnosis of
249
macroscopically appearing EFE tissue, to the unraveling of the mechanistic cause of EFE
250
formation, and the connection to clinical presentation of EFE tissue formation localized to areas
251
of flow disturbances due to valvar defects.
16
Discussion
252 253
In this study, we established that structural changes of EFE tissue toward a more
254
infiltrative growth into the underlying myocardium and less cellularity occurs with increasing
255
age. Furthermore, we describe for the first time that hemodynamic alterations in the way of jets
256
on the level of the mitral valve or regurgitant flow across an incompetent aortic valve promote
257
localized aggravated EFE growth and reoccurrence despite excessive surgical resection. “EFE-
258
like” tissue growth independent of an underlying HLHS diagnosis, shows the same pattern.
259
Following detailed analysis of this “EFE-like” tissue, we determined that the underlying
260
mechanism for development is EndMT which we had already reported in detail in patients with
261
EFE associated with HLHS (6). Our data further indicate that HLHS alone does not predispose
262
this patient cohort to EFE formation but that the underlying communality is the presence of
263
valvar defects represented as either mitral stenosis or aortic insufficiency. Both valvar defects
264
result in flow disturbances, displayed as jets across the valve onto localized areas of the LV
265
cavity which responds by endocardial thickening through subendocardial development of EFE
266
tissue. In HLHS patients, despite several surgical resections of this tissue, progression still occurs
267
unless the valvar defect is repaired.
268
Another novel finding of this study is that unlike previously suggested, EFE develops and
269
progresses as a distinct layer of fibrotic tissue which can be easily dissected from the underlying
270
myocardium (7). Following thorough examination of our resected tissue samples from HLHS
271
and non-HLHS patients, all EFE tissue samples contained some degree of muscle tissue.
272
Macroscopically this finding suggests infiltrative growth of EFE tissue into the underlying
273
myocardium which we confirm microscopically through MT staining. With increasing age,
274
subendocardial tissue growth takes on a more infiltrative pattern with collagen and elastin
17
275
bundles protruding into the adjacent myocardium. Thus, our data show that the main trigger for
276
the development and progression of EFE is flow disturbances, which might also occur in other
277
congenital heart diseases leading to macroscopically “EFE-like” tissue unrelated to the primary
278
diagnosis of HLHS.
279
In our EFE tissue samples we could show that localized response of the endocardium to
280
flow disturbances (e.g. shear stress) created EFE. It is well known that endothelial cells lining the
281
vasculature respond to alterations in shear stress by changing their shape and function (18) since
282
they are exposed to a large variety of biochemical and hemodynamic stimuli. Thus, endothelial
283
cells are required to be highly plastic which allows for adaptation to alterations in fluid shear
284
forces. Plasticity is essential to maintain homeostasis but pathological stimuli might alter this
285
process and contribute to disease through adverse plasticity which EndMT is an example of (19).
286
Areas exposed to disturbed blood flow and thus, alterations in shear stress generate a
287
microenvironment conducive for EndMT. As a consequence, activation of EndMT results in
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intimal hyperplasia with extracellular matrix remodeling (19,20). EFE has the appearance of
289
organized layers resembling degenerative intimal hyperplasia of arterial vessel walls. The details
290
on hemodynamic forces and their role in maintaining vascular integrity have been well studied in
291
the vasculature but little is known whether laminar shear stress is protective also for the
292
endocardial endothelial cells. However, it supports the idea that flow disturbances may play a
293
role in endocardial remodeling through EndMT driven by activation of transcription factors
294
which are actively involved in suppression of VE-cadherins and other endothelial specific
295
proteins. Furthermore, in an animal model of EFE formation which we had previously described
296
in detail, an association with alterations in mechanical forces including flow disturbances across
297
an incompetent aortic valve is associated with formation of EFE (12).
18
298
Functional indicators of EndMT in tissue are increased expression of collagens and
299
elastin, as well as production of enzymes degrading the extracellular matrix, such as matrix
300
metalloproteinase-2 (MMP)-2, MMP-9 or MMP-12 (elastase). The expression of mesenchymal
301
markers corresponds to a newly acquired ability to produce and remodel the extracellular matrix
302
(21). With regard to remodeling, activation of MMPs are important following the interruption of
303
cell-cell or cell-matrix contact. In our patient cohort, no defect in the degradative potential of the
304
extracellular matrix of the endocardium or myocardium was found since all MMPs (MMP12 and
305
MMP2/9) were active in all tissue samples and distribution was focused in areas of active
306
remodeling.
307
In summary, flow disturbances exposing endocardial endothelial cells to altered shear
308
forces are the main trigger for induction of EndMT leading to reoccurrence of EFE displaying a
309
more infiltrative growth pattern into the underlying myocardium. Supported by our data, we can
310
also conclude that these alterations are not limited to congenital cardiac pathologies associated
311
with HLHS but are related to severe valvar defects with jets onto the endocardial surface
312
appearing to respond by EFE thickening. Targeting EFE therapeutically with surgical resection
313
likely is not sufficient to address reoccurrence and prevent infiltrative growth. A more
314
comprehensive approach by addressing the underlying mechanism of EndMT through localized
315
or systemic therapy is warranted but requires more investigation.
19
316
Acknowledgment
317
We would like to thank Breanna Piekarski, BSN, MPH for contribution regarding IRB and
318
patient related organization, and collecting tissue samples.
20
References:
319 320
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Figure Legends
378 379
Table 1: Detailed information of consented patients with hypoplastic left heart syndrome
380
Data presented include age of patients, surgeries in chronological order, number of EFE
381
resections and localization of EFE tissue that was obtained for histological analysis.
382 383
Table 2: Detailed information of patients with diagnosis other than hypoplastic left heart
384
syndrome
385
In these patients, indication for surgical intervention were either mitral valve (MV) stenosis or
386
aortic valve (AV) regurgitation causing flow disturbances in the left ventricle. However, imaging
387
studies did not describe any endocardial abnormalities in the sense of endocardial fibroelastosis
388
(EFE) prior to surgery. EFE resection was performed following the cardiac surgeon’s ad hoc
389
decision intraoperatively. Age of the patients, diagnosis and localization of EFE resection is
390
presented.
391 392
Figure 1:
393
(A) Macroscopic appearance of resected endocardial fibroelastosis (EFE) tissue from the left
394
ventricle of patients with hypoplastic left heart syndrome (HLHS) is shown; left: tissue sample
395
facing the myocardium (red=muscle), right: sample side facing toward the ventricular lumen.
396
(B) A representative picture of an Elastin van Giesson stain shows the distribution of collagen
397
(blue) and elastin (black) and muscle (white-yellow) in EFE tissue samples.
398
(C) A summary of muscle-collagen-elastin distributions are shown where tissue samples were
399
grouped according to age (<1year, 1-5years, >5years). There is no significant difference in the
400
distribution within each specific age groups.
24
401 402
Figure 2:
403
(A) Representative in situ zymography of gelatinase activity and collagen in endocardial
404
fibroelastosis (EFE) tissue obtained from patients diagnosed with hypoplastic left heart
405
syndrome is shown. Gelatinase is shown in green, collagen in red and nuclei in blue.
406
(B) Representative in situ zymography of elastase activity and tropoelastin in in endocardial
407
fibroelastosis (EFE) tissue obtained from patients diagnosed with hypoplastic left heart
408
syndrome. Elastase is shown in green, tropoelastin in red and nuclei in blue.
409
(C and D) Summary of matrix metalloproteinase (MMP) activity (elastase on the left and
410
gelatinase on the right) per field of vision for different age groups (<1year, 1-5years, >5years) is
411
shown. There was no significant age-related difference in the balance of MMP activity to
412
collagen/elastin production.
413 414
Figure 3:
415
Representative sections of endocardial fibroelastosis (EFE) and “EFE-like” tissues are displayed.
416
Double-staining of endothelial cells with an endothelial cell marker (cluster of differentiation 31
417
- CD-31) in red and an fibroblast marker (α-smooth muscle actin – SMA) in green, or endothelial
418
cells with CD-31 (red) and nuclei-positive staining for transcription factors Twist and Slug/Snail
419
(green) is indicative of active endothelial-to-mesenchymal transition (EndMT). Nuclei are
420
stained in blue.
421
(A) EFE tissue from HLHS patients is positive for EndMT (CD-31/αSMA double-staining, white
422
arrows); (B) “EFE-like” tissue from non-HLHS patients is positive for EndMT (CD-31/αSMA
423
double-staining, white arrows); (C) EFE tissue and “EFE-like” tissue stained for the transcription
25
424
factors Twist (white arrows), (D) and Slug/Snail (white arrows), respectively, which are
425
indicative of active EndMT when co-localized with endothelial cell nuclei. (E) Normal rat heart
426
tissue and (F) human right ventricular tissue are negative for EndMT.
427 428
Figure 4:
429
As indicated by these representative left ventricular tissue samples stained with Masson’s
430
Trichrome, infiltrative growth patterns of collagen fibers (blue) into the underlying myocardium
431
(red) is associated with increasing age, (A) infants (<1year) (B) toddler and preschooler (1-
432
5years) (C) school-aged children (5-12years). The summary in (D) shows significantly higher
433
infiltrative growth in the oldest age group (mean±SEM, *p<0.05 versus school-aged children).
434 435
Figure 5:
436
Distribution of collagen and muscle tissue on Masson’s Trichrome stains is shown for a (A)
437
normal rat heart, (B) normal human tissue, (C) left ventricular (LV) tissue from a hypoplastic left
438
heart syndrome (HLHS)-patient, and (D) LV from non-HLHS patient. Muscle is stained in red
439
and collagen in blue where of the latter is not limited to the endocardial surface but grows
440
infiltratively into the underlying myocardium in (C) and (D).
441 442
Figure 6:
443
Endocardial fibroelastosis (EFE) tissue that infiltrates the myocardium stains positive for
444
endothelial-to-mesenchymal transition (EndMT). A representative section of EFE tissue was
445
stained with Masson’s Trichrome (blue: collagen fibers, red: myocardium) and correspondingly
446
on the left, the same areas were stained to identify active EndMT (green: fibroblast marker, α-
26
447
SMA; red: endothelial cell marker, CD31; blue: nuclei marker). Double-staining is indicative for
448
EndMT (white arrows) which is more pronounced in the transition zone between endocardial
449
thickening due to EFE tissue and the underlying myocardium.
450 451
Figure 7:
452
The analytic process from intraoperative diagnosis to mechanistic cause connected with clinical
453
presentation is described here. Macroscopically appearing endocardial fibroelastosis (EFE) tissue
454
is identified intraoperatively and resected for histological analysis. Immunohistochemical
455
staining establishes endothelial-to-mesenchymal transition (EndMT) as the underlying
456
mechanism for endocardial fibroelastosis (EFE) tissue formation. EFE formation is directly
457
localized to areas of flow disturbances due to valvar defects in the left ventricle.
458 459
Video:
460
This video shows a detailed view of the surgical procedure excising endocardial fibroelastosis
461
from the left ventricular cavity and mitral valve surface.