The Role of Scleraxis in Heart Valve Development and Disease

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2014-10-20
The Role of Scleraxis in Heart Valve Development
and Disease
Damien N. Barnette
University of Miami, [email protected]
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UNIVERSITY OF MIAMI
THE ROLE OF SCLERAXIS IN HEART VALVE DEVELOPMENT AND
DISEASE
By
Damien N. Barnette
A DISSERTATION
Submitted to the Faculty
of the University of Miami
in partial fulfillment of the requirements for
the degree of Doctor of Philosophy
Coral Gables, Florida
December 2014
©2014
Damien N. Barnette
All Rights Reserved
UNIVERSITY OF MIAMI
A dissertation submitted in partial fulfillment of
the requirements for the degree of
Doctor of Philosophy
THE ROLE OF SCLERAXIS IN HEART VALVE DEVELOPMENT AND DISEASE
Damien N. Barnette
Approved:
___________________________
Joy Lincoln, Ph.D.
Associate Professor of Pediatrics
Ohio State University
(Formerly Assistant Professor of
Molecular and Cellular
Pharmacology University of Miami)
____________________________
Danuta Szczesna-Cordary, Ph.D.
Professor of Molecular and Cellular
Pharmacology
___________________________
Michael Kim, Ph.D.
Assistant Professor of Molecular
and Cellular Pharmacology
____________________________
Vladlen Slepak, Ph.D.
Professor of Molecular and Cellular
Pharmacology
___________________________
Jae Lee, Ph.D.
Assistant Professor of Neurological
Surgery
____________________________
M. Brian Blake, Ph.D.
Dean of Graduate School
BARNETTE, DAMIEN N.
(Ph.D., Molecular and Cellular Pharmacology)
The Role of Scleraxis in Heart Valve Development
and Disease
(December 2014)
Abstract of a dissertation at the University of Miami.
Dissertation supervised by Professor Joy Lincoln.
No. of pages in text. (116)
Mitral valve prolapse (MVP) affects more than 2% of the population in the
United States, and is the most common cause of chronic mitral valve
regurgitation (MVR) in developed countries. It is estimated that MVP affects
more than 150 million people worldwide. When left untreated, MVP can lead to
severe MVR resulting in a myriad of cardiac complications. The mortality rate of
MVP patients with severe MVR is over 6% per year. Despite clinical significance,
there remains a lack of medical therapies for MVP, with surgical intervention
being the most effective treatment to date. Therefore, understanding normal
heart valve development, and how it is altered during disease, may provide novel
insights into new therapeutic targets. MVP associated with myxomatous mitral
valve degeneration (MMVD) is the most common cause of MVR requiring
surgery. Healthy mature heart valves are highly organized and composed of
stratified layers of elastins, collagens and proteoglycans that collectively provide
all of the necessary biomechanical properties for structure-function relationships
throughout life. In contrast, diseased valves that suffer from MMVD are
pathologically thickened and characterized by an abnormal abundance of
extracellular matrix (ECM) proteoglycans, which prevents the valves from closing
properly and leads to regurgitation and functional prolapse. Although the ECM
composition of mature tri-laminar valves has been well described, little is known
about the molecular mechanisms that establish and maintain these highly
organized structures. However, the etiology of MVP has historically been
associated with genetic connective tissue disorders including Marfan syndrome
(MFS). A deeper understanding of ECM regulation in normal valve development
will help elucidate conserve pathways in disease as a potential means of therapy.
In the current studies, we show that the basic helix-loop-helix (bHLH)
transcription factor Scleraxis (Scx) regulates ECM deposition, with a loss of Scx
being largely attributed to a significant decrease in the expression and
contribution of chondroitin sulfate proteoglycans (CSPGs) to the mature valve
leaflets. In addition, we determine that Scx is sufficient to promote CSPG
expression in both embryonic and mature valve cells. We further delineate that
canonical Tgfβ-Smad signaling positively regulates Scx and CSPG expression,
while activated MAPK attenuates this pathway in a Tgfβ-independent manner.
We
show
that
MAPK
activation
is
sufficient
to
stabilize
the
bHLH
activator/repressor Twist1, however conclude that Twist1 does not bind to or
transcriptionally regulate Scx. We also show that Scx is increased in a MFS
mouse model of MMVD, and overexpression can promote myxomatous
phenotypes in otherwise normal human mitral valve interstitial cells. Using this
model, we explore the idea that reduced Scx function may potentially rescue
myxomatous valve phenotypes in vivo.
Furthermore, we have identified
previously unappreciated protein-coding and non-protein-coding mRNAs that are
differentially expressed in the absence of Scx during valve remodeling stages.
We report an enrichment of mRNAs associated with processes related to gene
regulation and cellular development. Furthermore, bioinformatics analysis
predicted known (Tgfβ2) and novel (Onecut1) upstream regulators of Scx during
valve remodeling. In addition, we show that the loss of Scx leads to differential
changes in mRNA transcripts and alternative splicing of several genes. Together,
these findings provide insights into molecular signaling pathways that regulate
Scx, and identify novel genes and hierarchical networks that are regulated by
Scx during valve development, which may be altered in MMVD.
TABLE OF CONTENTS
Page
LIST OF FIGURES .............................................................................................. vi
LIST OF TABLES ............................................................................................... viii
ABBREVIATIONS............................................................................................... ix
Chapter 1. Introduction .................................................................................... 1
1.1 The developing heart valves and supporting structures ........................ 2
1.1.1 Microarchitecture of mature heart valves ........................................ 2
1.1.2 Heart valve development and regulation ......................................... 4
1.2 Heart valve disease ............................................................................... 7
1.2.1 Origins of valve disease during development ................................. 7
1.2.2 Myxomatous degeneration and Marfan syndrome .......................... 9
1.3 Tgfβ signaling in heart valve development and MMVD........................ 11
1.4 Scleraxis: function and regulation in development............................... 14
1.5 Hypothesis ........................................................................................... 16
Chapter 2. Methods ........................................................................................ 17
2.1 Mouse tissue collection ........................................................................ 17
2.2 Heart valve explant cultures................................................................. 18
2.3 Generation of adenovirus..................................................................... 18
2.4 Avian VP cell culture system................................................................ 19
2.5 Murine C3H10T1/2 and NIH3T3 cell lines ............................................. 20
2.6 Human mitral valve interstitial cell cultures .......................................... 20
2.7 Porcine VIC (pVIC) cultures ................................................................. 21
2.8 RNA isolation, cDNA synthesis, and quantitative PCR ....................... 21
2.9 Western blotting ................................................................................... 24
2.10 Immunofluorescence............................................................................ 26
2.11 RNA sequencing of atrioventricular canals from E.15.5 Scx-/- and
Scx+/+ embryos .................................................................................... 26
2.11.2 Tissue collection .......................................................................... 26
2.11.2 Sequence analyses and data processing ................................... 27
2.11.3 Principal component analysis ....................................................... 28
2.11.4 Venn diagram ............................................................................... 28
2.11.5 Clustering analysis ...................................................................... 29
2.11.6 Alternative splicing indexes .......................................................... 29
2.11.7 Pathway analyses ........................................................................ 30
2.12 Twist1 siRNA knockdown in C3H10T1/2 cells ....................................... 31
2.13 Chromatin immunoprecipitation (ChIP) ................................................ 31
2.14 Luciferase assays ................................................................................ 33
iii
Chapter 3. Role of Scleraxis in heart valve extracellular matrix… .................. 34
3.1 Proteoglycan expression is attenuated in heart valves from
embryonic and post natal Scx-/- mice ................................................... 37
3.2 Scx overexpression in embryonic VP cells and adult VICs leads to
increased CSPG expression ................................................................ 39
3.3 Scx and CSPG expression is positively regulated by Tgfβ2 ................ 41
3.4 MAPK signaling attenuates Tgfβ2-mediated Scx regulation ................ 43
3.5 MAPK signaling negatively regulates Scx in VP cells .......................... 45
3.6 Overexpression of Scx in mature human valve interstitial cells
promotes proteoglycans....................................................................... 47
3.7 Twist1 is stabilized by ERK activation and does not transcriptionally
repress Scx .......................................................................................... 48
3.8 Summary.............................................................................................. 50
Chapter 4. Scx regulation of novel target genes and molecular pathways ...... 52
4.1 Pairwise and clustering analysis distinguish E15.5 Scx-/- AVC
regions from controls ........................................................................... 54
4.2 Pathway analysis reveals differentially expressed mRNAs
associated with gene regulation and cellular development in AVCs
from E15.5 Scx-/- embryos ................................................................ 56
4.3 Exon abundance is significantly altered in the absence of Scx ........... 59
4.4 Summary.............................................................................................. 60
Chapter 5. The role of Scx in mouse models of MMVD ................................... 62
5.1 Scx is increased in valves from a MFS mouse model of MMVD ......... 64
5.2 Loss of Scx function rescues valve phenotypes in a MFS mouse
model of MMVD ................................................................................... 65
5.3 Summary.............................................................................................. 67
Chapter 6. Discussion .................................................................................... 69
6.1 Role of Scx signaling in ECM regulation during heart valve
development ........................................................................................ 70
6.1.1 Scx regulation of proteoglycan expression in heart valves............. 70
6.1.2 Signaling pathways regulating Scx in developing heart valves ...... 73
6.2 Implicating Scx function in mechanisms of myxomatous valve
phenotypes in MMVD........................................................................... 76
6.3 Gene networks regulated by Scx in remodeling heart valves .............. 78
6.4 Summary and working model of Scx signaling in heart valves ............ 83
6.5 Perspectives and clinical applications.................................................. 86
Supplemental Results ....................................................................................... 88
Appendix 1 ....................................................................................................... 90
Appendix 2 ....................................................................................................... 91
iv
Appendix 3 ....................................................................................................... 93
Appendix 4 ....................................................................................................... 96
REFERENCES. ................................................................................................ 99
v
LIST OF FIGURES
Figure 1
Heart valve development and disease ............................................... 5
Figure 2
Proteoglycan expression is reduced in atrioventricular canal regions
isolated from post natal Scx-/- mice ................................................. 38
Figure 3
ECM profile array of valve regions from Scx-/- and Scx+/+ post natal
mice ................................................................................................. 39
Figure 4
Scleraxis overexpression in avian VP cells and porcine valve
interstitial cells promotes chondroitin sulfate proteoglycan
expression........................................................................................ 40
Figure 5
Tgfβ2 regulates Scx expression in vitro and in vivo and promotes
chondroitin sulfate proteoglycan expression .................................... 42
Figure 6
MEK1 activation represses Tgfβ2-mediated Scx expression ......... 44
Figure 7
Activated MEK1 signaling represses Scx and chondroitin sulfate
proteoglycan expression in heart VP cells ....................................... 46
Figure 8
pERK1/2 stabilizes Twist1 protein .................................................... 48
Figure 9
Twist1 knockdown in C3H10T1/2 cells does not regulate Scx
expression ....................................................................................... 49
Figure 10 Twist1 does not directly bind Scx promoter ..................................... 50
Figure 11 Twist1 does not transactivation of Scx luciferase activity ................ 51
Figure 12 Loss of Scx function in remodeling heart valves leads to distinct
transcriptome profiles....................................................................... 55
Figure 13 Predicted upstream regulators of Scx in remodeling heart valves ... 58
Figure 14 Exon-level splicing indices of mRNAs affected by alternative
splicing events in Scx-/- samples at E15.5 ........................................ 60
Figure 15 Scx is increased in mitral valve regions from Fbn1 mutant mice ..... 64
Figure 16 Breeding diagram for generation of rescue and control mice .......... 65
Figure 17 Loss of Scx decreases proteoglycans in mitral valves from
Fbn1C1039G/+ mice ............................................................................ 66
vi
Figure 18 Loss of Scx rescues morphological MMVD phenotypes observed
in valves from Fbn1C1039G/+ mice ...................................................... 67
Figure 19 Tgfβ- and ERK-mediated regulation of Scx in normal and
diseased heart valves ..................................................................... 84
Supplemental Figure 1 Scx is increased in myxomatous mitral valves from
10-month old Fln-A deficient mice .................................................. 88
Supplemental Figure 2 Scx is increased in VICs from human patients with
myxomatous valve disease ............................................................. 89
vii
LIST OF TABLES
Table 1
List of primer sets for qPCR ............................................................ 22
Table 2
Antibodies used for Western blotting and Immunohistochemistry ... 25
Table 3
qPCR analysis to show fold changes in gene expression in AdVScx-FLAG infected human mitral VICs isolated from four donor
hearts, compared to AdV-GFP infected controls ............................. 47
Table 4
Mendelian ratios of P6.5 neonatal mice from Fbn1-/+;Scx-/+ intercross
breeding scheme ............................................................................. 68
viii
ABBREVIATIONS
AdV
AV
AVC
bHLH
BMP
BSA
caMEK1
cE
CSPG
CT
dp
DMSO
dnMEK1
E
EC
ECM
ERK
EMT
Fbn1
FGF
Fln-A
GFP
GO
HH St.
hMVIC
KEGG
MAPK
MEK
MFS
MMVD
MMVP
MVP
MVR
NICD
OFT
P
pVIC
PBS
PFA
qPCR
RPKM
Scx
SL
(p)Smad
adenovirus
atrioventricular
atrioventricular canal
basic helix loop helix
bone morphogenetic proteins
bovine serum albumin
constitutively active MEK 1
chicken embryonic day
chondroitin sulfate proteoglycan
cycle count threshold
dually phosphorylated
dimethyl suloxide
dominant negative MEK 1
embryonic day
endocardial cushion
extracellular matrix
extracellular signal-regulated kinase
epithelial-to-mesenchymal transformation
Fibrillin-1
fibroblast growth factor
Filamin-A
green fluorescent protein
gene ontology
Hamburger Hamilton stage
human mitral valve interstitial cell
Kyoto Encyclopedia of Genes and Genomes
mitogen-activated protein kinase
mitogen-activated protein kinase kinase
Marfan syndrome
myxomatous mitral valve degeneration/disease
myxomatous mitral valve prolapse
mitral valve prolapse
mitral valve regurgitation
Notch intracellular domain
outflow tract
postnatal day
porcine valve interstitial cell
phosphate buffered saline
paraformaldehyde
quantitative polymerase chain reaction
reads per kilo-base per million
Scleraxis
semilunar
(phosphorylated) small mother’s against decapentaplegic
ix
SMA
Tgf
VIC
VP
smooth muscle actin
transforming growth factor
valve interstitial cell
valve precursor
x
Chapter 1. Introduction
Heart valves allow for unidirectional blood flow during the cardiac cycle, which is
essential for proper cardiovascular function throughout life.
The mature
mammalian heart contains two sets of heart valves: the atrioventricular (AV)
valves which separate the atria from the ventricles, and the semilunar (SL) valves
which separate the ventricles from the great arteries (Anderson, Ho et al. 2000).
The AV valves include the mitral valve and the tricuspid valve, which contain
asymmetric leaflets that are connected to annuli on the hinge regions, and
fastened to the ventricles by chordae tendineae and papillary muscles on the free
ends (Anderson, Ho et al. 2000, Hinton and Yutzey 2011). The AV valves open
during diastole to allow the ventricles to fill with blood, and close during systole to
prevent blood from flowing back into the atria. The SL valves include the aortic
valve and the pulmonary valve, which open during ventricular systole to allow
blood to flow out of the heart into the systemic circulation (Anderson, Ho et al.
2000), and close during diastole to prevent blood from flowing back into the
ventricles (Boignard 2011). Together, proper opening and closing of AV and SL
valves allow for cooperative blood flow in one direction, while preventing
retrograde flow.
Valve dysfunction is characterized by improper blood flow as a result of
either incomplete opening (stenosis) and/or closing (regurgitation) of the valve
structures. In both instances, prolonged progression often leads to secondary
heart defects including ventricular dysfunction, and ultimately heart failure
(Davies, Moore et al. 1978, Jones, O'Kane et al. 2001, Otto 2007). Congenital
1
2
valve malformations have been estimated as high as 5% of live births, and
contribute to over 20,000 deaths annually (Rosamond, Flegal et al. 2008, LloydJones, Adams et al. 2010). Currently, there are no clinical treatments to prevent
the progression of valve disease, with surgical intervention as the only mode of
action to remedy severe valve malformations. However, valve replacement
surgery is a costly procedure, reaching approximately $1 billion per year in the
United States alone (Rajamannan, Gersh et al. 2003).
In addition, current
approaches are limited by the inability of prosthetic valves to grow, repair, and
remodel in vivo (Chavez and Cosgrove 1988, Supino, Borer et al. 2004). Despite
the prevalence of valve disease and surgical limitations, the etiology has not
been well defined and therefore therapeutic advancements have remained
limited. As increasing evidence suggests a genetic basis for latent valve disease
(Smith and Matthews 1955, Roberts 1970, Garg 2006), a more complete
understanding of the molecular mechanisms that regulate normal valve
development and maintenance may lead to new preventative treatments and/or
advancements in current valve intervention approaches to improve patient
outcomes.
1.1 The developing heart valves and supporting structures
1.1.1 Microarchitecture of mature heart valves
The mechanical forces exerted by blood and the myocardium drive the ability of
heart valves to open and close. The ability of heart valves to facilitate
unobstructed,
unidirectional
blood
flow
depends
congruently
on
their
3
microarchitecture and overall morphology. Mature valve leaflets are composed
of three stratified layers of specialized extracellular matrix (ECM) interspersed
within valve interstitial cells (VICs), overlaid by a layer of valve endothelial cells
(Hinton, Lincoln et al. 2006, Hinton and Yutzey 2011, Lincoln and Yutzey 2011).
VICs synthesize and secrete essential ECM, and are necessary to establish and
maintain the highly organized ECM microstructure for proper valve function. The
subsequent role of the ECM is to provide all the necessary biomechanical
properties to withstand constant changes in hemodynamic force during the
cardiac cycle. The ECM is compartmentalized into three connective tissue layers
that all function cooperatively for optimal biomechanics (Rabkin-Aikawa, Mayer et
al. 2005). The fibrosa layer of the valves is located furthest away from blood flow
and is predominantly comprised of parallel bundles of fibrillar collagens that are
circumferentially oriented to provide tensile stiffness (Missirlis and Armeniades
1977, Broom 1978, Kershaw, Misfeld et al. 2004, Sacks and Yoganathan 2007).
In contrast, the atrialis and ventricularis layer of AV and SL valves respectively is
adjacent to blood flow and comprised of filamentous elastic fibers that are
radially-oriented to allow for flexibility and extensibility (Schoen 1997, Vesely
1998). The spongiosa is the proteoglycan-rich middle layer, and serves as a
connection between the orthogonally arranged fibrosa and artialis/ventricularis
layers to provide compressibility and preserve valve integrity. The valve leaflets
are reinforced by supporting structures, including chordae tendinae and the valve
annulus. The chordae tendinae are external, collagen-rich tendinous cords that
connect the AV valve leaflets to the papillary muscles of the ventricular
4
myocardium. However, the cusps of the SL valves have internal support and are
anchored directly to the arterial root.
The annulus is composed primarily of
fibrous collagens and acts as a joist to oppose dispersion forces and provide
tissue stability. Additionally, there is tissue at the tips of the AV and SL valves
that ensures functional valve closure (Gross and Kugel 1931, Lincoln, Alfieri et al.
2004, Person, Klewer et al. 2005, Rabkin-Aikawa, Mayer et al. 2005). A balance
in biomechanical properties within the valve leaflets and supporting structures is
paramount for their proper function. The slightest imbalance in valve
microstructure often results in valvular malfunctions, highlighting the importance
of processes required for proper valve development.
1.1.2 Heart valve development and regulation
The embryonic vertebrate heart is a primitive heart tube composed of an
endocardial-endothelial cell layer surrounded by a myocardial cell layer (Fishman
and Chien 1997, Moorman and Christoffels 2003, Martinsen 2005).
Growth
factor signals emanating from the adjacent myocardium result in secretion of
gelatinous ECM between the two cell layers known as cardiac jelly (Lyons,
Pelton et al. 1990, Harrelson, Kelly et al. 2004, Plageman and Yutzey 2004, Ma,
Lu et al. 2005, Combs and Yutzey 2009). This production of ECM results in the
formation of hyaluronan-rich endocardial cushion (EC) swellings in the outflow
tract (OFT) and atrioventricular canal (AVC) regions of the looping heart, and is
the first evidence of valvulogenesis. Although ECs are nascent valve structures
at this stage, they act as physical barriers that direct blood flow through the
5
primitive heart tube and prevent backflow (Schroeder, Jackson et al. 2003).
Further signals from the myocardium induce endothelial-to-mesenchymal
transformation (EMT) (Krug, Runyan et al. 1985), when a subset of endothelial
cells lose cell-cell contact and migrate into the cardiac jelly to populate the ECs
with transformed mesenchymal cells (Fig.1A). Subsequently, the mesenchymal
valve precursor (VP) cells in the ECs proliferate and differentiate into cell lineage
types that contribute to valvuloseptal structures and adult VICs, and secrete
specialized ECM within the valvular compartments (Fig. 1B) (Hinton, Lincoln et
al. 2006). As the valve primordia grows and undergoes extensive remodeling,
the ECM is highly enriched in hyaluronan, versican, collagens and other
basement membrane components (Little and Rongish 1995, Hinton, Lincoln et al.
2006, Chakraborty, Cheek et al. 2008). The valve primordia elongates to form
thin valve leaflets composed of diversified cell types that secrete and organize
Figure 1. Heart Valve Development and Disease. (A) Endocardial cushions
(ECs) contain a subpopulation of VP cells. (B) Subsequently, ECs remodel,
marked by cell differentiation and ECM secretion to form valve primordia. (C)
As a result, mature valves and supporting structures are composed of highly
stratified layers of connective tissue: artialis (gray), spongiosa (light blue),
and fibrosa layer (yellow); along with supporting structures: chordate tendinae
(tan). (D) However, myxomatous valve disease is characterized by thickened,
proteoglycan-rich valve leaflets with disorganized extracellular matrix.
Diagram depicts mitral valve
6
specialized ECM into compartmentalized valve layers and supporting structures
(Fig. 1C, mitral valve shown) (Armstrong and Bischoff 2004, Martinsen 2005,
Hinton, Lincoln et al. 2006).
During the final stages of valve maturation,
proliferation and ECM production subside as VICs transition into a quiescent
state and the trilaminar structure of the valve is complete (Aikawa, Whittaker et
al. 2006, Hinton, Lincoln et al. 2006). Although the hallmark stages of valve
development are well defined, the signaling pathways and regulatory
components that control these processes are less clear.
Although the AVC cushions form approximately 1 day before the OFT
cushions, the cellular pathways that regulate valve development are generally
conserved during valvulogenesis (Camenisch, Schroeder et al. 2002, Delot
2003). Many studies suggest that bone morphogenetic protein (Bmp), a member
of the transforming growth factor-beta (Tgfβ) superfamily, acts as the major
signaling component that originate from the myocardium to initiate EC formation
and subsequent EMT (Combs and Yutzey 2009).
In addition, it has been
established that activation of the Tgfβ (Brown, Boyer et al. 1996, Nakajima,
Yamagishi et al. 2000) and Notch (Timmerman, Grego-Bessa et al. 2004)
pathways in endothelial cells is required for EMT processes in EC initiation.
Perturbations in these signaling pathways can lead to abnormal or absent EC
formation, resulting in a range of valvular and ventricular-septal defects
associated with severe cardiac malfunctions including embryonic heart failure
(Ma, Lu et al. 2005, Person, Klewer et al. 2005, Lincoln, Kist et al. 2007, Snider,
Hinton et al. 2008). Twist1 is a basic-helix-loop-helix (bHLH) transcription factor
7
highly expressed in ECs and is downregulated during heart valve remodeling
(Chakraborty, Cheek et al. 2008). Twist1 promotes cell proliferation and
migration during EC remodeling while inhibiting differentiation (Shelton and
Yutzey 2008). In addition, fibroblast growth factor (Fgf) signaling is involved in
valve primordia remodeling including cell proliferation (Sugi, Ito et al. 2003). The
proper function of these pathways and their associated proteins also requires the
appropriate ECM environment, as it has been shown that hyaluronan and
versican are required for EC formation (Camenisch and McDonald 2000). Once
the valves are fully mature, it is essential that cellular signaling and ECM
homeostasis be maintained to preserve the biomechanical integrity of the valves.
Abnormal expression and distribution of ECM proteins including collagens,
fibrillins, and elastins result in developmental valve defects (Fig. 1D).
Dysregulation of these pathways and cellular components in developing and
mature valves has also been associated with disease, highlighting conserved
signaling pathways in development and disease.
1.2 Heart valve disease
1.2.1 Origins of valve disease during development
Historically recognized as a latently acquired malformation, it has recently
emerged that valve disease occurring later in life has its origins in subtle
developmental abnormalities (Smith and Matthews 1955, Roberts 1970, Garg
2006). In addition, studies have shown that the majority of valve disease cases
involve malformed valve structures (Pomerance 1972, Passik, Ackermann et al.
8
1987, Hinton, Lincoln et al. 2006). This increasing evidence suggests that valve
disease may be attributed to slight aberrations in developmental processes,
which lead to a predisposition to valve disease over time. In contrast to healthy
valves, diseased valves are characterized by histopathological changes in ECM
distribution and composition, and VIC disarray (Hinton, Lincoln et al. 2006,
Hinton and Yutzey 2011).
Given the structure-function relationship in heart
valves, these alterations result in biomechanical insufficiencies that lead to the
inability for the valves to open or close properly during the cardiac cycle. At the
cellular level, valve disease is characterized by VIC activation associated with an
increase ECM and remodeling enzymes that are also expressed in VP cells
during development, including the re-expression of alpha-smooth muscle actin
(SMA) and Twist1 (Rabkin-Aikawa, Farber et al. 2004, Hinton, Lincoln et al.
2006, Liu, Joag et al. 2007). As it is currently, these valve histopathologies are
categorized into two phenotypic patterns: fibrotic changes and myxomatous
changes.
Fibrosis is characterized by collagen accumulation, elastin
fragmentation, and proteoglycan breakdown resulting in a stiff valve that is prone
to stenosis. This is often associated with a hardening of the connective tissue
and progressive fibrosis, with severe disease marked by calcification.
Conversely, myxomatous degeneration is characterized by collagen degradation,
elastin fiber fragmentation, and proteoglycan accumulation resulting in a floppy
valve that is prone to prolapse and regurgitation (Fig. 1D) (Hinton and Yutzey
2011). Valvular regurgitation is the most common manifestation of myxomatous
degeneration, and associated volume overload leads to atrial and ventricular
9
remodeling, chordal rupture, and congestive heart failure (Shah 2010, Guy and
Hill 2012).
Although this pattern of valve disease is not fully understood,
evidence suggests that myxomatous changes are a result of disruptions in
valvular ECM that induce signaling pathways conserved in development which
ultimately cause disease (Hinton and Yutzey 2011). As these pathogeneses are
elucidated, new light can be shed on the etiologies and regulatory mechanisms
of valve disease.
1.2.2 Myxomatous degeneration and Marfan syndrome
Myxomatous degeneration is a pathological weakening of the connective tissue,
and although various tissues in the body can develop histopathologic features,
the heart valves are among the most highly affected.
These myxomatous
changes lead to gross thickening and overall pathological weakening of the
valves that result in associated functional prolapse and regurgitation, primarily
observed in mitral valves (Tamura, Fukuda et al. 1998, Rabkin-Aikawa, Mayer et
al. 2005, Schoen 2005).
Myxomatous mitral valve degeneration (MMVD) is
characterized by ECM proteoglycan expansion (Olsen and Al-Rufaie 1980,
Kinsella, Bressler et al. 2004, Gupta, Barzilla et al. 2009), VIC activation (Hinton
and Yutzey 2011), elastin fragmentation (Akhtar, Meek et al. 1999), and
collagenous fibrosa layer attenuation or loss (Nasuti, Zhang et al. 2004) . These
changes in the valve apparatus lead to pathological weakening of the valve
biomechanics and functional mitral incompetence that result in prolapsed valve
10
leaflets that bulge back into the adjacent atria (Whittaker, Boughner et al. 1987,
Cosgrove and Stewart 1989, Takano, Miyamoto et al. 2005).
MMVD has generally been considered a genetic disorder of the
connective tissue, with genetic origins accounting for the majority of incidences in
the United States and Europe (Guy and Hill 2012); however its genetic
components in the human population have only been recently described in
emerging studies (Dietz, Cutting et al. 1991, Li, Toland et al. 1997, Loeys,
Schwarze et al. 2006). It is noteworthy to mention that the genetic components
of MMVD are in developmental connective tissue processes (Weiss, Mimbs et al.
1975, Guy and Hill 2012) and have often been associated with systemic
connective tissue disorders such as the Ehler’s Danlos syndrome, Stickler
syndrome, and most notably Marfan syndrome (MFS) (Liberfarb and Goldblatt
1986, Jones, O'Kane et al. 2001, Grau, Pirelli et al. 2007). A deeper
understanding of the molecular pathways involved in MMVD pathogenesis
associated connective tissue disorders is needed.
MFS is a common, systemic connective tissue disorder with an incidence
as high as 1 per 5,000 individuals in the United States (Nienaber and Von
Kodolitsch 1999, Pyeritz 2000). It is largely associated with mutations in Fibrillin1 (Fbn1) and a range of clinical manifestations including cardiac defects such as
mitral valve prolapse (MVP) and MMVD (Dietz, Cutting et al. 1991, Ng, Cheng et
al. 2004, Dietz, Loeys et al. 2005).
MMVD affects an estimated 5% of the
general population and 88% of MFS patients, and is the leading indication for
surgery and death in affected children (Wilcken and Hickey 1988, Freed, Levy et
11
al. 1999, van Karnebeek, Naeff et al. 2001, Avierinos, Gersh et al. 2002, Gould,
Sinha et al. 2012). Currently, there are no medical therapies or interventions to
prevent myxomatous degeneration in individuals predisposed to developing
MFS. This is largely attributed to the current lack of mechanistic understanding
of myxomatous valve pathogenesis, thereby limiting therapeutic advancements.
Understanding the molecular mechanisms that underlie valve pathology will
improve clinical outcomes for MMVD patients with both syndromic and nonsyndromic etiologies.
1.3 Tgfβ signaling in heart valve development and MMVD
Although the molecular mechanisms of MMVD are not fully understood, many
signaling pathways implicated in MMVD are conserved during valvulogenesis.
Mitral valve phenotypes observed in MFS are associated with changes in
developmental pathways that contribute to valvular ECM and overall valve
integrity, including Tgfβ signaling (Ng, Cheng et al. 2004, Judge and Dietz 2008).
In valve development, Tgfβ1 and Tgfβ2 are initially expressed during EC
formation and EMT processes, while Tgfβ3 is first expressed during remodelling
stages (Akhurst, Lehnert et al. 1990, Camenisch, Molin et al. 2002, Molin,
Bartram et al. 2003). Despite specific expression in AVCs and ECs, previous
studies using mouse models have shown that complete loss of Tgfβ1 and Tgfβ3
have no reported cardiac abnormalities (Garside, Chang et al. 2013). However,
Tgfβ2 null mice exhibit numerous cardiac defects that affect the OFT, AVC, septa
and aortic arch (Sanford, Ormsby et al. 1997, Bartram, Molin et al. 2001),
12
highlighting its critical role in valve development. In addition, valves from Tgfβ2-/mice display hypercellular ECs and abnormal valvular ECM composition (Azhar,
Runyan et al. 2009). Loss of the Tgfβ receptor TβRII has been shown to be
lethal at embryonic day (E) 10.5 as a result of yolk sac and hematpoiesis defects,
while more tissue specific loss in tyrosine kinase 2 (Tie2)-expressing cells leads
to lethality at E13 due to hypoplastic ECs and trabeculae defects (Sridurongrit,
Larsson et al. 2008). These studies suggest that the Tgfβ ligands and receptors
have very vital roles during the hallmark stages of embryonic valve development.
In healthy adult valves, VICs are relatively quiescent and help to maintain
the valve structure and matrix integrity. However when VICs are injured, Tgfβ
signaling plays a pivotal role in reactivating VICs and sustaining their activation to
promote valve repair (Walker, Masters et al. 2004, Liu and Gotlieb 2008).
Prolonged activation of VICs can result in abnormal ECM and alterations in
biomechanical properties of the valve, leading to increased susceptibility to valve
disease (Jian, Narula et al. 2003). Additionally, Tgfβ signaling is involved in
maintaining heart function by regulating fibrotic processes after injury
(Rosenkranz 2004, Xiao and Zhang 2008). Perturbations in Tgfβ signaling have
been associated with several valve disease states, suggesting aberrant
activation/inhibition of this pathway during valve development or homeostasis
may lead to disease later in life (Garside, Chang et al. 2013).
MMVD is often diagnosed in late stages and has been largely associated
with connective tissue disorders related to mutations in ECM genes (Devereux,
Brown et al. 1982, Hinton and Yutzey 2011). Increasing evidence suggests that
13
disruptions in valvular ECM induce signaling pathways that lead to maladaptive
remodeling and ultimately valve disease (Hinton and Yutzey 2011). Elucidating
the mechanism of MMVD pathogenesis in patients has been advanced by the
use of ECM-deficient mice models that recapitulate human disease (Dallas,
Miyazono et al. 1995, Isogai, Ono et al. 2003, Ng, Cheng et al. 2004). Previous
studies have shown that an established mouse model of MFS, which displays
MMVD phenotypes, is associated with disruptions in Fbn1 interactions with large
latent complexes and ECM proteins that result in increased Tgfβ signaling and
Tgfβ-responsive
genes,
including
regulators
of
remodeling
and
matrix
components (Ng, Cheng et al. 2004, Kern, Wessels et al. 2010). Similarly, loss
of function of ECM genes Elastin and Periostin results in altered Tgfβ signaling
associated with valve degeneration and functional defects (Snider, Hinton et al.
2008, Hinton, Adelman-Brown et al. 2010). Treatment with Tgfβ-neutralizing
antibodies or angiotensin II type 1 receptor blocker Losartan prevents the
development of MFS-related valve anomalies (Ng, Cheng et al. 2004, Habashi,
Judge et al. 2006). A link between Tgfβ and MMVD is also been demonstrated in
Loeys-Dietz syndrome, a similar disease to MFS that is caused by loss of
function mutations in Tgfβ receptor Types I or II, with associated paradoxical
increases in Tgfβ signaling as a result of compensatory signaling by other Tgfβ
receptor types (Dietz, Loeys et al. 2005). These models of MMVD are also
associated with increased activation of Tgfβ downstream targets small mother’s
against decapentaplegic 2 and 3 (Smad2/3). Interestingly, the Fbn1-deficient
MFS mouse model also displays increased activation of extracellular signal-
14
regulated kinases 1 and 2 (ERK1/2) that have been shown to be a principal
effector of Fbn1-dependent phenotypes (Habashi, Doyle et al. 2011). Although
the role of Tgfβ signaling has been studied in several models of MMVD, the
mechanisms have not been clearly defined.
Conserved pathways in
development and disease suggest regulatory genes involved in valvulogenesis
may mediate MMVD phenotypes.
1.4 Scleraxis: function and regulation in development
Scleraxis (Scx) is a member of the bHLH family of transcription factors that have
been shown to play critical roles in cell differentiation, connective tissue
development, and ECM organization (Schweitzer, Chyung et al. 2001,
Murchison, Price et al. 2007, Levay, Peacock et al. 2008). Scx was first detected
in the sclerotome compartment of somites and in mesenchymal cells of the limb
buds of mice during early development, and was initially reported as a regulator
of gene expression within cell lineages that give rise to cartilage and connective
tissues (Cserjesi, Brown et al. 1995, Brown, Wagner et al. 1999). Additionally,
Scx is highly expressed in progenitor cells that form ligaments, tendons, and
bronchial cartilage (Brent, Schweitzer et al. 2003, Dubrulle and Pourquie 2003).
Its expression pattern is particularly high at the interface of muscles and skeletal
primordial at E13.5, but becomes largely restricted to tendons by E15.5 (Asou,
Nifuji et al. 2002). Although Scx knockout mice are viable, they have significant
defects in load-bearing tendon formation (Murchison, Price et al. 2007). Scx-/animals exhibit striking disruptions in tendon differentiation in the dorsal flexure of
15
the forelimb, and have limited use of all paws. These mice also display reduced
function of their back muscles and movement their tails, with overall tendon
defects observed from E13.5. Scx-/- mice also show alterations in tendentious
matrix associated with a notable decrease in the number of collagen fibers, and
disorganization within the tendon matrix (Murchison, Price et al. 2007).
Scx is also expressed in other tissues of high mechanical demand,
including the heart valves (Lincoln, Alfieri et al. 2006, Levay, Peacock et al.
2008). It has previously been shown that Scx is expressed in a subpopulation of
mesenchyme cells within the ECs during valve development, and is first
detectable around E15.5 during stages of remodeling (Levay, Peacock et al.
2008). In E17.5 embryos null for Scx, valve cells display prolonged mesenchymal
cell phenotypes suggesting defects in VP cell differentiation and maturation. At
birth, mutant valves are thickened with highly unorganized ECM, and by juvenile
stages the valve leaflets are grossly malformed and display characteristics of
pathological fibrosis including excess collagen deposition (Levay, Peacock et al.
2008). Scx expression during valvulogenesis has been shown to correlate with
expression of Type II collagen and Tenascin (Lincoln, Alfieri et al. 2006, Zhao,
Etter et al. 2007), although the mechanism is not fully described. It has been
shown that Scx is upregulated in response to Tgfβ1 through canonical Smad
signaling in cardiac fibroblast cells (Espira, Lamoureux et al. 2009), while other
studies in somites have reported that balanced Scx expression is regulated by
modulation of ERK1/2 activity (Smith, Sweetman et al. 2005). Similarly, studies
in developing avian heart valves have shown that Fgf growth factors are
16
important regulators of Scx expression, associated with increased ERK1/2
activation (Lincoln, Alfieri et al. 2006). The signaling pathways that regulate Scx
also overlap with those altered in MMVD, yet Scx has not been linked to valve
disease in the human population to date.
Moreover, the potential role of Scx in
the initiation or progression of myxomatous phenotypes has not been explored.
1.5 Hypothesis
These studies explore the role of Scx in embryonic and adult heart valves and its
potential function in disease. We hypothesize that Scx is an essential gene that
regulates matrix proteins during normal valve development and is regulated by
conserved pathways in disease. The ability for Scx to regulate ECM proteins is
determined using established avian, murine, and porcine in vitro systems. In
addition, Scx loss of function is used to explore its role in vivo during embryonic
and mature heart valve stages. A whole genome approach is used to elucidate
novel genes and transcriptional networks that are regulated by Scx in remodeling
heart valves. Signaling pathways involved in valve disease, particularly MMVD
changes, are examined to determine their regulation of Scx expression using in
vitro and in vivo approaches. The function of Scx in MMVD phenotypes is
examined in a MFS mouse model of MMVD. Together these studies improve our
understanding of the signaling pathways that regulate Scx, identify novel targets
of Scx during valve development, as well as define a role for Scx in myxomatous
valve disease.
Chapter 2. Methods
2.1 Mouse tissue collection
Scx-/- and Scx+/+ littermate mice were generated as previously described
(Murchison, Price et al. 2007, Levay, Peacock et al. 2008), and collected at
E16.5, counting day E0.5 by evidence of a copulation plug. For histology, hearts
were dissected in 1X phosphate-buffered saline (PBS) and fixed in 4%
paraformaldehyde (PFA)/PBS overnight at 4°C. After fixation, hearts were
processed
for
paraffin
wax
embedding
and
sectioned
at
8µm
for
immunohistochemistry (IHC) as described (Levay, Peacock et al. 2008). In brief,
hearts were dehydrated through a graded ethanol series (25%, 50%, 75%, 95%)
and 100% butanol series and embedded in paraffin wax and sectioned using
Leica microtome. Sections were placed on Superfrost glass slides using a warm
water bath, allowed to dry overnight, and dehydrated through a graded xylene
series prior to IHC. Alternatively, AVC tissue was dissected from unfixed hearts
at postnatal day 1 (P1) and RNA was extracted using Trizol according to the
manufacturer’s instructions. Fbn1C1039G/C1039G, Fbn1C103G/+, and Fbn1+/+ (wild
type) mice were generated as described (Ng, Cheng et al. 2004) and RNA was
extracted from AVC tissue collected from P6.5 hearts (see below).
Tgfβ2-/-,
Tgfβ2-/+, and Tgfβ2+/+ mice were generated and genotyped as described (Azhar,
Brown et al. 2011) and RNA was extracted from whole hearts at E13.5. All
animal procedures were approved and performed in accordance with The
Nationwide Children’s Hospital Research Institute IACUC guidelines.
17
18
2.2 Heart valve explant cultures
Mitral and tricuspid valves were dissected from Scx+/-, and Scx+/+ (wild type) mice
at P1 and immediately cultured as floating explants on pore filters as previously
described (Huk, Hammond et al. 2013). In brief, mitral and tricuspid valves were
dissected from isolated P1 hearts under a microscope and placed on 10mm-wide
0.1-µm
pore
filters
(VCWP,
Millipore)
with
culture
media
(1%
Penicillin/Streptomycin, M199 media (Invitrogen) and 10% FBS (Invitrogen)) in 2cm culture dishes. Valve explants from each mouse were attached to a separate
filter. Four hours after time of culture, growth media supplemented with bovine
serum albumin (BSA) or 200pM Tgfβ2 was added to the floating cultures
(Lincoln, Alfieri et al. 2006) for 48 hours. Following treatment, RNA was collected
as described below.
2.3 Generation of adenovirus
Full length Scx was amplified from E14.5 mouse limb genomic DNA using PCR
primers designed to amplify the gene with the addition of a FLAG tag at the 5’
end: 5’-C TGG ATC CGC CAC CATG GAC TAC AAG GAC GAC GAT GAC AAA
TCC TCC GCC ATG CTG CGT TCA G and 3’-CGT GAA TTC TCA ACT TCG
AAT CGC CGT CTT TCT G.
The underlined sequence encodes the FLAG
protein sequence (DYKDDDDK). Resulting PCR product was purified using
GenEluteTM Gel Extraction Kit (Sigma) according to the manufacturer’s
instructions, and digested with Xho1 and EcoR restriction enzymes in Buffer D at
room temperature for 2 hours alongside the pShuttle-IRES-hrGFP-1 vector.
19
Digestion reactions were mixed and incubated at 37°C overnight. Ligation was
confirmed by agarose gel and sequencing, and adenoviral Scx-FLAG (AdV-ScxFLAG) was produced and tittered using the AdEasy-XL and AdEasy Viral Titer
Kit respectively according to the manufacturer’s instructions (Stratagene).
2.4 Avian VP cell culture system
Fertilized White Leghorn chicken eggs (Charles River Laboratories) were
incubated in high humidity at 38°C, and embryonic hearts were collected at
Hamburger Hamilton (HH) stage 25. Atrioventricular ECs were dissected away
from the adjacent myocardium using tungsten needles, collected in 200µL of
normal growth media, treated with 100µL of trypsin–EDTA (Invitrogen) at 37°C
for 5 min, and passed 3 times through a 25G × 1 ½ needle (Lincoln, Alfieri et al.
2006). Dissociated VP cells from 12 hearts were plated onto a 0.01% collagentreated two-well chamber slide (Labtek) and incubated for 3 days in the culture
media. Then, cells were infected with 1.5×109 PFU adenoviral green fluorescent
protein (AdV-GFP), 3.5×107 PFU adenoviral constitutively active MEK1 (AdVcaMEK1), or 8.5×108 PFU adenoviral dominant negative MEK1 (AdV-dnMEK1) in
serum-free media for a time-course of 4, 16, and 48 hours. Adenoviruses were
obtained from Dr. Jeff Molkentin, Cincinnati Children’s Hospital Medical Center
(Seven Hills Bioreagents) (Bueno, De Windt et al. 2000, Liang and Chen 2001).
For Scx gain-of-function studies, cultures were infected for 48 hours with AdVScx-FLAG or AdV-GFP control. For growth factor studies, cell cultures were
treated with 200pM Tgfβ2 (Sigma) or BSA vehicle control for 30 minutes or 48
20
hours in normal growth media. Following treatment, protein and RNA were
collected for western blot (see below) and quantitative polymerase chain reaction
(qPCR)(see below) or cell cultures were fixed with 4% PFA/PBS for 30 minutes
at room temperature for immunostaining (see below).
2.5 Murine C3H10T1/2 and NIH3T3 cell lines
C3H10T1/2 and NIH3T3 cells were obtained from the American Type Culture
Collection and maintained in growth media as recommended. 70% confluent
cultures were treated with 200pM Tgfβ2 or BSA vehicle control for 48 hours in
normal growth media. For MEK rescue studies, C3H10T1/2 cell cultures were
pre-treated with AdV-caMEK1, AdV-dnMEK1, or AdV-GFP for 6 hours in serumfree media (as described above). Following infection, media was removed and
replaced with normal growth media supplemented with Tgfβ2 or BSA for 48
hours. After treatments, RNA was collected or cells were fixed in 4% PFA/PBS
for 30 minutes at room temperature for immunostaining (see details below).
2.6 Human mitral valve interstitial cell (hMVIC) cultures
Mitral valve tissue was collected from four control patients rejected for
transplantation and three patients with myxomatous mitral valve prolapse
(MMVP) during elective surgery. hMVIC cultures were established and
maintained in serum-supplemented EBM media as described (Hulin, Deroanne et
al. 2012) and passaged to P7. Control cells were seeded in 6-well plates to
~70% confluency and infected with 4×108 PFU AdV-GFP (Seven Hills
21
Bioreagents) or 1.6×107 PFU AdV-Scx-FLAG in normal media for 48 hours and
RNA was extracted. The differences in these PFU values are based upon the
consistent
infection
efficiencies
of
76.17%±4.12%
(AdV-GFP)
and
78.27%±2.67% (AdV-Scx). Additionally, untreated hMVICs from control and
MMVP patients were plated for 48 hours and RNA was extracted to determine
basal gene expression.
2.7 Porcine VIC (pVIC) cultures
pVICs were isolated as previously described (Gould and Butcher 2010). In brief,
valves were isolated from juvenile pigs, swabbed to remove endothelial cell layer,
and immediately dissociated in 30mL of collagenase solution (600 U/mL) for 1218 hours with agitation. Cells were pelleted for 5 minutes at 1000 RPM,
resuspended in interstitial media, and incubated for 2 days. Purified cells were
then seeded on collagen-coated chamber slides to ~80% confluency. Cultures
were infected with previously mentioned concentrations of AdV-GFP or AdV-ScxFLAG in serum-free media, or Tgfβ2 or BSA vehicle control in normal growth
media for 48 hours. Following treatment, cultures were fixed with 4% PFA/PBS
for 30 minutes at room temperature and subjected to IHC and imaged (see
below).
2.8 RNA isolation, cDNA synthesis, and quantitative PCR
Total RNA was isolated using Trizol (Invitrogen) as previously described above
(Peacock, Levay et al. 2010). Briefly, tissue or cells was collected in 200µL of
22
Trizol reagent, and 40uL of chloroform was added to extract total RNA. The
aqueous phase was collected and allowed to precipitate overnight at -20°C in
100µL of isopropanol. RNA was pelleted, washed in 90% ethanol, air dried at
room temperature in a ventilated cell culture hood, and resuspended in 20uL of
RNA-free water. cDNA was generated from 200-300ng mRNA using high
capacity RNA-to-DNA kit according to manufacturer’s instructions (Applied
Biosystems).
1µl cDNA was subject to qPCR amplification (StepOne Plus,
Applied Biosystems) using SYBR Green FastMix (Applied Biosystems) with
specific primers targeting chicken, mouse, and human mRNAs listed in Table 1.
In addition, Taqman FastMix and probes (Applied Biosystems) were used to
target human, murine, and chicken Scx. Following qPCR analyses, the cycle
count threshold (Ct) for each gene of interest was normalized to a species
specific housekeeping gene (GAPDH chicken, L7 mouse, and 18S human), and
the ∆Ct and fold changes in experimental samples over controls were determined
(Peacock, Levay et al. 2010). All qPCR reactions were run on the StepOne Plus
Real-time machine using the manufacturer’s suggested program setting.
Statistically significant differences in gene expression levels were determined
using Student’s t-test or one-way ANOVA plus a post-hoc test as indicated using
at least 3 independent experiments, with p<0.05 considered significant.
Table 1. List of primer sets for qPCR
Gene
Sequence (5’ to 3’)
Perlecan
Mouse: F- 5’-GCT GCT AGC GGT GAC GCA TGG-3’
R: 5’-ACT GTG CCC AGG CGT CGG AA-3’
23
Lumican
Mouse: F: 5’-CTG ACC GAG TCC GTC GGT CCA-3’
R: 5’-CCG TCG AAG GAG CCG AGC TT-3’
Brevican
Mouse: F: 5’-CGA CAG TGC CAG CCA CGG TG-3’
R: 5’-GCC TGG CAA ACA TAG GCA GCG G-3’
Neurocan
Mouse: F: 5’-CGG CCT GAA TGA CCG GAC AGT
AGA-3’
R: 5’-CGC CCA CTC TCA TGT GCC ACC-3’
Decorin
Chicken: F: 5’-GCC ACG CGG TTC CAC CAG AA-3’
R: 5’-CAG CGG AAG GGG CAC ACT GG-3’
Mouse: F: 5’-GGT GTC AGC TGG ATG CGC TCA C-3’
R: 5’-TGC AGC CCA GGC AAA AGG GTT-3’
Human: F: 5′-CTG GGC TGG ACC GTT TCA AC-3’
R: 5′-GAT GGC ATT GAC AGC GGA AGG-3’
Biglycan
Mouse: F: 5’-TTA CTG ACC GCC TGG CCA TCC A-3’
R: 5’-TGC TTA GGA GTC AGG GGG AAG
CTG T-3’
Human: F: 5′-ACA CCA TCA ACC GCC AGA GTC-3’
R: 5′-GAC AGC CAC CGA CCT CAG AAG-3’
Aggrecan
Mouse: F: 5’-GCT GCC CCT GCC CCG TAA TG-3’
R: 5’-AGT CCG GCC CAC GTG TGA CT-3’
Human: F: 5′-TGC GTG GGT GAC AAG GAC AG-3’
R: 5′-CAA GGC GTG TGG CGA AGA AC-3’
Fibromodulin Mouse: F: 5’-CTG CCA CAT TCT CCA ACC CAA
GG-3’
24
R: 5’-AGG ACG GAG GCC CAC TGC ATT-3’
Human: F: 5′-GGC TGC TCT GGA TTG CTC TC-3’
R: 5′-CGG GTC AGG TTG TTG TGG TC-3’
Versican
Chicken: F: 5’-CGG CTG AGA GAG AAT GCC GCC-3’
R: 5’-TCC GGC TGG TTT GGT CGC CA-3’
Mouse: F: 5’-GCT GCC CCG AGC CTT TCT GG-3’
R: 5’-GCG CTT GGC CAC AGC ACC TC-3’
Human: F: 5′-ATC TGG ATG GTG ATG TGT TC-3’
R: 5′-AAT CGC ACT GGT CAA AGC-3’
Collagen Ia1
Human: F: 5′-CGT GGC AGT GAT GGA AGT GTG-3’
R: 5′-ACC AGC AGG ACC AGC GTT AC-3’
Collagen IIa1
Human: F: 5′-TGG AGC AGC AAG AGC AAG GAG-3’
R: 5′-CGT GGA CAG CAG GCG TAG G-3’
18S
Human: F: 5′-AAC GAT GCC AAC TGG TGA TGC-3’
R: 5′-CTC CTG GTG GTG CCC TTC C-3’
2.9 Western blotting
Cells were lysed in sample lysis buffer (1X SDS buffer, 62.5mM Tris pH 7.5, 1X
EDTA-free protease inhibitor cocktail (Roche)). 15-20µg of total protein for each
experimental sample was run on 12% Tris-Glycine SDS PAGE gel (BioRad) and
transferred to 0.45-µm nitrocellulose membranes (BioRad) at a constant 300mA
for 1.5 hrs. Membranes were blocked in 3% BSA (Millipore) in Tris-buffered
saline/Tween 20 (TBST) for 1hr, followed by overnight incubation at 4°C in 1.5%
25
BSA
with
antibodies
against
CS-56
(CSPG)
(Sigma),
actin/tubulin
(Chemicon/Millipore), dually phosphorylated ERK1/2 Thr202/Tyr204 (dpERK1/2)
(Cell Signaling), or phosphorylated Smad2 (pSmad 465/467)(Cell Signaling) at
the dilutions listed in Table 2. Membranes were washed 3 x TBST, and
incubation with anti-mouse- or anti-rabbit-horseradish peroxidase-conjugated
secondary antibody (1:15000, Cell Signaling) for 1 hour at room temperature.
Membranes were washed 3 x TBST and developed using Super Signal West
Femto Substrate (Pierce) and BioMax MR film (Eastman Kodak) with exposure
times from 30 seconds to 10 minutes. Band densities were calculated from at
least 3 biological replicates and normalized to loading controls using Image Pro
Plus software.
Table 2. Antibodies used for Western blotting and Immunohistochemistry
Antibody
Company
Type
Dilution
Secondary
Chondroitin sulfate
Sigma
Western blot
1:1000
Mouse HRP
IHC, paraffin
1:200
Alexa Donkey
anti-mouse
568
dpERK1/2
Cell Signaling
Western blot
1:1000
Mouse HRP
pSmad2
Cell Signaling
Western blot
1:1500
Rabbit HRP
Actin
Millipore
Western blot
1:5000
Mouse HRP
Tubulin
Cell Signaling
Western blot
1:500
Rabbit HRP
26
2.10 Immunofluorescence
Fixed cell cultures were washed twice in 1X PBS for 5 minutes and blocked (2%
horse serum, 2%BSA, 0.1% NP-40/PBS) for 1hr at room temperature. CS-56
antibody to detect CSPG expression was diluted (Table 2, Sigma) in 1:1 blocking
solution/PBS, and cells were incubated overnight at 4°C. Slides were washed 3 x
PBS and incubated with secondary antibody (1:400, 1 mg/ml, Molecular Probes)
for 1hr at room temperature in the dark. Cells were then washed, stained with
DAPI for 10 mins at room temperature and mounted in Vectorshield
(VectorLabs). Fluorescent immunoreactivity was visualized using Olympus BX60
microscope, and captured using CellSens imaging software. Immunoreactivity
was quantitated using Image Pro Plus software and the intensity sum of Alexa568 positive CSPGs, over the total number of DAPI-positive stained nuclei was
calculated with p<0.05 considered significant.
2.11 RNA sequencing of atrioventricular canals from E.15.5 Scx-/- and Scx+/+
embryos
2.11.1 Tissue collection
Scx-/- and Scx+/+ mice were generated as previously described and collected at
E15.5. AVC regions (containing mitral, tricuspid and aortic valves) from Scx+/+
(n=3) and Scx-/- (n=3) embryos were dissected from hearts and RNA extracted
using Trizol reagent (Invitrogen).
All animal procedures were approved and
performed in accordance with The Nationwide Children's Hospital Research
Institute IACUC guidelines.
27
2.11.2 Sequence analyses and data processing
Total RNA samples were sent to Ocean Ridge Biosciences (ORB, Palm Beach
Gardens, FL) for quality control analysis and processing. RNA concentrations
were determined by ribogreen fluorometry, and RNA integrity and purity
assessed using agarose gel electrophoresis. All samples reported a RNA
Integrity score of “I”, indicating intact RNA with strong ribosomal banding. Firstand second-strand cDNA was synthesized from purified RNA to construct the
DNA libraries.
The cDNA library for each sample was sequenced using the
Illumina HiSeqTM 2000 instrument and sequencing by synthesis (SBS)
technology (Illumina, San Diego, CA, USA). Tophat 1.4.1 software was used to
align the library reads to the UCSC Mouse (mm9) reference genome (>75%
efficiency), and annotated using Samtools v0.1.18. EasyRNASeq version 1.6
was used to count reads mapping within Ensembl version 66 exons and calculate
the normalized counts for each gene. Raw count files were annotated using data
from Ensembl Mouse version 66. Reads per kilo-base per million (RPKM) values
were calculated using easyRNASeq output, and automatically processed using
Perl version 5.10.1. RPKM values were filtered to retain genes with a minimum
of ~50 mapped reads in one or more samples.
The threshold of 50 mapped
reads is considered the Reliable Quantification Threshold, as RPKM values for a
gene represented by 50 reads should be reproducible in technical replicates. To
avoid reporting large fold changes due to random variation of counts from low
abundance mRNA, RPKM values equivalent to a count of ≤10 reads per gene
28
were replaced with the average RPKM value equivalent to 10 reads/gene across
all the samples in the experiment. One-way ANOVA was performed and foldchanges were calculated using R version 3.0 statistical computing software. If the
mean of both groups considered in a fold-change comparison were below the
Reliable Detection Threshold (50 reads/ gene), “NA” was reported. Significant
fold changes were considered with p-value <0.05. Integrative Genomics Viewer
was used to visually verify differential changes in a selection of random genes by
comparing the visual counts of the individual reads from alignments with the raw
counts.
2.11.3 Principal component analysis
Principal component analysis was conducted by Ocean Ridge Biosciences
to visualize separation of samples and overall correlation of gene expression. All
data in this RNA-seq study are available through the Gene Expression Ominbus
(http://www.ncbi.nlm.nih.gov/geo/), Accession Number GSE57423.
2.11.4 Venn diagram
All detectable genes in Scx-/- and Scx+/+ samples were selected for
representation in a Venn diagram. Genes were considered ‘undetectable’ if the
RPKM was below the Detection Threshold for the corresponding sample. If at
least one of the gene reads from a triplicate sample set was proven undetectable
while all gene reads in the comparative sample set was proven detectable, the
gene was considered uniquely expressed in that sample. If all gene reads from
29
both triplicate sample sets had detectable RPKM values above the Detection
Threshold, the gene was considered common amongst sample groups. Genes
with at least one triplicate below the Detection Threshold in both sample sets are
not represented in the Venn diagram.
2.11.5 Clustering analysis
Genes corresponding to differentially expressed transcript clusters were
selected for hierarchical clustering, with threshold criteria of p<0.05 in a one-way
ANOVA analysis. The 862 differentially genes were clustered using Cluster 3.0
software. The log2-transformed data was pre-processed by median centering,
and then hierarchically clustered using centered correlation as the similarity
metric, and average linkage as the clustering method.
2.11.6 Alternative splicing indexes
The normalized RPKM mapped to annotated UCSC exons were determined
using easyRNASeq software. The annotations for each gene were added from
Ensembl BioMart. The exon-level RPKM values were filtered in two steps. First,
exons were discarded if their corresponding genes did not reach the Reliable
Quantification Threshold (~50 reads/ gene) in at least one sample. Second,
exons were discarded if the exons were not detected (at least one read/ exon) in
at least one sample. Prior to calculating Splicing Indexes, the exon data was
adjusted such that RPKM values of ≤1 read/exon were replaced with the RPKM
that was equivalent to 1 read/exon, as calculated from an average of all samples
30
in the data set. The Splicing Indexes were calculated based on the formula:
exon RPKM/ gene RPKM. The Splicing Index value for a given exon and sample
was replaced with “NA” if the corresponding gene count was not reliably detected
(<50 reads/gene).
ANOVA and Tukey test were performed to determine
statistically significant differences in Scx-/- vs. Scx+/+ samples, and significance of
“NA” was reported for an exon if the Splicing Index of one or more samples was
set to “NA” due to low or absent gene level expression.
2.11.7 Pathway analyses
Identified differentially expressed genes were further analyzed for the
inclusion in gene ontology and pathways order to determine the distribution of
genes amongst functional biological processes. WebGestalt software (Vanderbilt
University) was utilized for a statistics-based pathway analysis to compare the
relative distribution of genes that met specific significance criteria to the
distribution of all detectable genes. Statistical significance is based on an
adjusted p value <0.05 for enrichment of genes meeting the selection criteria,
relative to the reference genes in specific pathways. The WebGestalt software
was used to query three pathway databases including KEGG, Wiki, and GO
pathways.
Additional analyses were performed using Ingenuity Pathway
Analysis software (IPA, Ingenuity Systems, Redwood City, CA, USA).
The
annotated genes were grouped into networks, functions, and/or canonical
pathways. The txt. files with gene IDs, fold change expression, and p values
were uploaded in the software, and genes were mapped into corresponding gene
31
objects in the Ingenuity Knowledge Base. Genes with fold changes >1.5 and p
values <0.05 were used to generate a network of focus genes into global
molecular networks and predicted upstream signaling pathways. Fisher's exact
test was used to identify the most significantly (p<0.05) altered biological
functions and/or diseases within the dataset.
2.12
Twist1 siRNA knockdown in C3H10T1/2 cells
Stealth siRNA oligonucleotides (oligos) were obtained, along with Lipofectamine
from Invitrogen. The sequences of the siRNA oligos for Twist1 were 5′-UGG
CGG CAA GGU ACA UCG ACU UCC U-3′ and 3′-AGG AAG UCG AUG UAC
CUG GCC GCC A-5′. The scrambled siRNA sequences were: 5′-CGA AUC CUA
AUG CUG CUC CCU ACU U-3′ and 3′-AAG UAG GGA GGA GCA UUA CCA
UUC G-5′. C3H10T1/2 cells were plated in 6-well plates at 90% confluency and
transfected with Block-IT fluorescent oligo, Lipofectamine 2000, and either Twist1
or Scramble siRNA oligos (Invitrogen) in serum-free media according to
manufacturer’s instructions. After 6 hours, media was replaced with complete
growth media and RNA collected after 24, 48, and 72 hours using standard Trizol
protocol.
2.13
Chromatin immunoprecipitation (ChIP)
Eight canonical E-box consensus sites were identified within promoter region of
the murine mmp15 gene (NC_000074.5; Chromosome: 8; Location: 8 D1; 8
45.5cM) and conservation between mouse and human was determined using the
32
basic local alignment search tool (NCBI blast). Twist1 binding to Scx was
evaluated in C3H10T1/2 cells (n=4). Protein/DNA complexes were cross-linked for
10 minutes in formaldehyde (Sigma) at a final concentration of 0.5%. Fixed tissue
was lysed and sonicated three times for 10 seconds at 1-minute intervals
(Ultrasonic cell disruptor; Microson). For ChIP, cell lysates were incubated with
Twist1 antibody (6µg; Sigma) and incubated overnight at 4°C with gentle rocking.
Immunoprecipitation with normal rabbit IgG was used as a negative control and
ChIPs performed according to the manufacturer’s instructions (EZ ChIP,
MilliPore). Immunoprecipited and input DNA were subjected to qPCR using the
following primers to amplify six E-box-rich regions within Scx: region 1 (Forward:
TCA CCT GTG TCA CTG GCT AGA GA; Reverse: CAG CTG CTG GAA GCC
TTC ACT CC), region 2 (Forward: ACC TGG GCA TAG CAG GGA CGC TC;
Reverse: TGT CCG TTG CCT CAG TGT CTC GC), region 3 (Forward: CTT
GGC CGA GGG AGT TTG GGG; Reverse: GCC TCG ATT TGT ATC TGT GCC
C), region 5 (Forward: TCA GAC TGT AGG GCC AAC CGT TG; Reverse: GAC
CTG TGG TCC CTC AAG CCT G), region 6 (Forward: GAA TTC ATC GTA CCA
TGC CAG G; Reverse: CTT CTG GGC ACT TGA GGC TGA TC), region 7
(Forward: CCC ATG AGT GCA CAC ACA CAC AC; Reverse: GCC TGG CCA
CAC CCT GTC TGA CT). Primers for region 4 were also included as a negative
control as canonical E-box sites were not identified within this region (Forward:
ACT GCG CTG CGC ACA CTC AT; Reverse: GGT CCC GAG TGG CAT GGT
TG), and Col1a2 was used as a positive control as Twist1 has previously been
shown to bind this region (Forward: ACC GAA GCC TGG AAA GTG TA;
33
Reverse: TCC CCA CCT ACT GTC CAA AC). Four independent ChIPs were
performed and significant enrichment of E-box regions using the Twist1 antibody
over IgG control as determined by qPCR and student’s t-test (p<0.05).
2.14
Luciferase assays
The 750-base-pair proximal promoter upstream of Scx ATG start site was cloned
into the pGL3-Luc vector (pGL3-ScxPro) as described above using Sac1 and
Kpn1 (Promega) sites. Luciferase assays were performed in C3H10T1/2 cells
plated at 2×105 per well in a 24-well plate 16–20 hours prior to transfection with
Lipofectamine reagent (Invitrogen) according to manufacturer's instructions. 200
ng of pGL3-ScxPro or pGL3 (200 ng/well) and empty pcDNA or pcDNA-Twist1
(200 ng/well) were co-transfected into each well, along with 20 ng of pGL4
(Renilla luciferase, Promega). All transfections were performed in 0.5mL
OptiMem for 4 hours before the addition of 0.5 mL DMEM (Sigma) supplemented
with 4 mM L-Glutamine and 10% FBS. Cell lysates were collected 24 hours
following transfection according to the manufacturer's instructions for dual
luciferase assays (Promega). Data is represented as an average percent of
luciferase activity of the pGL3-ScxPro co-transfected with pcDNA (set at 100%)
and normalized to pGL4 Renilla signal (n = 4).
Chapter 3. The Role of Scleraxis in Regulating Valvular Extracellular Matrix
Scx was first reported for its expression patterns in the developing somites
and limb buds of mice (Cserjesi, Brown et al. 1995). Additional studies have
shown that Scx can positively promotes the cell fate of mesenchymal precursor
cells (Schweitzer, Chyung et al. 2001, Edom-Vovard, Schuler et al. 2002, Brent,
Schweitzer et al. 2003, Brent and Tabin 2004, Shukunami, Takimoto et al. 2006).
In heart valves, Scx is expressed at low levels in VP cells during early EMT
stages; however when VP cells begin to differentiate during cushion remodeling,
Scx expression is dramatically increased and remains high throughout valve
maturation and adulthood (Levay, Peacock et al. 2008). Heart valves from Scx-/mice have defects in VP cell differentiation, marked by prolonged expression of
mesenchymal genes. In addition, Scx null mice have abnormally thick valves
with defects in ECM organization including collagen fragmentation (Levay,
Peacock et al. 2008), similar to those observed in affected tendons (Murchison,
Price et al. 2007). Known signaling pathways that regulate Scx are limited, with
previous reports describing only Tgfβ-Smad (Espira, Lamoureux et al. 2009) and
mitogen-activated protein kinase (MAPK) (Smith, Sweetman et al. 2005)
signaling as upstream regulators, in cardiac fibroblasts and developing somites
respectively. Tgfβ signaling has also been shown to contribute to hypercellular
ECs and abnormal valvular ECM composition (Azhar, Runyan et al. 2009),
similar to valve phenotypes of Scx null mice. Although these upstream signaling
pathways have been shown to play a role in Scx expression and ECM deposition
34
35
in other systems, their roles have not been demonstrated in heart valves and
direct regulators of Scx have not been determined.
Twist1, a bHLH transcription factor first identified as a critical regulator of
mesoderm formation and specification (Thisse, el Messal et al. 1987), is
expressed in precursor cells within the developing pharyngeal arches, limbs, and
ECs (Chen and Behringer 1995, Firulli, Redick et al. 2007, Chakraborty, Wirrig et
al. 2010, Lee and Yutzey 2011). Twist1 promotes the proliferation, migration, and
expression of nascent ECM within these precursor cell types during early
embryogenesis. Specifically during valvulogenesis, Twist1 is highly expressed at
E12.5 during early EC formation when VP cells are extremely proliferative and
unstructured ECM is predominately expressed (Chakraborty, Wirrig et al. 2010,
Lee and Yutzey 2011). During remodeling, Twist1 is dramatically reduced as ECs
begin to differentiate and express of more specialized ECM; and its expression
remains low in mature valves throughout life (Chakraborty, Wirrig et al. 2010, Lee
and Yutzey 2011). However, Twist1 is re-expressed in mature, diseased valves
including calcific (Chakraborty, Wirrig et al. 2010) and myxomatous (Cheek,
Wirrig et al. 2012) valve disease. It is unclear whether Twist1 regulates these
developmental and disease processes, however it has been shown that Twist1
can modulate expression of downstream target genes associated with
proliferation, migration, and ECM production (Lee and Yutzey 2011) through its
function as a transcriptional repressor (Vesuna, van Diest et al. 2008).
Upstream, it has also been shown that Tgfβ signaling can induce Twist1
expression (Cho, Jeong et al. 2013) and MAPK signaling can increase
36
phosphorylation and stabilization of Twist1 protein in cancer cells (Hong, Zhou et
al. 2011). However, whether Twist1 has an intermediate role in the Tgfβ-Scx
signaling axis to regulate ECM during valve development is unknown. Given
Twist1 and Scx have opposing expression patterns and both play important roles
in conserved signaling pathways and processes during development, we
hypothesized
that
Scx
regulates
valvular
ECM
components
including
proteoglycans, and Tgfβ-Smad signaling regulates Scx while activated ERK1/2
signaling stabilizes Twist1 protein levels to promote direct repression of Scx.
In this current study, we report that valve phenotypes observed in Scx-/mice are largely attributed to significant decreases in the expression and
contribution of chondroitin sulfate proteoglycans (CSPGs) to the mature valve
leaflets (Barnette, Hulin et al. 2013). To examine the mechanisms of Scxmediated CSPG regulation, we manipulated Scx function as well as canonical
and non-canonical Tgfβ signaling pathways in embryonic avian VP cells and
mature pVICs in vitro. Using these approaches, we show that Scx is sufficient to
promote CSPG expression in both embryonic and mature valve cells. In addition,
Scx overexpression in normal hMVICs results in a molecular profile similar that
observed in MMVD. We further delineate that canonical Tgfβ-Smad signaling
positively regulates Scx-mediated regulation of CSPGs, while activated MAPK
attenuates this pathway in a Tgfβ-independent manner. We determine that MEK
activation stabilizes Twist1 protein levels in avian VP cells, but does not bind to
or transcriptionally regulate Scx. Findings from this study provide new
mechanistic insights into the role of Scx in the regulation of CSPGs in healthy
37
valve leaflets, and raise interests in Scx function as a pathological regulator of
valve disease.
3.1 Proteoglycan expression is attenuated in heart valves from embryonic
and post natal Scx-/- mice
We have previously shown that Scx-/- mice develop valvular phenotypes
associated with alterations in connective tissue organization (Levay, Peacock et
al. 2008). As proteoglycans are highly abundant in valves, particularly within the
spongiosa, we examined their expression patterns were affected in Scx null mice
using a combination of qPCR (see Table 1) and IHC (see Table 2). In AVC
regions from P1 null mice, the expression of keratin sulfates (Lumican,
Fibromodulin) and CSPGs (Brevican, Neurocan, Decorin, Biglycan) was
significantly downregulated compared to wild type
(Scx+/+) controls. No
significant changes were observed in Perlecan (heparin sulfate proteoglycan),
Aggrecan or Versican CSPGs (Figure 2A). Additional IHC analysis using a panCSPG antibody (Table 2) revealed decreased and punctate expression patterns
of CSPGs within remodeling mitral valve leaflets of post natal Scx-/- pups (Figure
2B-C). Similar findings were observed in Scx-/- mice at E15.5 and 3 months of
age (data not shown). Normal extracellular CSPG immunoreactivity was
observed in regions where Scx is not normally expressed (atria shown in Figure
2D-E). In addition, ECM array studies show that AVC regions from Scx-/- have a
decrease in essential matrix components, relative to littermate controls (Figure
38
Figure 2. Proteoglycan expression is reduced in
atrioventricular canal regions isolated from post natal Scx-/mice. (A) qPCR analysis to show fold changes in proteoglycan
gene expression in AVC regions isolated from post natal Scx-/mice compared to wild type littermate controls using primer sets
listed in Table 1. * p<0.05 using Student’s t-test, n=4. (B-E) IHC to
detect CSPG (Table 2) expression (green) in mitral valves (arrows,
B, C) and atria (D, E) from post natal wild type (Scx+/+) (B, D) and
Scx-/- (C, E) mice. Blue indicates DAPI-stained cell nuclei, red
indicates wheat germ agglutinin staining (cell membranes). mv,
mitral valve.
3). These studies suggest that Scx is important for expression of proteoglycans
and ECM composition in developing heart valves.
39
Figure 3. ECM profile array of valve regions from Scx-/- and Scx+/+
post natal mice. qPCR to show decreases in all significantly altered
genes involved in ECM and cell adhesion. All genes are significantly
downregulated as p<0.05.
3.2 Scx overexpression in embryonic VP cells and adult VICs leads to
increased CSPG expression
Our in vivo data shows that loss of Scx leads to decreased expression of
proteoglycans including CSPGs (Figure 2). To determine if Scx gain of function is
sufficient to promote CSPG expression, we utilized established embryonic avian
VP and adult pVIC in vitro systems (Lincoln, Alfieri et al. 2006, Bosse, Hans et al.
2013). In the avian system, atrioventricular ECs are isolated away from the
adjacent myocardium of HH Stage 25 chick embryos, and mesenchyme cells
within the cushions are cultured as a monolayer in the absence of cell-cell
contact. At this stage, VP cells do not express high levels of Scx and are
considered undifferentiated (Lincoln, Alfieri et al. 2006). In the porcine model,
40
Figure 4. Scleraxis overexpression in avian VP cells and porcine
valve interstitial cells promotes chondroitin sulfate proteoglycan
expression. (A) Western blot analysis to show CSPG expression in
HH Stage 25 avian heart VP cell cultures following 48 hour infection
with AdV-Scx-FLAG (Scx-FLAG) or AdV-GFP (GFP). α-Tubulin was
used as a loading control. (B) Densitometry quantitation of Western
blot shown in (A), *=p<0.05. (C-D) Immunohistochemistry to detect
CSPG expression (red) in porcine VICs cultures infected with AdVGFP or AdV-Scx-FLAG. Blue indicates DAPI-positive cell nuclei. (E)
Quantitation of CSPG immunoreactivity shown in C-D normalized to
cell number per magnification field. *=p<0.05 using Student’s t-test,
n=3.
valve cells are isolated from juvenile pigs and are therefore considered mature
fibroblast-like interstitial cells. Using these embryonic and mature valve cell
culture systems, we overexpressed Scx by infecting with a GFP-labeled
adenovirus containing full-length, FLAG-tagged mouse Scx cDNA (AdV-ScxFLAG) for 48 hours. As a control, cells were infected with empty GFP-labeled
adenovirus (AdV-GFP). Consistent with our loss of function studies, gain of
function in vitro leads to increased CSPG expression as observed by western
blot analysis of CSPG expression in avian VP cells (Figure 4A-B) and
41
immunostaining in porcine VICs (Figure 4C-E). These studies demonstrate that
Scx is sufficient to promote CSPG expression in both embryonic and mature
valve cells in vitro.
3.3 Scx and CSPG expression is positively regulated by Tgfβ2
Previous studies have shown that Scx is positively regulated by Tgfβ signaling in
fibroblasts and tenocytes (Espira, Lamoureux et al. 2009, Lorda-Diez, Montero et
al. 2009, Bagchi and Czubryt 2012, Farhat, Al-Maliki et al. 2012, Mendias,
Gumucio et al. 2012), however conserved mechanisms in valves have not been
reported. To address this, avian VP cells were treated with 200pM Tgfβ2 for 48
hours and Scx expression was examined. As shown in Figure 5A, Scx is
increased 1.7-fold (±0.14) in Tgfβ2-treated VP cells compared to controls. This
pattern was also observed in similarly treated murine mesenchymal C3H10T1/2
(54.3-fold ±2.96) and fibroblast NIH3T3 (8.2-fold ±1.21) cell lines. In support of
the positive regulation of Scx by Tgfβ2 treatment, qPCR analysis shows
decreased Scx mRNA levels in hearts isolated from E13.5 Tgfβ2+/- and Tgfβ2-/mice (Figure 5B). To further determine if Tgfβ2-mediated Scx expression
promotes CSPG expression, immunostaining was performed in treated avian VP
cells (Figure 5C, D, G) and pVICs (Figure 5E, F, H). Consistent with Scx
overexpression studies (Figure 4), Tgfβ2 is sufficient to promote CSPG
expression in embryonic and mature valve cells. Mitral valve explants from P1
Scx+/+ and Scx+/- mice were also subjected Tgfβ2 treatment to examine the
requirement of Scx for Tgfβ2-mediated regulation of CSPGs. Of the CSPGs
42
Figure 5. Tgfβ2 regulates
Scx expression in vitro and
in vivo, and promotes
chondroitin
sulfate
proteoglycan expression.
(A) qPCR to show Scx fold
changes in avian VP cells,
and C3H10T1/2 and NIH3T3
cell lines treated with Tgfβ2
for 48 hours compared to
vehicle (n=3). (B) qPCR to
show Scx expression in
E13.5 hearts from Tgfβ2+/and Tgfβ2-/- mice compared
to Tgfβ2+/+ controls. (C-D)
IHC
to
detect
CSPG
expression (red) in avian VP
cells treated with Tgfβ2 or
vehicle. (E-F) IHC to detect
CSPG expression (red) in
porcine VIC cultures treated
for 48 hours 200pM Tgfβ2 or
vehicle. Blue indicates DAPIpositive cell nuclei. (G, H)
Quantitation
of
CSPG
immunoreactivity in avian VP
cells (C, D) and porcine VICs
(E, F). (*=p<0.05 using oneway ANOVA plus a post-hoc
test n=3.) (I) qPCR to show
fold changes in aggrecan
expression in valve explants
from Scx+/+ and Scx+/- PND1
pups treated with Tgfβ2
treatment or vehicle for 48
hours.
(*=p<0.05
Tgfβ2
versus PBS, #=p<0.05 Tgfβ2
treatment in Scx+/+ versus
Scx-/- using Students t-test,
n=3).
examined (Decorin, Lumican, Versican, Biglycan), only Aggrecan expression was
significantly increased in response to Tgfβ2 treatment and this was not observed
in Scx+/- treated explants (Figure 5I). Together, these data shows that Tgfβ-
43
mediated regulation of Scx is conserved in heart valves, and this pathway is
sufficient to promote CSPG expression.
3.4 MAPK signaling attenuates Tgfβ2-mediated Scx regulation
Studies have shown that Tgfβ treatment in myofibroblasts is mediated through
canonical Smad signaling, and Smad3 functionally interacts with Scx to regulate
activity of target genes including Col1a2 (Espira, Lamoureux et al. 2009, Bagchi
and Czubryt 2012). In this study we show that Tgfβ2 treatment in avian VP cells
increases pSmad2 expression after 30 minutes (Figure 6A). In addition to
Smads, it has been shown that Scx can also be regulated by MAPK signaling in
VP cells and developing somites (Smith, Sweetman et al. 2005, Lincoln, Alfieri et
al. 2006, Zhao, Etter et al. 2007). As Tgfβ can signal through non-canonical
MAPK pathways, we examined expression levels of dpERK1/2 as an indicator of
MAPK activity. By Western blot, significant changes in dpERK1/2 levels were not
observed following Tgfβ2 treatment, further suggesting that Smad is the
downstream effector of Tgfβ2 signaling that regulates Scx in our system.
However, when C3H10T1/2 cells were pre-treated for 6 hours prior to Tgfβ2
treatment with AdV-caMEK1 (Liang and Chen 2001), a known upstream effector
of ERK1/2, Scx expression was significantly attenuated compared to those pretreated with AdV-GFP (Figure 6B). Similar co-treatment with a AdV-dnMEK1
(Bueno, De Windt et al. 2000) had no effect on the ability of Tgfβ2 to promote
Scx expression. C3H10T1/2 cells were chosen for these studies as they exhibit
embryonic mesenchymal cell phenotypes similar to VP cells (Reznikoff, Bertram
44
et al. 1973). It therefore appears that Tgfβ2-Smad signaling positively regulates
Scx expression, and Tgfβ2-independent MAPK activity can repress this pathway.
Figure 6. MEK1 activation represses Tgfβ2-mediated Scx
expression. (A) Western blot analysis to show pSmad2 and
dpERK1/2 levels in avian VP cell cultures treated with 200pM Tgfβ2
for 30 minutes, compared to BSA vehicle controls. Actin was used
as a loading control (B) qPCR analysis to show Scx expression in
murine C3H10T1/2 cells pre-infected with AdV-GFP, AdV-caMEK1,
or AdV-dnMEK1 for 6 hours prior to 48-hour treatment with 200pM
Tgfβ2 or BSA vehicle control. *=p<0.05 vs. GFP+BSA, #=p<0.05
vs. GFP+Tgfβ2 using one-way ANOVA plus a post-hoc test.
45
3.5 MAPK signaling negatively regulates Scx in VP cells
Our data shows that MAPK signaling represses Tgfβ2-mediated regulation of
Scx. To examine if MAPK activity regulates Scx in the absence of exogenous
Tgfβ2, avian VP cells were subject to infection with AdV-caMEK1 and AdVdnMEK1 for 48 hours. As confirmed by western blot, 48-hour AdV-caMEK1 and
AdV-dnMEK1 treatment successfully increased and decreased dpERK1/2
respectively in VP cells (Figure 7A). Only one band was observed when
detecting dpERK1/2 Thr202/Tyr204, consistent with previous reports using the
same avian VP cell culture system (Krenz, Yutzey et al. 2005). To determine if
altered MEK1 (and therefore ERK1) activity effects Scx expression in VP cells, a
time course of AdV-caMEK1 and AdV-dnMEK1 treatments were performed. At
48 hours post infection, a significant increase in Scx expression was observed
with AdV-dnMEK1 treatment, while in contrast Scx was decreased following AdVcaMEK1 infection (Figure 7B). In addition to changes in Scx expression, AdVcaMEK1 treatments reduced CSPG expression, while AdV-dnMEK1 infections
increased levels as determined by western blot (Figures 7C-D) and IHC (Figures
7E-G) analysis. Collectively, these data demonstrate that in VP cells, MAPK
signaling negatively regulates Scx and CSPG expression, even in the absence of
active, exogenous Tgfβ signaling.
46
Figure 7. Activated MEK1 signaling represses Scx and
chondroitin sulfate proteoglycan expression in heart VP
cells. (A) Western blot analysis to show increased and
decreased dpERK1/2 levels in avian heart VP cells infected
for 48 hours with AdV-caMEK1 and AdV-dnMEK1
respectively, compared to AdV-GFP controls. (B) qPCR to
show fold changes in Scx expression in avian VP cells
following AdV-caMEK1 and AdV-dnMEK1 infection for 4, 16
and 48 hours, compared to AdV-GFP controls (n=4),
*=p<0.05. (C) Representative Western Blot to indicate CSPG
expression in avian VP cells following AdV-GFP, AdVcaMEK1 and AdV-dnMEK1 treatments for 48 hours. (D)
Densitometry quantitation of Western blot analysis in (C),
*=p<0.05 using one-way ANOVA plus a post-hoc test. (E-G)
Immunohistochemistry to detect CSPG expression in avian
VP cell cultures infected with AdV-GFP (E), AdV-caMEK1
(F) or AdV-dnMEK1 (G).
47
3.6 Overexpression of Scx in mature human valve interstitial cells
promotes proteoglycans
We have shown that Scx overexpression in avian VP cells and porcine VICs
promotes expression of CSPGs (Figure 4). To further extend this using a more
clinically relevant model system, we infected hMVICs isolated from donor hearts
(Hulin, Deroanne et al. 2012) with AdV-Scx-FLAG, and examined levels of
several proteoglycans and collagens abundantly expressed in human MMVD. As
expected with human samples, we observed variability in basal gene expression
across the four independent non-diseased samples. However, analysis showed a
consistent trend towards increased expression of Aggrecan, Biglycan, Decorin,
Fibromodulin, Type I and II collagen, and Versican in AdV-Scx-FLAG infected
samples compared to AdV-GFP controls (Table 3). This data shows that Scx
gain of function can promote molecular phenotypes associated with myxomatous
valve disease in otherwise healthy hMVICs.
Table 3. qPCR analysis to show fold changes in gene expression in
AdV-Scx-FLAG infected human mitral VICs isolated from four donor
hearts, compared to AdV-GFP infected controls. *p=<0.05
Aggrecan
Patient
102
1.18
Patient Patient Patient
104
106
110
0.58
2.59
2.66
Biglycan
1.98
1.44
0.94
1.12
1.37±0.46
Decorin
5.66
5.38
2.60
2.25
3.97±1.80
Fibromodulin
2.17
1.39
1.36
1.07
1.50±0.47
Type I Collagen
4.84
3.01
2.16
2.20
3.05±1.25
Type II Collagen
4.54
14.62
4.10
3.66
6.73±5.27*
Versican
2.50
2.47
1.56
0.69
1.81±0.86
Average
1.75±1.04
48
3.7 Twist1 is stabilized by ERK activation and does not transcriptionally
repress Scx
To determine whether ERK1/2 activation is able
to stabilize Twist1 protein levels in valve cells,
avian VP cell cultures were treated for 16 hours
with AdV-caMEK1 or AdV-GFP. We show that
Twist1 protein levels are increased in AdVcaMEK1 treated cultures compared to AdV-GFP
controls (Figure 8B). This was concluded to be a
phosphorylation event of Twist1 protein, and not
due to an increase in gene expression, as qPCR
results showed no change in Twist1 transcript
after 16-hour AdV-caMEK1 treatment (Figure
8A).
We hypothesized that Twist1 represses
Scx
expression
valvulogenesis.
in
Given
heart
the
valves
low
during
transfection
efficiency of avian VP cells, we performed Twist1
Figure
8.
pERK1/2
stabilizes Twist1 protein.
(A) q-PCR to show 16-hour
caMEK1 treatment does
not effect Twist1 gene
expression,
while
(B)
western blot shows Twist1
protein
levels
is
significantly increased with
caMEK1/2
treatment
compared to AdV-GFP
control.
loss of function studies in embryonic fibroblast C3H10T1/2 cells, as they exhibit
similar cellular phenotypes to VP cells and have improved transfection efficiency.
Twist1 knockdown using siRNA oligos (siRNA-Twist1) showed no change in Scx
expression compared to scramble control (Ctrl siRNA), despite 95% Twist1
knockdown efficiency (Figure 9). These findings were supported by ChIP studies
that show Twist1 does not bind any of the 6 regions containing 9 conserved E-
49
box sites (Figure 10A) within
the Scx promoter. This was
determined
by
no
enrichment
between
the
Twist1 and Rabbit IgG pulldown samples relative to the
negative control, Region 4
(Figure 10B). We observe a
Figure 9. Twist1 knockdown in C3H10T1/2 cells
does not regulate Scx expression. q-PCR to 4-fold enrichment in the
show unchanged Scx expression and Twist1
knockdown after 24, 48, and 72 hours Col2a1 promoter region that
transfection with oligos targeting Twist1 or nontargeting control. * denotes significance as has
previously
been
p<0.05.
reported to have consensus
E-box sites specific to Twist1 (Chakraborty, Wirrig et al. 2010). Dual luciferase
assays were performed in which a 750-base-pair proximal promoter of Scx was
cloned into the pGL3-Luc vector (pGL3-ScxPro) and co-transfected with empty
pcDNA or pcDNA-Twist1 vectors.
Twist1 lacks the ability to transcriptionally
repress pGL3-ScxPro when co-transfected with pcDNA-Twist1, compared to
empty-pcDNA control (Figure 11). Taken together, these studies determine
Twist1 is stabilized by ERK activation in avian VP cells, however Twist1 does not
bind to or transcriptionally repress Scx.
50
Figure 10. Twist1 does not directly bind Scx
promoter.
(A) Schematic to show nine conserved E-box sites
(red lines) upstream of Scx. Primer sets were
designed to amplify 7 regions of interest (gray bar
area): 6 containing all conserved E-box sites and 1
which does not. (B) ChIP was performed on
crossed-linked DNA from C3H10T1/2 cells using
Twist1 or Rabbit IgG antibodies. DNA was
subjected to qPCR with primer sets for the
corresponding regions of interest. Primers for
Col2a1 and Region 4 were used as positive and
negative controls respectively. * denotes
significance as p<0.05.
3.8
Summary
Here, we demonstrate that Scx is both necessary and sufficient for expression of
proteoglycans including CSPGs, in both embryonic and mature valve cells.
Similarly, Scx can promote a trend towards increased gene expression of
proteoglycans and collagens in hMVICs, thereby promoting MMVD-like
phenotypes.
Dissection of the molecular pathways previously reported to
regulate Scx in other systems reveals that Scx is regulated upstream by
51
canonical Tgfβ signaling and promotes CSPG expression in heart valves.
Further, we show that activated MAPK attenuates Tgfβ2-mediated Scx
expression, and represses Scx and CSPGs in the absence of exogenous Tgfβ2.
We determine Twist1 protein levels are stabilized by MAPK-ERK signaling in VP
cells, however does not act as a transcriptional regulator of Scx. Overall these
studies support a positive role for Tgfβ-Smad signaling in regulating Scx and
proteoglycan expression in embryonic and adult valves, and demonstrate this
signaling axis can be modulated by MAPK. Further, we have identified a
signaling pathway that when altered, could underlie MMVD pathogenesis
observed in the human population.
Figure 11 . Twist1 does not repress
transactivation of Scx luciferase activity.
C3H10T1/2 cells were co-transfected with pGL3ScxPro or empty pGL3, and pcDNA-Twist1 or empty
pcDNA for 48 hours. Twist1 does not suppress Scx
luciferase activity compared to empty pcDNA control,
and normalized to renilla.
Chapter 4. Scx Loss of Function in Remodeling Heart Valves
Mature heart valve leaflets and supporting structures are largely derived
from a population of mesenchymal precursor cells within ECs that form as a
result of EMT in the AVC and OFT regions (Combs and Yutzey 2009). Defects in
generating this pool of VP cells most often result in embryonic lethality and
therefore valvular phenotypes attributed to EC-related defects are not frequently
observed at birth. Once EMT is complete, valve cells proliferate and the ECs
remodel and elongate to form primitive valve primordia structures. During the
beginning stages of valve remodeling, VP cells lose their mesenchymal
phenotype and differentiate into VICs that mediate breakdown of the ECM within
the valve primordia and secrete specialized matrix components that will later
form the mature valve structures. In the mouse, valve remodeling begins around
E15.5 and continues until post natal stages. The process of EC formation has
been well studied, however the pathways and genes critical during valve
remodeling stages are less known. We have previously shown that Scx is not
expressed in developing heart valve structures until around E15.5 during stages
of valve remodeling (Levay, Peacock et al. 2008). In E17.5 embryos null for Scx,
mesenchymal phenotypes are prolonged in valve cells, suggesting defects in VIC
maturation. By birth, the valves are thickened and the ECM is highly
unorganized, and by juvenile stages the valve leaflets are grossly malformed and
display characteristics of pathological fibrosis including excess collagen
deposition. We previously showed that Scx plays an additional role in regulating
components of the valve ECM by promoting expression of proteoglycans
52
53
(Barnette, Hulin et al. 2013). Furthermore, it was shown that heart valves isolated
from a mouse model of MMVD have increased Scx expression In addition, we
explored Scx expression in VICs from patients with MMVD. Together, these
studies show Scx is required for proper ECM composition during valve
remodeling and it may play a role in MMVD disease states. Yet, the downstream
targets and functional role of Scx during valve remodeling remain largely
unknown.
As previous studies have collectively shown Scx regulates
proteoglycans in valve cells and is required for VP cell differentiation and ECM
organization, we hypothesized that loss of Scx during remodeling stages results
in decreased expression of ECM genes and changes in regulatory gene
networks involved in cellular differentiation.
Using a whole genome RNA-Seq approach, we have identified previously
unappreciated
protein-coding
and
non-protein-coding
mRNAs
that
are
differentially expressed during valve remodeling (Barnette, VandeKopple et al.
2014). Based on our previous studies, we were surprised to see that biological
processes and molecular functions associated with valvular ECM were not
significantly altered in Scx-/- embryos at E15.5. However, we report enrichment of
mRNAs
associated
with
processes
related
to
gene
regulation
(methyltransferases, DNA binding, nucleosomal binding, miRs, signaling) and
cellular
development
(cell
assembly
and
organization).
Furthermore,
bioinformatics analysis predicted known (Tgfβ2) and novel (Onecut1) upstream
regulators of Scx in the valves at this time during embryonic development. In
addition to changes in gene expression, splicing index analysis identified several
54
mRNAs affected by alternative splicing in the absence of Scx. Together, these
findings identify genes and hierarchical networks regulated by Scx in remodeling
heart valves, and provide insights into molecular and cellular processes that
when altered could lead to disease.
4.1
Pairwise and clustering analysis distinguish E15.5 Scx-/- AVC regions
from controls
As we have previously shown that Scx is highly expressed in heart valves from
E15.5 and required for formation of valvular structures (Levay, Peacock et al.
2008), examining differential gene expression in valves from null and control
mice could provide insights into the potential function of Scx during valve
remodeling. To do this, we performed global transcriptome analysis in the AVC
regions containing mitral, tricuspid and aortic valves isolated from E15.5 Scx-/(n=3) and Scx+/+ (n=3) hearts. Samples were subject to RNA-seq using Illumina
HiSeq 2000, following confirmation of a 1523.23 ± 58.68 fold decrease in Exon 1
expression in Scx-/- samples compared to controls (Murchison, Price et al. 2007).
Annotation from Ensembl and Reliable Quantification Threshold settings (50
RPKM) resulted in a total of 18,810 detectable genes. Pairwise comparisons
between Scx-/- and Scx+/+ sample groups were made and 362 mRNAs were
found to be uniquely expressed in Scx-/- samples, 885 in Scx+/+ control samples,
and 15,650 genes were commonly expressed in both sample sets (Figure 8A);
while 2,798 genes were categorized as ‘undetected’ after Detection Threshold
criteria. These observations suggest that a total of 1,247 genes (sum of unique
55
Figure 12. Loss of Scx function in remodeling heart valves
leads to distinct transcriptome profiles. (A) Venn diagram to
show the number of detectable protein-coding and non-protein
coding mRNAs that were unique and common to Scx+/+ and
Scx-/- samples. (B) Heat map to show hierarchical clustering of
differentially expressed genes (>1.5-fold change, p<0.05) in
Scx+/+ and Scx-/- samples.
genes) are regulated in a Scx-dependent manner in remodeling heart valve at
E15.5. The top 25 most differentially expressed protein-coding mRNAs in Scx-/samples are indicated in Appendix 1, and affected non-protein-coding genes are
shown in Appendix 2.
To further examine changes in gene expression, one-way ANOVA
analysis was performed to compare significant differences in the core 18,810
detectable gene expression profiles in Scx-/- samples and Scx+/+ controls. Of
these, a total of 862 genes were differently expressed with a p-value of <0.05.
645 genes were upregulated, while 217 genes were downregulated in Scx-/-
56
samples compared to controls. To visually represent commonality or variance in
the pattern of the 862 differentially expressed genes between the two sample
groups, hierarchical clustering and heat map analyses were performed. As
shown in Figure 8B, Scx-/- samples clustered differently from controls, suggesting
indifferent gene expression profiles.
4.2
Pathway analysis reveals differentially expressed mRNAs associated
with gene regulation and cellular development in AVCs from E15.5 Scx-/embryos
To determine the biological processes and molecular functions altered by the
loss of Scx in E15.5 heart valves, pathway analysis was performed. Of the 862
differentially expressed genes that met the criteria threshold (p-value <0.05), 300
showed a significant fold change >1.5. Of these 300, 238 (157 increased, 81
decreased) genes had annotated Entrez identification numbers and were
therefore were used for subsequent pathway analysis using Gene Ontology
(GO), KEGG, Wiki pathway analyses using Ingenuity IPA software. We found
that differentially expressed mRNAs in Scx-/- versus Scx+/+ samples are largely
associated with mechanisms related to gene regulation, vitamin A metabolism
and cellular development processes (Appendix 3). These include mRNAs
associated with methyltransferases, regulatory DNA binding, and Notch
signaling, all of which have been shown to regulate expression and function of
target genes. In addition, predicted changes in 9-cis- retinoic acid-, vitamin Aand retinoic acid-biosynthesis and metabolic processes were observed.
57
Significant changes in genes associated with cell development, cell morphology,
cellular assembly and organization, and cell death/survival suggest an additional
role for Scx in remodeling heart valves. Ingenuity IPA software was used to
predict upstream regulators of Scx. Based on differential gene expression
changes (fold change >1.5, p-value <0.05) 132 targets were predicted as
upstream regulators of Scx in this system, including the known regulator Tgfβ,
(Espira, Lamoureux et al. 2009, Bagchi and Czubryt 2012, Barnette, Hulin et al.
2013) which was ranked number 2 based on p-value (Figure 9A) and Onecut1
(Figure 9B), a member of the Cut homeobox family of transcription factors
involved in DNA binding that was ranked number 1. Together these bioinformatic
approaches have revealed previously unappreciated networks and processes
that are potentially mediated by Scx in remodeling heart valves.
To determine the biological processes altered during valve remodeling,
pathway analyses were performed on genes with fold changes >1.5 and p values
<0.05. A total of 300 differentially expressed genes met the aforementioned
criteria (194 increased, 106 decreased), 240 genes were mapped to the
WebGestalt database (158 increased, 82 decreased), and 238 genes had unique
Entrez IDs (157 increased, 81 decreased). These 238 differentially expressed
genes were analyzed for inclusion in GO, KEGG, and Wiki pathway analyses.
Based on the gene list for each pathway, 2 KEGG pathways, 2 Wiki pathways,
and 16 GO categories (6 biological processes and 10 molecular functions) were
significantly affected. Appendix 3 shows significantly altered KEGG and Wiki
pathways and GO categories with their associated genes and differential
58
changes in expression.
Of the affected molecular functions and biological
processes, gene regulation remained a consistent theme. Seven molecular
functions associated with methyltransferase activity were significantly altered in
Scx-/- valve regions compared to Scx+/+ littermate controls. In addition, gene
regulation related to DNA binding and nucleosomal DNA binding was significantly
changed.
Additionally, ‘Notch Signaling Pathway’ was the most significantly
altered (Wiki, p=1.00E-03) amongst the affected KEGG and Wiki pathways.
Together these studies suggest Scx regulates several processes involved in
regulating gene transcription of valve signaling pathways during remodeling.
Further pathways analysis was performed using IPA software to examine
altered gene networks and changes in molecular and cellular functions. A total of
300 differentially expressed genes with fold changes >1.5 and p value <0.05
were mapped into its corresponding gene object in the Ingenuity Knowledge
Base. The most significantly altered molecular/cellular functions included cellular
A
B
Figure 13. Predicted upstream regulators of Scx in remodeling heart
valves. Ingenuity software analysis of differential gene expression changes
(>1.5-fold change, p<0.05) in Scx-/- samples predict Tgfβ2 (A) and Onecut1
(B) as upstream regulators of Scx in AVC regions.
59
development, cell morphology, and cellular assembly, organization, and
compromise (Appendix 3). These molecular functions are associated with related
network functions including cellular and embryonic development, cell-to-cell
signaling, tissue development, connective tissue disorders, and cell morphology.
In addition, we analyzed gene networks to gain insights into those genes that
potentially regulate Scx. The top 2 predicted upstream regulators of Scx, Tgfβ2
and Onecut1, showed significant (p<0.05) correlative gene networks (Figure 13).
IPA analysis revealed a theme of cellular development alterations with loss of
Scx function that may be regulated by parallel upstream signaling pathways.
4.3
Exon abundance is significantly altered in the absence of Scx
To determine alterations in the abundance of individual exons of detectable
genes in Scx-/- samples, exon-level expression profiling was performed using
easyRNASeq. A total of 99 protein-coding genes were significantly alternatively
spliced (p<0.05), and are shown in Appendix 4. Figure 14 plots the splicing
indices of each exon (at least 10) for the top 8 most significantly spliced genes in
our samples, including Egfl7, Hdac6 and Rnf38. As indicated by the asterisks,
Scx-/- samples show distinct exon-specific expression profiles compared to
controls, and suggest a role for Scx in post-transcriptional events during valve
remodeling.
60
Figure 14. Exon-level splicing indices of mRNAs affected by alternative
splicing events in Scx-/- samples at E15.5. Splicing indices of the top 8 mRNAs
affected by changes in exon abundance (at least 10 exons) in the absence of
Scx. * indicate significant differences in exon abundance (p<0.05).
4.4
Summary
In these studies we have identified previously unappreciated protein-coding and
non-protein-coding mRNAs that are differentially expressed in the absence of
Scx during valve development. Based on previous studies, we expected to see
biological processes and molecular functions associated with valvular ECM, but
overall these processes were not significantly altered in Scx-/- embryos at E15.5.
However, we report enrichment of mRNAs associated with processes related to
methyltransferase, DNA binding, nucleosomal binding, miRNAs, signaling, and
cellular assembly and organization.
Furthermore, bioinformatics analysis
revealed Tgfβ2 and Onecut1 as upstream regulators of Scx. Splicing index
analyses show several genes affected by alternative splicing in the absence of
61
Scx. Together, these findings identify novel genes and hierarchical networks
regulated by Scx during valve remodeling, which may provide insights into
signaling pathways altered in disease.
Chapter 5. The Role of Scx in Mouse Models of MMVD
MFS is a common, systemic connective tissue disorder that affects
approximately 1 in 5000 individuals in the United States, including men and
women of all ethnic backgrounds (Nienaber and Von Kodolitsch 1999, Pyeritz
2000). MFS is largely associated with mutations in Fbn1 and results in cardiac
defects such as MVP and MMVD (Dietz, Cutting et al. 1991, Ng, Cheng et al.
2004, Dietz, Loeys et al. 2005). Diseased myxomatous mitral valves are
pathologically thickened and characterized by an abnormal abundance of
proteoglycans throughout the valve leaflet that prevent closure and lead to
functional prolapse and regurgitation (Olsen and Al-Rufaie 1980, Cosgrove and
Stewart 1989). Although some disease manifestations in MFS can be attributed
to a structural deficiency due to Fbn1 mutations, MMVD phenotypes are less
readily reconciled by structural protein dysfunction.
In addition to its role as a structural matrix protein, Fbn1 regulates Tgfβ
signaling which has been reported to be increased in MFS patients (Ng, Cheng
et al. 2004, Geirsson, Singh et al. 2012, Hulin, Deroanne et al. 2012). Similarly,
patients with mutations in TgfβR1 develop many phenotypic features of MFS
including MMVD and MVP (Loeys, Schwarze et al. 2006). Mice harboring a
missense mutation in Fbn1 (Fbn1C1039G) serve as an established mouse model of
MFS and develop myxomatous valve phenotypes associated with increased
proteoglycans by post natal day 6.5, but homozygous (Fbn1C1039G/C1039G) mice
die soon after as a result of aortic dissection (Ng, Cheng et al. 2004). However,
Fbn1C1039G/+ (heterozygous) animals do not die prematurely and develop similar
62
63
valve abnormalities as homozygous animals.
Like the human disease, Tgfβ
activity is increased in Fbn1C1039G/C1039G and Fbn1C1039G/+ mice and mitral valve
phenotypes can be rescued by treatment with Tgfβ neutralizing antibodies
between embryonic stages E14.5-E17.5 (Ng, Cheng et al. 2004); suggesting that
MFS-related MMVD begins during embryogenesis. However, the molecular cues
in the pathogenesis of MMVD are not clear.
Tgfβ-Smad signaling has previously been reported to be upstream of Scx
and regulates its expression in cardiac fibroblasts (Espira, Lamoureux et al.
2009). Heart valves from Scx-/- mice are abnormally thick with defects in VP cell
differentiation and ECM organization (Levay, Peacock et al. 2008) and
decreased proteoglycan expression throughout the valve leaflets (Barnette, Hulin
et al. 2013). In addition, our studies show Tgfβ-Smad signaling in embryonic and
adult valve cells positively regulate Scx (Barnette, Hulin et al. 2013). These
studies show Scx is required for proper valve structure and proteoglycan
composition and is regulated by signaling pathways associated with MMVD
including Tgfβ, suggesting it may act as a physiological regulator of mitral valve
morphology. However, Scx has yet to be implicated in valve disease
pathogeneses, therefore we hypothesized that Scx is increased in the Fbn1-MFS
mouse model of MMVD , and reduced Scx function will rescue observed MMVD
phenotypes.
We determine that Scx is increased in Fbn1C1039G mice at P6.5 when
MMVD phenotypes are observed, and its expression is likely conserved in other
models of MMVD. To examine the role of Scx in the Fbn1 model of MMVD, we
64
generated Fbn1C1039G/+;Scx-/+ and Fbn1C1039G/C1039G;Scx-/+ mice, which have
reduced Scx function, to serve as rescue models of MMVD phenotypes observed
in Fbn1 mutant mice.
We show these mice are vital at P6.5 stages although
genotypes
Scx
null
for
were
not
recovered.
Heart
valves
from
Fbn1C1039G/C1039G;Scx-/+ pups have reduced proteoglycan deposition and
improved valve morphology at P6.5 compared to Fbn1 mutants with normal Scx
function. These studies are the first to report Scx as a potential physiological
regulator of MMVD phenotypes.
5.1 Scx is increased in valves from a MFS mouse model of MMVD
Mice
carrying
a
homozygous
or
heterozygous knock-in mutation for
Fbn1 (Fbn1C1039G) serve as a model
for
MFS-associated
MMVD
and
develop myxomatous changes in mitral
valves by P6.5 (Ng, Cheng et al. 2004).
To determine if Scx expression is
altered in this established model of
MMVD, qPCR was performed on AVC
regions
isolated
Fbn1C1039G/C1039G and
mice.
from
Fbn1
P6.5
C1039G/+
Figure 15. Scx is increased in mitral
valve regions from Fbn1 mutant
mice. qPCR to show increased Scx
expression in AVC regions isolated
from P6.5 Fbn1C1039G/C1039G mice,
compared to wild type littermate
controls, N=4. *=p<0.05 using oneway ANOVA plus a post-hoc test.
As shown in Figure 15, Scx expression is significantly increased in
Fbn1C1039G/C1039G mice at P6.5 compared to controls. Preliminary data from 10-
65
Figure 16. Breeding diagram for generation of rescue and control mice.
Fbn1C1039G/+ and Scx-/+ mice were bred to generate Fbn1C1039G/+;Scx-/+ mice
to generate two experimental rescue models, Fbn1C1039G/+;Scx-/+ (green box)
and Fbn1C1039G/C1039G;Scx-/+ (red box); along with their respective littermate
controls (underlined).
month-old conditional knockout Fln-A mice (Sauls, de Vlaming et al. 2012,
Supplemental Figure 1) and hMVICs (Hulin, Deroanne et al. 2012, Supplemental
Figure 2) suggest this increase in Scx expression could be conserved across
models of MMVD. These studies show increased Scx in a mouse model of
MMVD, suggesting a potential cause-and-effect relationship between Scx and
MMVD phenotypes, however, warrants further examination.
5.2
Loss of Scx function rescues valve phenotypes in a MFS mouse
model of MMVD
To
examine
the
necessity
of
Scx
function
in
MMVD
phenotypes,
Fbn1C1039G/+;Scx-/+ and littermate control mice were generated by intercrossing
Fbn1C1039G/+;Scx-/+
mice
(Figure
16).
Viable
Fbn1C1039G/+;Scx+/+
mutant,
66
A
B
Fbn1
C1039G/+;
Scx
Fbn1
+/+
C1039G/+;
Scx
-/+
Figure 17. Loss of Scx decreases proteoglycans in mitral
valves from Fbn1C1039G/+ mice. Alican blue staining shows
mitral valves from 7-week old Fbn1C1039G/+;Scx-/+ mice (B)
have less proteoglycans and reduced leaflet length compared
to Fbn1C1039G/+;Scx-++ controls (A).
Fbn1C1039G/+;Scx-/+ rescue, and Fbn1+/+;Scx+/+ control P6.5 mice were generated
with anticipated deviations in Mendelian ratios in Scx-/- neonates as previously
described (Levay, Peacock et al. 2008) (Table 4). As Scx null mice also have
underlying valve phenotypes including thickened valves (Levay, Peacock et al.
2008)
and
ECM
proteoglycan
defects
(Barnette,
Hulin
et
al.
2013),
Fbn1C1039G/+;Scx-/+ mice serve as a rescue model to achieve loss of Scx function
without introducing new valve histopathological phenotypes. Alcian blue staining
on heart valves from P6.5 Fbn1C1039G/+;Scx-/+ rescue mice show decreased
proteoglycan deposition and reduced valve leaflet length compared to
Fbn1C1039G/+;Scx+/+ mutant mice (Figure 17). In addition H&E staining to assess
overall
valve
morphology
shows
a
reduction
in
valve
thickness
in
Fbn1C1039G/+;Scx-/+ mice compared to Fbn1C1039G/+;Scx+/+ mutants (Figure 18).
Further studies to define overall valve morphology including leaflet volume,
examine gene expression patterns, and functional rescue are necessary;
67
however, these studies show reduced Scx function partially rescues MMVD
phenotypes in a murine model of MMVD.
Figure 18. Loss of Scx rescues morphological MMVD
phenotypes observed in valves from Fbn1C1039G/+ mice. H&E
staining of mitral valves from P6.5 Fbn1C1039G/+;Scx-/+ mice (B)
show reduced valve thickness with the loss of Scx compared to
Fbn1C1039G/+;Scx+/+ mutants (A).
5.3
Summary
We show that mitral valve regions from Fbn1C1039G mutant mice have increased
Scx expression, which may be conserved in other models of MMVD including
Fln-A mutant mice, and MMVD patients. Further, we demonstrate that reduced
Scx function in the Fbn1-mutant mouse model of MFS is sufficient to partially
rescue the myxomatous valve phenotypes observed at P6.5, including reduced
proteoglycan deposition and decreased valve thickness and leaflet length.
Additional analysis of these rescue affects is necessary to elucidate a deeper
mechanism for Scx in this model of MMVD. However, these studies provide a
framework for examining the role of Scx in models of myxomatous degeneration,
and suggest Scx loss of function may improve mxomatous phenotypes observed
in various MMVD models.
68
Fbn1-/;Scx+/+
3/22
Fbn1-/;Scx-/+
0%
0/22
Fbn1+/+;
Scx-/-
12.5%
0%
0/22
Fbn1;Scx-/-
6.25%
0%
0/22
Fbn1-/;Scx-/-
Ratios
N
Table 4. Mendelian ratios of P6.5 neonatal mice from Fbn1-/+;Scx-/+ intercross breeding scheme
Fbn1+/+;
Scx-/+
2/22
13.6%
6.25%
/+
Fbn1;Scx+/+
1/22
9.1%
12.5%
/+
3/22
4.5%
6.25%
Fbn1;Scx-/+
12/22
13.6%
12.5%
/+
1/22
54.5%
12.5
Fbn1+/+;
Scx+/+
4.5%
25%
Expected
Ratios
6.25%
Chapter 6. Discussion
To date, studies defining the role of Scx have focused on connective tissues of
high mechanical demand; with initial work dedicated to tendons (Cserjesi, Brown
et al. 1995, Schweitzer, Chyung et al. 2001, Edom-Vovard, Schuler et al. 2002,
Brent and Tabin 2004, Shukunami, Takimoto et al. 2006), and more recent
studies extending to cardiac fibroblasts and heart valves (Levay, Peacock et al.
2008, Espira, Lamoureux et al. 2009, Bagchi and Czubryt 2012, Barnette, Hulin
et al. 2013). Interestingly, defects in ECM organization and cell differentiation are
commonly observed in all these affected structures in Scx null mice (Schweitzer,
Chyung et al. 2001, Levay, Peacock et al. 2008, Espira, Lamoureux et al. 2009,
Bagchi and Czubryt 2012, Barnette, Hulin et al. 2013), however the underlying
causes are not understood but likely conserved. In heart valves, these
phenotypes begin in the developing embryo and by birth valves are abnormally
thick and progressively worsen over time (Levay, Peacock et al. 2008). In order
to define the roles that Scx plays in connective tissue systems and identify how
loss of function gives rise to valve anomalies, we performed a host of in vitro and
in vivo studies to uncover the mechanism and function. Using these approaches,
we have identified previously unappreciated signaling pathways and downstream
targets that are regulated by Scx during development. Our studies have also
elucidated several mechanisms of Scx regulation that are conserved in
embryonic and adult valve cells, and implicated Scx in disease models on MMVD
including human patients. Together, these findings provide new insights into the
69
70
mechanisms of Scx function in heart valves that could have consequences in
disease pathogenesis.
6.1
Role of Scx signaling in ECM regulation during heart valve
development
6.1.1 Scx regulation of proteoglycan expression in heart valves
It is well described that an abnormal abundance of proteoglycans, including
CSPGs, are a histological hallmark of myxomatous valve disease, however
mechanisms that establish and maintain proteoglycan homeostasis in healthy
developing and mature valve structures have not been described. In this study
we identify the bHLH transcription factor Scx as a regulator of CSPG expression
in immature VP cells and mature VICs (Figures 2 and 4). In vitro, this is mediated
upstream by Tgfβ2-Smad signaling (Figures 5 and 6). In vivo, Tgfβ2 (and Tgfβ3)
is highly expressed in VICs from early remodeling stages (Molin, Bartram et al.
2003), consistent with Scx expression (Levay, Peacock et al. 2008). However,
Tgfβ1 is also sufficient to promote Scx in muscle and cardiac fibroblasts (Espira,
Lamoureux et al. 2009, Lorda-Diez, Montero et al. 2009, Bagchi and Czubryt
2012, Farhat, Al-Maliki et al. 2012, Mendias, Gumucio et al. 2012) and therefore
as a secretory growth factor, it is plausible that Tgfβ1 from surrounding valve
endothelial cells (Molin, Bartram et al. 2003) could act upon Scx in VICs in vivo.
Consistent with Tgfβ1 as a positive regulator of Scx, we show that Scx is reduced
in hearts from Tgfβ2-/- mice (Figure 5B). Interestingly, Tgfβ2-/- mice have valve
remodeling defects associated with leaflet thickening and increased proteoglycan
71
deposition by E18.5 (Azhar, Brown et al. 2011); contradictory to the proposed
mechanism presented from this study (Figure 5). However, VIC proliferation is
increased in Tgfβ2-/- mice from as early as E14.5, and therefore it is possible that
the overabundance of proteoglycans is secondary to increased cell number, and
independent of reduced, but not absent Scx expression (Figure 5B). Our data
shows that Brevican, Neurocan, Decorin, Biglycan and not Aggrecan are
significantly reduced in valves from Scx-/- mice (Figure 2). However, only
Aggrecan is significantly increased in response to Tgfβ2 treatment of post-natal
mitral valve explants (Figure 5). Although consistent with previous tendon studies
(Robbins, Evanko et al. 1997), this data does not completely elucidate how the
Tgfβ2-Scx signaling axis regulates proteoglycan gene expression in heart valves.
As Tgfβ2 signals through multiple pathways and Scx heterodimerizes with other
bHLH transcription factors to elicit its downstream effects (Cserjesi, Brown et al.
1995, Furumatsu, Shukunami et al. 2010), it is considered that similar to previous
findings in tendons and cardiac fibroblasts (Espira, Lamoureux et al. 2009,
Lorda-Diez, Montero et al. 2009, Bagchi and Czubryt 2012, Farhat, Al-Maliki et
al. 2012, Mendias, Gumucio et al. 2012) other Tgfβ effectors and downstream
cofactors including ERK1/2 and bHLH E-proteins may reconcile this differential
CSPG expression in the valves.
While direct target genes regulated by Scx in heart valves remain
unknown, Scx has previously been shown to regulate ECM matrix proteins in
other systems.
In developing chick limbs, Scx gain of function promotes
Tenascin and Tendomodulin; two glycoproteins highly expressed in tendons
72
(Edom-Vovard, Bonnin et al. 2001, Edom-Vovard, Schuler et al. 2002,
Shukunami, Takimoto et al. 2006). Although these studies have been informative
in identifying genes that change in response to Scx function, direct regulation
was not been reported. More recently, Czubryt and colleagues demonstrated
molecular interactions and transactivation of Scx with E-box sites within the
proximal promoter region of Col1a2 in cardiac fibroblasts (Bagchi and Czubryt
2012). This study also showed that Scx-mediated regulation of Col1a2 is induced
by Tgfβ1 signaling, and dependent on Smad3 (Bagchi and Czubryt 2012). In this
current study, the mechanism of how Scx regulates proteoglycans in VP and
mature interstitial cells is not yet clear. It is suggested that similar to Col1a2, Scx
regulates specific proteoglycan genes (Figure 2A) through identified conserved
E-box binding sites. Scx may not regulate the transactivation of CSPGs alone,
but form multi/hetero dimers with known bHLH co-regulators including E2A
proteins E12 and E47 (Espira, Lamoureux et al. 2009, Bagchi and Czubryt 2012).
Formation of the stratified valve structures begins in the embryo with
localized secretion of collagens, proteoglycans and elastins by VP cells within the
developing tri-laminar layers.
Perturbations in this process either during
development, or after birth can lead to alterations in ECM distribution, improper
valve biomechanics, and valve dysfunction.
In myxomatous valve disease,
changes in ECM abundance are associated with an abnormal increase in
proteoglycans (Gupta, Barzilla et al. 2009), and this is commonly observed in
patients with MFS. In mice null for Scx, ECM organization is perturbed and
valves are significantly thickened from as early as E16.5, with a decrease in cell
73
number (Levay, Peacock et al. 2008); however, as shown in Figure 2,
proteoglycans are reduced. Therefore, we speculate that thickening is the result
of observed collagen fiber fragmentation and increased collagen deposition
(Levay, Peacock et al. 2008) that may be reflective of a fibrotic valvulopathy.
The signaling pathways that regulate Scx during development and homeostasis
may mediate the causative histopathologies.
6.1.2 Signaling pathways regulating Scx in developing heart valves
Studies have shown that formation of highly organized valve structures is
dependent on the tight regulation of signaling pathways in a temporal and spatial
manner (Lincoln and Yutzey 2011). In this study we not only identify Tgfβ2-Smad
signaling as a positive regulator of Scx and CSPG expression in heart valves
(Figure 5), but show that MAPK signaling converges onto this pathway to have a
negative effect (Figures 6 and 7). As Tgfβ2 treatment does not affect ERK activity
(Figure 6A) and MEK1 regulates Scx in the absence of Tgfβ2 treatment (Figure
7B), it is likely that MAPK can function as a repressor of Scx in a Tgfβ2independent manner. Our findings show that direct activation of ERK1/2
negatively regulates Scx, while reduced ERK1/2 activity increases Scx (Figure
7B). In mesenchymal precursor cells of the developing somites the opposite is
observed, as active dpERK is crucial for Scx expression (Smith, Sweetman et al.
2005). However, increased ERK activity also induces expression of the dual
specificity phosphatase Mkp3. These studies introduce a negative feedback loop
pathway that appropriately downregulates ERK-induced Scx activation to restrict
74
its expression during precursor cell specification and differentiation (Smith,
Sweetman et al. 2005). In contrast to somites, increased Scx was not observed
in VP cells at 4, 16 or 48 hours following AdV-caMEK1 infection (Figure 7B), and
therefore we are doubtful that similar feedback mechanisms are conserved
between these two precursor cell populations. However, it cannot be excluded
that phosphatase activity is important for modulating ERK1/2 activity in valves in
order to regulate appropriate levels of Scx and establish formation of the
proteoglycan-rich spongiosa layer.
Findings in Figure 7 suggest that direct manipulation of MEK1 suppresses
Scx in the absence of exogenous Tgfβ2 signaling, however there are several
pieces of data to suggest that ERK1/2 kinase function does not directly regulate
Scx expression through protein phosphorylation events. Firstly, manipulation of
MEK1/2 leads to changes in Scx at the transcript level. Second, prediction
software did not reveal ERK1/2 phosphorylation sites within the Scx sequence.
Third, decreased Scx expression was not observed until 48 hours after AdvcaMEK1/2 treatment, which is longer than anticipated for a phosphorylation
event. It was therefore considered that dpERK1/2 could positively regulate a
repressor, or negatively regulate an activator of Scx in a signaling cascade
independent of Tgfβ activity. However, our data in Figure 6B also suggests that
dpERK1/2 attenuates Tgfβ2-Smad-mediated activation of Scx and therefore
when Tgfβ signaling is active, ERK1/2 converges onto this signaling pathway.
While it remains unclear how this occurs, crosstalk between MAPK and Smad
has been reported in Xenopus (Kretzschmar, Doody et al. 1997) and murine cell
75
lines (Hulin, Deroanne et al. 2012) through ERK-mediated phosphorylation of the
Smad linker region that has been shown to both suppress (Kretzschmar, Doody
et al. 1999) and increase (Hulin, Deroanne et al. 2012) transcriptional activation
of downstream target genes.
Given the likelihood of an intermediate regulator of the ERK-Scx signaling
pathway, we examined Twist1 as a direct upstream regulator of Scx, as it has
opposing expression patterns and is involved in similar developmental processes
during valvulogenesis. We show that MAPK signaling is sufficient to stabilize
Twist1 levels in heart valves (Figure 8); however Twist1 does not bind (Figure
10) or transcriptionally repress (Figure 11) Scx, and loss of function fails to
increase Scx expression (Figure 9).
Although avian VP cells and murine
C3H10T1/2 embryonic fibroblast cells have similar cellular phenotypes, it is
considered that Twist1 binding to the Scx promoter is a species- and/or cellspecific process. Additionally, the potential for Twist1 to transcriptionally repress
Scx could be less localized than the selected 750bp promoter, as Twist1 has
been previously shown to transcriptionally regulate target genes by binding to Ebox sites up to 15-20 Kb upstream of the transcriptional start site (Lee and
Yutzey 2011). Previous studies suggest Twist1 not only promotes proliferation
and migration but also ECM gene expression (Chakraborty, Wirrig et al. 2010),
which is inconsistent with its proposed role as a Scx repressor. As Twist1 does
not seem to function as a direct upstream regulator of Scx, further studies to
examine other regulators of Scx is necessary to elucidate its role in valve
development and disease.
76
6.2
Implicating Scx function in mechanisms of myxomatous valve
phenotypes in MMVD
Genetic causes of MFS (Fbn1 mutations) and the MFS-like condition Loeys-Dietz
syndrome (Tgfβ receptor 1/2 mutations) result in increased Tgfβ signaling (Dietz,
Cutting et al. 1991, Dietz, Loeys et al. 2005). Affected Fbn1C1039G mice (and
humans (Matt, Schoenhoff et al. 2009)) show significant increases in Tgfβ
signaling, and treatment with neutralizing antibodies during stages of embryonic
EC remodeling (E14.5-E17.5) rescues mitral valve defects (Ng, Cheng et al.
2004). This suggests that increased Tgfβ signaling underlies disease
pathogenesis, and MMVD has origins during valvulogenesis and in particular
stages of cushion remodeling. Interestingly, both Smad2/3 and Erk1/2 are
increased in Fbn1C1039G mice and MFS patients due to the paradoxical activation
of Tgfβ signaling (Holm, Habashi et al. 2011). In this study we observed only a
subtle, but significant decreased in Scx expression (~30%) in E13.5 hearts from
Tgfβ2+/- and Tgfβ2-/- mice (Figure 5). This could be attributed to compensation by
other Tgfβ ligands, but could also be the result of an imbalance in the regulation
of Scx by Erk1/2 and Smad2/3.
The role of Scx in MMVD has not been reported, yet we have identified a
signaling pathway that when altered, could underlie myxomatous mitral valve
pathogenesis observed in the human population. Although alterations in Scx
expression or mutation in its gene has not been established in human MMVD,
these studies show that Scx expression is increased in a MFS mouse model of
MMVD (Figures 11), which may be conserved in other non-syndromic MMVD
77
models (Supplemental Data). Reduced Scx function in the MFS model of MMVD
resulted in an overall reduction in proteoglycan deposition, and decreased valve
leaflet length and thickness during stages of initial MMVD phenotypes (Figures
14 and 15); however, no rescue in functional defects at adulthood. As these
studies only preliminarily examine the function of Scx in this model, it is
considered that more depth examinations may unveil subtle rescue effects that
may have not been appreciated. However, it is noteworthy to highlight that
MMVD can exist in a stable form without significant physiological consequences,
which may contribute to the lack of any functional changes that may be
observed.
Interestingly, both Fbn1 mutant and Fln-A deficient models are
associated with increased Tgfβ signaling (Ng, Cheng et al. 2004, Sauls, de
Vlaming et al. 2012); recapitulating observations made in valves surgically
removed from MMVD patients at the time of replacement surgery (Akhtar, Meek
et al. 1999, Radermecker, Limet et al. 2003, Gupta, Barzilla et al. 2009). This
suggests a role for Scx in myxomatous phenotypes in human patients. However,
it is important that these studies include in-depth analyses of activated pathways
and downstream targets to define the molecular signals that may attribute to the
morphological changes observed. Given both Fbn1 and Fln-A mouse models are
related to syndromic and non-syndromic origins of myxomatous degeneration
respectively, these studies also suggest that the Scx signaling pathway may play
a conserved role in various etiologies of MMVD in the human population. These
studies set the groundwork to purposefully explore the role of Scx in models of
MMVD.
78
6.3
Gene networks regulated by Scx in remodeling heart valves
Many of our studies have examined the role of Scx in ECM remodeling in
the context of myxomatous degeneration and disease. As increasing evidence
suggests that valve malformations observed in adult patients has origins
stemming from embryonic development (Dietz, Cutting et al. 1991, Kuivaniemi,
Tromp et al. 1997, Li, Toland et al. 1997), we aimed to elucidate the role of Scx
at initial developmental valve remodeling to uncover new regulatory networks
that may be altered in disease. Of the 862 differentially expressed genes
identified in E15.5 Scx-/- embryos, 645 (74.8%) were upregulated, therefore
suggesting that similar to other bHLH proteins, Scx largely functions as a
transcriptional repressor. However, it is unclear which downstream stream genes
are direct or indirect targets of Scx; and as Scx has not previously been shown to
be a repressor of its target genes, further gain of functions studies will provide
more evidence of its function. While repressive function of Scx on target DNA is
suggestive, this study has identified additional functions of Scx in mediating gene
regulation (Barnette, VandeKopple et al. 2014). Based on differential gene
expression changes, processes associated with methyltransferase activity were
significantly affected in the absence of Scx (Appendix 3). This includes
decreases in Dot1l (0.66-fold) and Mll2 (0.52-fold) which mediate methylation of
histones to silence genes (Singer, Kahana et al. 1998, Feng, Wang et al. 2002,
Milne, Briggs et al. 2002, Janzen, Hake et al. 2006), therefore fitting with the
overall increased gene expression in this study. In addition, several miRNAs
79
were significantly decreased (miR432 (0.09-fold), miR-700 (0.1-fold), miR-692-1
(0.35-fold)) which could also contribute to relieved post-transcriptional gene
repression. However, using Panther and Target Scan software we were unable
to identify conserved seed sequences for these miRNAs in predicted target
genes that were increased in Scx-/- embryos. Therefore, this suggest that these
Scx-dependent miRs are either acting indirectly on the increased gene set, or
their decrease in expression is independent of the differential gene expression
findings. In contrast to decreased miRs, miR-758, miR-134 and miR-27b were
significantly increased.
Interestingly miR-758 is predicted to bind conserved
seed regions within Collagen type 4 alpha 1, a basement membrane collagen
type that was found to be significantly decreased (0.67-fold, p=5.29E-03) in Scx-/embryos.
We also used Ingenuity Pathway Analysis software to predict potential
upstream regulators of Scx, including the mostly highly ranked Onecut1 (Figure
9B), a transcription factor previously shown to play roles in regulating gene
expression (Yamamoto, Matsuoka et al. 2013). Onecut1, also known as
hematocyte nuclear factor 6 (Hnf6), is highly expressed in the liver where it
regulates gene transcription. However, Onecut1 is also expressed in the heart
and somites, similar to Scx, and has been shown to play a role in cell fate
decisions in precursor cells by inhibiting Tgfβ signaling in hepatocytes and biliary
cells (Clotman, Jacquemin et al. 2005). Interestingly, Onecut1 has been shown
to regulate cell-matrix adhesion and cell migration (Margagliotti, Clotman et al.
2007) and differentiation in the liver (Jacquemin, Durviaux et al. 2000), which are
80
the key cellular events occurring during heart valve remodeling when Scx
expression is first initiated. The role of Onecut1 in heart valves have not been
determined, however previous roles for Onecut1 in other systems suggest
Onecut1 may have a unique regulatory role in Scx transcription in the valves, as
Scx is not highly enriched in the liver.
Although Tgfβ signaling has been
previously shown to regulate Scx expression, no direct regulators of Scx
transcription has been established, however the Onecut1 may directly regulate
Scx through its DNA binding functions.
Our analysis also revealed significant decreases in Dll4 (0.61-fold) and
Ncor2 (0.46-fold), which led to associated changes in Notch signaling as
determined by Gene Ontology. While Notch is an important player in valve
development and disease (MacGrogan, Luna-Zurita et al. 2011), a specific role in
valve remodeling or associations with Scx have not been made. Interestingly in
data not shown, Scx was unable to increase activity of the Notch intracellular
domain (NICD) in porcine VICs, and therefore we can only speculate that the
Notch signaling pathway may indirectly regulate of Scx. These findings, based
on
RNA-seq
and
bioinformatics
analyses,
have
identified
previously
unappreciated roles for Scx in the regulation of gene expression. As previous
studies have shown that valve development requires tight control of growth
factors, transcription factors, and ECM proteins (Combs and Yutzey 2009),
unveiling possible epigenetic events regulated by Scx could provide important
new insights into disease mechanisms.
81
In remodeling heart valves around E15.5, VP cells are transitioning from a
‘primitive’ mesenchyme cell phenotype towards an activated VIC phenotype. This
is characterized by loss of mesenchyme cell markers and maintenance of SMA,
an established marker of activated VICs (Schoen 2008). As a myofibroblast-like
cell, activated VICs exhibit an organized actin cytoskeleton and express focal
adhesion proteins (Schoen 2008, Li, Goodwin et al. 2013). As mentioned,
mesenchyme cell markers are persistently expressed in valves isolated from Scx/-
embryos at E17.5, suggesting defects in VIC activation (Levay, Peacock et al.
2008). In an activated state, VICs mediate remodeling of the valve connective
tissue, which is tightly controlled and required for embryonic development.
However, adult VICs are quiescent and therefore abnormal activation propagates
pathogenic remodeling, leading to disease (Rabkin, Aikawa et al. 2001, Schoen
2008). In this study, mesenchyme cell markers were not increased in Scx-/embryos at E15.5 contrary to observations made in E17.5 Scx-/- embryos, and
this discrepancy may be due to differences in the time points examined in our
two studies. However, we do see significant changes in several genes
associated with assembly and maintenance of the actin cytoskeleton at E17.5.
These include Phactr1 (0.55-fold), Plectin (0.66-fold), Fap (1.65-fold), Actn4
(0.75-fold), Parvb (0.40-fold), in addition to mRNAs that regulate cell adhesion
and migration (Efna5) associated with cellular assembly and organization
processes (Appendix 3). Therefore, it is considered that Scx may play a
significant role in mediating activated VIC phenotypes, which is not only essential
82
for valve development but also in the initiation of disease processes in adult
valves.
Previous work has shown that Scx plays a major role in regulating ECM
gene expression and organization in heart valves, tendons and cardiac
fibroblasts (Schweitzer, Chyung et al. 2001, Levay, Peacock et al. 2008, Espira,
Lamoureux et al. 2009, Bagchi and Czubryt 2012, Barnette, Hulin et al. 2013).
However to our surprise, molecular processes and biological functions
associated with connective tissue were not significantly altered in Scx-/- embryos
at E15.5, although we did observe differential expression of ECM-related genes.
In addition, Tgfβ was predicted as an upstream regulator of Scx (Figure 9A),
which has previously been shown to mediate Scx-dependent expression of ECM
genes (Espira, Lamoureux et al. 2009, Bagchi and Czubryt 2012, Barnette, Hulin
et al. 2013). In these studies, we also show that heart valves from post-natal
Scx-/- mice have decreased proteoglycan content (Figure 2) (Barnette, Hulin et al.
2013), and previous studies by The Czubryt group showed regulation of Col1a2
by Scx in cardiac fibroblasts isolated from adult rats (Espira, Lamoureux et al.
2009). Therefore, we speculate that Scx-mediated regulation of the ECM is
temporal and most important after birth in the valves and myocardium.
These studies have shed light on several new roles for Scx in remodeling
heart valves that could be applied to other connective tissue systems. In addition,
we have generated a profile of protein-coding and non-protein-coding mRNAs
whose expression is dependent upon Scx function. Many of these are associated
with gene regulation and cellular development functions, however it is not yet
83
clear which genes are directly, or indirectly regulated by Scx. Nonetheless,
creating this transcriptome has not only provided a comprehensive list of mRNAs
expressed in healthy remodeling heart valves (Scx+/+), but given a direction for
future studies to identify how defects during embryonic development cause valve
disease after birth or later in life.
6.4 Summary and working model of Scx signaling in heart valves
We show that Scx plays a role in gene regulation and cellular processes
including DNA binding, methyltransferases, and microRNAs.
In addition, we
have predicted Oncecut1 as a potential upstream regulator of Scx transcription.
We show that Scx is required for proper expression proteoglycans in hearts
valves, and canonical Tgfβ signaling positively regulates Scx and proteoglycan
expression in embryonic and adult valve cells. However, we determined that
ERK1/2 activation represses that ability of Tgfβ signaling to regulate Scx and
proteoglycan expression, and this signaling represses Scx in the absences of
exogenous Tgfβ. Findings from these studies have uncovered a new role for Scx
in heart valve development, homeostasis, and disease.
These studies were
performed in multiple model organisms including avian, murine, porcine, and
human species, using both in vitro and in vivo approaches. As Scx has been
identified in the avian system that allows for adequate amounts of tissue at the
same embryonic time point, we are able to use this in vitro culture system to
elucidate the signaling mechanism in valve cells. The murine model allows for us
to examine Scx signaling in vivo and ex vivo at both embryonic and adult stages
84
Figure 19. Tgfβ- and ERK-mediated regulation of Scx
in normal and diseased heart valves. We postulate
that in normal valves (Top) dpERK1/2 activates an
intermediate transcriptional repressor of Scx and inhibits
the Tgfβ pathway at the Smad level. Subsequently, Scx
promotes optimal expression of regulatory genes during
embryonic remodeling that help to establish and maintain
ECM throughout adulthood. However in myxomatous
valves (Bottom), increased Tgfβ2 causes a surge in
Smad2 activation and results in increased Scx and excess
production of ECM proteoglycans which compromises
valve integrity and function.
of valve development.
However valve leaflets from mice do not provide
adequate cells for in vitro cultures, therefore we used a porcine model for
functional experiments to determine the role of Scx in adult valve cells.
Unfortunately, assessing Scx expression in the porcine model comes at a
85
disadvantage, as the Scx gene has not be characterized in this species.
Examining the Scx signaling pathway in valve cells from human patients allows
for a clinically relevant model and suggests the proposed signaling pathway may
be conserved in humans. Using these various models allowed us to develop a
working model (Figure 19) for the Scx signaling pathway in development and
disease; however as a caveat, it is considered Scx may function in different
mechanisms across these species.
The general conclusions of studies suggest in normal heart valves, basal
Tgfβ2 levels bind to TgfβRII receptors to activate and phosphorylate receptorregulated effector Smad2. Activation of Smad2 allows its complex formation with
Smad4, which translocate to the nucleus, binds to Smad sequences within the
promoter of target gene Scx to transcriptionally regulate its expression.
To
modulate Scx expression, activated ERK1/2 signaling is able to converge on the
Tgfβ pathway by potentially inhibiting Smad2/Smad4 complex translocation to the
nucleus, and thus reducing Scx expression.
As MAPK signaling has been
predicted to regulate Oncecut1, we hypothesize that during valve development
Oncecut1 is activated by dpERK1/2 to regulate Scx and cellular proliferation and
differentiation (Figure 19, top). However in MMVD, there is an increase in free
Tgfβ2 due to changes in hemodynamic flow and/or defects in critical ECM
components such as Fbn1. This increase in Tgfβ signaling results in an
overwhelming activation of Smad2 that increases Scx levels, and subsequent
misexpression of proteoglycans (Figure 19, bottom). Proteoglycan deposition
eventually leads to functionally insufficient valves that present as regurgitation.
86
Although these signaling events are completely defined, further elucidation of this
working model may lead to a deeper understanding of valve signaling in
development and disease to better advance therapeutic treatments.
6.5
Perspectives and clinical applications
The incidence of valve disease remains high in numbers, however effective
treatments remain relatively limited to surgical intervention. Clinical indications for
valve replacement include valve insufficiency, ventricular dysfunction, or exercise
intolerances in asymptomatic patients (Hinton and Yutzey 2011). Bioprosthetic
valve replacement has become the intervention of choice however lack the ability
to grow and remodel in vivo over time, ultimately resulting in failure. The longterm goal is to generate therapeutic alternatives that bypass the need for highrisk, costly open-heart surgery. As valve disease is often associated with
changes conserved in development, therapeutic strategies focused on markers
of these activated pathways may improve management for patients with valve
disease. Early identification of valve disease will allow for prompt intervention,
rather than treating aggressive, late-stage disease. With emerging studies
suggesting that valve disease is associated with ECM alterations, it seems
promising to focus future studies on the genes and signaling pathways
responsible for establishing and maintaining proper valve connective tissue.
However, the complex mechanism of these genes and pathways require a more
complete understanding.
87
Identifying signaling pathways that mediate onset or progression of
myxomatous valve disease is critical for the development of new treatments.
Although approaches in animal models have focused on inhibiting Tgfβ signaling,
clinical trials using pan Tgfβ-neutralizing antibodies cannot be readily translated
to treatment with human disease in the absence of FDA-approved humanized
antibodies that block Tgfβ (Judge, Rouf et al. 2011).
As there is extensive
interaction between Tgfβ and angiotensin II signaling, studies examining
angiotensin II blockers as potential therapeutics for MFS have been inconclusive
and aimed at other cardiovascular defects outside of valve disease (Judge, Rouf
et al. 2011).
In severe cases of MVP, β-adrenergic receptor blockers are
commonly used in clinical practice; however, these therapies do not treat valve
pathologies, only help to ameliorate the secondary ventricular defects that result
from valve insufficiency. Repairing valvular connective tissue at end-stage
degeneration seems an extreme challenge, and it is therefore advantageous to
explore more specific developmental and homeostatic pathways to prevent or
reduce progression.
Elucidating these mechanisms will provide a deeper
understanding of valve disease pathogenesis, and novel therapeutics to improve
clinical outcomes.
Supplemental Results
We have examined mitral valves from Filamin-A (Fln-A) deficient mice, as it has
previously shown that Fln-A regulates Tgfβ signaling (Sasaki, Masuda et al.
2001), and loss of function in humans and mice are associated with MMVD
phenotypes and functional regurgitation (Norris, Moreno-Rodriguez et al. 2010,
Lardeux, Kyndt et al. 2011, Sauls, de Vlaming et al. 2012). A pilot study (N=1) in
mitral valves from 10-month-old conditional knockout Fln-A mice (Sauls, de
Vlaming et al. 2012) shows a potentially small increase in Scx expression
compared to control (Supplemental Figure 1).
Supplemental Figure 1. Scx is increased in
myxomatous mitral valves from 10-month old FlnA deficient mice. qPCR to show increased Scx
expression in mitral valves isolated from eight 10month old mice, compared to wild type littermate
controls, N=1.
88
89
Supplemental Figure 2. Scx is
increased in VICs from human
patients
with
myxomatous
valve disease. qPCR to show
changes in Scx expression in
hMVICs isolated from human
patients with MMVD compared to
hMVICs
from
control
nondiseased hearts, N=3.
We performed a preliminary study in hMVICS (Hulin, Deroanne et al. 2012) to
investigate the Scx transcript levels in normal patients compared to those with
MMVD. We observe two out of three human patients with MMVD trended toward
subtle increases in Scx, while the third sample showed a decrease
(Supplemental Figure 2).
Cst9
Ly6i
Gene
melan-A
cystatin 9
lymphocyte antigen 6 complex, locus I
Description
5.78
6.78
8.01
11.01
Fold Change
2.16E-02
1.13E-02
2.76E-04
7.98E-03
p-value
Appendix 1: Top 25 most differentially expressed protein-coding mRNAs (>1.5-fold change, p<0.05) in
Scx-/- AVC samples compared to Scx+/+ controls.
Mlana
DnaJ (Hsp40) homolog, subfamily B, member 7
1.05E-02
Dnajb7
1.02E-02
1.46E-02
4.62
6.27E-03
5.76
3.64
4.10E-02
aldo-keto reductase family 1, member B7
PARK2 co-regulated
3.61
1.67E-02
Akr1b7
Rnase1
GATS protein-like 3
2.35
4.88E-02
3.09E-02
Pacrg
RNA pseudouridylate synthase domain containing 1
2.35
3.85E-02
2.60E-03
Gatsl3
RAD54 homolog B (S. cerevisiae)
3.10
2.58E-02
5.69
Rpusd1
FtsJ homolog 2 (E. coli)
3.10
5.10
Rad54b
natriuretic peptide type C
2.96
glutamine repeat protein 1
Ftsj2
parvalbumin
ribonuclease, RNase A family, 1 (pancreatic)
Nppc
apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3
Glrp1
Pvalb
SH3/ankyrin domain gene 3
parvin, beta
tRNA methyltransferase 61 homolog A (S. cerevisiae)
alanine and arginine rich domain containing protein
C1q and tumor necrosis factor related protein 2
myeloid cell nuclear differentiation antigen
0.42
0.42
0.41
0.40
2.56
2.68
2.69
2.89
2.25E-02
2.93E-02
7.25E-03
1.81E-02
1.60E-02
3.28E-02
2.51E-02
3.70E-02
Apobec3
Parvb
multiple EGF-like-domains 10
Mnda
Shank3
H2.0-like homeobox
Hlx
camello-like 1
0.31
0.29
3.43E-02
1.67E-02
Trmt61a
Aard
C1qtnf2
Megf10
Cml1
UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 4
B3galt4
90
Description
Non-coding Micro RNA
microRNA 758
microRNA 134
microRNA 27b
microRNA 692-1
microRNA 700
microRNA 432
Non-coding nuclear/nucleolar RNA
Small nucleolar RNA U13
Small nucleolar RNA SNORD29
Small nucleolar RNA, C/D box 99
Small nucleolar RNA SNORA32
Small nucleolar RNA, C/D box 104
Small nucleolar RNA SNORA42/SNORA80 family
U6 spliceosomal RNA
Small nucleolar RNA SNORD101
Small nucleolar RNA SNORA32
Small nucleolar RNA, C/D box 93
U6 spliceosomal RNA
Small nucleolar RNA SNORD59
Small nucleolar RNA SNORA9
Small nucleolar RNA, C/D box 95
Small nucleolar RNA SNORA25
Small nucleolar RNA, H/ACA box 21
Small nucleolar RNA SNORA55
37.71
29.97
29.62
19.22
16.73
7.96
6.64
6.39
5.80
5.41
4.31
3.93
0.46
0.37
0.36
0.24
0.15
56.98
8.39
6.24
0.35
0.10
0.09
Fold Change
2.55E-04
2.33E-02
4.85E-03
1.06E-03
2.90E-02
8.92E-03
3.14E-02
4.72E-02
4.76E-02
2.62E-02
3.81E-02
1.41E-02
1.20E-02
3.67E-02
3.11E-03
1.72E-02
2.52E-02
2.19E-03
1.07E-03
5.88E-03
3.69E-02
1.29E-02
4.20E-02
p-Value
Appendix 2: Differentially expressed non-protein coding mRNAs (>1.5-fold change, p<0.05) in Scx-/AVC samples compared to Scx+/+ controls
Gene
Mir758
Mir134
Mir27b
Mir692-1
Mir700
Mir432
SnoU13
Snord29
Snord99
Snora32
Snord104
Snora42
U6
Snord101
Snora32
Snord93
U6
Snord59
Snora9
Snord95
Snora25
Snora21
Snora55
91
92
Gene
Snora84
Snord32a
2310010G23Rik
Gm14261
Gm15506
Gm17246
C630043F03Rik
Gm16765
Gm17639
A930012L18Rik
4930513N10Rik
Gm10524
mt-Tv
Description
Non-coding nuclear/nucleolar RNA
Small nucleolar RNA SNORA84
Small nucleolar RNA SNORD32a
Non-coding nuclear/nucleolar RNA
RIKEN cDNA 2310010G23 gene
predicted gene 14261
predicted gene 15506
predicted gene, 17246
RIKEN cDNA C630043F03 gene
predicted gene, 16765
predicted gene, 17639
RIKEN cDNA A930012L18 gene
RIKEN cDNA 4930513N10 gene
predicted gene 10524
Mitochondrial RNA
mitochondrially encoded tRNA valine
0.36
6.60
3.62
3.41
2.26
2.25
0.42
0.41
0.37
0.35
0.22
0.03
N/A
Fold Change
9.78E-03
1.04E-02
1.35E-02
2.19E-02
1.78E-02
2.98E-02
1.44E-02
4.23E-02
4.21E-02
6.56E-03
2.52E-02
1.12E-02
4.90E-02
p-Value
GO:0042904
GO:0042905
GO:0035238
GO:0002138
GO.0016102
GO:0016114
GO:0042363
GO:0042573
GO:0004028
GO:0001758
GO:0005112
GO:0044212
GO:0001067
GO:0000975
Bioinformatics
Source and
Associated
Pathway Number
GO:0016278
GO:0018024
GO:0016279
GO:0042056
GO:0008276
GO:0008170
GO:0008757
0.00006
0.00006
0.00006
0.00020
0.00020
0.00040
0.00009
0.00130
0.00040
0.00040
0.0007
0.0071
0.0071
0.0071
0.0005
0.0005
0.0005
0.0009
0.0022
0.0017
0.0058
p-value
Aldh1a1
Aldh1a2
Dll4
Ncor2
Junb
Hlx
Nfatc2
Ncor2
Mll2
Dot1l
Mll2
Wbp7
Gene
2.29
1.52
0.61
0.46
0.49
0.42
0.53
0.46
0.52
0.66
0.52
0.54
Fold
Change
Appendix 3: Bioinformatics pathway analysis to predict molecular functions and biological processes
significantly affected by the loss of Scx in heart valves at E15.5.
GO, Gene Ontology.
Molecular function/ Biological process
Lysine N-methyltransferase activity
Histone lysine N-methyltransferase
Protein lysine N-methyltransferase activity
Histone methyltransferase activity
Protein methyltransferase activity
N-methyltransferase activity
S-adenosylmethionine-dependent
methyltransferase activity
Transcription regulatory region DNA binding
Regulatory region nucleic acid binding
Regulatory region DNA binding
Notch binding
9-cis-retinoic acid biosynthesis process
9-cis-retinoic acid metabolic process
Vitamin A biosynthetic process
Retinoic acid biosynthetic process
Diterpenoid biosynthetic process
Terpenoid biosynthetic process
Fat-soluble vitamin biosynthetic process
Retinoic acid metabolic process
3-chloroallyl aldehyde dehydrogenase activity
Retinal dehydrogenase activity
93
94
Delta-Notch Signaling Pathway
Notch Signaling Pathway
Notch Signaling Pathway
Pentose and glucuronate
interconversions
Cysteine and methionine metabolism
Secondary active transmembrane
transporter activity
Nucleosomal DNA binding
Ingenuity: IPA
Wiki
Wiki
KEGG:04330
KEGG:00040
KEGG:00270
GO:0015291
GO:0031492
3.26E-04
0.0146
0.0010
0.0090
0.0079
0.0027
0.0122
0.0006
Cellular Development
Hmgn5
Hmgn3
AI317395
Slc9a8
Slc16a4
Mpst
Trdmt1
Cdo1
Akr1b7
Aldh1a1
Dll4
Ncor2
Maml1
Dll4
Ncor2
Maml1
Dll4
Ncor2
Maml1
Fam20c
Fas
Gpc4
Hspg2
Junb
Lrp5
Maml1
Ncor2
Nfatc2
Nod1
Nppc
Nucb2
1.60
1.61
0.64
0.66
0.54
1.74
1.59
1.64
5.76
2.29
0.61
0.46
0.64
0.61
0.46
0.64
0.61
0.46
0.64
0.57
1.76
0.52
0.55
0.56
0.49
0.56
0.64
0.46
0.53
0.51
3.10
95
Cell Death and Survival
Cellular Compromise
Cellular Assembly and Organization
Cell Morphology
Ingenuity: IPA
Ingenuity: IPA
Ingenuity: IPA
Ingenuity: IPA
1.25E-03
4.52E-04
4.52E-04
4.52E-04
Efna5
Fas
Fas
Plec
Fas
Plec
Maml1
Plec
0.55
1.76
1.76
0.66
1.76
0.66
0.64
0.66
Appendix 4: Exon-level alternative splicing in atrioventricular canal
regions isolated from E15.5 Scx-/- vs. Scx+/+ embryos
Exon Start (bp) Exon End (bp) Exon Rank Gene Name Fold Change P Value 5,111,849 87,372,880 87,372,880 55,130,777 55,130,777 140,375,019 140,375,019 46,647,855 38,591,025 40,763,850 150,537,925 150,537,925 107,562,474 95,522,628 107,562,471 107,562,468 107,562,454 12,013,059 12,013,059 40,763,850 120,937,961 33,301,930 52,007,483 46,039,654 28,133,553 28,133,553 107,562,430 6,328,229 107,562,418 168,501,642 168,501,642 36,855,561 52,007,485 26,444,063 40,763,850 132,595,765 5,111,974 87,372,968 87,372,968 55,130,819 55,130,819 140,375,076 140,375,076 46,647,949 38,591,055 40,763,954 150,538,078 150,538,078 107,562,501 95,523,710 107,562,501 107,562,501 107,562,501 12,013,147 12,013,147 40,763,963 120,938,165 33,301,995 52,007,546 46,039,740 28,133,640 28,133,640 107,562,501 6,328,324 107,562,501 168,501,718 168,501,718 36,855,688 52,007,546 26,444,081 40,764,016 132,595,827 73 4 5 12 13 3 3 1 4 1 4 2 1 5 1 1 1 2 2 1 3 10 1 8 11 9 1 3 1 10 10 32 1 1 1 11 Syne1 Cib1 Cib1 Uimc1 Uimc1 Plekha5 Plekha5 Sfxn2 Tmem39a Arl4a Camta1 Camta1 0610037L13Rik Stk25 0610037L13Rik 0610037L13Rik 0610037L13Rik Tmem67 Tmem67 Arl4a Tmem56 Depdc5 Vrk3 9130011E15Rik Shkbp1 Shkbp1 0610037L13Rik Zfp78 0610037L13Rik Atp9a Atp9a 4932438A13Rik Vrk3 Egfl7 Arl4a Pfkfb2 0.01 37.14 37.14 72.89 72.89 110.96 110.96 0.05 29.89 26.01 0.11 0.11 715.64 1.40 647.90 590.73 420.39 0.03 0.03 24.69 11.25 34.04 30.49 0.02 0.04 0.04 281.57 14.02 241.34 65.71 65.71 2.48 31.06 20.35 16.86 0.02 1.24E-­‐06 2.04E-­‐06 2.04E-­‐06 2.21E-­‐06 2.21E-­‐06 3.34E-­‐06 3.34E-­‐06 6.13E-­‐06 6.42E-­‐06 7.09E-­‐06 9.18E-­‐06 9.18E-­‐06 9.33E-­‐06 9.91E-­‐06 1.00E-­‐05 1.06E-­‐05 1.35E-­‐05 1.36E-­‐05 1.36E-­‐05 1.44E-­‐05 1.48E-­‐05 1.65E-­‐05 1.69E-­‐05 1.70E-­‐05 1.79E-­‐05 1.79E-­‐05 1.82E-­‐05 1.99E-­‐05 2.03E-­‐05 2.04E-­‐05 2.04E-­‐05 2.10E-­‐05 2.12E-­‐05 2.16E-­‐05 2.39E-­‐05 2.41E-­‐05 96
97
130,276,576 88,419,289 97,976,675 97,976,675 97,381,647 20,781,508 144,781,553 34,752,577 34,752,577 138,190,587 100,896,825 53,392,617 59,784,116 59,784,116 25,521,212 76,037,108 125,095,596 57,644,746 53,392,602 53,392,602 25,315,687 76,280,631 144,781,564 49,593,355 4,127,698 88,912,959 129,196,296 129,196,296 37,362,797 63,981,830 134,719,377 51,828,108 44,171,845 7,508,485 95,438,142 95,438,142 128,009,490 90,288,216 48,738,203 114,667,923 114,667,923 69,863,619 160,684,068 34,999,159 130,276,616 88,419,443 97,976,966 97,976,966 97,381,696 20,781,508 144,781,746 34,752,672 34,752,672 138,190,623 100,896,873 53,392,686 59,784,377 59,784,377 25,521,396 76,037,239 125,095,748 57,645,458 53,392,686 53,392,686 25,315,799 76,280,724 144,781,746 49,593,526 4,127,739 88,915,876 129,196,404 129,196,404 37,362,948 63,982,279 134,719,469 51,828,147 44,171,924 7,508,507 95,438,277 95,438,277 128,009,645 90,288,306 48,738,329 114,668,004 114,668,004 69,863,744 160,684,149 34,999,281 5 1 2 2 2 1 1 14 7 6 3 1 3 7 20 1 8 2 4 4 1 9 1 3 3 1 8 3 7 5 26 2 2 26 5 11 21 16 1 3 4 1 4 2 Psph Arhgef2 Ripk4 Ripk4 Polr2c Arhgap21 Odf2l Phf19 Phf19 Camsap2 Smn1 Kif3a Pemt Pemt Cacna1h Kit Cdt1 Hand1 Kif3a Kif3a BC029214 Ttc37 Odf2l Mocs1 Tmem134 Trabd Hdac1 Hdac1 Wfs1 Cbr4 Gtf2i Nmi Rnf38 Hdac6 Lpcat2 Lpcat2 Erbb3 Ptprj AW146154 Cmtm7 Cmtm7 Gtf2a2 Bmx Tirap 0.34 10.44 0.10 0.10 20.17 323.81 2.59 0.12 0.12 70.15 53.23 28.22 0.30 0.30 0.09 25.00 8.73 5.42 31.25 31.25 20.94 76.55 2.61 10.24 19.68 1.36 0.24 0.24 17.12 1.63 0.56 82.42 0.05 20.17 0.02 0.02 21.25 33.63 29.42 1.41 1.41 32.13 32.28 24.21 2.42E-­‐05 6.50E-­‐05 6.57E-­‐05 6.57E-­‐05 6.61E-­‐05 6.77E-­‐05 6.84E-­‐05 6.86E-­‐05 6.86E-­‐05 7.89E-­‐05 7.99E-­‐05 8.08E-­‐05 8.20E-­‐05 8.20E-­‐05 8.81E-­‐05 8.83E-­‐05 9.02E-­‐05 9.13E-­‐05 9.17E-­‐05 9.17E-­‐05 9.19E-­‐05 9.19E-­‐05 9.64E-­‐05 9.66E-­‐05 9.69E-­‐05 9.73E-­‐05 9.74E-­‐05 9.74E-­‐05 9.78E-­‐05 9.81E-­‐05 1.01E-­‐04 1.01E-­‐04 2.66E-­‐05 2.93E-­‐05 2.99E-­‐05 2.99E-­‐05 3.21E-­‐05 3.32E-­‐05 3.64E-­‐05 3.72E-­‐05 3.72E-­‐05 3.99E-­‐05 4.34E-­‐05 4.39E-­‐05 98
76,037,135 56,210,311 56,210,311 51,201,373 51,201,373 38,335,433 76,182,426 33,756,609 33,280,794 150,574,778 81,609,040 92,042,024 55,890,650 156,827,419 134,392,682 136,266,797 128,983,603 134,942,605 156,827,421 59,784,116 59,784,116 25,521,212 76,037,108 125,095,596 57,644,746 53,392,602 53,392,602 25,315,687 76,280,631 144,781,564 49,593,355 4,127,698 88,912,959 129,196,296 129,196,296 37,362,797 63,981,830 134,719,377 51,828,108 76,037,239 56,210,408 56,210,408 51,201,675 51,201,675 38,335,525 76,182,471 33,756,854 33,280,954 150,574,969 81,609,139 92,042,102 55,890,777 156,827,491 134,392,949 136,266,914 128,983,707 134,942,742 156,827,491 59,784,377 59,784,377 25,521,396 76,037,239 125,095,748 57,645,458 53,392,686 53,392,686 25,315,799 76,280,724 144,781,746 49,593,526 4,127,739 88,915,876 129,196,404 129,196,404 37,362,948 63,982,279 134,719,469 51,828,147 12 2 5 4 3 3 2 1 4 1 11 3 15 5 3 11 16 12 2 3 7 20 1 8 2 4 4 1 9 1 3 3 1 8 3 7 5 26 2 Kit Ubxn6 Ubxn6 Gp49a Gp49a Ttc26 Maf1 Heg1 Depdc5 Entpd6 Zc3h7b Parm1 D630037F22Rik Dsn1 Tmem57 Tbc1d8b Enpep Hsd3b7 Dsn1 Pemt Pemt Cacna1h Kit Cdt1 Hand1 Kif3a Kif3a BC029214 Ttc37 Odf2l Mocs1 Tmem134 Trabd Hdac1 Hdac1 Wfs1 Cbr4 Gtf2i Nmi 29.40 1.59 1.59 0.07 0.07 38.61 55.66 5.83 2.41 0.06 0.05 0.02 0.03 53.59 0.06 38.88 37.55 0.05 54.46 0.30 0.30 0.09 25.00 8.73 5.42 31.25 31.25 20.94 76.55 2.61 10.24 19.68 1.36 0.24 0.24 17.12 1.63 0.56 82.42 4.49E-­‐05 4.49E-­‐05 4.49E-­‐05 4.70E-­‐05 4.70E-­‐05 4.74E-­‐05 4.75E-­‐05 4.88E-­‐05 5.06E-­‐05 5.09E-­‐05 5.36E-­‐05 5.39E-­‐05 5.55E-­‐05 5.67E-­‐05 5.74E-­‐05 5.91E-­‐05 5.99E-­‐05 6.01E-­‐05 6.24E-­‐05 8.20E-­‐05 8.20E-­‐05 8.81E-­‐05 8.83E-­‐05 9.02E-­‐05 9.13E-­‐05 9.17E-­‐05 9.17E-­‐05 9.19E-­‐05 9.19E-­‐05 9.64E-­‐05 9.66E-­‐05 9.69E-­‐05 9.73E-­‐05 9.74E-­‐05 9.74E-­‐05 9.78E-­‐05 9.81E-­‐05 1.01E-­‐04 1.01E-­‐04 REFERENCES
Aikawa, E., P. Whittaker, M. Farber, K. Mendelson, R. F. Padera, M. Aikawa and
F. J. Schoen (2006). "Human semilunar cardiac valve remodeling by activated
cells from fetus to adult: implications for postnatal adaptation, pathology, and
tissue engineering." Circulation 113(10): 1344-1352.
Akhtar, S., K. M. Meek and V. James (1999). "Immunolocalization of elastin,
collagen type I and type III, fibronectin, and vitronectin in extracellular matrix
components of normal and myxomatous mitral heart valve chordae tendineae."
Cardiovasc Pathol 8(4): 203-211.
Akhurst, R. J., S. A. Lehnert, A. Faissner and E. Duffie (1990). "TGF beta in
murine morphogenetic processes: the early embryo and cardiogenesis."
Development 108(4): 645-656.
Anderson, R. H., S. Y. Ho and A. E. Becker (2000). "Anatomy of the human
atrioventricular junctions revisited." Anat Rec 260(1): 81-91.
Armstrong, E. J. and J. Bischoff (2004). "Heart valve development: endothelial
cell signaling and differentiation." Circ Res 95(5): 459-470.
Asou, Y., A. Nifuji, K. Tsuji, K. Shinomiya, E. N. Olson, P. Koopman and M. Noda
(2002). "Coordinated expression of scleraxis and Sox9 genes during embryonic
development of tendons and cartilage." J Orthop Res 20(4): 827-833.
Avierinos, J. F., B. J. Gersh, L. J. Melton, 3rd, K. R. Bailey, C. Shub, R. A.
Nishimura, A. J. Tajik and M. Enriquez-Sarano (2002). "Natural history of
asymptomatic mitral valve prolapse in the community." Circulation 106(11): 13551361.
Azhar, M., K. Brown, C. Gard, H. Chen, S. Rajan, D. A. Elliott, M. V. Stevens, T.
D. Camenisch, S. J. Conway and T. Doetschman (2011). "Transforming growth
factor Beta2 is required for valve remodeling during heart development." Dev Dyn
240(9): 2127-2141.
Azhar, M., R. B. Runyan, C. Gard, L. P. Sanford, M. L. Miller, A. Andringa, S.
Pawlowski, S. Rajan and T. Doetschman (2009). "Ligand-specific function of
transforming growth factor beta in epithelial-mesenchymal transition in heart
development." Dev Dyn 238(2): 431-442.
99
100
Bagchi, R. A. and M. P. Czubryt (2012). "Synergistic roles of scleraxis and
Smads in the regulation of collagen 1alpha2 gene expression." Biochim Biophys
Acta 1823(10): 1936-1944.
Barnette, D. N., A. Hulin, A. S. I. Ahmed, A. C. Colige, M. Azhar and J. Lincoln
(2013). "Tgfβ-Smad and MAPK signaling regulate scleraxis and proteoglycan
expression in heart valves." J Mol Cell Cardiol.
Barnette, D. N., M. VandeKopple, Y. Wu, D. A. Willoughby and J. Lincoln (2014).
"RNA-Seq Analysis to Identify Novel Roles of Scleraxis during Embryonic Mouse
Heart Valve Remodeling." PLoS One 9(7): e101425.
Bartram, U., D. G. Molin, L. J. Wisse, A. Mohamad, L. P. Sanford, T.
Doetschman, C. P. Speer, R. E. Poelmann and A. C. Gittenberger-de Groot
(2001). "Double-outlet right ventricle and overriding tricuspid valve reflect
disturbances of looping, myocardialization, endocardial cushion differentiation,
and apoptosis in TGF-beta(2)-knockout mice." Circulation 103(22): 2745-2752.
Boignard, A. (2011). "[Heart valve diseases, from physiology to diagnosis]." Rev
Infirm(173): 14-17.
Bosse, K., C. P. Hans, N. Zhao, S. N. Koenig, N. Huang, A. Guggilam, S.
LaHaye, G. Tao, P. A. Lucchesi, J. Lincoln, B. Lilly and V. Garg (2013).
"Endothelial nitric oxide signaling regulates Notch1 in aortic valve disease." J Mol
Cell Cardiol 60: 27-35.
Brent, A. E., R. Schweitzer and C. J. Tabin (2003). "A somitic compartment of
tendon progenitors." Cell 113(2): 235-248.
Brent, A. E. and C. J. Tabin (2004). "FGF acts directly on the somitic tendon
progenitors through the Ets transcription factors Pea3 and Erm to regulate
scleraxis expression." Development 131(16): 3885-3896.
Broom, N. D. (1978). "The observation of collagen and elastin structures in wet
whole mounts of pulmonary and aortic leaflets." J Thorac Cardiovasc Surg 75(1):
121-130.
Brown, C. B., A. S. Boyer, R. B. Runyan and J. V. Barnett (1996). "Antibodies to
the Type II TGFbeta receptor block cell activation and migration during
atrioventricular cushion transformation in the heart." Dev Biol 174(2): 248-257.
101
Brown, D., D. Wagner, X. Li, J. A. Richardson and E. N. Olson (1999). "Dual role
of the basic helix-loop-helix transcription factor scleraxis in mesoderm formation
and chondrogenesis during mouse embryogenesis." Development 126(19):
4317-4329.
Bueno, O. F., L. J. De Windt, K. M. Tymitz, S. A. Witt, T. R. Kimball, R. Klevitsky,
T. E. Hewett, S. P. Jones, D. J. Lefer, C. F. Peng, R. N. Kitsis and J. D.
Molkentin (2000). "The MEK1-ERK1/2 signaling pathway promotes compensated
cardiac hypertrophy in transgenic mice." EMBO J 19(23): 6341-6350.
Camenisch, T. D. and J. A. McDonald (2000). "Hyaluronan: is bigger better?" Am
J Respir Cell Mol Biol 23(4): 431-433.
Camenisch, T. D., D. G. Molin, A. Person, R. B. Runyan, A. C. Gittenberger-de
Groot, J. A. McDonald and S. E. Klewer (2002). "Temporal and distinct TGFbeta
ligand requirements during mouse and avian endocardial cushion
morphogenesis." Dev Biol 248(1): 170-181.
Camenisch, T. D., J. A. Schroeder, J. Bradley, S. E. Klewer and J. A. McDonald
(2002). "Heart-valve mesenchyme formation is dependent on hyaluronanaugmented activation of ErbB2-ErbB3 receptors." Nat Med 8(8): 850-855.
Chakraborty, S., J. Cheek, B. Sakthivel, B. J. Aronow and K. E. Yutzey (2008).
"Shared gene expression profiles in developing heart valves and osteoblast
progenitor cells." Physiol Genomics 35(1): 75-85.
Chakraborty, S., E. E. Wirrig, R. B. Hinton, W. H. Merrill, D. B. Spicer and K. E.
Yutzey (2010). "Twist1 promotes heart valve cell proliferation and extracellular
matrix gene expression during development in vivo and is expressed in human
diseased aortic valves." Dev Biol 347(1): 167-179.
Chavez, A. M. and D. M. Cosgrove (1988). "Surgery for mitral prolapse." Herz
13(6): 400-404.
Cheek, J. D., E. E. Wirrig, C. M. Alfieri, J. F. James and K. E. Yutzey (2012).
"Differential activation of valvulogenic, chondrogenic, and osteogenic pathways in
mouse models of myxomatous and calcific aortic valve disease." J Mol Cell
Cardiol 52(3): 689-700.
102
Chen, Z. F. and R. R. Behringer (1995). "twist is required in head mesenchyme
for cranial neural tube morphogenesis." Genes Dev 9(6): 686-699.
Cho, K. H., K. J. Jeong, S. C. Shin, J. Kang, C. G. Park and H. Y. Lee (2013).
"STAT3 mediates TGF-beta1-induced TWIST1 expression and prostate cancer
invasion." Cancer Lett 336(1): 167-173.
Clotman, F., P. Jacquemin, N. Plumb-Rudewiez, C. E. Pierreux, P. Van der
Smissen, H. C. Dietz, P. J. Courtoy, G. G. Rousseau and F. P. Lemaigre (2005).
"Control of liver cell fate decision by a gradient of TGF beta signaling modulated
by Onecut transcription factors." Genes Dev 19(16): 1849-1854.
Combs, M. D. and K. E. Yutzey (2009). "Heart valve development: regulatory
networks in development and disease." Circ Res 105(5): 408-421.
Cosgrove, D. M. and W. J. Stewart (1989). "Mitral valvuloplasty." Curr Probl
Cardiol 14(7): 359-415.
Cserjesi, P., D. Brown, K. L. Ligon, G. E. Lyons, N. G. Copeland, D. J. Gilbert, N.
A. Jenkins and E. N. Olson (1995). "Scleraxis: a basic helix-loop-helix protein
that prefigures skeletal formation during mouse embryogenesis." Development
121(4): 1099-1110.
Dallas, S. L., K. Miyazono, T. M. Skerry, G. R. Mundy and L. F. Bonewald (1995).
"Dual role for the latent transforming growth factor-beta binding protein in storage
of latent TGF-beta in the extracellular matrix and as a structural matrix protein." J
Cell Biol 131(2): 539-549.
Davies, M. J., B. P. Moore and M. V. Braimbridge (1978). "The floppy mitral
valve. Study of incidence, pathology, and complications in surgical, necropsy,
and forensic material." Br Heart J 40(5): 468-481.
Delot, E. C. (2003). "Control of endocardial cushion and cardiac valve maturation
by BMP signaling pathways." Mol Genet Metab 80(1-2): 27-35.
Devereux, R. B., W. T. Brown, R. Kramer-Fox and I. Sachs (1982). "Inheritance
of mitral valve prolapse: effect of age and sex on gene expression." Ann Intern
Med 97(6): 826-832.
103
Dietz, H. C., G. R. Cutting, R. E. Pyeritz, C. L. Maslen, L. Y. Sakai, G. M. Corson,
E. G. Puffenberger, A. Hamosh, E. J. Nanthakumar, S. M. Curristin and et al.
(1991). "Marfan syndrome caused by a recurrent de novo missense mutation in
the fibrillin gene." Nature 352(6333): 337-339.
Dietz, H. C., B. Loeys, L. Carta and F. Ramirez (2005). "Recent progress towards
a molecular understanding of Marfan syndrome." Am J Med Genet C Semin Med
Genet 139C(1): 4-9.
Dubrulle, J. and O. Pourquie (2003). "Welcome to syndetome: a new somitic
compartment." Dev Cell 4(5): 611-612.
Edom-Vovard, F., M. Bonnin and D. Duprez (2001). "Fgf8 transcripts are located
in tendons during embryonic chick limb development." Mech Dev 108(1-2): 203206.
Edom-Vovard, F., B. Schuler, M. A. Bonnin, M. A. Teillet and D. Duprez (2002).
"Fgf4 positively regulates scleraxis and tenascin expression in chick limb
tendons." Dev Biol 247(2): 351-366.
Espira, L., L. Lamoureux, S. C. Jones, R. D. Gerard, I. M. Dixon and M. P.
Czubryt (2009). "The basic helix-loop-helix transcription factor scleraxis regulates
fibroblast collagen synthesis." J Mol Cell Cardiol 47(2): 188-195.
Farhat, Y. M., A. A. Al-Maliki, T. Chen, S. C. Juneja, E. M. Schwarz, R. J.
O'Keefe and H. A. Awad (2012). "Gene expression analysis of the pleiotropic
effects of TGF-beta1 in an in vitro model of flexor tendon healing." PLoS One
7(12): e51411.
Feng, Q., H. Wang, H. H. Ng, H. Erdjument-Bromage, P. Tempst, K. Struhl and
Y. Zhang (2002). "Methylation of H3-lysine 79 is mediated by a new family of
HMTases without a SET domain." Curr Biol 12(12): 1052-1058.
Firulli, B. A., B. A. Redick, S. J. Conway and A. B. Firulli (2007). "Mutations within
helix I of Twist1 result in distinct limb defects and variation of DNA binding
affinities." J Biol Chem 282(37): 27536-27546.
Fishman, M. C. and K. R. Chien (1997). "Fashioning the vertebrate heart: earliest
embryonic decisions." Development 124(11): 2099-2117.
104
Freed, L. A., D. Levy, R. A. Levine, M. G. Larson, J. C. Evans, D. L. Fuller, B.
Lehman and E. J. Benjamin (1999). "Prevalence and clinical outcome of mitralvalve prolapse." N Engl J Med 341(1): 1-7.
Furumatsu, T., C. Shukunami, M. Amemiya-Kudo, H. Shimano and T. Ozaki
(2010). "Scleraxis and E47 cooperatively regulate the Sox9-dependent
transcription." Int J Biochem Cell Biol 42(1): 148-156.
Garg, V. (2006). "Molecular genetics of aortic valve disease." Curr Opin Cardiol
21(3): 180-184.
Garside, V. C., A. C. Chang, A. Karsan and P. A. Hoodless (2013). "Coordinating Notch, BMP, and TGF-beta signaling during heart valve development."
Cell Mol Life Sci 70(16): 2899-2917.
Geirsson, A., M. Singh, R. Ali, H. Abbas, W. Li, J. A. Sanchez, S. Hashim and G.
Tellides (2012). "Modulation of transforming growth factor-beta signaling and
extracellular matrix production in myxomatous mitral valves by angiotensin II
receptor blockers." Circulation 126(11 Suppl 1): S189-197.
Gould, R. A. and J. T. Butcher (2010). "Isolation of valvular endothelial cells." J
Vis Exp(46).
Gould, R. A., R. Sinha, H. Aziz, R. Rouf, H. C. Dietz, 3rd, D. P. Judge and J.
Butcher (2012). "Multi-scale biomechanical remodeling in aging and genetic
mutant murine mitral valve leaflets: insights into Marfan syndrome." PLoS One
7(9): e44639.
Grau, J. B., L. Pirelli, P. J. Yu, A. C. Galloway and H. Ostrer (2007). "The
genetics of mitral valve prolapse." Clin Genet 72(4): 288-295.
Gross, L. and M. A. Kugel (1931). "Topographic Anatomy and Histology of the
Valves in the Human Heart." Am J Pathol 7(5): 445-474 447.
Gupta, V., J. E. Barzilla, J. S. Mendez, E. H. Stephens, E. L. Lee, C. D. Collard,
R. Laucirica, P. H. Weigel and K. J. Grande-Allen (2009). "Abundance and
location of proteoglycans and hyaluronan within normal and myxomatous mitral
valves." Cardiovasc Pathol 18(4): 191-197.
105
Guy, T. S. and A. C. Hill (2012). "Mitral valve prolapse." Annu Rev Med 63: 277292.
Habashi, J. P., J. J. Doyle, T. M. Holm, H. Aziz, F. Schoenhoff, D. Bedja, Y.
Chen, A. N. Modiri, D. P. Judge and H. C. Dietz (2011). "Angiotensin II type 2
receptor signaling attenuates aortic aneurysm in mice through ERK antagonism."
Science 332(6027): 361-365.
Habashi, J. P., D. P. Judge, T. M. Holm, R. D. Cohn, B. L. Loeys, T. K. Cooper,
L. Myers, E. C. Klein, G. Liu, C. Calvi, M. Podowski, E. R. Neptune, M. K.
Halushka, D. Bedja, K. Gabrielson, D. B. Rifkin, L. Carta, F. Ramirez, D. L. Huso
and H. C. Dietz (2006). "Losartan, an AT1 antagonist, prevents aortic aneurysm
in a mouse model of Marfan syndrome." Science 312(5770): 117-121.
Harrelson, Z., R. G. Kelly, S. N. Goldin, J. J. Gibson-Brown, R. J. Bollag, L. M.
Silver and V. E. Papaioannou (2004). "Tbx2 is essential for patterning the
atrioventricular canal and for morphogenesis of the outflow tract during heart
development." Development 131(20): 5041-5052.
Hinton, R. B., J. Adelman-Brown, S. Witt, V. K. Krishnamurthy, H. Osinska, B.
Sakthivel, J. F. James, D. Y. Li, D. A. Narmoneva, R. P. Mecham and D. W.
Benson (2010). "Elastin haploinsufficiency results in progressive aortic valve
malformation and latent valve disease in a mouse model." Circ Res 107(4): 549557.
Hinton, R. B., Jr., J. Lincoln, G. H. Deutsch, H. Osinska, P. B. Manning, D. W.
Benson and K. E. Yutzey (2006). "Extracellular matrix remodeling and
organization in developing and diseased aortic valves." Circ Res 98(11): 14311438.
Hinton, R. B. and K. E. Yutzey (2011). "Heart valve structure and function in
development and disease." Annu Rev Physiol 73: 29-46.
Holm, T. M., J. P. Habashi, J. J. Doyle, D. Bedja, Y. Chen, C. van Erp, M. E.
Lindsay, D. Kim, F. Schoenhoff, R. D. Cohn, B. L. Loeys, C. J. Thomas, S.
Patnaik, J. J. Marugan, D. P. Judge and H. C. Dietz (2011). "Noncanonical
TGFbeta signaling contributes to aortic aneurysm progression in Marfan
syndrome mice." Science 332(6027): 358-361.
106
Hong, J., J. Zhou, J. Fu, T. He, J. Qin, L. Wang, L. Liao and J. Xu (2011).
"Phosphorylation of serine 68 of Twist1 by MAPKs stabilizes Twist1 protein and
promotes breast cancer cell invasiveness." Cancer Res 71(11): 3980-3990.
Huk, D. J., H. L. Hammond, H. Kegechika and J. Lincoln (2013). "Increased
dietary intake of vitamin A promotes aortic valve calcification in vivo." Arterioscler
Thromb Vasc Biol 33(2): 285-293.
Hulin, A., C. F. Deroanne, C. A. Lambert, B. Dumont, V. Castronovo, J. O.
Defraigne, B. V. Nusgens, M. A. Radermecker and A. C. Colige (2012).
"Metallothionein-dependent up-regulation of TGF-beta2 participates in the
remodelling of the myxomatous mitral valve." Cardiovasc Res 93(3): 480-489.
Isogai, Z., R. N. Ono, S. Ushiro, D. R. Keene, Y. Chen, R. Mazzieri, N. L.
Charbonneau, D. P. Reinhardt, D. B. Rifkin and L. Y. Sakai (2003). "Latent
transforming growth factor beta-binding protein 1 interacts with fibrillin and is a
microfibril-associated protein." J Biol Chem 278(4): 2750-2757.
Jacquemin, P., S. M. Durviaux, J. Jensen, C. Godfraind, G. Gradwohl, F.
Guillemot, O. D. Madsen, P. Carmeliet, M. Dewerchin, D. Collen, G. G.
Rousseau and F. P. Lemaigre (2000). "Transcription factor hepatocyte nuclear
factor 6 regulates pancreatic endocrine cell differentiation and controls
expression of the proendocrine gene ngn3." Mol Cell Biol 20(12): 4445-4454.
Janzen, C. J., S. B. Hake, J. E. Lowell and G. A. Cross (2006). "Selective di- or
trimethylation of histone H3 lysine 76 by two DOT1 homologs is important for cell
cycle regulation in Trypanosoma brucei." Mol Cell 23(4): 497-507.
Jian, B., N. Narula, Q. Y. Li, E. R. Mohler, 3rd and R. J. Levy (2003).
"Progression of aortic valve stenosis: TGF-beta1 is present in calcified aortic
valve cusps and promotes aortic valve interstitial cell calcification via apoptosis."
Ann Thorac Surg 75(2): 457-465; discussion 465-456.
Jones, J. M., H. O'Kane, D. J. Gladstone, M. A. Sarsam, G. Campalani, S. W.
MacGowan, J. Cleland and G. W. Cran (2001). "Repeat heart valve surgery: risk
factors for operative mortality." J Thorac Cardiovasc Surg 122(5): 913-918.
Judge, D. P. and H. C. Dietz (2008). "Therapy of Marfan syndrome." Annu Rev
Med 59: 43-59.
107
Judge, D. P., R. Rouf, J. Habashi and H. C. Dietz (2011). "Mitral valve disease in
Marfan syndrome and related disorders." J Cardiovasc Transl Res 4(6): 741-747.
Kern, C. B., A. Wessels, J. McGarity, L. J. Dixon, E. Alston, W. S. Argraves, D.
Geeting, C. M. Nelson, D. R. Menick and S. S. Apte (2010). "Reduced versican
cleavage due to Adamts9 haploinsufficiency is associated with cardiac and aortic
anomalies." Matrix Biol 29(4): 304-316.
Kershaw, J. D., M. Misfeld, H. H. Sievers, M. H. Yacoub and A. H. Chester
(2004). "Specific regional and directional contractile responses of aortic cusp
tissue." J Heart Valve Dis 13(5): 798-803.
Kinsella, M. G., S. L. Bressler and T. N. Wight (2004). "The regulated synthesis
of versican, decorin, and biglycan: extracellular matrix proteoglycans that
influence cellular phenotype." Crit Rev Eukaryot Gene Expr 14(3): 203-234.
Krenz, M., K. E. Yutzey and J. Robbins (2005). "Noonan syndrome mutation
Q79R in Shp2 increases proliferation of valve primordia mesenchymal cells via
extracellular signal-regulated kinase 1/2 signaling." Circ Res 97(8): 813-820.
Kretzschmar, M., J. Doody and J. Massague (1997). "Opposing BMP and EGF
signalling pathways converge on the TGF-beta family mediator Smad1." Nature
389(6651): 618-622.
Kretzschmar, M., J. Doody, I. Timokhina and J. Massague (1999). "A mechanism
of repression of TGFbeta/ Smad signaling by oncogenic Ras." Genes Dev 13(7):
804-816.
Krug, E. L., R. B. Runyan and R. R. Markwald (1985). "Protein extracts from
early embryonic hearts initiate cardiac endothelial cytodifferentiation." Dev Biol
112(2): 414-426.
Kuivaniemi, H., G. Tromp and D. J. Prockop (1997). "Mutations in fibrillar
collagens (types I, II, III, and XI), fibril-associated collagen (type IX), and networkforming collagen (type X) cause a spectrum of diseases of bone, cartilage, and
blood vessels." Hum Mutat 9(4): 300-315.
Lardeux, A., F. Kyndt, S. Lecointe, H. L. Marec, J. Merot, J. J. Schott, T. Le
Tourneau and V. Probst (2011). "Filamin-a-related myxomatous mitral valve
108
dystrophy: genetic, echocardiographic and functional aspects." J Cardiovasc
Transl Res 4(6): 748-756.
Lee, M. P. and K. E. Yutzey (2011). "Twist1 directly regulates genes that promote
cell proliferation and migration in developing heart valves." PLoS One 6(12):
e29758.
Levay, A. K., J. D. Peacock, Y. Lu, M. Koch, R. B. Hinton, Jr., K. E. Kadler and J.
Lincoln (2008). "Scleraxis is required for cell lineage differentiation and
extracellular matrix remodeling during murine heart valve formation in vivo." Circ
Res 103(9): 948-956.
Li, D. Y., A. E. Toland, B. B. Boak, D. L. Atkinson, G. J. Ensing, C. A. Morris and
M. T. Keating (1997). "Elastin point mutations cause an obstructive vascular
disease, supravalvular aortic stenosis." Hum Mol Genet 6(7): 1021-1028.
Li, N., R. L. Goodwin and J. D. Potts (2013). "Zyxin regulates cell migration and
differentiation in EMT during chicken AV valve morphogenesis." Microsc
Microanal 19(4): 842-854.
Liang, C. C. and H. C. Chen (2001). "Sustained activation of extracellular signalregulated kinase stimulated by hepatocyte growth factor leads to integrin alpha 2
expression that is involved in cell scattering." J Biol Chem 276(24): 21146-21152.
Liberfarb, R. M. and A. Goldblatt (1986). "Prevalence of mitral-valve prolapse in
the Stickler syndrome." Am J Med Genet 24(3): 387-392.
Lincoln, J., C. M. Alfieri and K. E. Yutzey (2004). "Development of heart valve
leaflets and supporting apparatus in chicken and mouse embryos." Dev Dyn
230(2): 239-250.
Lincoln, J., C. M. Alfieri and K. E. Yutzey (2006). "BMP and FGF regulatory
pathways control cell lineage diversification of heart valve precursor cells." Dev
Biol 292(2): 292-302.
Lincoln, J., R. Kist, G. Scherer and K. E. Yutzey (2007). "Sox9 is required for
precursor cell expansion and extracellular matrix organization during mouse
heart valve development." Dev Biol 305(1): 120-132.
109
Lincoln, J. and K. E. Yutzey (2011). "Molecular and developmental mechanisms
of congenital heart valve disease." Birth Defects Res A Clin Mol Teratol 91(6):
526-534.
Little, C. D. and B. J. Rongish (1995). "The extracellular matrix during heart
development." Experientia 51(9-10): 873-882.
Liu, A. C. and A. I. Gotlieb (2008). "Transforming growth factor-beta regulates in
vitro heart valve repair by activated valve interstitial cells." Am J Pathol 173(5):
1275-1285.
Liu, A. C., V. R. Joag and A. I. Gotlieb (2007). "The emerging role of valve
interstitial cell phenotypes in regulating heart valve pathobiology." Am J Pathol
171(5): 1407-1418.
Lloyd-Jones, D., R. J. Adams, T. M. Brown, M. Carnethon, S. Dai, G. De Simone,
T. B. Ferguson, E. Ford, K. Furie, C. Gillespie, A. Go, K. Greenlund, N. Haase, S.
Hailpern, P. M. Ho, V. Howard, B. Kissela, S. Kittner, D. Lackland, L. Lisabeth, A.
Marelli, M. M. McDermott, J. Meigs, D. Mozaffarian, M. Mussolino, G. Nichol, V.
L. Roger, W. Rosamond, R. Sacco, P. Sorlie, T. Thom, S. Wasserthiel-Smoller,
N. D. Wong and J. Wylie-Rosett (2010). "Heart disease and stroke statistics-2010 update: a report from the American Heart Association." Circulation 121(7):
e46-e215.
Loeys, B. L., U. Schwarze, T. Holm, B. L. Callewaert, G. H. Thomas, H. Pannu,
J. F. De Backer, G. L. Oswald, S. Symoens, S. Manouvrier, A. E. Roberts, F.
Faravelli, M. A. Greco, R. E. Pyeritz, D. M. Milewicz, P. J. Coucke, D. E.
Cameron, A. C. Braverman, P. H. Byers, A. M. De Paepe and H. C. Dietz (2006).
"Aneurysm syndromes caused by mutations in the TGF-beta receptor." N Engl J
Med 355(8): 788-798.
Lorda-Diez, C. I., J. A. Montero, C. Martinez-Cue, J. A. Garcia-Porrero and J. M.
Hurle (2009). "Transforming growth factors beta coordinate cartilage and tendon
differentiation in the developing limb mesenchyme." J Biol Chem 284(43): 2998829996.
Lyons, K. M., R. W. Pelton and B. L. Hogan (1990). "Organogenesis and pattern
formation in the mouse: RNA distribution patterns suggest a role for bone
morphogenetic protein-2A (BMP-2A)." Development 109(4): 833-844.
110
Ma, L., M. F. Lu, R. J. Schwartz and J. F. Martin (2005). "Bmp2 is essential for
cardiac cushion epithelial-mesenchymal transition and myocardial patterning."
Development 132(24): 5601-5611.
MacGrogan, D., L. Luna-Zurita and J. L. de la Pompa (2011). "Notch signaling in
cardiac valve development and disease." Birth Defects Res A Clin Mol Teratol
91(6): 449-459.
Margagliotti, S., F. Clotman, C. E. Pierreux, J. B. Beaudry, P. Jacquemin, G. G.
Rousseau and F. P. Lemaigre (2007). "The Onecut transcription factors HNF6/OC-1 and OC-2 regulate early liver expansion by controlling hepatoblast
migration." Dev Biol 311(2): 579-589.
Martinsen, B. J. (2005). "Reference guide to the stages of chick heart
embryology." Dev Dyn 233(4): 1217-1237.
Matt, P., F. Schoenhoff, J. Habashi, T. Holm, C. Van Erp, D. Loch, O. D. Carlson,
B. F. Griswold, Q. Fu, J. De Backer, B. Loeys, D. L. Huso, N. B. McDonnell, J. E.
Van Eyk, H. C. Dietz and T. A. C. C. Gen (2009). "Circulating transforming
growth factor-beta in Marfan syndrome." Circulation 120(6): 526-532.
Mendias, C. L., J. P. Gumucio and E. B. Lynch (2012). "Mechanical loading and
TGF-beta change the expression of multiple miRNAs in tendon fibroblasts." J
Appl Physiol (1985) 113(1): 56-62.
Milne, T. A., S. D. Briggs, H. W. Brock, M. E. Martin, D. Gibbs, C. D. Allis and J.
L. Hess (2002). "MLL targets SET domain methyltransferase activity to Hox gene
promoters." Mol Cell 10(5): 1107-1117.
Missirlis, Y. F. and C. D. Armeniades (1977). "Ultrastructure of the human aortic
valve." Acta Anat (Basel) 98(2): 199-205.
Molin, D. G., U. Bartram, K. Van der Heiden, L. Van Iperen, C. P. Speer, B. P.
Hierck, R. E. Poelmann and A. C. Gittenberger-de-Groot (2003). "Expression
patterns of Tgfbeta1-3 associate with myocardialisation of the outflow tract and
the development of the epicardium and the fibrous heart skeleton." Dev Dyn
227(3): 431-444.
111
Moorman, A. F. and V. M. Christoffels (2003). "Development of the cardiac
conduction system: a matter of chamber development." Novartis Found Symp
250: 25-34; discussion 34-43, 276-279.
Murchison, N. D., B. A. Price, D. A. Conner, D. R. Keene, E. N. Olson, C. J.
Tabin and R. Schweitzer (2007). "Regulation of tendon differentiation by scleraxis
distinguishes force-transmitting tendons from muscle-anchoring tendons."
Development 134(14): 2697-2708.
Nakajima, Y., T. Yamagishi, S. Hokari and H. Nakamura (2000). "Mechanisms
involved in valvuloseptal endocardial cushion formation in early cardiogenesis:
roles of transforming growth factor (TGF)-beta and bone morphogenetic protein
(BMP)." Anat Rec 258(2): 119-127.
Nasuti, J. F., P. J. Zhang, M. D. Feldman, T. Pasha, J. S. Khurana, J. H.
Gorman, 3rd, R. C. Gorman, J. Narula and N. Narula (2004). "Fibrillin and other
matrix proteins in mitral valve prolapse syndrome." Ann Thorac Surg 77(2): 532536.
Ng, C. M., A. Cheng, L. A. Myers, F. Martinez-Murillo, C. Jie, D. Bedja, K. L.
Gabrielson, J. M. Hausladen, R. P. Mecham, D. P. Judge and H. C. Dietz (2004).
"TGF-beta-dependent pathogenesis of mitral valve prolapse in a mouse model of
Marfan syndrome." J Clin Invest 114(11): 1586-1592.
Nienaber, C. A. and Y. Von Kodolitsch (1999). "Therapeutic management of
patients with Marfan syndrome: focus on cardiovascular involvement." Cardiol
Rev 7(6): 332-341.
Norris, R. A., R. Moreno-Rodriguez, A. Wessels, J. Merot, P. Bruneval, A. H.
Chester, M. H. Yacoub, A. Hagege, S. A. Slaugenhaupt, E. Aikawa, J. J. Schott,
A. Lardeux, B. S. Harris, L. K. Williams, A. Richards, R. A. Levine and R. R.
Markwald (2010). "Expression of the familial cardiac valvular dystrophy gene,
filamin-A, during heart morphogenesis." Dev Dyn 239(7): 2118-2127.
Olsen, E. G. and H. K. Al-Rufaie (1980). "The floppy mitral valve. Study on
pathogenesis." Br Heart J 44(6): 674-683.
Otto, C. M. (2007). "Valvular heart disease: focus on women." Cardiol Rev 15(6):
291-297.
112
Passik, C. S., D. M. Ackermann, J. R. Pluth and W. D. Edwards (1987).
"Temporal changes in the causes of aortic stenosis: a surgical pathologic study
of 646 cases." Mayo Clin Proc 62(2): 119-123.
Peacock, J. D., A. K. Levay, D. B. Gillaspie, G. Tao and J. Lincoln (2010).
"Reduced sox9 function promotes heart valve calcification phenotypes in vivo."
Circ Res 106(4): 712-719.
Person, A. D., S. E. Klewer and R. B. Runyan (2005). "Cell biology of cardiac
cushion development." Int Rev Cytol 243: 287-335.
Plageman, T. F., Jr. and K. E. Yutzey (2004). "Differential expression and
function of Tbx5 and Tbx20 in cardiac development." J Biol Chem 279(18):
19026-19034.
Pomerance, A. (1972). "Pathogenesis of aortic stenosis and its relation to age."
Br Heart J 34(6): 569-574.
Pyeritz, R. E. (2000). "The Marfan syndrome." Annu Rev Med 51: 481-510.
Rabkin, E., M. Aikawa, J. R. Stone, Y. Fukumoto, P. Libby and F. J. Schoen
(2001). "Activated interstitial myofibroblasts express catabolic enzymes and
mediate matrix remodeling in myxomatous heart valves." Circulation 104(21):
2525-2532.
Rabkin-Aikawa, E., M. Farber, M. Aikawa and F. J. Schoen (2004). "Dynamic and
reversible changes of interstitial cell phenotype during remodeling of cardiac
valves." J Heart Valve Dis 13(5): 841-847.
Rabkin-Aikawa, E., J. E. Mayer, Jr. and F. J. Schoen (2005). "Heart valve
regeneration." Adv Biochem Eng Biotechnol 94: 141-179.
Radermecker, M. A., R. Limet, C. M. Lapiere and B. Nusgens (2003). "Increased
mRNA expression of decorin in the prolapsing posterior leaflet of the mitral
valve." Interact Cardiovasc Thorac Surg 2(3): 389-394.
Rajamannan, N. M., B. Gersh and R. O. Bonow (2003). "Calcific aortic stenosis:
from bench to the bedside--emerging clinical and cellular concepts." Heart 89(7):
801-805.
113
Reznikoff, C. A., J. S. Bertram, D. W. Brankow and C. Heidelberger (1973).
"Quantitative and qualitative studies of chemical transformation of cloned C3H
mouse embryo cells sensitive to postconfluence inhibition of cell division."
Cancer Res 33(12): 3239-3249.
Robbins, J. R., S. P. Evanko and K. G. Vogel (1997). "Mechanical loading and
TGF-beta regulate proteoglycan synthesis in tendon." Arch Biochem Biophys
342(2): 203-211.
Roberts, W. C. (1970). "The congenitally bicuspid aortic valve. A study of 85
autopsy cases." Am J Cardiol 26(1): 72-83.
Rosamond, W., K. Flegal, K. Furie, A. Go, K. Greenlund, N. Haase, S. M.
Hailpern, M. Ho, V. Howard, B. Kissela, S. Kittner, D. Lloyd-Jones, M.
McDermott, J. Meigs, C. Moy, G. Nichol, C. O'Donnell, V. Roger, P. Sorlie, J.
Steinberger, T. Thom, M. Wilson and Y. Hong (2008). "Heart disease and stroke
statistics--2008 update: a report from the American Heart Association Statistics
Committee and Stroke Statistics Subcommittee." Circulation 117(4): e25-146.
Rosenkranz, S. (2004). "TGF-beta1 and angiotensin networking in cardiac
remodeling." Cardiovasc Res 63(3): 423-432.
Sacks, M. S. and A. P. Yoganathan (2007). "Heart valve function: a
biomechanical perspective." Philos Trans R Soc Lond B Biol Sci 362(1484):
1369-1391.
Sanford, L. P., I. Ormsby, A. C. Gittenberger-de Groot, H. Sariola, R. Friedman,
G. P. Boivin, E. L. Cardell and T. Doetschman (1997). "TGFbeta2 knockout mice
have multiple developmental defects that are non-overlapping with other
TGFbeta knockout phenotypes." Development 124(13): 2659-2670.
Sasaki, A., Y. Masuda, Y. Ohta, K. Ikeda and K. Watanabe (2001). "Filamin
associates with Smads and regulates transforming growth factor-beta signaling."
J Biol Chem 276(21): 17871-17877.
Sauls, K., A. de Vlaming, B. S. Harris, K. Williams, A. Wessels, R. A. Levine, S.
A. Slaugenhaupt, R. L. Goodwin, L. M. Pavone, J. Merot, J. J. Schott, T. Le
Tourneau, T. Dix, S. Jesinkey, Y. Feng, C. Walsh, B. Zhou, S. Baldwin, R. R.
Markwald and R. A. Norris (2012). "Developmental basis for filamin-A-associated
myxomatous mitral valve disease." Cardiovasc Res 96(1): 109-119.
114
Schoen, F. J. (1997). "Aortic valve structure-function correlations: role of elastic
fibers no longer a stretch of the imagination." J Heart Valve Dis 6(1): 1-6.
Schoen, F. J. (2005). "Cardiac valves and valvular pathology: update on function,
disease, repair, and replacement." Cardiovasc Pathol 14(4): 189-194.
Schoen, F. J. (2008). "Evolving concepts of cardiac valve dynamics: the
continuum of development, functional structure, pathobiology, and tissue
engineering." Circulation 118(18): 1864-1880.
Schroeder, J. A., L. F. Jackson, D. C. Lee and T. D. Camenisch (2003). "Form
and function of developing heart valves: coordination by extracellular matrix and
growth factor signaling." J Mol Med (Berl) 81(7): 392-403.
Schweitzer, R., J. H. Chyung, L. C. Murtaugh, A. E. Brent, V. Rosen, E. N. Olson,
A. Lassar and C. J. Tabin (2001). "Analysis of the tendon cell fate using
Scleraxis, a specific marker for tendons and ligaments." Development 128(19):
3855-3866.
Shah, P. M. (2010). "Current concepts in mitral valve prolapse--diagnosis and
management." J Cardiol 56(2): 125-133.
Shelton, E. L. and K. E. Yutzey (2008). "Twist1 function in endocardial cushion
cell proliferation, migration, and differentiation during heart valve development."
Dev Biol 317(1): 282-295.
Shukunami, C., A. Takimoto, M. Oro and Y. Hiraki (2006). "Scleraxis positively
regulates the expression of tenomodulin, a differentiation marker of tenocytes."
Dev Biol 298(1): 234-247.
Singer, M. S., A. Kahana, A. J. Wolf, L. L. Meisinger, S. E. Peterson, C. Goggin,
M. Mahowald and D. E. Gottschling (1998). "Identification of high-copy disruptors
of telomeric silencing in Saccharomyces cerevisiae." Genetics 150(2): 613-632.
Smith, D. E. and M. B. Matthews (1955). "Aortic valvular stenosis with coarctation
of the aorta, with special reference to the development of aortic stenosis upon
congenital bicuspid valves." Br Heart J 17(2): 198-206.
115
Smith, T. G., D. Sweetman, M. Patterson, S. M. Keyse and A. Munsterberg
(2005). "Feedback interactions between MKP3 and ERK MAP kinase control
scleraxis expression and the specification of rib progenitors in the developing
chick somite." Development 132(6): 1305-1314.
Snider, P., R. B. Hinton, R. A. Moreno-Rodriguez, J. Wang, R. Rogers, A.
Lindsley, F. Li, D. A. Ingram, D. Menick, L. Field, A. B. Firulli, J. D. Molkentin, R.
Markwald and S. J. Conway (2008). "Periostin is required for maturation and
extracellular matrix stabilization of noncardiomyocyte lineages of the heart." Circ
Res 102(7): 752-760.
Sridurongrit, S., J. Larsson, R. Schwartz, P. Ruiz-Lozano and V. Kaartinen
(2008). "Signaling via the Tgf-beta type I receptor Alk5 in heart development."
Dev Biol 322(1): 208-218.
Sugi, Y., N. Ito, G. Szebenyi, K. Myers, J. F. Fallon, T. Mikawa and R. R.
Markwald (2003). "Fibroblast growth factor (FGF)-4 can induce proliferation of
cardiac cushion mesenchymal cells during early valve leaflet formation." Dev Biol
258(2): 252-263.
Supino, P. G., J. S. Borer, A. Yin, E. Dillingham and W. McClymont (2004). "The
epidemiology of valvular heart diseases: the problem is growing." Adv Cardiol 41:
9-15.
Takano, H., Y. Miyamoto, Y. Sawa, N. Fukushima, G. Matsumiya, T. Fujita and
H. Matsuda (2005). "Successful mitral valve replacement in a patient with EhlersDanlos syndrome type VI." Ann Thorac Surg 80(1): 320-322.
Tamura, K., Y. Fukuda and V. J. Ferrans (1998). "Elastic fiber abnormalities
associated with a leaflet perforation in floppy mitral valve." J Heart Valve Dis 7(4):
460-466.
Thisse, B., M. el Messal and F. Perrin-Schmitt (1987). "The twist gene: isolation
of a Drosophila zygotic gene necessary for the establishment of dorsoventral
pattern." Nucleic Acids Res 15(8): 3439-3453.
Timmerman, L. A., J. Grego-Bessa, A. Raya, E. Bertran, J. M. Perez-Pomares, J.
Diez, S. Aranda, S. Palomo, F. McCormick, J. C. Izpisua-Belmonte and J. L. de
la Pompa (2004). "Notch promotes epithelial-mesenchymal transition during
cardiac development and oncogenic transformation." Genes Dev 18(1): 99-115.
116
van Karnebeek, C. D., M. S. Naeff, B. J. Mulder, R. C. Hennekam and M.
Offringa (2001). "Natural history of cardiovascular manifestations in Marfan
syndrome." Arch Dis Child 84(2): 129-137.
Vesely, I. (1998). "The role of elastin in aortic valve mechanics." J Biomech
31(2): 115-123.
Vesuna, F., P. van Diest, J. H. Chen and V. Raman (2008). "Twist is a
transcriptional repressor of E-cadherin gene expression in breast cancer."
Biochem Biophys Res Commun 367(2): 235-241.
Walker, G. A., K. S. Masters, D. N. Shah, K. S. Anseth and L. A. Leinwand
(2004). "Valvular myofibroblast activation by transforming growth factor-beta:
implications for pathological extracellular matrix remodeling in heart valve
disease." Circ Res 95(3): 253-260.
Weiss, A. N., J. W. Mimbs, P. A. Ludbrook and B. E. Sobel (1975).
"Echocardiographic detection of mitral valve prolapse. Exclusion of false positive
diagnosis and determination of inheritance." Circulation 52(6): 1091-1096.
Whittaker, P., D. R. Boughner, D. G. Perkins and P. B. Canham (1987).
"Quantitative structural analysis of collagen in chordae tendineae and its relation
to floppy mitral valves and proteoglycan infiltration." Br Heart J 57(3): 264-269.
Wilcken, D. E. and A. J. Hickey (1988). "Lifetime risk for patients with mitral valve
prolapse of developing severe valve regurgitation requiring surgery." Circulation
78(1): 10-14.
Xiao, H. and Y. Y. Zhang (2008). "Understanding the role of transforming growth
factor-beta signalling in the heart: overview of studies using genetic mouse
models." Clin Exp Pharmacol Physiol 35(3): 335-341.
Yamamoto, K., T. A. Matsuoka, S. Kawashima, S. Takebe, F. Kubo, T.
Miyatsuka, H. Kaneto and I. Shimomura (2013). "A novel function of Onecut1
protein as a negative regulator of MafA gene expression." J Biol Chem 288(30):
21648-21658.
Zhao, B., L. Etter, R. B. Hinton, Jr. and D. W. Benson (2007). "BMP and FGF
regulatory pathways in semilunar valve precursor cells." Dev Dyn 236(4): 971980.