Characterization of Changes in Extracellular Matrix and ... · Andrea Victoria Kwong Master of...
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Characterization of Changes in Extracellular Matrix and Cellular Phenotypes in Early Calcific Aortic Valve Disease by Andrea Victoria Kwong A thesis submitted in conformity with the requirements for the degree of Master of Applied Science in Biomedical Engineering Institute of Biomaterials and Biomedical Engineering University of Toronto © Copyright by Andrea Victoria Kwong 2014
Transcript of Characterization of Changes in Extracellular Matrix and ... · Andrea Victoria Kwong Master of...
Characterization of Changes in Extracellular Matrix and Cellular Phenotypes in Early Calcific Aortic Valve Disease
by
Andrea Victoria Kwong
A thesis submitted in conformity with the requirements for the degree of Master of Applied Science in Biomedical Engineering
Institute of Biomaterials and Biomedical Engineering University of Toronto
© Copyright by Andrea Victoria Kwong 2014
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Characterization of Changes in Extracellular Matrix and Cellular
Phenotypes in Early Calcific Aortic Valve Disease
Andrea Victoria Kwong
Master of Applied Science in Biomedical Engineering
Institute of Biomaterials and Biomedical Engineering
University of Toronto
2014
Abstract
Calcific aortic valve disease (CAVD) is a prevalent disease associated with severe clinical
outcomes and without an effective medical treatment. While advanced disease is well-
characterized, there is an unmet scientific need to understand the active pathobiological
processes that promote eventual calcification and stenosis of the valve. Using a porcine model of
early CAVD, histological staining was used to identify the changes that occur in the extracellular
matrix (ECM) with a focus on proteoglycan content. Putatively more advanced lesions were
morphologically dense with increased biglycan, decreased hyaluronan, and higher ApoB score
and Sox9 fraction. These microenvironmental changes were used to delineate populations of
valve interstitial cells (VICs) for microarray analysis. Differentially upregulated genes from the
top of lesions and non-lesion fibrosa were related to lipid accumulation, inflammatory response,
osteochondrogenesis, and ECM remodeling. An improved understanding of early CAVD
microenvironment and VIC phenotypes lays the foundation to identify novel therapeutic targets.
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Acknowledgments
First and foremost, I would like to thank Dr. Craig Simmons for giving me this opportunity and
providing his guidance and support through the past few years. I feel extremely lucky to have
had such an approachable, patient, and enthusiastic supervisor, who is an all-around good guy. I
cannot imagine completing this thesis without Craig’s helpful insights and words of
encouragement, which always managed to breathe new life into my experiments when seemingly
nothing was working. I thoroughly enjoyed completing this project under Craig’s supervision.
I am also grateful to have been part of the Cellular Mechanobiology lab. Specifically, I would
like to thank Krista for showing me the ropes (and the joys of histology) from my first day in the
lab, Mark for being cynical with me and showing me how to do valve and RNA isolations, and
Zahra for being the best lab mom, helping me with just about everything in the lab. I would also
like to thank everyone else in the lab, past and present, for making it such a fun and positive
environment to come to every day. I have created fond memories over the last three years and
will miss all the laughs and shenanigans.
Outside of the lab, there are a number of people who provided their technical expertise to help
me complete my project. I would like to thank Brent Steer from the Marsden lab for teaching me
how to use the cryostat and laser capture microdissection system and Patrick Yau, Carl Virtanen,
Gurbaksh Basi, and Natalie Stickle from the Ontario Cancer Institute Genomics Centre for
helping me set up, process, and analyze the microarrays. I also very much appreciate the support
and insight of my committee members: Dr. Michelle Bendeck, Dr. Rita Kandel, and Dr. Eli
Sone.
Last, but not least, I would like to thank my friends and family for their support and
encouragement through these tough but satisfying years. We did it!
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Table of Contents
Acknowledgements ....................................................................................................................... iii
Table of Contents .......................................................................................................................... iv
List of Tables ............................................................................................................................... vii
List of Figures ............................................................................................................................. viii
Chapter 1 – Thesis Motivation and Overview ............................................................................... 1
1.1 Motivation ..................................................................................................................... 1
1.2 Thesis Overview ............................................................................................................ 3
Chapter 2 – Literature Review ....................................................................................................... 4
2.1 Normal Aortic Valve Structure and Function ............................................................... 4
2.1.1 Function and Macrostructure of the Healthy Aortic Valve ........................................ 4
2.1.2 Microstructure of the Health Aortic Valve ................................................................. 4
2.1.2.1 Extracellular matrix components ................................................................... 5
2.1.2.2 Cellular components ...................................................................................... 7
2.2 Calcific Aortic Valve Disease ....................................................................................... 7
2.2.1 Histopathology of Human CAVD .............................................................................. 8
2.2.1.1 Early CAVD lesions ....................................................................................... 8
2.2.1.2 Advanced CAVD lesions ............................................................................... 9
2.2.1.2.1 Lipid accumulation .......................................................................... 10
2.2.1.2.2 Inflammatory processes ................................................................... 10
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2.2.1.2.3 Extracellular matrix remodeling ...................................................... 11
2.2.1.2.4 Calcification ..................................................................................... 13
2.2.2 Osteochondrogenic VIC changes ............................................................................. 13
2.2.3 Proteoglycans and glycosaminoglycans in CAVD ................................................... 15
2.2.3.1 Implications of the role of PGs/GAGs from atherosclerosis ....................... 16
2.2.3.2 Localization and function of specific PGs/GAGs in CAVD ....................... 18
2.2.4 Porcine models of calcific aortic valve disease ........................................................ 21
2.2.4.1 A porcine model of early calcific aortic valve disease ................................ 22
Chapter 3 – Hypotheses and Objectives ...................................................................................... 24
Chapter 4 – Proteoglycan and glycosaminoglycan content in lesions of early CAVD ............... 25
4.1 Introduction ................................................................................................................. 25
4.2 Materials and Methods ................................................................................................ 26
4.2.1 Porcine model of early calcific aortic valve disease ....................................... 26
4.2.2 Porcine leaflet handling .................................................................................. 27
4.2.3 Histological and immunohistochemical staining ............................................ 27
4.2.4 Data retrieval and statistical analyses ............................................................. 29
4.3 Results ......................................................................................................................... 32
4.3.1 HF/HC diet alters lesion ECM morphology with temporal differences ......... 32
4.3.2 Lesions that are ApoB-positive and contain Sox9-expressing cells display
unique PG/GAG composition .......................................................................... 34
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4.3.3 Lesions with dense morphology display more advanced characteristics of
CAVD .............................................................................................................. 37
4.4 Discussion .................................................................................................................... 39
4.4.1 Biglycan may be involved in lipid retention and chondrogenesis in early
lesions of CAVD ............................................................................................. 39
4.4.2 Hyaluronan may play a protective role in early lesion pathogenesis .............. 40
4.4.3 Early CAVD lesions demonstrate further ECM remodeling with distinct
morphological characteristics .......................................................................... 41
4.5 Conclusion ................................................................................................................... 43
Chapter 5 – Phenotypes of valve interstitial cells in lesions of early CAVD .............................. 44
5.1 Introduction ................................................................................................................. 44
5.2 Materials and Methods ................................................................................................ 45
5.2.1 Frozen valve leaflet section preparation ......................................................... 45
5.2.2 Histological and immunohistochemical identification of lesions and samples of
interest ............................................................................................................. 46
5.2.3 Laser capture microdissection ......................................................................... 47
5.2.4 RNA isolation, amplification, and microarray analysis .................................. 48
5.2.5 Data processing and statistical analyses .......................................................... 49
5.2.6 Venn diagram analysis .................................................................................... 50
5.3 Results ......................................................................................................................... 50
5.3.1 Sample characterization .................................................................................. 50
5.3.2 Lesion and non-lesion VIC differential gene expression ................................ 52
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5.3.3 Differential gene expression of VICs within lesion areas ............................... 53
5.3.4 Differential gene expression of VICs from specific lesion areas and non-lesion
areas .......................................................................................................................... 56
5.4 Discussion .................................................................................................................... 58
5.5 Conclusion ................................................................................................................... 61
Chapter 6 – Conclusions and Future Work .................................................................................. 62
6.1 Conclusions ................................................................................................................. 62
6.2 Future Work ................................................................................................................. 63
6.2.1 Further characterization of ECM changes in early CAVD lesions ................. 63
6.2.2 Validation and pathway analysis of microarray results .................................. 64
6.2.3 In vitro studies of biglycan influence on VIC function .................................. 64
6.2.4 Mechanistic studies of hyaluronan interaction with VICs ................................ 65
References .................................................................................................................................... 66
Appendix A. Supplemental Data ................................................................................................. 84
A.1 Myofibroblast detection in porcine valve lesions ....................................................... 84
A.2 Porcine model diet formulation .................................................................................. 86
A.3 PG/GAG scoring validation and analyses .................................................................. 88
A.4 Specific PG/GAG-rich lesions display distinct morphological characteristics .......... 89
A.5 RNA and cDNA quality control before microarray analysis ..................................... 90
A.6 Quality control plots for microarray analyses ............................................................ 94
A.7 Hierarchical clustering of repeated-measures ANOVA results .................................. 96
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Appendix B. Protocols ................................................................................................................. 98
B.1 Valve leaflet histological processing for paraffin-embedded leaflets ........................ 98
B.2 Movat’s pentachrome staining for formalin-fixed, paraffin-embedded sections ...... 100
B.3 (Immuno)histochemistry for formalin-fixed, paraffin-embedded sections .............. 102
B.4 Image processing ...................................................................................................... 106
B.5 Valve leaflet histological processing for OCT-embedded leaflets ........................... 109
B.6 Movat’s pentachrome staining for frozen OCT-embedded sections ........................ 110
B.7. Immunohistochemistry staining for frozen OCT-embedded sections ..................... 112
B.8. Laser capture microdissection protocol ................................................................... 114
B.9. RNA isolation protocol ............................................................................................ 118
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List of Tables
Table 2.1. Transcription factors upregulated in CAVD and their roles in chondrogenic and/or
osteogenic processes .................................................................................................................... 15
Table 5.1. Expression levels of select macrophage-specific markers .......................................... 51
Table 5.2. Select differentially expressed genes between lesion (top and bottom) and non-lesion
areas ............................................................................................................................................. 53
Table 5.3. Select lipid-related genes that are upregulated in the top of lesions ........................... 54
Table 5.4. Select immune-related genes that are upregulated in the top of lesions ..................... 55
Table 5.5. Select ECM remodeling-related genes that are upregulated in the top of lesions ...... 56
Table 5.6. Select differentially expressed genes that are upregulated in the top of lesions
compared to non-lesion areas ....................................................................................................... 57
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List of Figures
Figure 2.1. Healthy aortic valves are composed of a heterogeneous tri-layered structure ............ 6
Figure 2.2. PG/GAG staining intensities within and surrounding calcified nodules ................................ 12
Figure 4.1. Qualitative classification of lesion morphology ........................................................ 29
Figure 4.2. Semi-quantative scoring system for ApoB and PG/GAG content ............................ 31
Figure 4.3. HF/HC diet alters lesion morphology with temporal differences ............................. 33
Figure 4.4. Temporal changes in lesion ECM composition ......................................................... 34
Figure 4.5. Relationship between ApoB and PG/GAG score in lesion areas .............................. 35
Figure 4.6. PG-rich lesions are associated with putative chondrogenesis ................................... 36
Figure 4.7. Relationship between Sox9 fraction and PG/GAG score in lesion areas .................. 37
Figure 4.8. Dense and diffuse lesion characteristics .................................................................... 38
Figure 5.1. Cryosectioning slide schematic for each porcine sample .......................................... 46
Figure 5.2. Lesions of interest for differential gene expression analysis ..................................... 48
Figure 5.3. Distribution of differentially expressed transcripts in lesion and non-lesion areas ... 52
Figure 5.4. Transcript expression in lesion areas ......................................................................... 52
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Chapter 1
1 Thesis Overview
1.1 Motivation
Calcific aortic valve disease (CAVD) is the most common valve disease in North America and
Europe [1]. In the United States, valvular heart diseases account for over 22,000 deaths per year
with CAVD comprising almost 15,000 of those deaths annually [2]. CAVD covers a wide
spectrum of pathological changes, including aortic valve sclerosis and the more advanced form
of aortic stenosis. In 1997, the Cardiovascular Health Study (CHS) reported that early sclerosis
affects 26% of the population over 65 years of age, while late stenosis is present in 2% of this
same population [3]. The incidence almost doubles for individuals over the age of 85, in which
sclerosis is present in 48% and stenosis in 4% [3]. In a follow-up study with the same
participants, the percentage of individuals with stenosis remained the same, but those with
sclerosis increased to 29%, indicating an increase in the prevalence of CAVD [4, 5]. Along with
an increasing prevalence, CAVD is known to be associated with several poor clinical
consequences [4]. Progression from sclerosis to stenosis affects 9% of elderly patients [5] and is
associated with an 80% five-year risk of progression to heart failure, valve replacement, or death
[4].
With a high prevalence and negative clinical outcomes, there are still no effective medical
treatments for CAVD. Currently, the only medical intervention is surgical replacement of the
valve, which has its disadvantages due to the invasiveness of surgery and problems with regards
to durability and lifelong anti-coagulant administration with biological and mechanical valves,
respectively [6]. Although the cholesterol-lowering statins have been explored as a potential
therapy because of their success in treating atherosclerosis, randomized controlled studies have
yet to show their effectiveness in the treatment of valve disease [7-9]. An improved
understanding of CAVD pathogenesis is essential to satisfy the unmet need for effective medical
treatments.
Once thought to be a disease caused by general “wear-and-tear”, CAVD progression is now
recognized as an active process hallmarked by changes in the organization, composition and
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mechanical properties of the valve extracellular matrix (ECM) [10]. This results in thickening
and stiffening of the leaflet, ultimately leading to obstruction of blood flow and impaired cardiac
function. Specifically, lesions and calcification predominantly occur on the aortic side of leaflets
[10, 11]. This side-specific pathosusceptibility is not fully understood, but may give insight into
the regulatory mechanisms involved in CAVD progression. Advanced valve disease has been
studied extensively, and is well-characterized by ECM disorganization, phenotypic changes in
valvular interstitial cells (VICs), lipid infiltration, inflammation, and calcification [12, 13]. In
contrast, early pathological alterations in the cellular and ECM composition of the aortic valve
and their role in disease initiation and progression are poorly understood. As such, the study of
early CAVD pathogenesis will provide a more complete picture of whole disease progression.
A feature of CAVD is aberrant regional expression of proteoglycans (PGs) and
glycosaminoglycans (GAGs), suggesting roles for PGs/GAGs in CAVD pathogenesis. In normal
valve tissue, PGs/GAGs are a major component of only the middle spongiosa layer, but in
advanced disease, increases in the PGs, biglycan, decorin, and versican, as well as the non-
sulfated GAG hyaluronan, have been observed surrounding calcified nodules in the fibrosa layer
[14]. Overall, the role of PGs and GAGs in CAVD has not been extensively studied. It is
suspected they may aid in the initiation of disease by retaining lipids and binding macrophages
as they do in atherosclerosis [15].
PG-rich lesions similar to those seen in the fibrosa layer of human diseased aortic valves [16, 17]
have been recapitulated recently in a porcine model of early aortic valve disease by Sider et al
[18, 19]. These lesions were observed before the appearance of lipid deposition, myofibroblasts,
certain inflammatory cells, and osteoblasts, suggesting that PG lesion formation is an initial step
that occurs before inflammation and VIC activation. Moreover, these early lesions were softer
than the collagen-rich healthy fibrosa, and PG content was positively correlated with expression
of Sox9, a chondrogenic transcription factor. Structural and compositional changes in the valve
ECM can alter VIC fate and function [20, 21] and therefore, soft PG-rich matrices may be
permissive to chondrogenic VIC differentiation. PGs may also indirectly alter VIC phenotypes
by mediating lipoprotein retention and the production of oxidized lipid byproducts that induce
inflammation and calcification. Using our porcine model of early aortic valve disease, I
addressed these issues in my thesis in two complementary studies that (1) examined the
localization of specific PGs/GAGs within early lesion areas using immunohistochemistry; and
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(2) examined VIC phenotypes in altered lesion environments by microarray analysis. These
studies provide new insights into the characteristics and pathobiological processes in early
CAVD.
1.2 Thesis Overview
This thesis is organized into six chapters. Chapter 1 provides the motivation for and overview of
the thesis. Chapter 2 presents a literature review of the topics relevant to this thesis, including
basic aortic valve structure and function and the current understanding of CAVD with a focus on
histopathology, osteochondrogenic processes, and the potential role of proteoglycans in early
disease progression. Chapter 3 states the hypotheses and objectives of this study. Chapter 4
describes the temporal changes of specific proteoglycans and hyaluronan, as well as the
relationship of these extracellular matrix components with markers of lipid retention and putative
chondrogenesis. Chapter 5 describes the lipid-, immune-, osteochondrogenic-, and ECM
remodeling-related phenotypic changes in valve interstitial cells within these lesion areas.
Chapter 6 summarizes the results and provides recommendations for future work.
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Chapter 2
2 Literature Review
2.1 Normal Aortic Valve Function and Structure
2.1.1 Function and Macrostructure of the Healthy Aortic Valve
Located between the left ventricle and the aorta, the aortic valve (AV) is responsible for
preventing backflow of oxygenated blood as it leaves the heart and enters the systemic
circulation. The AV is typically made up of three semilunar cusps or leaflets: the right coronary,
the left coronary, and the noncoronary, which are named according to their relationship to the
coronary ostia. Along the top of each leaflet is the free edge or lannula. Each leaflet attaches to
the aortic wall in a crescent-shaped manner starting from the ends of the free edge, known as the
commissures, and along the basal attachment. Behind each leaflet on the outflow side are dilated
indentations in the aortic root known as the sinuses of Valsalva, which are important for creating
the necessary conditions for valve closure. The non-coapting middle region of the valve leaflet is
known as the belly.
The AV opens and closes approximately 40 million times a year and 3 billion times within an
average lifetime [22]. Valve mechanics largely depend on the changes in hemodynamic forces
and pressure gradients that occur throughout the cardiac cycle. When the left ventricle contracts
during systole, high pressure accelerates oxygenated blood outward from the heart and pushes
the leaflets open. As the left ventricle relaxes in diastole, vortices form in the sinuses of
Valsalva, which stretch the leaflets and cause them to seal along the line of coaptation, allowing
the left ventricle to fill. Apposition of the fibrous nodule of Arantius in the middle of each free
edge ensures complete closure of the valve during diastole. Failure of these mechanisms can lead
to often serious health complications.
2.1.2 Microstructure of the Healthy Aortic Valve
Healthy human aortic valve leaflets are normally composed of three distinct layers: the aortic-
side fibrosa, the middle spongiosa, and the ventricular-side ventricularis (Figure 2.1). The layers
of the valve are heterogeneous: the fibrosa layer is primarily composed of collagen, the
spongiosa is mostly PGs and GAGs, and the ventricularis contains elastin fibers in addition to
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collagen [22, 23]. The two major cell components residing in the valve are valvular endothelial
cells (VECs) and valvular interstitial cells (VICs).
2.1.2.1 Extracellular matrix components
Proper valve function is highly dependent on the complex microstructure of the ECM. Normal
valves are predominantly composed of type I collagen and significant amounts of type III
collagen. Collagen type I is found mainly in the fibrosa layer oriented circumferentially, while
collagen type III is ubiquitously spread throughout the three layers in a less organized fashion
[24, 25]. Collagen fibers give mechanical strength to the valve, allowing expansion during valve
closure in diastole and providing strength to the ventricularis backbone by transferring load to
the aortic root wall [24-26].
Elastin is mostly found in the ventricularis layer, typically found in sheets that stretch radially
from the base of the leaflet to the line of coaptation [25, 27]. Overall, elastin fibers provide
flexibility to the leaflets, allowing repeated deformation and reformation as the valve opens and
closes. In the fibrosa layer, elastin forms honeycomb-like structures, suggesting that they may be
linked and mechanically coupled to collagen fibers. It is believed that the primary role of elastin
fibers involves maintaining valve architecture by returning collagen fibers to their resting
crimped state between loading cycles [24, 27, 28].
Proteoglycans are another critical ECM component and consist of a core protein covalently
linked to at least one GAG chain. GAG chains are repeating disaccharide units that contribute a
negative charge to the PG core and consequently, are an important contributor to PG function
[29-31]. The major types of PGs/GAGs in the valve are biglycan, decorin, versican, and
hyaluronan. Biglycan and decorin are both small, leucine-rich PGs and consist of chondroitin
sulfate and/or dermatan sulfate GAG chains, while versican is a large chondroitin sulfate PG.
Hyaluronan is a type of GAG and is unique because it is the only GAG that exists freely in the
body, separate from a PG chain. Total GAG composition in the valve is approximately 55%
hyaluronan, 30% chondroitin-4-sulfate/chondroitin-6-sulfate, and 15% dermatan sulfate [32, 33].
Although PGs/GAGs are predominantly found in the spongiosa layer, they are also present in the
other valve layers. Histological studies demonstrate unique PG/GAG localization patterns in
each of the valve layers. Decorin and biglycan are ubiquitously found throughout the valve
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layers, but most strongly expressed in the elastin-rich ventricularis layer, suggesting they may
play a role in maintaining leaflet tension and elastogenesis [26, 28]. These small, leucine-rich
PGs also frequently co-localize with collagen, suggesting a role in collagen fibrillogenesis [14,
34]. The large chondroitin sulfate PG versican and non-sulfated GAG hyaluronan are most
abundant in the spongiosa, where they are thought to provide resistance to cyclic compression
and provide lubrication to the outer fibrosa and ventricularis layers by keeping the spongiosa
properly hydrated [14, 26]. Overall, it is believed PGs are necessary for stable assembly of the
ECM and functional cell-ECM interactions.
Figure 2.1. Healthy aortic valves are composed of a heterogeneous tri-layered structure. Movat’s
pentachrome staining (blue = proteoglycan, yellow = collagen, red = cytoplasm, black = elastin/nuclei) of
a porcine aortic valve that is representative of the tri-layered structure in humans. Scale bar = 100 µm.
The valvular ECM architecture is not only critical in maintaining the functional mechanics of the
cardiac cycle, but is also a crucial component that transduces micromechanical forces into
molecular changes that mediate normal valve cell function and biology. Valve dysfunction
occurs when there is disruption of the tri-layered valve structure, resulting from maladaptive
ECM protein regulation, changes in quantity and distribution of ECM components, and
expression of ECM components usually only involved in development or osteochondrogenesis
Fibrosa
Spongiosa
Ventricularis
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[35]. Such environmental changes likely affect valve cell phenotype and differentiation, which
are known to be affected by both biomechanical and microenvironmental cues [36, 37].
2.1.2.2 Cellular components
The primary cell types in the aortic valve are: VECs, which form an outer monolayer lining the
surface of the leaflet, and VICs, which are found ubiquitously throughout the leaflet. VECs are
most likely indirectly involved in maintaining valve homeostasis and ECM remodeling by
regulating permeability and adhesiveness to inflammatory cells, and interacting with circulating
cells and local VICs through paracrine signaling [38]. VECs demonstrate phenotypes that are
side-specific [39] and different from vascular endothelial cells [22].
VICs are a heterogeneous population of fibroblasts, with less than 5% as myofibroblasts and
smooth muscle cells [23, 40-42]. Adult human VICs in situ are generally quiescent, expressing
low levels of alpha-smooth muscle actin (α-SMA) and matrix metalloproteinases (MMPs) [22,
23]. The main function of VICs is to maintain normal valve structure and function by remodeling
and repairing the ECM. They are strongly attached to and synthesize ECM, expressing matrix-
degrading enzymes and their inhibitors, which remodel collagen as well as other ECM
components [43].
2.2 Calcific aortic valve disease
Calcific aortic valve disease (CAVD) is the most common valve disease in North America and
Europe [1], comprising almost 15,000 of deaths annually in the United States [2]. CAVD covers
a wide spectrum of pathological changes, including aortic valve sclerosis, where the valve leaflet
thickens and stiffens without functional impairment, and the more advanced form of aortic
stenosis, where functional impairment is present. Currently, CAVD occurs in over 25% of the
population over the age of 65 years and its prevalence is rising [3, 4]. Moreover, it is associated
with many negative clinical outcomes, including a 40% increased risk of myocardial infarction
and a 50% increased risk of cardiovascular death [4]. Progression from sclerosis to stenosis
affects 9% of elderly patients [5] and is associated with an 80% five-year risk of progression to
heart failure, valve replacement, or death [4]. Currently, the only medical intervention is surgical
replacement of the valve. The disadvantages of invasive surgery, durability of biological
replacement valves, and lifelong anti-coagulant therapy with mechanical replacement valves [6]
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warrant an improved understanding of CAVD pathogenesis to satisfy the unmet need for
effective medical treatments.
For decades, CAVD was thought to be a disease caused by general “wear and tear” [44]. Now,
disease progression is recognized as an active process, hallmarked by changes in the
organization, composition and mechanical properties of valve ECM [10]. Specifically, lipid
infiltration, alterations in VIC phenotype, inflammation, and calcification occur, and are
mediated by pathways normally involved in valve development and bone and cartilage
metabolism [4, 10-13, 45]. Lesions and calcification predominantly occur in the fibrosa layer of
valve leaflets [10, 11]. This side-specific pathosusceptibility is not fully understood, but may
give insight into the regulatory mechanisms involved in CAVD progression. These changes
result in thickening and stiffening of the valve leaflets, ultimately leading to obstruction of blood
flow and impaired cardiac function. Much of what is known about CAVD stems from studies of
advanced or late-stage valve disease. In contrast, there remains a large gap in our understanding
of early changes. As such, further study of initial pathological mechanisms will vastly improve
our understanding of whole disease progression.
2.2.1 Histopathology of human CAVD
2.2.1.1 Early CAVD lesions
Valvular lesions likely arise due to endothelial dysfunction and/or disruption from high
mechanical forces or low shear stress [39, 46]. Such disturbed flow occurs on the aortic side of
the leaflet [39, 47, 48] where these lesions predominantly form. This side-specific
pathosusceptibility may provide clues to disease etiology. VECs on the fibrosa side of healthy
valves display a side-specific phenotype that is permissive to calcification, expressing genes that
promote or permit skeletal development and vascular calcification [39]. In addition, the aortic-
side endothelium also expresses compensatory protective mechanisms against inflammation and
lesion initiation. Even following a two-week hypercholesterolemic diet, this protective
endothelial phenotype persists on the fibrosa in pigs [49]. Further lending to this notion of side-
specific pathosusceptibility, using fluorescently tagged low density lipoprotein (LDL), it was
shown that the fibrosa side of the valve has a markedly enhanced potential to bind LDL
compared to the ventricularis side in healthy porcine aortic valves [50].
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Early human CAVD lesions are characterized by focal subendothelial thickening on the fibrosa
side of the leaflet. The most easily identifiable feature of these lesions is displacement of the
elastic lamina, which often appears fragmented and/or reduplicated [17]. The basement
membrane beneath the endothelium also often appears thin, frayed reduplicated and/or absent
[11]. Forming between the endothelial layer and displaced elastic lamina, early lesions
accumulate protein, lipid, inflammatory cell infiltrate, and extracellular mineralization [11, 17].
While variable levels of collagen and elastin accumulate in early CAVD lesions [11, 17], there is
an overall increase in the three major valve PGs [16]. Localization patterns suggest defined roles
for specific PGs, as biglycan and decorin tend to be present in areas where versican is absent
[16].
Neutral lipid accumulation in early human lesions has been detected by oil red O staining [11,
17, 51]. In contrast to histologically normal regions, lesions and their adjacent fibrosa
accumulate apolipoprotein (Apo) B, Apo(a), and ApoE [17]. Spatial comparisons suggest that
most of the extracellular neutral lipids are related either to plasma-derived or locally produced
apolipoproteins. Since LDL has been observed in early lesions [52], the similar distribution
patterns of ApoB and Apo(a) [17] suggest that both LDL and lipoprotein(a) are deposited. ApoE,
which is largely present in atherosclerotic lesions, is often found in valvular regions with ApoB
and Apo(a), but is also present extracellularly, without ApoB and within macrophages. Since
macrophages increase their secretion of ApoE in response to intracellular cholesterol loading,
much of this ApoE may be produced locally [17].
In early lesions, the presence of inflammatory cells is highly variable with macrophages in only
25-65% of lesions [12, 53] and T-lymphocytes less often [11]. When present, macrophages are
commonly located near the surface of the lesion, while the earliest forms of calcification occur in
a stippled pattern deeper near the base [11, 17]. Importantly, however, early lesions can form in
the absence of macrophages, T-lymphocytes, and calcification [53]. The layered appearance of
lesion features supports the active progression of disease pathogenesis.
2.2.1.2 Advanced CAVD lesions
As CAVD progresses, features observed in early disease, including loss of elastic lamina, protein
accumulation, lipid accumulation, and inflammation, are more pronounced, leading to more
marked thickening of the fibrosa and active tissue calcification.
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2.2.1.2.1 Lipid accumulation
Areas of lipid accumulation have also been identified by oil red O staining in late-stage diseased
valves [11, 17, 51], as well as the presence of ApoB, Apo(a), and ApoE [17]. Since ApoB is
present in the absence of Apo(a), the distribution of which is more restricted to the central
regions of the fibrosa, it is likely that both LDL and Lp(a) are deposited.
Oxidized LDL (oxLDL) is also present in stenotic aortic valves, localizing to subendothelial
regions on the fibrosa side with calcium deposition and co-localizing with ApoB and neutral
lipid [51, 54, 55]. OxLDL is both cytotoxic and can stimulate inflammatory activity. Specifically
in the valve, it has been shown to stimulate calcification in valve fibroblasts in vitro [56].
Stenotic valves with high oxLDL scores also have a significantly higher density of leukocytes,
macrophages, and T-lymphocytes compared to valves with lower oxLDL scores [54]. As well, in
diseased valves oxLDL co-localizes with lipoprotein lipase (LPL), which itself localizes in cell
dense areas with abundant macrophages and is associated with valve tissue remodeling.
Sequestration of lipids and oxidative transformation may initiate recruitment of inflammatory
cells, which may further promote the retention of lipids in a cyclic manner [55]. Moreover,
stimulation of aortic VICs with oxLDL has been shown to increase phosphate inorganic
transporter 1 (Pit1) and bone morphogenic protein 2 (BMP2), indicating a potential role for
lipids in osteogenic VIC differentiation and calcification [57].
2.2.1.2.2 Inflammatory processes
In advanced CAVD lesions, an increase in cellularity, including non-foam cell macrophages,
foam cell macrophages, and T-lymphocytes, is present [11, 17, 51, 58-64]. Macrophages are
present in 59-75% [11, 60] and lymphocytes in 75-90% [11, 65] of stenotic valves. Macrophages
and T-lymphocytes are commonly observed near calcium deposits in the subendothelium of the
fibrosa layer [58, 66], co-localizing with areas of neutral and oxidized lipid [51]. Mast cells [62,
67, 68] and B-lymphocytes [67] are also present in stenotic valves. In addition, the presence of a
variety of inflammatory mediators indicates active inflammatory processes. Overall, there is an
increased expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα)
[51, 63, 64], interleukin-1 [63] and -2 [58], matrix metalloproteinases (MMP-1, -2, -3, -9, -12)
[61, 63, 66, 69, 70] and complement activation [71, 72]. Oxidative stress may also play a role, as
11
gradients of superoxide and hydrogen peroxide are present with the highest levels occurring in
and near calcified areas [73].
The infiltration of inflammatory cells may create a pro-fibrotic environment involving the renin-
angiotensin system [74]. Increased levels of angiotensin-converting enzyme (ACE) [62, 75],
mast cell cathepsin G and chymase [62], and their enzymatic product, angiotensin II (AngII)
[75], are present. Interestingly, angiotensin II type 1 receptor (AT-1R) is only expressed by valve
fibroblasts in lesions [62, 75], suggesting the reaction of fibroblasts to angiotensin II is blunted
until they express this major pathogenic receptor for AngII [10]. The activation of AT-1R can
increase the production of oxidants and PGs [29, 75].
Endothelial cells also show evidence of inflammatory processes. Adhesion between endothelial
cells and circulating leukocytes is a key initial event in recruiting and transmigrating leukocytes
to sites of inflammation. Those found in valves with CAVD have increased levels of vascular
cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) [76, 77], and
endothelial selectin (E-selectin) compared to healthy valves [76]. In addition, diseased patients
exhibit elevated levels of E-selectin [76], which is thought to reflect systemic inflammatory
conditions.
2.2.1.2.3 Extracellular matrix remodeling
In advanced disease, the ECM experiences continued remodeling [78]. Similar to early disease
histopathology, distributions of the three major valve PGs, biglycan, decorin and versican, and
the GAG hyaluronan are altered in stenotic valves. In the main report published to date, stenotic
aortic valves removed during valve replacement surgery were immunostained for PGs/GAGs and
analyzed, with focus on expression in and around calcified nodules that were categorized as large
or small, which the authors interpreted as mature or early stage, respectively [14]. Biglycan and
decorin expression within larger calcified nodules was lower than in the regions immediately
surrounding the larger nodules, the regions within and surrounding smaller “pre-nodules”, and
the normal fibrosa, but there were no other significant differences in expression levels of
biglycan or decorin (Fig. 2.2). Versican and hyaluronan expression was lower within larger
nodules than in the immediate surrounding regions and lower within smaller “pre-nodules” than
in the regions surrounding larger nodules, but otherwise not significantly different regionally.
12
Figure 2.2. Proteoglycan and glycosaminoglycan staining intensities within and surrounding
calcified nodules. p<0.05 compared to Nod Surr. *p<0.05 compared to Fibrosa.
ǂp<0.05 compared to
Prenod. Nod Ctr=innermost 1/3 of the large nodule. Nod Edge=outer 1/3 of the large nodule. Nod
Surr=tissue immediately surrounding the large nodule. Prenod=prenodule. Prenod Surr=tissue
immediately surrounding the prenodule. Error bars = SEM. Adapted with permission from reference [14]
© Elsevier.
Changes in collagen composition and localization also occur, lending to the fibrotic
characteristics observed in advanced CAVD [77]. Dense fibrotic areas are present with
disorganization of collagen fibers in the fibrosa layer. In addition, there is an increase in the
synthesis of type I procollagen, the precursor of the major collagen component of normal valves,
but total collagen is significantly decreased in calcified valve leaflets compared to healthy
control valve leaflets. This suggests that throughout the entire valve there is an overall increased
turnover of type I collagen in stenosis, where degradation exceeds production [79]. Both
collagen II [13, 78] and X [78] are also observed in human adult CAVD, supporting the role of
active osteochondrogenic mechanisms underlying calcification in CAVD. In particular, localized
areas of collagen X expression are observed close to heavily calcified areas of the valve [13].
The disorganization of collagen bundles can also be attributed to the increased presence of
MMPs [69] and their tissue inhibitors (TIMPs) [63, 69]. Fragmented and disorganized elastin
13
fibers are reported in areas of prominent calcification and initial mineral deposition. This may be
due to increases in elastolytic enzymes, such as MMP-12, which has been detected in its active
form in these areas of ECM alteration, initial mineral deposition, and calcification. Areas of
collagen and elastin fragmentation are both suggested to be potential nidi for calcium deposition
[66].
2.2.1.2.4 Calcification
Ultimately, the valve tissue reaches a stage where it forms calcified nodules, primarily made of
amorphous calcium phosphate [67, 80, 81], and transforms from its natural pliant state to one
that is more rigid and unable to close properly. Calcification co-localizes with areas of lipid
deposition [11, 51] deeper within the lesion [11], and tends to be absent in areas devoid of lipid
[51].
In stenotic valves, calcific nodules are commonly found co-localizing with vasculature [66, 67,
77, 82]. Due to their thinness, diffusion of oxygen from the surfaces of healthy aortic valve
leaflets is sufficient to support the valve’s metabolic needs. When present, which occurs
sparsely, microvasculature is typically found at the base of the valve cusp and within the
ventricularis and spongiosa layers [83, 84]. The presence of neoangiogenesis co-localized with
calcification in diseased states suggests that it may also aid in endochondral ossification [67].
2.2.2 Osteochondrogenic VIC changes
While dystrophic calcification seems to be the major mechanism in heavily calcified valves [85],
heterotopic bone formation also contributes to local valvular calcification [67], indicating that
both passive and active processes are at work. Thirteen percent of calcified aortic valves
demonstrate mature lamellar bone formation with hematopoetic elements and active bone
remodeling, where there is both osteoblastic bone formation and osteoclastic bone resorption
[67]. Earlier stages of osteogenesis are also observed in the form of endochondral bone
formation [66, 67, 86], the process by which long bones are created from the replacement of
bone matrix over a cartilage template. In addition to the presence of bone itself, cartilage is
observed in human stenotic valves independently and with bone [86]. In endochondral
osteogenesis, chondrocytes differentiate from MSCs and as they proliferate, produce collagen
type II, IX and XI, and sulfated GAGs. Further differentiation of the chondrocytes results in
14
hypertrophic cells that produce collagen type X. The ECM eventually mineralizes, chondrocytes
apoptose, and the calcified cartilage template is infiltrated by capillaries and replaced by bone
[87]. These areas of endochondral ossification in the valve have been described as the
transformation of cartilage into lamellar bone through a zone of provisional calcification [86].
Consistent with this notion of bone formation, several chondrogenic and osteogenic transcription
factors are upregulated in human stenotic valves [88] (Table 2.1). Interestingly, along with their
involvement in cartilage and/or bone formation, many of these transcription factors are also
involved in valve development [13]. Non-calcific, pediatric diseased valves, which may
represent an earlier disease stage, demonstrate an upregulation of chondrogenic pathways with
increases in sex determining region Y box 9 (Sox9), myocyte enhancer factor 2C (Mef2c),
Twist-related protein 1 (Twist1), and muscle segment homeobox 2 (Msx2) [13]. In calcified
adult diseased valves, these chondrogenic transcription factors are also upregulated [13], along
with several other osteogenic transcription factors, including Runt-related transcription factor 2
(Runx2) [13, 80, 89], phosphorylated Smads (p-Smad) 1/5/8 [13], nuclear factor of activated T-
cells cytoplasmic 1 (NFATc1) [89], and osterix (Osx) [89].
Of note to studies of early disease, Sox9-expressing cells were observed in early lesions in a
porcine model of CAVD [19]. Sox9 is important for the differentiation of the chondrocyte
lineage and for the expression of genes characteristic of cartilage, such as collagen 2a1 (Col2a1)
[88, 90, 91]. In teratomas derived from homozygous Sox9 mutant embryonic stem cells, no
cartilage forms although the usual tissue type varieties for teratomas are present, indicating that
the transcription factor plays an essential role in chondrogenesis [92]. Bone morphogenic protein
2 (BMP-2), which is also detected in human stenotic valves [67, 93], increases the expression of
Sox9, as well as its downstream response genes such as Col2a1 [94]. Other mediators of Sox9
upregulation include fibroblast growth factors [95] and hedgehog signaling pathways [94].
15
Table 2.1. Transcription factors upregulated in CAVD and their roles in chondrogenic and/or
osteogenic processes
Transcription Factor Role in chondrogenesis and/or osteogenesis References
Sox9 Chondrocyte lineage differentiation
Expression of ECM genes characteristic of cartilage
[90], [91]
Mef2c Chondrocyte maturation
Bone formation
[96]
Twist1 Osteoblast differentiation inhibitor and chondrogenesis promoter in
osteoblast progenitors
[97]
Msx2 Proliferation of osteogenic progenitor cells
Bone and cartilage formation
[98], [99]
Runx2 Maturation of osteoblasts
Regulation of osteogenic ECM genes (e.g. collagen X and
osteocalcin)
[100], [101]
NFATc1 Regulation of osteoclast differentiation
Osteoblastic bone formation
[102], [103]
Osterix Osteoblast differentiation
Osteocalcin expression
Bone formation
[104]
In addition, the early CAVD porcine model by Sider et al. positively correlated the expression of
Sox9 with soft PG-rich matrices [18, 19]. In other tissues, expression of Sox9 is stiffness-
dependent and is linked to proteoglycan accumulation. In response to TGF-β1 stimulation,
murine chondrocytes express Sox9 and Col2a1 in a stiffness-sensitive manner [105]. In the
absence of biochemical factors, MSCs cultured on soft substrates (~1 kPa) express higher mRNA
levels of Sox9 and Col2a1 and accumulate PGs in cell aggregates compared to on stiffer
substrates (~15-150 kPa) [106]. As well, the trio of Sox5, Sox6, and Sox9 induces production of
a PG-rich matrix by MSCs [107]. Whether there is a causal relationship between PGs and
osteochondrogenic processes though has yet to be elucidated in valve disease.
2.2.3 Proteoglycans in calcific aortic valve disease
Recently, changes in the abundance and distribution of PG/GAG content in valve leaflets have
garnered interest. In healthy valve tissue, the most common PGs/GAGs are a major component
of the middle spongiosa layer. In advanced CAVD, increases in the PGs biglycan, decorin and
versican, and the non-sulfated GAG hyaluronan are observed in the area immediately
16
surrounding calcified nodules in the fibrosa layer [14]. Studies of early and advanced CAVD
indicate they may play a crucial role in disease progression.
The formation of PG-rich lesions, similar to those seen in humans [11, 16], are observed in
porcine [18] and mouse (unpublished data) diet-induced models of CAVD. In our swine model
by Sider et al. [18, 19] and in human valves with early disease [11, 17], these PG lesions are
observed before the appearance of myofibroblasts, macrophages, dendritic cells, and significant
lipid accumulation. I corroborated the absence of myofibroblasts by both immunoperoxidase and
immunofluorescence methods in the swine model (Appendix A.1). This suggests that PG lesion
formation is an initial step in CAVD pathogenesis, occurring before VIC activation,
inflammation and lipid retention. Further, early PG-rich lesions tend to be softer compared to
normal fibrosa, which may be more permissive to chondrogenesis. As discussed above,
expression of chondrogenic markers appears to be stiffness-dependent [105, 106]. Putative
chondrogenesis is supported by the increased presence of Sox9-positive cells observed in early
porcine CAVD lesions [18].
2.2.3.1 Implications of the role of proteoglycans from atherosclerosis
CAVD shares several risk factors with atherosclerosis, including hypertension,
hypercholesterolemia, smoking, male gender, diabetes, chronic renal disease and older age [3,
11]. Consequently, it is believed CAVD may have similar pathobiological processes as
atherosclerosis. In early lesions, characteristics shared by both diseases include displaced elastic
lamina, lipid infiltration and involvement of inflammatory cells [11]. Furthermore, the initiating
factor for progression to CAVD and atherosclerosis seems to involve endothelial injury at sites
of low shear and high tensile stress.
Although the role of PGs/GAGs in CAVD has not been extensively studied, it is suspected they
may aid in the initiation of disease by retaining lipids and binding macrophages, as they do in
atherosclerosis [15, 108]. Although recognized by the Council of American Heart Association as
normal intima, diffuse intimal thickening (DIT), which consists of PGs, elastin and smooth
muscle cells (SMCs), is thought by some to be a precursor of atherogenesis, as it consistently
presents in atherosclerosis-prone arteries and not in atherosclerosis-resistant arteries [108, 109].
The predominant PGs in these atherosclerosis-prone arteries are biglycan and versican, while
17
atherosclerosis-resistant arteries are thin and enriched in decorin [110, 111]. Decorin follows a
similar distribution pattern to biglycan in DIT, but has far fewer positively stained areas [109].
DIT occurs before lipoprotein deposition [15] with only a small number of macrophages present
and with no evidence of neovascularization [108]. The response-to-retention hypothesis suggests
that a predisposing factor, such as mechanical stress or cytokines, stimulates the local synthesis
of PGs that have a high binding affinity for lipoproteins [108]. Once these atherogenic ApoB-
containing lipoproteins enter the arterial intima, it is thought that they are retained by PGs [112].
The resulting lipoprotein-PG complexes are more susceptible to modifications, such as oxidation
and aggregation, which lead to uptake by macrophages to form foam cells. As well, the resulting
oxidized lipids may promote further production of PGs that have a high affinity to lipoproteins
[15].
In atherosclerosis, PGs bind lipoproteins through ionic interaction via their negatively charged
GAG chains, which can be mediated by accessory molecules such as lipoprotein lipase (LPL)
[15, 113-115]. The lipid-binding capacity of PGs, which relates to GAG chain length and
sulfation, contributes to the retention of lipoproteins in the intima [116-119]. The most common
PG in the vascular ECM is versican, followed by biglycan and decorin. In vitro studies have
shown that of the three major PGs, versican has the greatest potential to bind lipoproteins
because of its high number of LDL binding sites [111, 113]. In contrast, in vivo studies show that
biglycan and decorin most commonly co-localize with LDL in early atherosclerotic lesions [15,
112]. Overexpression of human biglycan by rat SMCs results in production of an ECM with
greater high-affinity lipoprotein binding [120]. Furthermore, pre-lesion biglycan was localized in
a similar distribution to lipids in the early phase of atherosclerotic lesions [15], suggesting that
biglycan may play an important role in the very initial stages of lipid deposition. Decorin may
play a role in linking lipoproteins to collagen in atherosclerosis, as it has been shown to link LDL
with collagen type I in vitro and co-localize with collagen and ApoB in atherosclerotic lesions in
vivo [108]. A transgenic mouse model of human ApoB100, which expressed PG-binding
defective LDL, receptor-binding defective LDL, or wild-type LDL further validated the
importance of PG binding to LDL in the initial stages of atherosclerosis [121]. Mice fed an
atherogenic diet were less susceptible to atherosclerotic lesion formation if they had PG-binding
defective LDL instead of wild-type LDL. Moreover, mice with different LDLs demonstrated no
18
difference in susceptibility to oxidation and macrophage uptake. Therefore, reduced
atherogenesis was likely due to reduced PG-binding ability.
Despite certain similarities between atherosclerosis and CAVD, implications drawn from
atherosclerosis research must be taken with a grain of salt. Less than 40% of patients with
CAVD have clinically significant coronary atherosclerosis [122]. In addition, although statins are
widely used to effectively treat atherosclerosis, randomized controlled studies have yet to show
their effectiveness in the treatment of valve disease [7-9]. Evidently, some distinct processes are
involved in CAVD. In contrast to atherosclerosis, there is no SMC involvement in CAVD [11].
As well, since lipoproteins are observed in the adjacent fibrosa, it is evident that in CAVD, early
lesions are not confined to the area bound by the elastic lamina, as they are in vascular disease.
Although some insight into CAVD progression may arise from studies of atherosclerosis,
differences between the diseases warrant further individual study of CAVD progression.
2.2.3.2 Localization and function of specific PGs/GAGs in CAVD
PGs/GAGs exhibit spatial heterogeneity in both early and advanced CAVD lesions. In early
lesions, versican tends to be absent in areas with biglycan and decorin [16]. Surrounding and
within calcified nodules in the fibrosa layer of stenotic valves, spatially heterogeneous changes
in the major valve PGs, as well as hyaluronan, are observed with dependence on nodule size [14]
(Figure 2.2). In stenotic leaflets, PGs and hyaluronan were found to have the greatest expression
in areas directly surrounding calcified nodules. Within smaller calcified nodules, termed
“prenodules” and interpreted to be less advanced, and surrounding regions, decorin and biglycan
are significantly more abundant compared to within and surrounding larger calcified nodules.
Biglycan and decorin may accumulate in early nodule formation, but as the nodule becomes
more mineralized, be more involved in remodeling the tissue surrounding nodules. Within
prenodules, versican and hyaluronan are negatively correlated with biglycan and decorin,
suggesting they may be less involved in early nodule formation. Their presence surrounding
nodules and prenodules suggests they may be more involved in remodeling areas surrounding
mineralized tissue.
Insight into the localization of specific PGs/GAGs involved in early CAVD will aid the
understanding of the pathobiological changes that result in calcification and stenosis.
Proteoglycan function and categorization is largely determined by the composition of its GAG
19
chains, which can differ in sulfation pattern, disaccharide composition, and chain length [29].
Decorin and biglycan are both small leucine-rich PGs that are composed of one and two GAG
chains, respectively, from chondroitin sulfate and/or dermatan sulfate. Versican is a large
chondroitin sulfate PG, which occurs in four isoforms: V0, V1, V2, and V3. Hyaluronan is a
non-sulfated GAG and instead, interactions occur largely through receptors such as CD44,
receptor for hyaluronan-mediated motility (RHAMM or CD168), and hyaluronan receptor for
endocytosis (HARE).
Proteoglycans potentially play a direct and indirect role in lipid retention during the early stages
of CAVD. Glycosaminoglycan chains contain negatively charged sulfate groups and carboxylic
groups, which allow PGs to interact with positively charged lysine and arginine residues, such as
those on apolipoproteins. Multiple LDL particles can bind to a single GAG chain [123, 124]. Of
the major valve PGs, decorin has one GAG chain, biglycan has two GAG chains, and versican
isoforms range up to 23 GAG chains [125]. In valve lesions though, apolipoproteins have been
observed to co-localize with biglycan and decorin [16, 17]. Using LDL affinity columns, it was
shown that decorin and biglycan are major mediators of lipid retention in porcine aortic valves
[50]. Structural properties of GAG chains that may influence apolipoprotein binding include
GAG chain length and sulfation pattern. In vitro studies demonstrate that PG binding affinity for
LDL is augmented with increasing GAG chain length [126]. Interestingly, GAG chains bound to
PG core proteins show higher affinity binding to LDL compared to free chains due to
thermodynamic considerations of molecular rigidity [117]. Several factors have been shown to
cause elongation of chondroitin sulfate chains, including TGFβ and oxLDL [127]. Subtle
changes in sulfation are also thought to be able to alter the ionic interactions of GAGs with
apolipoproteins. For example, in comparison to 4-sulfated GAGs, 6-sulfated GAGs are more
sterically accessible to the binding sites on LDL [29]. These PG-LDL interactions are largely
electrostatic in nature, as denaturing agents, SDS, and urea resulted in little, if any, eluent
following salt elution. Further, proteoglycans, specifically decorin, can act as bridging molecules
to mediate LDL-collagen interactions [50]. In addition to direct ionic interactions with lipids,
proteoglycan GAG chains are able to bind lipoproteins via bridging molecules, including LPL
[126] and ApoE [128].
Lipid retention then allows modifications of lipoproteins by enzymes, such as, hepatic lipase
(LIPC), phospholipid transfer protein (PLTP) and LPL [112], which can result in further lipid
20
retention and inflammation. In valve disease, biglycan induces increased expression of
phospholipid transfer protein (PLTP) by VICs via Toll-like receptor 2 (TLR2) [129] and decorin
has been shown to co-localize with LPL [55]. In these studies, biglycan and decorin were also
found to co-localize with oxLDL, which is associated with inflammation [54] and has been
implicated in osteogenic VIC differentiation [57]. Interestingly, biglycan has been shown to
contribute to the pro-osteogenic effect of oxLDL on human aortic VICs [130]. Stimulation of
VICs with oxLDL increases expression of biglycan, which in its soluble form can induce the
expression of BMP2 and ALP via TLR2.
The small leucine-rich PGs biglycan and decorin are also known to mediate collagen
fibrillogenesis [14, 131] and sequester TGF-β [14, 34]. The latter is particularly significant, as
TGF-β1 has been shown to induce stiffness-dependent VIC differentiation to chondrogenic or
myofibroblastic phenotypes [37, 132, 133] and stimulate expression of MMPs [134], which
mediate further ECM remodeling in disease. In addition, TGF-β1 has been shown to induce PG
core protein synthesis and GAG chain elongation in porcine VICs. Interestingly, PGs synthesized
in response to TGF-β1 demonstrate enhanced LDL binding [126].
Studies involving hyaluronan thus far demonstrate both potential protective and pathogenic roles
in the aortic valve. In stenotic human aortic valves, hyaluronan abundance is inversely related to
the magnitude of observed fibrosa layer calcification [135]. As well, VICs treated with
hyaluronan have suppressed calcified nodule formation, indicating it may have a protective role
[21]. This is consistent with other cell types, where hyaluronan attenuates the cellular response to
TGF-β1 [136]. In atherosclerosis though, hyaluronan has the ability to retain lipids [137] and is
involved in the accumulation and activation of inflammatory cells [138, 139], indicating it may
also play a role in lipid retention and chronic inflammation during CAVD. The presence of
hyaluronidase-1 and hyaluronan synthases co-localized with differentiation markers of brown fat
cells and chondrogenesis within and surrounding calcified nodules suggests that turnover of this
GAG has a role in disease progression [140].
The role of PGs in mineralization processes is not clear, but their GAG components, particularly
chondroitin-4-sulfates, have the capacity to bind calcium and interact with hydroxyapatite [141].
While the direct pathobiological roles of PGs/GAG in valve calcification have not yet been fully
explored, it is suspected that they may aid in lipid retention, which may initiate a cascade of
21
inflammation, osteogenesis and/or apoptosis, leading to calcification. Overall, an improved
understanding of the changes that occur in the ECM, particularly specific PGs/GAGs, as well as
the effect these changes in lesion microenvironment have on VICs, is required to define their
contribution to early CAVD progression.
2.2.4 Porcine models of calcific aortic valve disease
Due to the availability of human stenotic valves upon valve replacement, late-stage CAVD is
well-characterized. Knowledge of the initiating events involved in CAVD has been limited
though by the difficulty of retrieving suitable samples that represent the desired disease stage and
that are controlled for by confounding factors in human autopsy or transplant patients.
Consequently, the use of animal models is necessary to satisfy the unmet need to uncover the
pathobiological processes involved in CAVD.
The animals most commonly used in the study of CAVD are mouse, rabbit, and swine [142].
Swine are thought to be excellent models for the study of atherosclerosis [143, 144] and recently,
of CAVD [142, 145] because of their many similarities to humans. Unlike mice, swine have tri-
layered aortic valve leaflets, which is an important feature considering the pathosuceptibility of
the fibrosa side to forming CAVD lesions. Swine also spontaneously develop atherosclerotic,
human-like lesions, a process that is accelerated by high-fat/high-cholesterol diets [146]. In
contrast, wild-type mice fed standard diets do not exhibit spontaneous calcification [145] and
usually require diet and/or genetic predisposition to induce advanced disease [147-150]. Rabbits
also do not develop spontaneous atherosclerotic lesions and have a significantly different lipid
metabolism compared to humans [143]. As a result, they usually require very high cholesterol
levels to induce advanced disease [151-153]. When fed an atherogenic diet, swine exhibit similar
lipid profiles [154] and lipoprotein metabolism [146, 155] as humans. Furthermore, swine and
human genomes are comparable in size and homologous in sequence and chromosomal structure,
making porcine models useful for genomic studies [145]. Swine have not been extensively used
in CAVD research though, mainly due to their large size, which creates limitations because of
cost and maintenance issues.
Studies of CAVD in swine show that they develop human-like lesions when fed a
hypercholesterolemic diet [18, 39, 49]. Distinct areas of subendothelial lipid accumulation and
early calcific nodules are present on the fibrosa side at two weeks and moreso, at six months [39,
22
49]. At both time points, no frank inflammation is present [49]. This diet also induces a side-
specific protective phenotype in fibrosa side VECs that is anti-calcific, anti-apoptotic, and anti-
inflammatory.
2.2.4.1 A porcine model of early calcific aortic valve disease
Recently, a porcine model was developed by Sider et al. to gain further insights into the early
ECM changes that occur with CAVD [18, 19]. This porcine model successfully mimics many
characteristics of early human CAVD, which are enhanced by hypercholesterolemia. Moreover,
the diets achieved cholesterol levels for both control and experimental groups that are
comparable to normal humans and those with familial hypercholesterolemia, respectively.
The fibrosa side of these valve leaflets forms human-like early CAVD lesions, the most
advanced of which develop after being fed HF/HC diet for 2 or 5 months. Lesions are composed
primarily of PGs with varying amounts of collagen and elastin, which are laid down between the
endothelial cell layer and the displaced, fragmented and/or reduplicated elastic lamina on the
fibrosa side of the valve. Often, these changes in the elastic lamina are the first visible sign that
there is disruption of the normal valve microstructure. These lesions occur in the absence of
myofibroblasts, osteoblasts, macrophages, and dendritic cells, indicating that they, indeed,
represent an early stage of valve disease.
Lesions from pigs fed the HF/HC diet also have a greater presence of ApoB, Sox9-positive cells,
and Msx2-positive cells compared with pigs fed normal chow, suggesting a role in lipid retention
and putative osteochondrogenesis. Although it has been proposed that lipid retention is an initial
step of valve disease, ApoB was present in only 28% of all lesions, suggesting that it is preceded
by PG deposition. According to layer-specific stiffness analysis using micropipette aspiration,
these early lesions also have a tendency to be softer than normal fibrosa. In addition, soft onlays
contained more PG and less fibrillar collagen compared to normal fibrosa. It has been suggested
that this soft, PG-rich microenvironment is permissive to increased Sox9 expression by resident
VICs, but this causal relationship has yet to be elucidated.
An important limitation of this model is that no calcification was observed up to the time points
studied. Although markers associated with early osteogenic differentiation were present, this
does not definitively support initiation of mineralization. Still, diabetic swine have been shown
23
to develop early limited valvular calcification over similar time periods [39, 49] and other swine
on an atherogenic diet formed atherosclerotic lesions with calcification [146], indicating the
potential for cardiovascular calcification in swine. Nevertheless, the similarities with early
human disease suggest that this porcine model allows for critical insights into the initial stages of
lesion formation and sclerosis.
24
Chapter 3
3 Hypotheses and Objectives
3.1 Hypotheses
There are temporal changes in the amount of specific proteoglycans and glycosaminoglycans
(PGs/GAGs) in early PG-rich lesions as calcific aortic valve disease (CAVD) progresses. In
particular, it is hypothesized that levels of biglycan, decorin, and versican increase and
hyaluronan decrease with increasing time and administration of a high-fat/high-cholesterol diet.
Further, it is hypothesized that PGs/GAG found in more advanced lesions associate with lipid
retention and Sox9-expressing cells. With these changes in the lesion microenvironment, there
are phenotypic differences between valve interstitial cells (VICs) in lesions versus the normal
fibrosa.
3.2 Objectives
Using a porcine model of early CAVD:
(1) To quantify the amount of specific PGs/GAG in early CAVD lesions using
immunohistochemistry;
(2) To define correlations between PG/GAG content in early CAVD lesions with lipid
retention and Sox9-expressing cells using immunohistochemistry; and
(3) To characterize the phenotypic differences between VICs in the lesion and healthy
fibrosa using microarrays.
25
Chapter 4
4 Proteoglycan and glycosaminoglycan content in lesions of early CAVD
4.1 Introduction
Once thought to be a disease of passive degeneration [44], calcific aortic valve disease (CAVD)
is currently recognized as an active process in which cellular and extracellular matrix (ECM)
changes result in thickening and stiffening of the valve leaflets, which ultimately lead to
obstruction of blood flow and impaired cardiac function [10]. Due to the availability of suitable
tissue samples from aortic valve replacement, advanced disease has been extensively studied and
is characterized by ECM disorganization, fibrosis, and calcification [10, 67]. Still, CAVD
persists as a prevalent disease with poor clinical consequences and no effective medical therapy
[3, 4]. Consequently, studies of early disease processes are essential to better understand disease
progression and to find novel targets for CAVD treatment.
Aortic valve leaflets demonstrate a side-specific pathosusceptibility, preferentially forming
CAVD lesions on the fibrosa side of the valve. Early lesions are characterized by areas of focal
subendothelial thickening with displacement of the elastic lamina, which often appears
fragmented and/or reduplicated, and a thin, frayed, reduplicated, or absent basement membrane
[11, 17]. This subendothelial thickening often accumulates lipid, inflammatory cell infiltrate,
extracellular mineralization, and protein [11, 17]. Lipoproteins are commonly found within these
lesions [11, 17] and often co-localize with PGs [16, 52]. The presence of macrophages in these
early lesions tends to vary drastically from none to substantial [11, 53], but when present, occur
near the surface of the lesion or in a stippled pattern near the base of the earliest calcified lesion
[11, 17]. This layered appearance further supports the notion of the active progression of CAVD
[11].
Early studies of CAVD have been hampered by the lack of suitable human tissue samples and
well-characterized animal models. Recently though, studies of early CAVD in swine showed that
they develop human-like lesions when fed an atherogenic diet [18, 39, 49]. Pigs are considered to
be excellent models for studies of atherosclerosis [143, 144], and recently, of CAVD [142, 145].
26
Compared to humans, they have (1) similar lipid profiles [154] and metabolism [146, 155]; (2)
comparable genome size and homology [145]; and (3) develop atherosclerotic-like lesions with
age, which is accelerated with HF/HC diet [146].
The porcine model developed by Sider et al. [18, 19] is able to successfully mimic several
characteristics of early CAVD lesions seen in humans [11, 17]. These lesions form before the
appearance of myofibroblasts, significant lipid accumulation, significant inflammatory cell
infiltrate, and calcification, indicating that they represent a very early disease stage. Interestingly,
while collagen and elastin content varies, PGs appear as the primary component of accumulation
within these lesions. Proteoglycans are typically found in the spongiosa layer of healthy aortic
valves, but accumulations have been observed in both early sclerotic [16] and advanced stenotic
valves [14]. From this porcine model of early CAVD, it is suggested that PG accumulation
precedes lipoprotein retention, since the formation of PG-rich lesions often occurs without any
detectable lipoprotein deposition. Nevertheless, ApoB deposition and Sox9 and Msx2 expression
scores are greater in PG-rich lesion than non-lesion areas, suggesting a role for PGs in lipid
retention and putative osteochondrogenesis.
In advanced CAVD, increases in the PGs biglycan, decorin, and versican, and the non-sulfated
GAG hyaluronan have been observed in the area immediately surrounding calcified nodules in
the fibrosa layer [14]. This chapter seeks to elucidate the localization patterns of these specific
PGs/GAGs in the aforementioned early porcine model of CAVD.
4.2 Materials and Methods
4.2.1 Porcine model of early calcific aortic valve disease
The porcine model developed by Sider et al. [18, 19] was used (Appendix A.2). Male Yorkshire
barrows were fed either a normal or high-fat/high-cholesterol (HF/HC) diet for 2- or 5-months
with 6 swine per group. The control grower corn/soybean diet consisted of 15% protein, 75%
carbohydrate, and 10% fat (kcal%) at 3.51 kcal/g. The experimental grower diet consisted of
15% protein, 53% carbohydrate, and 32% fat at 4.02 kcal/g, due to the addition of 12% lard and
1.5% cholesterol. Two-month control and two-month experimental pigs (start weight: 59-65 kg)
were fed the control diet at 3.51 kcal/g and experimental diet at 4.02 kcal/g, respectively, for 65
days. Five-month control pigs (start weight: 14-16 kg) were initially fed a standard
27
corn/soybean-based starter III diet, due to their young age, containing 19% protein, 71%
carbohydrate, and 10% fat at 3.51 kcal/g until ~40 kg followed with the control grower
corn/soybean diet up until 155 days. Five-month experimental pigs (start weight: 14-16 kg) were
fed the starter III diet supplemented with an additional 12% lard and 1.5% cholesterol,
containing 19% protein, 49% carbohydrate, and 32% fat at 4.01 kcal/g until ~40 kg followed
with the experimental grower diet up until 155 days.
The experimental diet was fed at 87% of the mass of the control diet to provide isocaloric intake.
Feed was adjusted weekly by weight to achieve a growth rate of ~0.75 kg/day (actual average:
0.88 kg/day) for the 2 month pigs and ~0.70 kg/day (actual average: 0.62 kg/day) for the 5 month
pigs. Swine, weighing 106-126 kg, were sacrificed by electric shock and bleed out, according to
standard abattoir practices.
The protocol was approved by the University of Guelph and University of Toronto Animal Care
Committees. All animals were housed individually and treated in accordance with the
recommendations of the Guide for the Care of Laboratory Animals published by the United
States National Institute of Health [156].
4.2.2 Porcine leaflet handling
In total, 24 right coronary leaflets (6 per group) were harvested within 2 hours of sacrifice and
frozen in 10% dimethyl sulfoxide (DMSO) in RPMI medium in a 1°C freezing container and
stored at -180°C. After 6-7 months, leaflets were then thawed for 5 min in a 37°C bath and
DMSO was removed with three 10 min PBS with Ca+2
and Mg+2
doublings on ice. Leaflets were
then fixed in 10% neutral buffered formalin for 48 hours at room temperature and stored in 70%
ethanol at 4°C for 1-2 months. Due to technical difficulties, one 2-month control leaflet was not
processed. Right coronary leaflets were segmented along the circumferential midline, embedded
in paraffin, and cut into 5 µm sections according to routine procedure (Appendix B.1).
4.2.3 Histological and immunohistochemical staining
Radial centre sections of the valve leaflet were examined for PG/GAG content, as they provide a
good representation of whole valve leaflet composition. Sections were stained by Movat’s
pentachrome (Electron Microscopy Sciences, Hatfield, PA, USA) to identify collagen (yellow),
PGs (blue), cytoplasm, muscle (red), elastin, and cell nuclei (black) components (Appendix B.2).
28
This staining also allowed for visualization of the three valve leaflet layers. Elastin staining by
resorcin-fuchsin (Electron Microscopy Sciences) was also used to highlight reduplication and/or
displacement of the elastic lamina.
Staining for biglycan, decorin, versican, and hyaluronan was performed on serial centre sections
to correlate spatial expression of specific PGs/GAG. In addition ApoB and Sox9 staining was
completed to correlate lipid deposition and an early chondrogenic marker with PG/GAG
expression. Biglycan (anti-biglycan; goat polyclonal, 20 µg/mL, sc-27936, Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA), decorin (anti-decorin; rabbit polyclonal, 10 µg/mL,
sc-22753, Santa Cruz Biotechnology Inc.), versican (anti-versican; rabbit polyclonal, 1.3 µg/mL,
16770002, Novus Biologicals, Littleton, CO, USA), ApoB (anti-ApoB; sheep polyclonal, 3.6
µg/mL, AHP214, AbD Serotec, Raleigh, NC, USA) and Sox9 (anti-Sox9; rabbit polyclonal, 3
µg/mL, ab26414, Abcam, Cambridge, MA, USA) were detected immunohistochemically, while
hyaluronan was detected using biotinylated hyaluronan-binding protein (bovine nasal cartilage,
1.25 µg/mL, 385911, Calbiochem, Gibbstown, NJ, USA) (Appendix B.3).
Briefly, IHC began by melting the wax on the sections at 60°C for 30 min. This was followed by
deparaffinization in xylene and rehydration in graded ethanol baths. Antigen retrieval for Sox9
staining involved incubation in Tris-EDTA for 30 min in a 98°C water bath, followed by cooling
in room temperature water for 20 min. All other IHC staining protocols utilized antigen retrieval
using 1 µg/µl Trypsin-CaCl2 (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) for 30 min in
a 37°C water bath. Endogenous peroxidases were then blocked with 3% H2O2/methanol for 10
min. Non-specific staining was blocked using the appropriate 1% serum buffer for 45 min prior
to an hour-long incubation with primary antibody or binding protein. Samples, with the
exception of those for hyaluronan staining, were then incubated with biotin-labeled secondary
(anti-goat, anti-sheep, or anti-rabbit; Vector Laboratories, Burlington, ON, Canada) in 2%
specific serum (rabbit, horse, or goat) for 30 min. All samples were incubated with avidin-biotin-
peroxidase conjugate (Vectastain Elite ABC Kit, Vector Laboratories) for 30 min.
Positive staining was visualized following a 5 min incubation in Vector NovaRED (Vector
Laboratories), followed by Vector Hematoxylin QS (Vector Laboratories) counterstaining.
PBS/Tween was used to wash between steps. For all staining protocols except hyaluronan,
negative controls involved no primary and IgG controls (Santa Cruz Biotechnology Inc. or R&D
29
Systems, Minneapolis, MN, USA). For hyaluronan staining, negative controls involved no
primary controls and sections pre-treated with 110.25 units/mL hyaluronidase (Streptomyces
hyalurolyticus, H1136, Sigma-Aldrich Canada Ltd.) and stored in PBS at 4°C until use.
4.2.4 Data retrieval and statistical analyses
PG-rich lesions were identified using Movat’s pentachrome. For each lesion, images of the serial
sections stained with Movat’s pentachrome, and for the PGs/GAG of interest, ApoB, Sox9, and
elastin were layered in Adobe Photoshop so that lesion areas overlapped. Displaced elastic
lamina, as identified by elastin staining, was used to trace out and isolate only the lesion area in
each of the layered images.
Quantitative data were retrieved from these lesion images using algorithms in ImageJ, which
calculated total stained area based on hue, saturation, and brightness thresholds (Appendix B.4).
The total area of each lesion was calculated by subtracting white space area from the total image
area. These values were used to calculate percent PG/GAG staining of total lesion area. For the
Movat’s pentachrome images, a similar algorithm was used to separate and quantify: (1)
proteoglycans, (2) collagen, and (3) cytoplasm, elastin, and nuclei. For each lesion, cell density
was quantified. Furthermore, each lesion was qualitatively described as morphologically dense
or diffuse (Figure 4.1). These criteria allowed for characterization of whole lesion areas.
Figure 4.1. Qualitative classification of lesion morphology. (A) Dense and (B) diffuse proteoglycan-
rich lesions stained with Movat’s pentachrome staining (blue = proteoglycans, yellow = collagen, black =
nuclei/elastin, red = cytoplasm). Scale bar = 80 µm.
Localized analysis of lesion areas was performed by dividing each lesion into grids with 100 µm
x 100 µm grid cells. Each grid cell was scored on a semi-quantitative scale based on staining
A B
30
intensity and area for ApoB and PG/GAG (Figure 4.2). For grid cells with ApoB positive areas,
PG/GAG score was assessed only within the lesion area with positive ApoB staining (i.e., in co-
localized ApoB and PG/GAG areas within the lesion in a grid cell). For grid cells without ApoB
positive areas, PG/GAG score was designated based on the entire lesion area present in the grid
cell. The proportion of Sox9-positive cells was also quantified in each grid cell to elucidate any
relationship between PG/GAG type and putative chondrogenesis. For Sox9 analyses, PG/GAG
scores were assigned based on staining observed in the entire grid cell. Using a grading system
based partially on staining intensity has limitations. For example, technical issues such as section
thickness and staining performed on different days could alter levels of staining intensity. To
validate this PG/GAG scoring system though, scores were normalized and combined for each
lesion and compared to the data for percent PG/GAG staining of total lesion area. These results
strongly correlated for each PG/GAG, suggesting that technical variations in staining intensity
did not have a major influence on scoring (Appendix A.3). Cell density was also measured by
counting all cells in the images stained with Resorcin Fuchsin and dividing the cell count by the
total lesion area.
Due to the small sample size (n=50), the Shapiro-Wilk Test was used to assess the normality of
lesion data sets to determine whether parametric tests or non-parametric equivalents would be
used for analyses. Firstly, percent staining of total lesion area was compared for PG/GAG and
the Movat’s pentachrome separation variables between swine from the different time points and
diet groups using one-way ANOVA with Tukey’s post-hoc test or Kruskal-Wallis Test with
Mann-Whitney post-hoc tests. Bonferroni corrections (significance: p<0.0083) were applied to
these Mann-Whitney post-hoc tests to account for multiple comparisons. Changes in cell density
were also compared between groups using one-way ANOVA followed by Tukey’s post-hoc
tests. Secondly, localized areas from all lesions were characterized using Kruskal-Wallis tests
and Mann-Whitney post-hoc tests to compare PG/GAG score with ApoB score and Sox9
fraction. To account for multiple comparisons, Bonferroni corrections (significance: p<0.005)
were applied to post-hoc tests. Lastly, morphologically dense and diffuse lesions were compared
for PG/GAG composition, ApoB score and Sox9 fraction using Mann-Whitney tests. In addition,
the frequency of morphologically dense and diffuse lesions was compared between different time
points and diet groups using Fisher’s exact test. All statistical analyses were performed on IBM
SPSS Statistics (version 20.0.0) with significance level p<0.05, unless otherwise stated.
31
0 1 2 3 4
Ap
oB
Big
lyca
n
De
corin
Ve
rsic
an
Hya
luro
na
n
Figure 4.2. Semi-quantative scoring system for ApoB and PG/GAG content. Sample images for each
scoring category (0-4).
32
4.3 Results
4.3.1 HF/HC diet alters lesion ECM composition with temporal differences
In total, 50 PG-rich lesions were identified with Movat’s pentachrome staining and ranged from
36 to 376 µm in thickness. Analysis of lesion PG/GAG content demonstrated significant
differences in PG composition between the time point and diet groups for each of the PGs/GAG
(p<0.05) according to one-way ANOVA and Kruskal-Wallis (Figure 4.3). Differences, in
particular, were observed with the HF/HC diet compared to normal chow at two months.
Specifically, increases in biglycan (p<0.001) and decreases in hyaluronan (p=0.009) with the
HF/HC diet were noted at this time point. After five months on the HF/HC diet, lesions
expressed less biglycan (p<0.001), decorin (p=0.003) and versican (p<0.001) than at two months.
There were no significant differences based on the Bonferroni-corrected p-value of 0.0083
between the control groups at two months and five months for any of the PGs/GAG (biglycan:
p=0.101, decorin: p=0.010, versican: p=0.017, hyaluronan: p=0.999). These trends involving
biglycan and decorin were recapitulated in analyses of PG/GAG lesion scores (Appendix
A.3).Additionally, while there was apparent variability in the presence of each of the PGs, some
moderate level of hyaluronan remained largely consistent within most lesion areas.
Between the time point and diet groups, for swine fed the HF/HC diet, lesions from 5-month pigs
had increased proportions of collagen (p<0.001) and decreased proportions of nuclei, cytoplasm,
and elastin (p<0.001) compared to lesions from 2-month pigs (Figure 4.4). Overall, cell density
remained consistent between the time point and diet groups, but was less in the 5-month control
versus 5-month experimental lesions (p=0.036).
33
MP Biglycan Decorin Versican Hyaluronan
s2
Mo
nth
Co
ntr
ol
HF
/HC
5 M
onth
Co
ntr
ol
HF
/HC
Figure 4.3. HF/HC diet altered lesion PG composition with temporal differences. HF/HC = high-fat/high-
cholesterol diet. MP = Movat’s pentachrome (blue = proteoglycan, yellow = collagen, black = nuclei/elastin, red =
cytoplasm/muscle). Error bars = SEM. *p<0.0083
* * *
*
*
34
Figure 4.4. Temporal changes in lesion ECM composition. Movat’s pentachrome staining revealed
increases in collagen and decreases in “other” (nuclei, cytoplasm, and elastin) between the 2-month and
5-month HF/HC diet lesions. HF/HC = high-fat, high-cholesterol. Error bars = SEM.
4.3.2 Lesion areas that contain ApoB and Sox9-expressing cells display unique PG/GAG composition
ApoB staining was typically subendothelial, but there were a few cases of staining deeper within
the lesion. Lesion areas were semi-quantitatively scored for ApoB and spatially correlated to
PG/GAG content to characterize the relationship between specific PG/GAG presence and lipid
retention in early valve disease. ApoB scores were compared between scores for each PG/GAG
using Kruskal Wallis tests, followed by Mann-Whitney post-hoc tests. For biglycan and decorin,
there was no difference in ApoB score between areas with low scores (0 to 2) of these PGs.
ApoB scores also did not differ between the higher scoring (3 and 4) areas for biglycan
(p=0.378) and decorin (p=0.012). Overall, increases in biglycan and decorin score were
associated with increased ApoB score (p<0.004) (Figure 4.5A,C). Areas with no versican had
higher ApoB scores than areas with versican (p<0.002), but in general, varying versican scores
did not differ in ApoB score (Figure 4.5E). Between all hylauronan scores, there was no
difference in ApoB score (Figure 4.5G).
35
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
Figure 4.5. Relationship between ApoB and PG/GAG score in lesion areas. Increased apolipoprotein B
(ApoB) severity was associated with higher scores of biglycan and decorin. Error bars = SEM. *p<0.005
Overall, average ApoB scores for all levels of each PG/GAG were low (> 1.7). When the
frequencies of PG/GAG scores were sorted by ApoB score, it was evident that there was
immense variability in PG/GAG scores when there was no ApoB present (Figure 4.5). Still, the
*
*
*
36
trends observed from Kruskal Wallis tests were visible with the skew in frequencies of PG/GAG
scores with changing ApoB scores.
Sox9-positive cells were often observed near the base of early CAVD lesions (Figure 4.6).
Within lesion areas, the fraction of Sox9-expressing cells and area PG/GAG score were used to
identify the relationship between PG/GAG expression and indications of early chondrogenesis
based on previous findings by Sider et al [19] (Figure 4.7). Overall, there was no difference in
Sox9 fraction between lesion areas with low biglycan scores (0 and 1). Sox9 fraction also did not
differ between the higher scoring (3 and 4) areas for biglycan (p=0.822). Compared to low
biglycan scoring areas, there was an increase in fraction of Sox9-positive cells within high
biglycan scoring areas (p<0.002). For decorin and versican, there were largely no differences in
proportion of Sox9-positive cells between PG scores. With higher hyaluronan score, there was a
decrease in Sox9-positive cell fraction (p<0.002).
Figure 4.6. PG-rich lesions are associated the presence of Sox9-expressing cells(arrows). MP =
Movat’s pentachrome (blue = proteoglycan, yellow = collagen, black = nuclei/elastin, red =
cytoplasm/muscle). Scale bar = 100 µm.
MP
Sox9
37
(A)
(B)
(C)
(D)
Figure 4.7. Relationship between Sox9 fraction and PG/GAG score in lesion areas. Error bars = SEM.
*p<0.005.
4.3.3 Lesions with dense morphology display distinct characteristics compared to those with diffuse morphology
Dense and diffuse lesion morphologies (Figure 4.1) were also characterized based on PG/GAG
content, ApoB lesion score, and lesion Sox9-positive cell fraction (Figure 4.8). Overall,
compared to lesions with a diffuse morphology, dense lesions expressed higher levels of
biglycan (p<0.001) and lower levels of hyaluronan (p=0.003). On average, dense lesions also had
higher ApoB scores (p=0.002) and a higher proportion of Sox9-expressing cells (p=0.006).
Dense lesions were more common in swine fed the HF/HC diet compared to normal chow at the
two-month time point (p=0.011). There was no difference in the frequency of dense and diffuse
lesions between swine fed the HF/HC diet compared to normal chow at the 5-month time point
and between swine from the 2- and 5-month time points fed the HF/HC diet.
*
*
*
38
(A)
(B)
(C)
(D)
Figure 4.8. Dense and diffuse lesion characteristics and frequency with time points and diets.
Morphologically distinct lesions were compared based on (A) specific proteoglycan and
glycosaminoglycan (PG/GAG) content; (B) apolipoprotein B (ApoB) score; and (C) Sox9 fraction in
dense and diffuse lesions. (D) The number of dense lesions was compared between time points and diet
groups. Error bars = SEM. *p<0.05, **p<0.0125
*
*
*
*
**
39
4.4 Discussion
The availability of suitable human aortic valve samples has limited the study of early CAVD.
The porcine model developed by Sider et al. [18, 19] successfully mimics several traits seen in
humans [11, 17] and allows for the study of the initial stages of disease pathogenesis, which
satisfies an unmet scientific need for a disease without an effective medical therapy. Valve
leaflets from this porcine model demonstrated the presence of lesions that accumulated PGs, a
feature that has been seen in calcified aortic valves [14] and early disease stages [16]. Here, the
quantification of PG/GAG content in the early CAVD lesions of these porcine valves, as well as
their association with lipid retention and putative chondrogenesis markers, was elucidated.
4.4.1 Biglycan may be involved in lipid retention and chondrogenesis in early lesions of CAVD
Notably, increases in biglycan with HF/HC diet were evident in lesions at the two-month time
point. Previously, biglycan has been observed in and surrounding small calcified nodules with
greater abundance than in and around larger calcified nodules [14]. Our observations reinforce
the supposition that biglycan is involved in early nodule formation. Often, in early nodules and
small calcified nodules from human aortic valves, biglycan is observed to co-localize with
decorin [14, 16]. At the two-month time point, decorin content also increased in lesions from
HF/HC diet-fed pigs, but this was not statistically significant. Within localized lesion areas,
increased biglycan score was also associated with increasing ApoB score and Sox9 fraction,
indicating that its role in early CAVD pathogenesis may involve lipid retention and early
chondrogenesis consistent with previous observations by Sider et al [19].
Biglycan has been observed to co-localize with apolipoproteins in human valves [16, 17] and
recently, was shown to mediate lipid retention in porcine aortic valves likely by binding LDL
lysine residues with its negatively charged GAG chains [50]. Certain PGs, particularly decorin,
have also been shown to act as accessory molecules to mediate LDL retention on collagen [50,
157]. While biglycan does bind to collagen in vitro [158], it is unknown whether it plays any role
in LDL-collagen interactions. In vitro studies show that biglycan is able to sequester TGF-β [14,
34], which in turn is able to stimulate VIC expression of PGs that have enhanced binding to LDL
[126]. The likely role of biglycan in LDL deposition as an initiating factor in CAVD is
unsurprising, as it would mimic findings from early human atherosclerosis [15].
40
Lipid retention, whether directly or indirectly mediated by biglycan, allows modifications of
lipoproteins, which can perpetuate a cycle of additional lipid retention and inflammation. In
CAVD, biglycan induces the expression of phospholipid transfer protein (PLTP) by VICs
through interactions with Toll-like receptor 2 (TLR2) [129]. In atherosclerosis, PLTP promotes
modification of ApoAI, which increases its binding affinities for PGs [159] and may reduce the
ability of modified HDLs to perform reverse cholesterol transport [129, 159]. In human stenotic
valves, biglycan and ApoB also co-localize with oxidized LDL (oxLDL) [129], the accumulation
of which is strongly associated with increased inflammatory activity and tissue remodeling [51,
54]. Thus, the retention of lipoproteins by biglycan within valve lesions may promote enzyme-
mediated modification and oxidation by reactive oxygen intermediates or radicals generated by
cellular enzymes to promote the initiation of CAVD progression.
As well as an involvement in early lipid retention, biglycan may play a role in chondrogenic
processes in CAVD. Within lesion areas, increased Sox9 fraction was also associated with higher
biglycan scores. The transcription factor Sox9 is observed in both non-calcified pediatric and
calcified adult diseased valves [13]. Expression of Sox9, as well as other chondrogenic
transcription factors such as Twist1, Mef2c in putatively early and late-stage calcified valves,
may promote the production of a cartilage-like matrix that induces VIC activation in CAVD
pathogenesis [13]. Further, the soft microenvironment of these PG-rich lesions [18] may also
promote chondrogenic differentiation of VICs when stimulated by TGF-β [160], which can be
sequestered by biglycan [14, 34]. Soluble biglycan contributes to the pro-osteogenic effects of
oxLDL on VICs by increasing the expression of BMP2 and ALP through TLR2 [130]. Thus as
CAVD progresses, the role of biglycan in lesion formation may begin with chondrogenic
pathways that eventually lead to osteochondrogenic processes, resulting in the formation of
cartilage and bone that is often seen in stenotic valves [67, 86].
4.4.2 Hyaluronan may play a protective role in early CAVD lesion pathogenesis
In contrast to biglycan, hyaluronan content showed a decreasing trend with presumed lesion
severity. At two months, pigs fed the HF/HC diet demonstrated diminished hyaluronan content
when compared to pigs fed the normal chow diet. The decrease in hyaluronan with these more
advanced lesions suggests that the non-sulfated GAG plays a protective role during the early
stages of CAVD progression. In vitro, VICs interacting with hyaluronan through CD44
41
demonstrate suppressed calcified nodule formation [21]. These results are recapitulated in vivo,
where the abundance of hyaluronan is inversely related to the magnitude of fibrosa calcification
[135]. Further, native porcine valve leaflets treated with anti-CD44 increase expression of ALP
and Runx2, all markers of VIC dysfunction [21]. At this early stage of disease progression
though, where calcification is not yet observed [19], hyaluronan may play a protective role
inhibiting lipid retention and putative chondrogenesis, since hyaluronan demonstrated an inverse
relationship with ApoB score and Sox9-positive cell fraction. Within the valve, hyaluronan
turnover and/or removal have been associated with hypoxia, osteogenesis, apoptosis, and cell
proliferation [21, 140], but the relationship of the GAG with lipid retention and chondrogenesis
has not been explored.
4.4.3 Early CAVD lesions demonstrate further ECM remodeling with distinct morphological characteristics
With pigs fed the HF/HC diet, levels of biglycan, decorin, and versican decreased between the
two- and five-month time points. Interestingly, although there were changes in specific PG/GAG
content, total PG content was not altered. This is explained in part by hyaluronan content being
unchanged within most lesion areas (Appendix A.3), with hyaluronan consistently comprising
~65% of the whole lesion proteoglycan area in lesions from pigs fed the HF/HC diet (Figs. 4.3
and 4.4), regardless of time on the diet. Further, while biglycan, decorin, and versican levels in
the HF/HC lesions decreased from two- to five-month time points, there was greater separation
(i.e., less co-localization) of areas positively stained for versican versus those positively stained
for biglycan and decorin in five-month HF/HC lesions compared to two-month HF/HC lesions.
This is consistent with reports from advanced stenotic lesions in which biglycan- and decorin-
stained areas co-localize but do not overlap with versican-stained areas [16]. In addition,
increases in collagen were observed between the two- and five-month HF/HC groups, suggesting
that as disease progresses there may be further remodeling of the ECM, where PGs are replaced
by other ECM components within the lesion. In contrast, previous studies of advanced CAVD
demonstrated decreased total collagen content within whole, calcified valves [79]. The
differences likely reflect different phases of remodeling (early fibrotic vs. late calcific), plus the
more localized and lesion-specific analysis in this study (vs. whole leaflet analysis).
Furthermore, comparisons of dense and diffuse lesions showed that morphological differences
may be useful in identifying early lesion stages. Compared to diffuse lesions, dense lesions had
42
greater biglycan content and lower levels of hyaluronan. In these valve centre sections, dense
lesions had higher ApoB scores and increased proportions of Sox9-positive cells, which
recapitulates a previous study analyzing lesions in non-centre sections [18, 19]. These
observations indicate that lesions may progress from a more diffuse morphology to become
dense, accumulating biglycan, losing hyaluronan, and developing a lipid-retaining and putatively
chondrogenic profile in the process.
Dense lesions were found to be more common in swine fed the HF/HC diet compared to normal
chow diet after 2-months. Surprisingly, there was no propensity for dense lesion morphologies in
HF/HC diet swine compared to normal chow diet swine at 5-months, nor in 5-month swine
compared to 2-month swine fed the HF/HC diet, as might be expected if dense lesions
represented a more advanced morphology. Comparisons between swine fed for 2- and 5-months
on HF/HC diet may be affected by differences in when HF/HC diet was administered. Five-
month HF/HC swine began the atherogenic diet at an earlier age than two-month HF/HC swine.
This may confound temporal comparisons compared to if swine began HF/HC diet at the same
age with five-month swine receiving this diet for a longer period of time. It is also possible that
diffuse and dense lesions do not represent progressive stages of early lesion formation, but
entirely different early lesion types.
Interestingly, large dense PG-rich lesions occasionally appeared morphologically cartilaginous
with lipid near the surface and Sox9-expressing cells near the base of the lesion [18]. This
layered appearance is similar to the stratified appearance of macrophages in non-calcified and
early calcified lesions [11, 17] and provides further evidence of the active pathological processes
at work in early lesions. Proteoglycan, specifically biglycan, accumulation may promote the
retention of lipids and chondrogenesis, which eventually activate inflammatory pathways and
calcification.
This is the first instance when specific PGs/GAG were characterized within early CAVD lesions.
Previous studies of advanced CAVD lesions demonstrated the presence of biglycan and decorin
and absence of versican and hyaluronan immediately surrounding small calcified nodules [14].
This is consistent in part with observations in our early porcine model of CAVD, where increases
in biglycan and decreases in hyaluronan were observed within lesion areas. In contrast, previous
studies also showed that larger and possibly more advanced calcified nodules were surrounded
43
by versican and hyaluronan, but not biglycan and decorin [14]. These characteristics of advanced
stenotic valves do not correlate with the observed PG/GAG changes observed in our model,
suggesting that there may be multiple shifts in ECM remodeling during CAVD progression. For
example, biglycan may increase in early CAVD lesions to promote lipid retention and eventual
early calcified nodule formation, but may be replaced by other components when calcification
becomes more severe. Furthermore, hyaluronan may play a protective role early on in CAVD
progression, as suggested by its absence in lesions in our early porcine model and around smaller
calcified nodules, but may play a role in continued mineralization of larger calcified nodules.
This also supported by changes observed in collagen content. In our early porcine model of
CAVD, we saw increases in total collagen content whereas in advanced stenotic valves, total
collagen content decreases [79]. While early lesions eventually lose PG and accumulate collagen,
in advanced lesions this collagen may become calcified. Overall, these observations are
suggestive of the continued ECM remodeling that occurs throughout CAVD progression.
4.5 Conclusion
The presence and role of PGs is only a recently studied component of CAVD. In a previous
study using this porcine model, the formation of PG-rich lesions in the absence of lipid
deposition, macrophages, osteoblasts, or myofibroblasts suggested an important role for this
ECM component in early CAVD stages. Through this histological study, possible roles of
specific PG/GAGs were elucidated. In particular, the accumulation of biglycan and loss of
hyaluronan within early lesions may play a role in lipid retention and putative chondrogenesis,
due to their association with ApoB and Sox9, respectively.
44
Chapter 5
5 Phenotypes of valve interstitial cells in lesions of early calcific aortic valve disease
5.1 Introduction
Focal and layer-dependent susceptibility to calcific aortic valve disease (CAVD) suggests that
the valve is composed of a heterogeneous population of cells that are phenotypically different
based on their microenvironments. This has been determined in valvular endothelial cells
(VECs), which on the fibrosa side have enhanced anti-oxidative and calcification-permissive
characteristics compared to their ventricularis-side counterparts [39]. The spatial heterogeneity
of VECs also contributes to valvular interstitial cell (VIC) function, which is partly regulated by
VEC paracrine signaling [161-163]. Within the population of VICs, there is a subpopulation of
mesenchymal progenitor cells with adipogenic, chondrogenic, osteogenic, and myofibrogenic
potential [164]. Pathological cells, which are mostly myofibroblasts [41] and osteoblasts [67,
80], likely arise from these progenitors [20].
In addition to VEC paracrine signaling, VIC function is influenced by mechanical factors,
biochemical stimuli, and extracellular matrix (ECM) cues. VICs are shielded from the disturbed
hemodynamics on the fibrosa side and undisturbed shear stress conditions on the ventricularis
side, that likely affect VEC side-dependent pathosuceptibility. Instead, VICs experience
mechanical deformation through interactions with the ECM, which itself deforms as the valve
opens and closes. Pathological stretching of valve leaflets increases the expression of proteolytic
enzymes, pro-inflammatory proteins, and differentiation markers that are upregulated in diseased
states [165-167]. These changes are often seen in the fibrosa layer [165, 166], which is stiffer in
comparison to the ventricularis layer [168-170]. Within the individual valve layers, micropipette
aspiration indicates the presence of distinct soft and stiff regions [170]. Extracellular matrix
stiffness may directly affect VIC function, but also modulates VIC responses to biochemical
factors. For example, when grown in osteogenic differentiation media, VIC differentiation into
osteoblasts with the formation of bone nodules preferentially occurs on softer (~20 kPa)
substrates, while stiffer (>100 kPa) substrates promote differentiation into myofibroblasts [36,
45
133]. Further, myofibroblast differentiation of VICs induced by transforming growth factor beta
1 (TGF-β1) only occurs on stiff substrates in a β-catenin dependent manner [37].
Myofibroblasts and osteoblasts are commonly observed in late stages of disease, but rarely in the
early stages. Changes in the valve microenvironment still occur, as ECM disorganization is a
major characteristic of both early and late stages of CAVD. Early valve disease is identified by
focal subendothelial thickening on the fibrosa side of the leaflet with accumulations of
proteoglycan, lipid, inflammatory cell infiltrate, and extracellular mineralization [11, 17]. Late-
stage valve disease continues with these ECM changes, but with greater severity, resulting in
fibrosis and calcification. Recently, a porcine model of early CAVD by Sider et al. [18, 19]
demonstrated the formation of proteoglycan (PG)-rich lesions on the pathosuceptible fibrosa side
of the valve. At this early stage of disease progression, myofibroblasts, osteoblasts, and
significant inflammatory infiltrate were absent, but lipid retention and putative chondrogenesis
were evidenced by the presence of ApoB and Sox9-expressing cells.
Micromechanical testing demonstrated that these PG-rich lesions are softer than adjacent non-
lesion fibrosa [18]. In addition, ApoB positive areas are often present immediately beneath the
subendothelium and Sox9-expressing cells are commonly observed at the base of these lesions.
These observations demonstrate that there are not only differences between lesion and non-lesion
areas, but spatially within lesions as well. Previous studies of native valve cell phenotype using
microarrays have only profiled VICs from the entire leaflet, effectively ignoring the putative
heterogeneity between lesion and non-lesion areas and within lesions [129, 171]. The focal
nature of valve disease and sensitivity of VICs to their changing microenvironment warrant the
study of VIC phenotype with a spatially directed approach. This chapter compares VIC
phenotypes in early CAVD lesion areas and non-lesion fibrosa.
5.2 Materials and Methods
5.2.1 Frozen valve leaflet section preparation
Left coronary valve leaflets from the early porcine model by Sider et al. [18, 19] were also used
for this chapter (see 4.2.1). Leaflets frozen in optimal cutting temperature (OCT) compound were
chosen for this objective, as they allow retrieval of higher quality RNA from laser capture
microdissection (LCM) than formalin-fixed, paraffin-embedded sections [172] (Appendix B.5).
46
VICs were isolated and compared for differential gene expression in lesion and non-lesion areas.
Lesion areas included those (1) at the top half of the lesion, and (2) at the bottom half of the
lesion, where Sox9-expressing cells are often present [18]. Non-lesion areas were those far away
from lesions, but still within the fibrosa layer.
Figure 5.1. Cryosectioning slide schematic for each porcine sample. Serial sections for staining (MP =
Movat’s pentachrome; Sox9) and for RNA isolation (LCM = laser capture microdissection) allowed for
identification and tracking of lesion morphology during LCM. For each pig, four sets of slides (denoted
below the bracket) were prepared followed by two additional staining slides.
Eight-micrometer radial centre sections from the left coronary valve leaflets were cryosectioned
using the Leica CM 3050S cryostat in Dr. Philip Marsden’s Lab (Li Ka Shing Research Institute,
St. Michael’s Hospital, Toronto). Sections were mounted on charged slides to maximize
adherence of leaflet sections to slides during staining and of undesired tissue areas to slides
during LCM. For each pig sample, four sets of slides were prepared followed by two additional
staining slides. Each set contained serial sections on two staining slides and three LCM slides
(Figure 5.1). Each slide for staining had two tissue sections and each slide for LCM had four
tissue sections. Slides for LCM were kept at -80°C until use. Slides for staining were kept at
room temperature overnight to allow sections to better adhere and subsequently stored at -80°C
until staining.
5.2.2 Histological and immunohistochemical identification of lesions and samples of interest
According to Sider et al. [18, 19] and results from Chapter 4, lesions from two-month HF/HC
samples and dense lesions were most commonly associated with higher Sox9-positive cell
fraction. Therefore, dense lesions from two month HF/HC samples were used to probe VIC
phenotypic changes more broadly by microarray analysis. PG-rich lesions were identified on
sections stained with Movat’s pentachrome, using a modified protocol optimized for frozen
MP Sox9 MP Sox9 LCM LCM LCM
x4
47
sections (Appendix B.6). Lesions were further narrowed down by Sox9 (anti-Sox9, rabbit
polyclonal, 2 µg/mL, ab3697, Abcam) immunohistochemical staining, which identified those
with putatively chondrogenic cells (Appendix B.7). Briefly, IHC began by warming the slides at
room temperature for 30 min. During this time, acetone was pre-cooled at -20°C for the
subsequent acetone fixation for 10 min at room temperature. Endogenous peroxidases were then
blocked with 3% H2O2/methanol for 10 min. Non-specific staining was blocked using 1% goat
serum buffer for 30 min prior to an hour-long incubation with primary antibody. Samples were
then incubated with biotin labeled anti-rabbit secondary (Vector Laboratories, Burlington, ON,
Canada) with 1.5% goat serum for 30 min. All samples were incubated with avidin-biotin-
peroxidase conjugate (Vectastain Elite ABC Kit, Vector Laboratories) for 30 min. Positive
staining was visualized following a 5 min incubation in Vector NovaRED (Vector Laboratories),
followed by Vector Hematoxylin QS (Vector Laboratories) counterstaining. PBS/Tween was
used to wash between steps. Negative controls involved no primary and IgG controls (R&D
Systems, Minneapolis, MN, USA). In total, four pig samples from the 2-month HF/HC group
were selected based on largest lesion size and presence of putatively chondrogenic cells, as
designated by Movat’s pentachrome and Sox9 staining, respectively (Figure 5.2).
5.2.3 Laser capture microdissection
Serial sections intended for LCM were quickly stained immediately prior to microdissection
using Arcturus Histogene Staining Solution (Life Technologies, Burlington, ON, Canada). All
three slides in each LCM set were stained and microdissected in one batch within an hour. For
each set, VICs were isolated with the Arcturus PixCell IIe (Li Ka Shing Research Institute) from
the areas of interest in the following order: (1) top of lesion; (2) bottom of lesion; and (3) non-
lesion areas. The smallest laser spot size, 7.5 µm, was used to ensure accurate capture of regions
of interest. The power of the pulse ranged from 60-80 mW and the duration of each pulse from
30.0-35.0 msec. The LCM environment was regulated by a dehumidifier, which maintained the
relative humidity below 35% in the room. For each area of interest, VICs were captured on
separate CapSure Macro LCM Caps (Life Technologies). For each pig, all nine microdissected
tissue samples were capture on the same day to reduce technical variability. Following 30 min
incubation in extraction buffer from the PicoPure RNA Isolation Kit (Life Technologies) at
42°C, samples were stored at -80°C until RNA isolation (Appendix B.8).
48
(A)
(B)
(C)
(D)
Figure 5.2. Lesions of interest for differential gene expression analysis. The largest dense lesions (arrows)
from four pigs fed the HF/HC diet for two-months (labeled by numerical identifiers) were distinguished by
Movat’s pentachrome (MP) staining.
5.2.4 RNA isolation, amplification, and microarray analysis
RNA was isolated from all nine microdissected samples in a set at the same time using the
PicoPure RNA Isolation Kit (Life Technologies). DNase treatment was performed using a
RNase-free DNase Set (Qiagen, Gaithersburg, MD, USA). All samples were eluted with 11 µL
of elution buffer (Appendix B.9). After isolation, replicates of the areas of interest from each set
were pooled.
Samples were sent to the Ontario Cancer Institute Genomics Centre (Toronto, ON, Canada) for
downstream processing and statistical analysis. RNA quality was analyzed using a 2100
Bioanalyzer (Agilent Technologies Canada Inc., Mississauga, ON, Canada). All samples had
RNA integrity numbers between 2.3-7.0 and concentrations between 242-816 pg/µL (Appendix
A.5). RNA was amplified using the Ovation Pico Whole Transcript Amplification Kit (NuGen,
11107 11212
12509 11907
49
San Carlos, CA, USA). The resulting cDNA was also run on the 2100 Bioanalyzer for quality
control (Appendix A.5). Profiles indicated suitable cDNA samples with curves that plateaued
around 200-500 nucleotides with some high molecular weight products around 500-1000
nucleotides. Amplified samples were run on the GeneChip 1.0ST Porcine Array (Affymetrix,
Santa Clara, CA, USA), where a total of 19 124 probe sets are represented.
5.2.5 Data processing and statistical analyses
Data was checked for overall quality using R (v2.15.3) with the Bioconductor framework and the
Array Quality Metrics package. After importing the data, a number of graphs were generated to
ascertain if there were any potential problems or outliers (Appendix A.6). A boxplot of
unprocessed log-intensity distributions and a histogram of the density of log-intensities
demonstrated similar probe intensities between arrays. The histogram of log-intensities was also
uni-modally distributed, which suggests the absence of artifacts that affect certain areas of the
arrays. This was recapitulated by 2D plots of expression characteristics (predicted by Probe
Level Model (PLM) estimates) at their array positions. Each array demonstrated homogeneous
colour coded values, implying that there was no spatial bias within the arrays. Relative Log
Expression (RLE) boxplots were constructed by calculating ratios between the expression of a
probe set and the median expression of this probe set across all arrays of the experiment. The
boxes were similar in range and centred close to 0, which is expected assuming only relatively
few genes are differentially expressed. Boxplots of Normalized Unscaled Standard Error (NUSE)
values indicated that array quality was satisfactory, as distributions centred around 1 (low quality
distributions centre around 1.1) and each array had a similar global spread. Finally, a correlation
coefficient for each pair of arrays was qualitatively presented on a coloured matrix. The minimal
coefficient value across all arrays was 0.90, indicating that there is homogeneity among array
intensities. Overall, all samples passed quality control and were included in subsequent analysis.
Data was imported into GeneSpring v12.6 for analysis. During import, the data was normalized
using a robust multiarray average (RMA) 16 normalization followed by a “per probe” median-
centred normalization, which are standard for Affymetrix ST arrays. All data analysis and
visualization were performed on log2 transformed data. To approximate the level of macrophage
contamination, non-median centred intensity averages for macrophage markers were categorized
as: (1) not expressed (<100); (2) lowly expressed (100-500); (3) moderately expressed (500-
50
4000); and (4) highly expressed (>4000) (personal communication with Natalie Stickle, UHN
Microarray Centre). These values roughly correlated to the 25th
, 80th
, 95th
, and 99th
percentiles of
intensity levels, respectively. The average expression value for all transcripts was below the 75th
percentile of intensities.
For subsequent analyses, data were filtered to remove the confounding effect of probes that
showed no signal. Probes above the 20th
percentile of intensities from any of the groups of
interest were allowed to pass through this filtering. The final set contained 15 057 probe sets.
Repeated Measures ANOVA with a Benjamini-Hochberg false discovery rate (FDR) corrected
p<0.05 did not show any significant probe sets. Therefore, the repeated measures ANOVA was
repeated with an uncorrected p-value cut-off of 0.05 (Appendix A.7). The significant results of
this repeated measures ANOVA (1246 probesets) were analyzed by Tukey’s post-hoc tests. To
interpret these results, the fold change was calculated for each pair of interest and a cut-off of 1.5
fold up or down was applied. Benjamini and Yekutieli corrected (p<0.3) hypergeometric tests
were applied to look for enriched Gene Ontology (GO) categories that overlapped. Due to the
large number of entries and lack of complete porcine annotations, an exhaustive analysis of the
complete gene list is beyond the scope of this study. In some cases where GO terms were
missing, GO terms of homologous genes from Homo sapiens were used. Differentially expressed
genes with putative significance to CAVD pathology were identified and discussed with a focus
on enriched GO biological processes.
5.2.6 Venn diagram analysis
Venn diagram analysis was performed to determine genes that were modulated commonly in
different regions. Two-way Venn diagrams were created using the list of differentially expressed
entries from top vs. fibrosa and bottom vs. fibrosa Tukey’s post hoc tests and VENNY online
software (http://bioinfogp.cnb.csic.es/tools/venny/index.html, BioinfoGP Bioinformatics for
Genomics and Proteomics CNB-CSIC, Madrid, Spain) [173].
5.3 Results
5.3.1 Sample characterization
Valve interstitial cells (VICs) were isolated from the top of lesions, bottom of lesions, and
normal fibrosa of pigs fed the HF/HC diet for two months. Previously, the formation of PG-rich
51
lesions at the two-month time point in this porcine model of CAVD were found to occur in the
absence of macrophages, as identified by immunohistochemical staining [18]. Markers of these
inflammatory cells expressed at a low level (roughly <500 intensity level) or were not expressed
(<100 intensity level), confirming the absence of these possible cell contaminants in the
differential gene expression analyses (Table 5.1).
Table 5.1. Expression levels of select macrophage-specific markers
Gene Name Top of Lesion Bottom of Lesion Normal Fibrosa
EMR1 76.2 84.1 136.1
CD14 118.2 111.7 133.4
LOC100520753 (CD68) 171.0 189.9 488.0
SIGLEC-1 (CD169) 187.5 208.2 196.4
ITGAM (Mac-1) 167.2 176.8 151.7
FCGR1A (CD64) 67.5 66.9 98.6
CD80 92.3 117.3 131.5
CD86 271.9 278.5 572.3
Average median centred values
Differential gene expression by cells from the (1) top half of early lesions, (2) bottom half of
early lesions, and (3) non-lesion fibrosa was assessed. Repeated Measures ANOVA found 1246
differentially expressed sequences (p<0.05). Tukey’s post-hoc tests (p<0.05) found that between
groups there were 525 differentially expressed probe sets between the top and bottom of lesions
(TvsB), 832 differentially expressed probe sets between the top of lesion and non-lesion areas
(TvsF), and 176 differentially expressed probe sets between bottom of lesion and non-lesion
areas (BvsF). When a fold change cutoff of 1.5 was applied to these post-hoc results, 156 TvsB,
215 TvsF, and 18 BvsF differentially expressed transcripts remained (Figure 5.3).
52
(A)
(B)
(C)
Figure 5.3. Distribution of differentially expressed transcripts in lesion and non-lesion areas. (A) Top of lesion vs.
bottom of lesion; (B) Top of lesion vs. normal fibrosa; (C) Bottom of lesion vs. normal fibrosa.
5.3.2 Lesion and non-lesion VIC differential gene expression
Differentially expressed genes common between TvsF and BvsF comparisons identified
transcripts that were differentially expressed between the fibrosa and throughout the lesion. With
a fold change cutoff of 1.5, only 7 total entities were identified. Without fold change cutoffs,
TvsF and BvsF comparisons identified 82 transcripts, 39 upregulated and 43 downregulated, that
were differentially expressed in VICs throughout the lesion area compared to VICs from normal
fibrosa (Figure 5.4). Analysis of GO terms for transcripts differentially expressed throughout the
lesion found only a few enriched biological processes with putative relevance to valve disease,
such as immune response and regulation of apoptosis (Table 5.2).
(A)
(B)
Figure 5.4. Transcript expression in lesion areas. Blue regions represent transcripts differentially expressed
between top of lesion and normal fibrosa VICs (TvsF). Yellow regions represent transcripts differentially
expressed between bottom of lesion and normal fibrosa VICs (BvsF). Union regions represent transcripts that
are differentially expressed throughout the lesion vs. the fibrosa. [UP] = upregulated. [DOWN] =
downregulated. No fold change cut-off was applied.
53
Table 5.2. Select differentially expressed genes between lesion (top and bottom) and non-lesion
areas
Gene Name L/F Regulation GO Biological Process
IL1R1 Up Cytokine-mediated signaling pathway
LOC100516004 Up Lipopolysaccharide biosynthetic process
IL16 Up Induction of positive chemotaxis
NFKB2 Up Toll-like receptor signaling pathwayǂ
SERBP1 Up Regulation of apoptotic processǂ
LAMTOR5 Up Negative regulation of apoptotic processǂ
EIF4E Up Cytokine-mediated signaling pathwayǂ
LIPG Down Lipid metabolic process
HBP15/L22 Down Alpha-beta T cell differentiation
RCAN2 Down Calcium-mediated signaling
SMAD6 Down Immune response, negative regulation of apoptotic
process, negative regulation of BMP signaling
pathway, transforming growth factor beta receptor
signaling pathway, cell substrate adhesion
IGF1R Down Immune response, positive/negative regulation of
MAPK cascade, negative regulation of apoptotic
process
AKAP6 Down cAMP-mediated signaling, cellular response to
cytokine stimulus
TTC8 Down Fat cell differentiation
CRY Down Oxidation-reduction process, fatty acid metabolic
process
No fold change cutoff was applied. L/F = regulation in whole lesion area compared to non-lesion fibrosa cells. GO =
Gene Ontology. IL1R1, interleukin 1 receptor type I; LOC100516004, cyclin-dependent kinases regulatory subunit
1-like; IL16, interleukin 16; NFKB2, nuclear factor of kappa light polypeptide gene enhancer in B-cells 2; SERBP1,
SERPINE1 mRNA binding protein 1; LAMTOR5, late endosomal/lysosomal adaptor, MAPK and MTOR activator
5; EIF4E, eukaryotic translation initiation factor 4E; LIPG, endothelial lipase; HBP15/L22, heparin-binding protein;
RCAN2, regulator of calcineurin 2; SMAD6, SMAD family member 6; IGF1R, insulin-like growth factor 1
receptor; AKAP6, A kinase (PRKA) anchor protein 6; TTC8, tetratricopeptide repeat domain 8; CRY, CRY protein.
ǂ Incomplete GO biological process terms from homologous Homo sapiens genes
5.3.3 Differential gene expression of VICs within lesion areas
Comparisons from the TvsB group were used to examine differences in VIC gene expression
within spatially distinct areas of the lesion. With a fold change cutoff of 1.5, there were 156
differentially expressed genes between the top and bottom of lesion areas. Benjamini Yekutieli
corrected (p<0.3) hypergeometric tests were used to look for enriched GO categories. Sequences
associated with a wide range of biological processes were altered in the different VIC
environments.
54
Of the 35 downregulated transcripts, only 18 had GO biological process information. Genes
involved in mesenchymal cell development, mesenchymal cell differentiation, and cardiac
epithelial to mesenchymal transition, namely HEY1, ERBB4-like, and WNT16-like, were
enriched and downregulated in the top of lesions compared to the bottom of lesions. Of the 121
upregulated transcripts, 73 had GO biological process data. Upregulated GO biological processes
that are of putative relevance to early CAVD included those involved in lipid-related processes
(Table 5.3) and immune response (Table 5.4). The presence of increased lipid-related genes in
the top of lesions recapitulates the commonly observed presence of ApoB-positive areas in the
subendothelial regions of lesions (Chapter 4). In addition, several enriched GO molecular
function terms involved lipid-related processes, such as lipid binding, lipase activity, and
lipoprotein particle remodeling.
Table 5.3. Select lipid-related genes that are upregulated in the top of lesions
Gene Name T/B Fold Change T/F Fold Change GO biological process
CD36 5.84 4.92 LDL particle mediated signaling, lipid
localization, positive regulation of lipid storage
SMPDL3A 5.65 9.39 Lipid catabolic process
PLA2G7 4.65 5.10 LDL particle remodeling, lipid catabolic process
APOE 3.93 4.27 Lipid transport, LDL particle remodeling, lipid
catabolic process
MSR1 3.74 Lipoprotein transport, cholesterol transport,
positive regulation of cholesterol storage
LPL 3.03 2.99 Lipid catabolic process
LIPA 2.84 2.64 Lipid metabolic process
ABCA1 2.25 2.84 Lipid localization
IL18 1.96 2.22 Lipopolysaccharide mediated signaling pathway
FZD4 1.51 Lipid transport, lipid localization
T/B = top of lesion cells relative to bottom of lesion cells. T/F = top of lesion cells relative to non-lesion fibrosa
cells. GO = Gene Ontology. CD36, fatty acid translocase; SMPDL3A, sphingomyelin phosphodiesterase acid-like
3A; PLA2G7, phospholipase A2 group VII; APOE, apolipoprotein E; MSR1, macrophage scavenger receptor 1;
LPL, lipoprotein lipase; LIPA, lipase A; ABCA, ATP-binding cassette sub-family A member 1; IL18, interleukin
18; FZD4, frizzled family receptor 4.
55
Table 5.4. Select immune-related genes that are upregulated in the top of lesions
Gene Name T/B Fold Change T/F Fold Change GO biological process
CXCL14 6.47 Immune response
CTSS 3.27 2.90 Adaptive immune response
GPR183 3.12 Immune response, leukocyte activation,
lymphocyte activation
CCL20 2.53 4.00 Immune response
CD83 2.13 Positive regulation of lymphocyte activation,
positive regulation of leukocyte activation,
positive regulation of T cell activation
IL8 2.04 1.67 Cell chemotaxis, Inflammatory response
IL18 1.96 2.22 Positive regulation of lymphocyte activation,
positive regulation of leukocyte activation,
positive regulation of T cell activation,
SLA-DQA1 1.80 Positive regulation of lymphocyte activation,
positive regulation of leukocyte activation,
positive regulation of T cell activation
TNFAIP8 1.77 1.44 Negative regulation of apoptotic process
ICAM1 1.71 1.74 Leukocyte cell-cell adhesion
SELP 1.62 1.59 Positive regulation of leukocyte migration
SLA-DQB1 1.61 Positive regulation of lymphocyte activation,
positive regulation of leukocyte activation,
positive regulation of T cell activation
VAV1 1.57 1.48 Leukocyte activation, lymphocyte activation
T/B = top of lesion cells relative to bottom of lesion cells. T/F = top of lesion cells relative to non-lesion fibrosa
cells. GO = Gene Ontology. CXCL14, chemokine (C-X-C motif) ligand 14; CTSS, cathepsin S; GPR183, G-protein
coupled receptor 183; CCL20, chemokine (C-C motif) ligand 20; CD83, B-cell activation protein; IL8, interleukin-
8; IL18, interleukin-18; SLA-DQA1, major histocompatibility antigen SLA-DQA; IL2RG, interleukin-2 receptor
gamma; TNFAIP8, tumor necrosis factor alpha-induced protein 8; SELP, selectin P; SLA-DQB1, major
histocompatibility antigen SLA-DQB1; VAV1, vav 1 guanine nucleotide exchange factor.
ECM remodeling is hallmark of both early and late stages of CAVD pathogenesis. Overall, GO
biological process analyses did not identify ECM remodeling as enriched. Using fold change
cutoffs and Tukey’s post hoc test results though, VIC expression of genes involved in ECM
disassembly and organization were found to be upregulated within the top of lesions compared to
the bottom of lesions (Table 5.5).
Further examination of differentially expressed transcripts found other pathways that may be
involved in early disease pathogenesis. In this early model of CAVD, Sox9-expressing cells are
commonly observed in the bottom of lesions, suggesting chondrogenesis may be involved in
CAVD progression of these areas. Although not found to be differentially expressed between
56
valve areas, SOX9 was moderately to highly expressed in all samples analyzed (expression
values > 1200). Analysis of gene expression though showed upregulation of osteopontin in the
top of lesions compared to the bottom of lesions (T/B fold change =4.07), suggesting that
osteogenic processes may also be occurring at this early stage of disease.
Table 5.5. Select ECM remodeling-related genes that are upregulated in the top of lesions
Gene Name Gene Symbol T/B Fold Change T/F Fold Change
Cathepsin S CTSS 3.27 2.90
ADAM metallopeptidase domain 28 ADAM28 3.04 3.03
Cathepsin Z CTSZ 2.26 2.21
Matrix metalloproteinase 9 MMP9 1.96 1.81
Hyaluronidase 2 HYAL2 1.80 1.93
Matrix metalloproteinase 14 MMP14 1.64 1.77
Chondroitin sulfate synthase 1 CHSY1 1.52 2.04
Metalloproteinase inhibitor 1 TIMP1 1.52 1.67
T/B = top of lesion cells relative to bottom of lesion cells. T/F = top of lesion cells relative to non-lesion fibrosa
cells.
5.3.4 Differential gene expression of VICs from specific lesion areas and non-lesion areas
With a fold change cutoff of 1.5, 18 total transcripts were differentially expressed between the
bottom of lesions and non-lesion fibrosa. Of these transcripts, only eight were fully annotated.
Unsurprisingly, no GO categories were enriched from this comparison. Using the same fold
change criteria, 215 differentially expressed genes were identified between VICs from the top of
lesions and normal fibrosa. Many (42%) of the transcripts from the TvsF comparison were also
identified in TvsB (Figure 5.5). Notably, the majority of aforementioned genes upregulated and
with putative relevance to CAVD in the TvsB comparison are also upregulated in the TvsF
comparison (Tables 5.3, 5.4, 5.5). Similarly, enriched GO terms that are upregulated in TvsF
include those involved in lipid-related processes, immune and inflammatory response, and ECM
remodeling.
57
(A)
(B)
Figure 5.4. Transcript expression in lesion areas. Blue regions represent transcripts differentially expressed
between top of lesion and normal fibrosa VICs (TvsF). Yellow regions represent transcripts differentially
expressed between top and bottom of lesion VICs (TvsB). Union regions represent transcripts that are
differentially expressed in the top of the lesion compared to other areas. [UP] = upregulated. [DOWN] =
downregulated.
Examining the top of lesions on an individual gene basis, there were other transcripts that were
differentially expressed within these areas. Oxidative stress seemed to play a greater role in
disease progression in these areas. Oxidized LDL (oxLDL) is able to bind scavenger receptors,
such as CD36 and lectin-type oxidized low density lipoprotein receptor 1 (LOX1), which were
upregulated in the top of lesions compared to other lesion areas (T/F fold change: 4.92 and 3.87,
respectively). As well, several genes relevant to osteochondrogenic pathways were upregulated
in the top of lesions (Table 5.6). Although toll-like receptor 4 (TLR4) is not categorized under an
osteochondrogenic biological process, stimulation of these receptors upregulates the expression
of osteogenic factors in VICs [174].
Table 5.6. Select differentially expressed genes that are upregulated in the top of lesions compared
to non-lesion areas.
Gene Symbol T/F Fold Change GO Biological Processes
SPP1 4.37 Biomineral tissue development, ossification
SULF1 2.43 Cartilage development, bone development, positive
regulation of BMP signaling pathway
CHSY1 2.04 Negative regulation of ossification
HYAL2 1.93 Cartilage development, hyaluronan catabolic process
TLR4 1.76 Inflammatory response, toll-like receptor signaling pathway
SLC20A1 1.52 Phosphate-containing compound metabolic process
T/B = top of lesion cells relative to non- lesion cells. T/F = top of lesion cells relative to non-lesion fibrosa cells. GO
= Gene Ontology. SPP1, osteopontin; SULF1, extracellular Sulf-1; CHSY1, chondroitin sulfate synthase 1; HYAL2,
hyaluronidase 2; TLR4, toll-like receptor 4; SLC20A1, solute carrier family 20 member 1. GO = Gene Ontology.
58
5.4 Discussion
Studies of early CAVD using this porcine model demonstrate the accumulation of PG-rich
lesions on the fibrosa side of valve leaflets without the presence of significant myofibroblast,
osteoblast, and inflammatory cell infiltration, as assessed by immunohistochemical expression of
phenotypic markers. Differences in stiffness between lesion and non-lesion areas and distinct
regions of lipid retention and putative chondrogenesis within lesions indicate that there are
varying microenvironments within the valve leaflet. Here, we examined the spatial variability of
VIC phenotypes within lesion and non-lesion areas.
Firstly, we sought to distinguish lesion and non-lesion VIC phenotypes by finding similar
differentially expressed genes in the TvsF and BvsF comparisons. With a fold change cutoff of
1.5, few genes were identified within this category and consequently, did not identify enriched
GO terminologies. Without fold change cutoffs, 82 differentially expressed transcripts between
the whole lesion and non-lesion fibrosa were identified. These genes fell under broad GO
biological processes, but included some that signify possible immune response and apoptotic
processes occurring throughout the lesion. The small number of differentially expressed genes
between the whole lesion and non-lesion areas may be explained by heterogeneity within the
lesion or limitations of the LCM technology. Lesions from this porcine model of early CAVD
commonly demonstrated ApoB positive areas immediately below the subendothelium at the top
of lesions and Sox9-expressing cell at the base of lesions. This spatial variability within lesions
was reflected in the comparatively higher number of differentially expressed genes between the
top and bottom of lesion VICs.
In total, there were 156 differentially expressed genes between the top and bottom of lesion areas
when a fold change cut-off of 1.5 was applied. GO analysis of these genes identified
mesenchymal cell differentiation and proliferation genes, HEY1, ERBB4, and WNT16, as
enriched and downregulated in the top half of lesions. HEY1 and ERBB4 are involved in normal
valve development processes, which are thought to be involved in valve disease [175]. HEY1 is
a target gene in Notch signaling that plays a critical cooperative role with HEYL in epithelial to
mesenchymal transition of endocardial cells to promote endocardial cushion development [176].
Relevant to valve disease, downregulation of HEY1 transcription occurs with inhibition of Notch
signaling and is associated with activation of osteogenic markers and increased calcified nodule
59
formation [150]. ERBB4 encodes an enzyme in the epidermal growth factor receptor (EGFR)
subfamily, which is required for semilunar valve development [177]. Expression of ERBB4 is
associated with left ventricular outflow defects, such as aortic valve stenosis, but its mechanism
in disease progress is not yet known [178]. Conditional knockout of NOTCH1 in mice has been
shown to alter the expression of neuregulin 1, a ligand of ERBB4, suggesting that there may be a
link between NOTCH and EGFR pathways [178, 179]. Although the non-canonical WNT16 has
not been studied in developing or diseased valves, it has been found to regulate the expression of
certain Notch ligands [180].
In the top of lesions, lipid-related, immune response, and ECM remodeling processes were also
enriched, but upregulated compared to bottom of lesion VICs. Many of these differentially
upregulated transcripts were also identified when comparing gene expression in the top of
lesions with non-lesion fibrosa areas. Overall, comparisons of the areas of interest demonstrated
that bottom of lesion VICs were more similar to non-lesion VICs than top of lesion VICs. With a
fold change cutoff of 1.5, only 18 transcripts were found to be differentially expressed in the
BvsF comparison. This raises the issue of the technical limitations of LCM in accurately
separating the top and bottom halves of lesion VICs. Although the smallest laser spot size was
used, it was difficult to capture lesion areas in precise halves while avoiding VECs. For all
samples, the top was captured first. In smaller lesions, this may have collected more than just
desired top half of the lesion, resulting in the capture of non-lesion VICs when attempting to
collect bottom of lesion VICs. This undoubtedly would affect the reliability of comparisons
involving bottom of lesion VICs. With this in mind, the remainder of discussion will focus on
differentially expressed genes between the top lesion and non-lesion areas.
In total, 162 genes were upregulated and 53 genes were downregulated in the top of lesions
compared to non-lesion fibrosa. Of relevance to CAVD, pathways involving lipid-, immune-,
and ECM remodeling-related genes were upregulated in the top of lesions, as well as transcripts
that are associated with osteochondrogenesis. When present, ApoB-positive areas were often
present near the top of lesions. In these areas, genes involved with lipid metabolism and
localization may promote early CAVD progression. Lipoprotein lipase (LPL) is thought to
sequester lipid to recruit macrophages, which in turn can produce more LPL and encourage
further lipid retention. The increase of LPL transcripts within lesion areas indicates that these
processes may be at work in early disease progression. Scavenger receptors, such as CD36 and
60
LOX1, were also upregulated in the top of lesions and can increase the cellular uptake of
modified lipoproteins, namely oxLDL. The accumulation of oxLDL has been associated with
inflammation and calcification in valve disease [54, 56]. The increase of phospholipase A2 group
VII (PLA2G7) mRNA expression in these same areas provides a possible link between the
accumulation of oxLDL, inflammatory response, and mineralization. PLA2G7 encodes an
enzyme that can metabolize oxLDL into lysophosphatidylcholine (LPC), which is a powerful
inflammatory metabolite [181]. In isolated VICs, LPC elevates the expression of phosphate-
related proteins, including sodium-dependent phosphate cotransporter 1 (Pit1) and osteopontin
[182]. These are encoded by SLC20A1 and SPP1, respectively, and were also differentially
upregulated in top of lesion VICs. Within early lesions, Pit1 and osteopontin may induce
phosphate-mediated apoptosis and mineralization of VICs, as they have been shown to do in
vitro [183]. Other genes with relevance to osteochondrogenic processes include SULF1,
HYAL2, and CHSY1, which have roles in chondrocyte development and ossification.
In contrast to the immunostaining results, SOX9 mRNA expression was not found to be
differentially expressed between regions, either within a lesion or between a lesion and the
normal fibrosa. Instead, SOX9 expression was uniformly high in all areas, which is consistent
with observations in swine [18] and mice [184] that Sox9-positive cells are present throughout
the valve. The discrepancy between the IHC and microarray results with regards to differential
SOX9 expression between lesion and non-lesion areas may be due to differential regulation of
transcript vs. protein expression or that the microarray experiments had insufficient statistical
power to detect regional differences. The latter issue could be addressed by regional analysis of
SOX9 expression by qPCR, perhaps with additional samples to increase the sample size and
statistical power.
Previously, PG-rich lesions were also shown to form in the absence of macrophages and
dendritic cells [18]. These observations were reinforced by the examination of inflammatory cell
markers, which were not expressed or expressed lowly. Still, genes involved in chemotaxis and
positive regulation of inflammatory cell activation were differentially upregulated in the top of
lesions. Greater expression of toll-like receptor 4 (TLR4) has been associated with pro-
inflammatory and pro-osteogenic responses [185, 186]. For example, TLR4 has been shown to
stimulate expression of intracellular adhesion molecule (ICAM1) and osteopontin [186, 187].
Upregulation of both TLR4 and ICAM1 at the top of lesions indicate that interactions between
61
these genes may promote leukocyte infiltration at this early stage of disease progression [186].
Interactions between the upregulated TLR4 and SPP1 genes also suggest pro-calcific pathways at
work. In minimally and heavily calcified stenotic valves, osteopontin is always present and
varies in proportion to calcium deposition [81]. Increased expression of osteopontin at the top of
lesions suggests that in early disease, it may act as a promoter of initial calcification.
Analysis of differentially expressed genes in the top of lesions also supports the notion of further
ECM remodeling. Increased expression of the elastolytic cathepsin S may be involved during the
initial stages in fragmenting and reduplicating the elastic lamina and/or during disease
progression in neoangiogenesis [188, 189]. In addition, upregulation of matrix
metalloproteinases (MMPs) -9 and -14, as well as metalloproteinase inhibitor 1 (TIMP1) may be
other sources of ECM remodeling, particularly changes in collagen, at this stage of lesion
development. Candidates responsible for the changes in PG/GAG content within lesions are also
indicated by the increased expression of hyaluronidase 2 (HYAL2) and chondroitin sulfate
synthase 1 (CHSY1). Interestingly, the differential expression of these genes corresponds with
the results from Chapter 4 where increases in biglycan and decreases in hyaluronan were
observed in lesions at this diet and time point.
5.5 Conclusion
Here, differential gene expression between lesion and non-lesion VICs was examined. Spatial
differences in VIC phenotypes between top of lesion and non-lesion areas were evident. In
particular, genes involved in lipid retention and metabolism, immune response, ECM
remodeling, and mineralization were identified as differentially upregulated in the top of lesion
VICs. While these findings do not demonstrate causality, they suggest correlative links between
valve microenvironment and VIC phenotype in early CAVD progression for further
investigation. Although histological studies demonstrate spatially different areas within lesions
with respect to lipid retention and putative chondrogenesis, few phenotypic differences were
observed between the top and bottom portions of the lesions, likely due to a combination of
small lesion size and limitations of LCM technology. Although a more detailed analysis of
microarray results was hampered by the lack of complete annotations for the porcine array, these
data nonetheless provide new insights into phenotypic changes in VICs and pathobiological
processes that occur early in valve disease.
62
Chapter 6
6 Conclusions and Future Work
6.1 Conclusions
The prevalence and lack of effective medical treatments for CAVD expose an unmet scientific
need to improve our understanding of disease progression. Early CAVD changes are an untapped
area of study that would vastly improve our understanding of how the disease progresses to
result in calcified and stenotic valves and may uncover novel therapeutic targets. A porcine
model of early CAVD was developed by Sider et al., revealing the accumulation of PG-rich
lesions without myofibroblast, osteoblast, and inflammatory cell infiltration [18, 19]. In many
cases, these lesions were observed in the absence of ApoB staining, suggesting proteoglycan
accumulation occurs before lipid retention. Using histological and microarray analyses, we
sought to further characterize the lesion ECM with a focus on PG/GAG content, as well as the
VIC phenotypes contributing to and resulting from these changes in valve microenvironment.
In this thesis, putatively advanced early lesions were characterized as morphologically dense and
having greater biglycan, but less hyaluronan content. Chondroitin sulfate synthase 1 synthesizes
a GAG component of biglycan and hyaluronidase 2 degrades hyaluronan. Interestingly, in VICs
from the top of lesions their respective genes, CHSY1 and HYAL2, were upregulated compared
to those from non-lesion fibrosa. Although histological analyses did not reveal distinct changes
in total PG, collagen, and other (nuclei, cytoplasm, elastin) within lesions, upregulation of
mRNA of elastolytic proteases (cathepsins) and enzymes involved in collagen turnover (MMPs
and TIMPs) recapitulate that active ECM remodeling is occurring. This is also supported by the
time-dependent decrease in biglycan, decorin, and versican with atherogenic diet and contrasting
severity of morphologically diffuse and dense lesions.
In early lesion areas, increases in biglycan score and decreases in hyaluronan were associated
with higher ApoB scores and Sox9 fraction. Biglycan may play a role in early lipid retention and
putative chondrogenesis, while hyaluronan may have a more protective role. The accumulation
of lipid, whether directly or indirectly mediated by biglycan, may allow modification of
lipoproteins, which could initiate a cycle of further lipid retention and inflammation. Within
63
lesion areas, several genes involved in lipid metabolic processes and lipid storage were
upregulated compared to non-lesion fibrosa areas. Interestingly, both biglycan and ApoB have
been shown to co-localize with oxLDL [129]. The upregulation of scavenger receptors within
lesion areas, suggest that cellular uptake of modified lipoproteins may occur and promote the
inflammatory response and calcification attributed to advanced stages of CAVD. PLA2G7,
SLC20A1, and SPP1 genes were also found to be upregulated within lesions and may connect
the oxidative transformation of accumulating lipids to mineralization. Furthermore, several genes
related to inflammatory and osteochondrogenic processes were differentially expressed between
top of lesion and non-lesion fibrosa.
The results from our histological and microarray analyses provide important insights into the
ECM and cellular changes that occur in early CAVD progression. Although our characterization
does not identify causative relationships, the correlational connections gained provide a stepping
stone for a better understanding of CAVD with the ultimate goal of finding an effective
therapeutic target.
6.2 Future Work
6.2.1 Further characterization of ECM changes in early CAVD lesions
Characterization of lesion collagen in relation to specific PG/GAG content would explore
another potential aspect of the role of PGs in disease progression. Biglycan and decorin are both
known to mediate collagen fibrillogenesis [14, 131], which could alter the biomechanical
properties of the lesion microenvironment. ROI analysis of Movat’s pentachrome collagen
staining with PG/GAG score could provide a general sense of collagen and specific PG/GAG
localization. Movat’s pentachrome sections could also be analyzed with an orientation-
independent birefringence imaging system (PolScope) to identify the amount of fibrillar collagen
present within lesion ROIs to determine its relationship with specific PGs/GAG.
To further complement the results from Chapter 4, IHC for chondroitin sulfate synthase 1 and
hyaluronidase 2 within lesions would allow for the examination of spatial, temporal, and diet-
related correlations with specific PG/GAGs. Spatial localization of these enzymes with the
chondroitin sulfate PGs biglycan and decorin, and hyaluronan would indicate potential players
responsible for the temporal and diet-related PG/GAG changes. Using Movat’s pentachrome
64
staining, correlations between MMP/TIMPs with collagen content and the elastolytic cathepsin S
with elastin would provide further insight into the ECM remodeling processes at work.
6.2.2 Validation and pathway analysis of microarray results
Examination of select differentially expressed genes using quantitative polymerase chain
reaction (qPCR) would validate results from the microarray analyses. In addition, IHC staining
would validate these results at the protein level. Transcripts of interest for validation include the
ECM remodeling genes (CHYS1, HYAL2, CTSS, MMP9, TIMP1) and genes discussed above
that may connect lipid retention to more advanced CAVD processes (CD36, LOX1, PLA2G7,
SLC20A1, SPP1).
In order to take full advantage of the hypothesis-generating power of microarrays, gene
expression network analysis can be used to reveal the transcriptional regulatory pathways. This
would require full annotation of the porcine arrays based on BLAST to cross-reference porcine
sequences to the human genome. These annotations would allow us to use readily available
pathway analysis tools, such as Database for Annotation, Visualization and Integrated Discovery
(DAVID), to better understand the transcriptional pathways involved in early CAVD lesion
formation.
6.2.3 In vitro studies of biglycan influence on VIC function
Both IHC and microarray analyses provide correlational results from which further causative
hypotheses can be generated. Early CAVD lesions from this porcine model were softer than
normal fibrosa [18] and demonstrated spatial and temporal PG/GAG content changes. From
these results, we hypothesize soft lesions microenvironments that are rich in biglycan and
deficient in hyaluronan contribute to VIC osteochondrogenic differentiation. This may occur via
a direct matricellular effect in which the fate of adherent VICs is modulated by the mechanical
and biochemical properties of the early lesion ECM. To elucidate the effect of PG/GAG content
and lesion micromechanical environment, it is proposed that 3D polyethylene glycol (PEG)
hydrogels be used to mimic normal fibrosa tissue (elastic modulus 11 - 22 kPa; collagen
adhesion peptides) and early lesions (elastic modulus ~ 5 kPa; incorporating soluble biglycan).
Freshly isolated porcine VICs would be cultured within the hydrogels in DMEM with 10% fetal
bovine serum. To determine the effect of soluble biglycan on the expression of SOX9,
65
SLC20A1, SPP1, RUNX2, and COL10A1, cells would be treated with recombinant biglycan
(0.05, 0.10, and 0.20 µg/mL) for 48 hours. Gene expression would be measured by qPCR, while
protein expression would be assayed using immunoblotting with quantification by gel
densitometry. Another non-exclusive mechanism by which early lesion PGs may contribute to
VIC pathological differentiation is through their retention of LDL and presentation of oxidized
LDL to VICs. This hypothesized mechanism is supported in part by two recent studies [126,
190], but these studies used standard tissue culture plates that do not mimic the soft and distinct
mechanical environments in the lesion vs. fibrosa. Thus, the effect of oxLDL on VIC expression
of osteochondral genes and proteins could be tested in the hydrogel models proposed above.
6.2.4 Mechanistic studies of hyaluronan interaction with VICs
In our study, hyaluronan was suggested to have a protective role in early CAVD, as it was
associated with areas that had lower ApoB scores and Sox9 fractions. Similarly, in a study of
advanced, calcified aortic valves, hyaluronan turnover was associated with markers of hypoxia
and ossification [140]. Positive expression of HA binding receptors RHAMM and HARE are
observed surrounding calcified nodules, while CD44 is not strongly correlated with calcification.
Hyaluronan binding to each of these three receptors has been associated with the activation and
propagation of signaling pathways, such as ERK 1 and 2 [191, 192]. Interestingly, it has been
established that hyaluronan plays different roles depending on its molecular weight [193-195]. In
vitro treatment of VICs with different molecular weights of soluble hyaluronan coupled with
inhibition of RHAMM, HARE, and CD44 receptors could help to elucidate the mechanisms by
which hyaluronan mediates its protective role in early CAVD lesions. Specifically, this would be
achieved by monitoring calcified nodule formation and quantification of markers involved in
VIC dysfunction, such as αSMA, by Western blotting.
66
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Appendices
A. Supplemental Data
A.1 Myofibroblast detection in porcine valve lesions
Previously, myofibroblast absence in early PG-rich lesions was detected using
immunoperoxidase methods by Sider et al. (2013). Specifically, no myofibroblasts were
observed at two months, while only few were observed at the base of lesions from the five-
month time point. Immunofluorescence (IF) provides greater signal sensitivity and was used to
corroborate the results from immunoperoxidase staining.
Materials and Methods
Formalin-fixed, paraffin-embedded radial centre sections from the early porcine model by Sider
et al. (2013) were previously stained for alph-smooth muscle actin (αSMA) using
immunoperoxidase methods. For direct comparison, serial sections were used for IF staining.
Briefly, IF staining began by melting the wax on sections at 60°C for 30 min. This was followed
by deparaffinization in xylene and rehydration in graded ethanol baths. Antigen retrieval
involved incubation in 1 µg/µl Trypsin-CaCl2 (Sigma-Aldrich) for 30 min in a 37°C water bath.
Non-specific staining was blocking using 10% goat serum buffer for 45 min prior to an hour-
long incubation with anti-αSMA primary antibody (mouse monoclonal, 4 µg/mL, ab7817,
Abcam). Samples were then incubated for 30 min with Alexa Fluor 568 goat anti-mouse
antibody (A-11004, 20 µg/mL, Life Technologies, Burlington, ON) diluted in 10% goat serum
buffer. Nuclei were stained with Hoechst (33258, 1µg/mL, Sigma-Aldrich) for 5 min.
PBS/Tween was used to wash between steps. Negative controls involved no primary and mouse
IgG controls (Santa Cruz).
Results and Discussion
PG-rich lesions of interest were visualized using Movat’s pentachrome. IF staining did not
identify any additional lesions with αSMA-positive cells that were not previously detected by
immunoperoxidase staining (Figure A.1). In the few lesions that did demonstrate areas of
positive αSMA, staining was spatially similar between immunoperoxidase and IF stained serial
sections.
85
(A)
(B)
(C)
(D)
(E)
(F)
Figure A.1. Immunoperoxidase (IP) and immunofluorescent (IF) staining of alpha-smooth muscle actin
(αSMA). IF did not identify αSMA positive lesions in addition to those found by IP staining (A,C,E). Staining
patterns of positive areas were similar in IP and IF staining (B,D,F). MP = Movat’s pentachrome (blue =
proteoglycan, yellow = collagen, black = nuclei/elastin, red = cytoplasm). Scale bar = 60µm.
MP MP
αSMA IP αSMA IP
αSMA IF αSMA IF
86
A.2 Porcine model diet formulation
The porcine model was designed and prepared by Sider et al. (2013). Barrows were supplied by
the Arkell Swine Research Station, University of Guelph. The porcine diet was formulated based
on the standard Guelph University Arkell Swine Research diets and simplified to a corn and
soybean base. All diets were balanced for protein, carbohydrate, fat, digestible lysine, calcium,
and available phosphates. Diets are isocaloric between control and experimental diets when
experimental diets are fed at 87% of control diet levels. Protein kcal% between control and
experimental diets were equalized, as were carbohydrate + fat kcal% between diets. The starter
diets were fed up until swine reached ~40kg, at which point they were fed the grower diets until
the study ended. All diets were verified by Dr. C. Kees De Lange [Animal and Poultry Science,
University of Guelph]. All diet components were supplied by Arkell other than the cholesterol
[Research Diets, Inc., New Brunswick, NJ, USA], and liquid lard [Quality Meat Packers, Ltd.,
Toronto, ON, Canada]. Diets were milled at the Arkell Research Feed Mill, University of
Guelph.
Table A.1. Control starter III diet formulation
87
Table A.2. Experimental starter III (12% lard, 1.5% cholesterol) diet formulation
Table A.3. Control grower diet formulation
Table A.4. Experimental grower (12% lard, 1.5% cholesterol) diet formulation
88
A.3 PG/GAG scoring validation and analyses
Lesions stained for biglycan, decorin, versican, and hyaluronan were divided into regions of
interest (ROIs) to allow for localized PG/GAG correlations. Lesions images were divided into
grids with 100µm x 100µm ROIs to allow for more localized analysis of lesion areas. Each ROI
was semi-quanitatively scored for each PG/GAG and ApoB (Figure 3.2). To validate the results,
a lesion score was calculated and correlated to its corresponding percent PG/GAG staining of
total lesion (Figure A.2). Each ROI score was normalized to the fraction of lesion area in each
cell. The lesion score was calculated by adding all normalized ROI scores in a lesion and
dividing it by the total lesion area.
Results
According to Spearman’s correlation, the lesion scores for each PG/GAG strongly correlated
with the percent PG/GAG staining of total lesion (p<0.001).
(A)
(B)
(C)
(D)
Figure A.2. Semi-quantiative scores strongly correlate with PG/GAG staining of total lesion area
percentages.
89
Using Kruskal Wallis tests with Mann-Whitney post-hoc tests with Bonferroni corrections
(p<0.0083), comparisons of PG/GAG lesion scores between pig time points and diet groups
yielded similar results to comparisons of percent area staining of PG/GAGs (Chapter 4). At two
months, lesions from the HF/HC diet have increased biglycan scores compared to lesions from
the normal chow diet (p<0.001). Furthermore, in HF/HC pigs, lesions from 5-months have lower
lesion scores of biglycan (p<0.001) and decorin (p=0.001) than those from 2-months.
Figure A.3. HF/HC diet alters lesion PG score with temporal differences. HF/HC = high-fat/high-
cholesterol diet. Error bars = SEM. *p<0.0083
A.4 Specific PG/GAG-rich lesions display distinct morphological
characteristics
Lesions rich in each PG/GAG were analyzed for association with ApoB-positive areas and Sox9-
expressing cells using Pearson’s Chi-Square Test or Fisher’s Exact Test. The latter was used in
the place of the former if one of the groups had a count of less than five. “Rich” lesions were
defined as those that express at least 45% PG/GAG staining of the total lesion area, as this is
approximately one-half standard deviations (±12.5%) above the mean percent PG/GAG staining
of the total lesion area for all PGs/GAG together (31.9%). ApoB positive lesions were defined as
those containing areas with a minimum score of one. Sox9-positive lesions were identified as
those with over ten percent Sox9 fraction. Spearman’s rank order correlation was also used to
correlate levels of percent staining of total lesion area between each of the PGs/GAG.
* *
*
90
Results and Discussion
Lesions categorized by those rich with and those not rich with specific PGs/GAG were
characterized by several factors, including dense/diffuse morphology, ApoB presence/absence,
presence/absence of Sox9-expressing cells, using Pearson’s Chi-Square Test and Fisher’s Exact
Test (Table A.4). Biglycan-rich lesions (n=11) demonstrated distinct characteristics compared to
lesions that do not express high levels of biglycan. Firstly, they were primarily found in lesions
from swine fed the HF/HC diet (p=0.016). Furthermore, the majority of biglycan-rich lesions
were morphologically dense (p=0.001). Association with ApoB-positive regions (p=0.017) and
Sox9-expressing cells (p=0.009) was another common trait of these lesions. Similar to biglycan,
lesions rich in decorin (n=14) and versican (n=25) were more commonly observed in two month
old swine (p=0.008 and p=0.003, respectively). Decorin-rich lesions were also more commonly
associated with ApoB-positive regions (p=0.029), suggesting they may also play a role in lipid
retention. Interestingly, versican-rich lesions were also often biglycan-rich, and vice versa
(p=0.037). Hyaluronan-rich lesions (n=16) were less commonly observed in swine fed the
HF/HC diet (p=0.009). Further, these lesions were also less commonly associated with ApoB-
positive regions (p=0.035).
Table A.4. Qualitative categorization of specific PG/GAG-rich lesions
HF/HC
Diet
Two-Month
Time Point
Dense
Morphology
ApoB
+
Sox9
+
Richness in other
PG/GAGs
Biglycan-rich 10/11* 11/11* 10/11* 8/11* 11/11* 6/11 decorin-rich
9/11 versican-rich*
2/11 hyaluronan-rich
Decorin-rich 9/14 13/14* 8/14 9/14* 9/14 6/14 biglycan-rich
9/14 versican-rich
5/14 hyaluronan-rich
Versican-rich 16/25 21/25* 14/25 10/25 18/25 9/25 biglycan-rich*
9/25 decorin-rich
9/25 hyaluronan-rich
Hyaluronan-rich 5/16* 12/16* 4/16 3/16* 11/16 2/16 biglycan-rich
5/16 decorin-rich
9/16 versican-rich
*p<0.05, compared to all lesions that are not rich in the specified PG/GAG
A.5 RNA and cDNA quality control before microarray analysis
Valve interstitial cells (VICs) were isolated from dense lesions from four 2-month high-fat/high-
cholesterol (HF/HC) pigs. Using laser capture microdissection (LCM), three areas of interest
91
were isolated from each sample: (1) top of the lesion; (2) bottom of the lesion; and (3) normal
fibrosa. RNA and cDNA quality were analyzed using a 2100 Bioanalyzer (Agilent Technologies
Canada Inc., Mississauga, ON, Canada) following RNA isolation and amplification, respectively.
RNA quality was examined using RNA integrity numbers (RINs) and noting the presence of
distinct peaks for 18S and 28S ribosomal subunits. Partial digestion was present in these RNA
samples. cDNA profiles indicated curves that plateaued around 200-500 nt with some high
molecular weight products around 500-1000 nt.
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
Figure A.4. Bioanalyzer results for top of lesion samples. RNA (A,C,E,G) and cDNA (B,D,F,H)
profiles indicate suitable samples for downstream microarray analysis. Pig Identifiers: (A) and (B) =
11107; (C) and (D) = Pig 11212; (E) and (F) = 12509; (G) and (H) = 11907. RIN = RNA Integrity
Number.
RIN = 5.4
RIN = 5.1
RIN = 6.2
RIN = 7.0
92
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
Figure A.4. Bioanalyzer results for bottom of lesion samples. RNA (A,C,E,G) and cDNA (B,D,F,H)
profiles indicate suitable samples for downstream microarray analysis. Pig Identifiers: (A) and (B) =
11107; (C) and (D) = Pig 11212; (E) and (F) = 12509; (G) and (H) = 11907. RIN = RNA Integrity
Number.
RIN = 4.9
RIN = 2.3
RIN = 5.0
RIN = 6.8
93
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
Figure A.4. Bioanalyzer results for normal fibrosa samples. RNA (A,C,E,G) and cDNA (B,D,F,H)
profiles indicate suitable samples for downstream microarray analysis. Pig Identifiers: (A) and (B) =
11107; (C) and (D) = Pig 11212; (E) and (F) = 12509; (G) and (H) = 11907. RIN = RNA Integrity
Number.
RIN = 6.1
RIN = 5.7
RIN = 3.6
RIN = 5.1
94
A.6 Quality control plots for microarray analyses
(A)
(B)
Figure A.5. (A) Boxplot of unprocessed log-intensity distributions and (B) histogram of the density of log-
intensities between arrays. Individual arrays were labeled numerically for the bottom of lesion, non-lesion areas,
and top of lesion, respectively, for the samples 11107 (1-3), 11212 (4-6), 11907 (7-9), and 12509 (10-12).
(A)
(B)
Figure A.6. (A) Relative log expression value and (B) normalized unscaled standard error value comparisons
between arrays, which were labeled numerically for the bottom of lesion, non-lesion areas, and top of lesion,
respectively, for the samples 11107 (1-3), 11212 (4-6), 11907 (7-9), and 12509 (10-12).
11
log in
tensity
array
array
norm
aliz
ed u
nscale
d e
rror
rela
tive lo
g e
xpre
ssio
n
array
95
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
(I)
(J)
(K)
(L)
Figure A.7. 2D images of raw probe intensity measurement for each array. Bottom of lesion, non-lesion, and top of
lesion arrays, respectively, correspond to (A-C) for sample 11107, (D-F) for sample 11212, (G-I) for sample 11907, and
(J-L) for sample 12509.
96
Figure A.8. Coloured correlation coefficient matrix. The color key indicates correlation between array
intensities, which are labeled numerically for the bottom of lesion, non-lesion areas, and top of lesion,
respectively, for the samples 11107 (1-3), 11212 (4-6), 11907 (7-9), and 12509 (10-12).
97
A.7 Hierarchical clustering of repeated-measures ANOVA results
12
50
9T
11
10
7T
11
21
2T
11
90
7T
11
90
7F
12
50
9F
11
90
7B
12
50
9B
11
21
2B
11
21
2F
11
10
7B
111
07F
Figure A.5. Hierarchical clustergram. One thousand forty-six transcripts identified by repeated measures
ANOVA (uncorrected p-value<0.05) as being significantly regulated among the three valve regions of interest
were clustered using unsupervised clustering analysis. A Pearson-centred correlation was used as a distance
metric with average linkage rules. Pig samples are categorized by their numerical identifiers and valve regions
are indicated by T=top of lesion, B=bottom of lesion, and F=non-lesion fibrosa. The relative expression for
each sample at a given probe is reflected by its color intensity (green=downregulated, red=upregulated).
Valve area
Sample ID
98
B. Protocols
B.1 Valve leaflet histological processing for paraffin-embedded
leaflets
Purpose: To prepare FFPE samples for histological and immunohistological processing
Materials:
Ethanol (standard lab grade)
Xylene
dH2O
10% neutral buffered formalin
Water bath
Tweezers
Paraffin-embedding station
20 mL scintillation vials
Microtome
Embedding cassettes (M506-2; Simport,
Beloeil, QC, Canada)
Tissue Path disposable base molds
(22038217; 15x5x5; Fisher Scientific)
TBS Poly/FIN H-PF Paraffin
Slides (12-550-15; Fisherbrand
Superfrost Plus)
Microtome blades (819-LP; Leica
Microsystems, Concord, ON, Canada)
Slide warmer
Procedure:
Fixation
1. Place leaflet in 10% neutral buffered formalin (NBF) for 48 hrs at room temperature (10x
volume of tissue) in a scintillation vial
2. Pour off NBF and rinse twice in 70% ethanol (ETOH)
3. Fill vial with 70% ETOH and keep at 4°C until embedding
*any segmentation of the sample prior to embedding is done at this point
Dehydration and Infiltration
4. 95% ETOH 2x 30 min
5. 100% ETOH 2x 30 min
6. Xylene 2x 1 hr
7. Fill vial with paraffin wax 2x 1 hr (at 60°C)
a. Leave vial lid loose and place samples in last paraffin change into fume hood at
room temperature overnight to allow the wax to solidify and any excess xylene to
evaporate. This is done if there are many samples for embedding
99
Embedding
8. Melt samples in batches in a 60°C water bath just prior to embedding
9. Dispense a little wax into the base mold and position sample with cut face touching the
base of the mold in the desired orientation using hot tweezers.
10. Touch the mold to cold surface and allow the wax to cool and slightly thicken to hold the
sample in place. Do not allow wax to totally solidify or fracture plane will occur between
wax layers.
11. Place cassette over mold and fill with wax.
12. Place on cold plate until wax is solidified.
13. Remove wax from mold using a scalpel to carefully release the sample. Samples can be
placed at -20°C prior to this to shrink the wax and help release the sample.
14. Store samples at 4°C.
Sectioning
15. Pre-heat water bath filled with ddH2O at 46°C (with no bubbles) and slide warmer at
40°C.
16. Take sample from fridge and place in microtome.
17. Cut 5 µm thick ribbons of section and transfer to clean, flat surface.
18. Cut ribbon into sets of two sections and use histology marker to number each set,
allowing multiple sets to be placed in the water bath.
19. Transfer sections to 30% ETOH room temperature bath for 1-2 min
20. Use a slide to lift samples out and slowly place into water bath. The ETOH treatment
causes the sample to stretch out when it contacts the water.
21. When sample is fully stretched out, slowly lift a side up under the section so that sections
adhere to the slide.
22. Gently flick the slide or use a Kimwipe to dry the slide and remove the majority of water
from under the section and place slide on slide warmer for 24-48 hrs or in a 40°C oven to
evaporate all water.
23. Store slides at 4°C until staining.
100
B.2 Movat’s pentachrome staining for formalin-fixed, paraffin-
embedded sections
Purpose: To identify extracellular matrix components of FFPE valve tissue
Materials:
Alcian blue 1% (EMS 26385-01)
Alkaline Alcohol (EMS 26385-02)
Orcein, 0.2% (EMS 26385-03)
Hematoxylin Alcoholic, 5% (EMS
26385-04)
Ferric Chloride, 10% (EMS 26385-05)
Lugol’s Iodine (EMS 26385-06)
Woodstain Scarlet-Acid Fuchsin
Working Solution (EMS 26385-07)
Acetic Acid, 0.5% (EMS 26385-08)
Phosphotungstic Acid, 5% (EMS
26385-09)
Alcoholic Saffron, 6% (EMS 26385-10)
Bouin’s Solution (Sigma HT10132)
Ethanol
Staining dishes and racks (EMS 0312-
20)
*EMS = Electron Microscopy Sciences
Procedure: *all solutions at room temperature unless otherwise stated
1. Xylene 3x 3 min
2. 100% ETOH 3x 2 min
3. 95% ETOH 2x 2 min
4. 70% ETOH1 2x 2 min
5. 50% ETOH 2x 2 min
6. dH2O 2x 2 min
7. Bouin’s Solution in 50°C water bath – mordants tissue
*important to wash well after to remove picric acid deposits
1 hr
8. dH2O 2x 5 dips
9. Running tap water 10 min
10. dH2O dip
11. Alcian Blue 1% - stains mucosubstances blue 25 min
12. dH2O 2x 5 dips
13. Alkaline Alcohol in 56°C water bath – converts alcian blue to
monastral fast blue, which is insoluble
10 min
14. Running tap water 10 min
15. dH2O dip
16. Orcein-Verhoeff Working Solution – stains elastic fibers and nuclei 2 hr
101
black **protect from light**
Immediately before use, combine in order:
1) Orcein 0.2% - 125 mL
2) Alcoholic Hematoxylin 5% - 40 mL
3) Ferric Chloride 10% - 25 mL
4) Lugol’s Iodine – 25 mL
17. dH2O 2x 5 dips
18. Ferric Chloride 2% - differentiate in solution until the elastic fibers
contrast sharply with the background **protect from light**
50 mL 10% ferric chloride + 200 mL dH2O
1.5 min
19. Running tap water 3 min
20. dH2O dip
21. Woodstain Scarlet-Acid Fuschin – stains fibrin intense red and
muscle red; cytoplasm, collagen, and ground substances will all be
red after this step
1 min
22. dH2O 2x 5 dips
23. Acetic Acid 0.5% 30 sec
24. Phosphotungstic Acid 5% - well-differentiated sections demonstrate
colorless collagen and blue-green mucopolysaccharides; it removes
red stain from collagen and ground substance
20 min
25. Acetic Acid 0.5% 30 sec
26. 100% ETOH 3x 1 min
27. Alcoholic Saffron – stains collagen and reticular fibers yellow 8 min
28. 100% ETOH 3x 2 min
29. Xylene 3x 3 min
30. Mount with non-aqueous mounting media
Results:
Nuclei = black
Cytoplasm = red
Elastic fibers = purple/black
Collagen/bone = yellow
Mucopolysaccharides = blue/green
Muscle = red
102
B.3 (Immuno)histochemistry for formalin-fixed, paraffin-
embedded sections
Purpose: To determine the presence, extent, and pattern of proteoglycans/glycosaminoglycan,
Sox9, and ApoB within FFPE valve leaflets.
Materials:
PBS (-/-) diluted to 1x in ddH2O
Ethanol (histological grade)
ddH2O
Xylene
Triton (Sigma: T8532)
Water bath
Trypsin CaCl2 (Sigma: T7168)
Tween (Sigma: P1379)
Methanol
Hydrogen Peroxide
Oven
Humidity Chamber
PAP Pen
Cover slips
VectaStain Universal/Standard Elite
ABC Kit (Vector Labs: PK-6200/PK-
6100)
Vector NovaRED (Vector Labs: SK-
4800)
Hematoxylin (Vector Hematoxylin QS,
H-3404)
Synthetic mounting media (Harleco
Krystalon; 64969-71; EMD Millipore,
Billerica, MA)
Antibodies
Primary antibodies
Polyclonal sheep anti-Apolipoprotein B (ABR (Cedarlane): AHP214, 2.15 mg/mL)
Polyclonal rabbit anti-Sox9 (Abcam: ab26414, 0.6 mg/mL)
Polyclonal goat anti-biglycan (Santa Cruz: sc-27936, 0.2 mg/mL)
Polyclonal rabbit anti-decorin (Santa Cruz: sc-22753, 0.2 mg/mL)
Polyclonal rabbit anti-versican (Novus Biologicals: 16770002, 1 mg/mL)
Secondary antibodies
Biotinylated horse anti-mouse/rabbit (Universal ABC kit, Vector Labs: PK-6200)
Biotinylated rabbit anti-goat (Vector Labs: BA-5000)
Biotinylated rabbit anti-sheep (Vector Labs: BA-6000)
Biotinylated goat anti-rabbit (Vector Labs: BA-1000)
103
Blocking sera
Horse serum (Universal ABC kit, Vector Labs: PK-6200)
Rabbit serum (Sigma: R9133), heat inactivated at 56°C for 30 min
Goat serum (Sigma: G9023), heat inactivated at 56°C for 30 min
IgG negative controls
Normal rabbit IgG (R&D Systems: AB-105-C, 1 mg/mL)
Normal sheep IgG (Santa Cruz: sc-2717)
Normal goat IgG (R&D Systems: AB-108-C, 1 mg/mL)
Table B.1. Immunohistochemistry summary
Primary Antibody Vector Labs Kit Antigen Retrieval Secondary
Antibody
Control Tissue
Antibody Concentration
ApoB 3.6 µg/mL Standard Trypsin-CaCl2 anti-sheep Bone
Sox9 3 µg/mL Standard Tris EDTA anti-rabbit Cartilage
Biglycan 20 µg/mL Standard Trypsin-CaCl2 anti-goat Heart Valve
Decorin 10 µg/mL Universal Trypsin-CaCl2 anti-rabbit Heart Valve
Versican 1.3 µg/mL Universal Trypsin-CaCl2 anti-rabbit Heart Valve
Protocol for Standard Immunohistochemistry:
1. Bake slides, 30 min, 60°C oven
2. Dewax/rehydration
a. Xylene 3x 5 min
b. 100% ETOH 3x 5 min
c. 95% ETOH 1x 5 min
d. 70% 1x 5 min
e. ddH2O 1x 5 min
3. Antigen retrieval
a. Enzymatic antigen retrieval: 120µL Trypsin-CaCl2 (1mg/ml) + 120mL ddH2O at
37°C in water bath for 30 min
b. Heat mediated antigen retrieval: Tris-EDTA Buffer (10 mM Tris, 1 mM EDTA,
0.05% Tween, pH 9.0) at 98°C in water bath for 20 min, then cooled in room
temperature water for 20 min, and rinsed in ddH2O
4. Wash – PBS/0.05% Tween 3x 3 min
5. Peroxidase block – 3% H2O2/Methanol (10.5mL 30% H2O2 + 94.5mL methanol) for 10
min at room temperature
104
6. Wash – PBS/0.05% Tween 2x 3 min
7. Keep sections hydrated with PBS, while outlining sections with a PAP pen
8. Serum block at room temperature in humidity chamber for 45 min
a. 1 drop (Universal kit = 50µL or Standard kit = 75 µL) of appropriate stock serum
+ 5mL PBS
9. Primary antibody at room temperature in humidity chamber for 1 hr
a. Antibody is diluted to appropriate concentration in 0.3% TritonX-100 in PBS
b. 0.3% TritonX-100: 3 µL Triton + 1 mL PBS
c. Only Triton/PBS or IgG of primary on negative controls
10. Wash – PBS/0.05% Tween 4x 3 min
11. Secondary antibody at room temperature in dark humidity chamber for 30 min
a. 1 drop (Universal kit = 50µL or Standard kit = 75 µL) of appropriate stock serum
+ 2.5mL PBS + 1 drop (Universal kit = 50 µL or Standard kit = 12.5 µL) of
appropriate biotinylated antibody
12. Wash – PBS/0.05% Tween 4x 3 min
13. VectaStain ABC at room temperature in dark humidity chamber for 30 min
a. 1 drop Reagent A + 2.5 mL PBS + 1 drop Reagent B (allow to stand for 30 min at
room temperature in the dark before use)
14. Wash – PBS/0.05% Tween 2x 3 min
15. Vector NovaRED at room temperature in the dark for 5 min
a. 5 mL dH2O
b. 50 µL Reagent #1 mix well
c. 50 µL Reagent #2 mix well
d. 45 µL Reagent #3 mix well
e. 75 µL hydrogen peroxide solution mix well
16. Wash – dH2O 1x 3 min
17. Rinse slides in tap water
18. Counterstain with Vector Hematoxylin QS on slide for 10 sec
a. Rinse hematoxylin off slides with tap water (until water becomes colourless)
19. Dehydration
a. 70% ETOH 1x 5 min
b. 95% ETOH 1x 5 min
c. 100% ETOH 2x 5 min
105
d. Xylene 2x 5 min
20. Mount coverslips onto slides with synthetic mounting media
Protocol for Hyaluronan Staining:
Additional Materials
Hyaluronidase from Streptomyces hyalurolyticus (Sigma: H1136, 882 U/vial)
o Reconstitute in 4mL of 20 mM sodium phosphate buffer (77 mM sodium
chloride, 0.01% BSA, pH 7.0) – resulting concentration: 220.5 U/mL
Hyaluronan-binding protein (Calbiochem: 385911, 500 µg/mL)
Control Slide Preparation
1. Bake slides at 60°C for 30 min
2. Dewax/rehydration
a. Xylene 3x 5 min
b. 100% ETOH 3x 5 min
c. 95% ETOH 1x 5 min
d. 70% 1x 5 min
e. ddH2O 1x 5 min
3. Treat slides with hyaluronidase in PBS – 100 U/mL at 37°C for 4 hrs
4. Store slides at 4°C in PBS until use (*add into slide set at peroxidase block – step 3)
Histochemistry Protocol
1. Bake slides, 30 min, 60°C oven
2. Dewax/rehydration
a. Xylene 3x 5 min
b. 100% ETOH 3x 5 min
c. 95% ETOH 1x 5 min
d. 70% 1x 5 min
e. ddH2O 1x 5 min
3. Peroxidase block – 3% H2O2/Methanol (10.5mL 30% H2O2 + 94.5mL methanol) for 10
min at room temperature
4. Wash – PBS/0.05% Tween 2x 3 min
5. Keep sections hydrated with PBS, while outlining sections with a PAP pen
6. 3% BSA block at room temperature for 45 min
106
7. Incubate with 110.25 U/mL biotinylated hyaluronan-binding protein at room temperature
in humidity chamber for 1 hr
a. 250 µL biotinylated hyaluronan-binding protein (220.5 U/mL) + 250 µL PBS
8. Wash – PBS/0.05% Tween 4x 3 min
9. VectaStain ABC at room temperature in dark humidity chamber for 30 min
a. 1 drop Reagent A + 2.5 mL PBS + 1 drop Reagent B (allow to stand for 30 min at
room temperature in the dark before use)
10. Wash – PBS/0.05% Tween 2x 3 min
11. Vector NovaRED at room temperature in the dark for 5 min
a. 5 mL dH2O
b. 50 µL Reagent #1 mix well
c. 50 µL Reagent #2 mix well
d. 45 µL Reagent #3 mix well
e. 75 µL hydrogen peroxide solution mix well
12. Wash – dH2O 1x 5 min
13. Rinse slides in tap water
14. Counterstain with Vector Hematoxylin QS on slide for 10 sec
a. Rinse hematoxylin off slides with tap water (until water becomes colourless)
15. Dehydration
a. 70% ETOH 1x 5 min
b. 95% ETOH 1x 5 min
c. 100% ETOH 2x 5 min
d. Xylene 2x 5 min
16. Mount coverslips onto slides with synthetic mounting media
B.4 Image processing
Purpose: To separate proteoglycan (PG) staining areas from Nova Vector Red (NVR) images
taken using the Aperio ScanScope XT Scanner at the Princess Margaret Hospital (PMH)
Advanced Optical Microscope Facility.
Materials:
ImageJ (version 1.47v)
107
G. Landini’s Colour Thresholding v1.9 plugin
ImageJ Calculator Plus plugin
Colour Separation:
//Thresholds for use with NVR PG images taken at PMH
//Adapted from macros created by Krista Sider
dir1 = getDirectory("Choose SOURCE Directory ");
dir2 = getDirectory("Choose MASK Directory ");
dir3 = getDirectory("Choose INVERSE mask Directory ");
list = getFileList(dir1);
setBatchMode(true);
format = "Measurements"
format2 = "8-bit TIFF"
run("Set Measurements...", "area limit display redirect=None decimal=3");
run("Options...", "iterations=1 black edm=Overwrite count=1");
run("Colors...", "foreground=white background=black selection=yellow");
run("Options...", "iterations=1 black count=1");
//Open Image to set scale then close - when commented out does measurement in pixels
open(dir1+list[0]);
//Removes Scale
run("Set Scale...", "distance=0 known=0 pixel=1 unit=pixel global");
close();
//Runs NVR Separation 1
for (j=0; j<list.length; j++) {
//Open Image
open(dir1+list[j]);
titleSample = getTitle;
//gets length of title and removes ".tif" from the file name
newname = substring(titleSample, 0, lengthOf(titleSample)-4);
run("Threshold Colour");
// Colour Thresholding v1.9-------
// Autogenerated macro, single images only!
// G Landini 2/Feb/2008.
//
// This only works with Black background and White foreground
min=newArray(3);
108
max=newArray(3);
filter=newArray(3);
a=getTitle();
run("HSB Stack");
run("Convert Stack to Images");
selectWindow("Hue");
rename("0");
selectWindow("Saturation");
rename("1");
selectWindow("Brightness");
rename("2");
//Hue Values
min[0]=35;
max[0]=220;
filter[0]="stop";
//Saturation Values
min[1]=15;
max[1]=255;
Mask Area Measurement:
//Created Jan 2012 by Krista Sider
dir1 = getDirectory("Choose SOURCE Mask Directory ");
dir2 = getDirectory("Choose RESULTS Directory ");
list = getFileList(dir1);
setBatchMode(true);
format = "Measurements"
run("Set Measurements...", "area limit display redirect=None decimal=3"); //measures area
run("Options...", "iterations=1 black edm=Overwrite count=1");
//Open Image to set scale then close
open(dir1+list[0]);
//Removes Scale
run("Set Scale...", "distance=0 known=0 pixel=1 unit=pixel global");
close();
// Measures the NVR Mask Area
for (i=0; i<list.length; i++) {
109
//Open Masked Image
open(dir1+list[i]);
//Convert to 8-bit image so can measure
run("8-bit");
//Make a Threshold for Measurement
setAutoThreshold();
//Measure Threshold Area
run("Measure");
close();
}
B.5 Valve leaflet histological processing for OCT-embedded
leaflets
Purpose: To prepare frozen, OCT-embedded samples for histological and immunohistological
processing
Materials:
RNaseZap and DNA Zap
RNase/DNase-free H2O
RNase-free PBS
Petri dishes
Moulds
OCT (VWR: 25608-930)
Kimwipes
Dry ice
Scissors
Tweezers
Liquid nitrogen
Ziploc bag
Protocol: **ensure all surfaces, solutions, and equipment are RNase/DNA-free
OCT-Embedding and Cryopreservation
1. Pour RNase-free PBS into petri dishes.
2. Fill bottom of mould with OCT.
3. Rinse leaflet and syringe needle in RNase-free PBS in one of the petri dishes.
a. The side of the leaflet facing the hub of the needle = aortic side
b. The side of the leaflet facing the tip of the needle = ventricular side
110
4. Remove the leaflet from the needle onto a dry petri dish, ensuring they are in the same
orientation. Cut the leaflet in half in the radial direction with scissors.
5. Rinse each leaflet half in PBS again, leaving them in PBS until they are ready to be
placed in the mould.
6. On a dry petri dish, lightly blot one leaflet half dry with Kimwipes.
7. Place that leaflet half in the mould partly filled with OCT with the aortic side up and the
midline towards the end of the mould without extra plastic.
a. Align the leaflet so it is flat, straight and aligned parallel to the edge of the mould.
b. Leave some room between the midline side of the leaflet and the edge of the
mould to allow for blade alignment during cryosectioning.
8. Top up the mould with OCT until it forms a small meniscus.
9. Place a labelled paper tag into the side with extra plastic.
10. Touch the bottom of the mould to liquid nitrogen for at most 60 seconds.
a. Just lower the mould until you can hear the liquid nitrogen boiling.
11. Once the OCT is completely white and hard (check the centre of the mould), remove
from liquid nitrogen.
12. If the OCT has bubbled and/or the valve tissue is still exposed on top of the mould, add
more OCT into the space that has formed and again, touch the bottom of the mould to
liquid nitrogen until the new OCT is white and hard.
13. Place mould with leaflet in a sealed Ziploc bag directly on dry ice for temporary storage.
14. Store leaflets at -80°C long term.
B.6 Movat’s pentachrome staining for frozen OCT-embedded
sections
Purpose: To identify extracellular matrix components of frozen OCT-embedded valve tissue
Materials:
Alcian blue 1% (EMS 26385-01)
Alkaline Alcohol (EMS 26385-02)
Orcein, 0.2% (EMS 26385-03)
Hematoxylin Alcoholic, 5% (EMS
26385-04)
Ferric Chloride, 10% (EMS 26385-05)
Lugol’s Iodine (EMS 26385-06)
Woodstain Scarlet-Acid Fuchsin
Working Solution (EMS 26385-07)
Acetic Acid, 0.5% (EMS 26385-08)
Phosphotungstic Acid, 5% (EMS
26385-09)
111
Alcoholic Saffron, 6% (EMS 26385-10)
Bouin’s Solution (Sigma HT10132)
Ethanol
Staining dishes and racks (EMS 0312-
20)
*EMS = Electron Microscopy Sciences
Procedure: *all solutions at room temperature unless otherwise stated
1. Warm slide box to room temperature before opening slides 30 min
2. Neutral buffered formalin 10 min
3. Running Tap water 3 min
4. Bouin’s Solution in 50°C water bath – mordants tissue
*important to wash well after to remove picric acid deposits
1 hr
5. dH2O 2x 5 dips
6. Running tap water 10 min
7. dH2O dip
8. Alcian Blue 1% - stains mucosubstances blue 50 min
9. dH2O 2x 5 dips
10. Alkaline Alcohol in 56°C water bath – converts alcian blue to
monastral fast blue, which is insoluble
10 min
11. Running tap water 10 min
12. dH2O dip
13. Orcein-Verhoeff Working Solution – stains elastic fibers and nuclei
black **protect from light**
Immediately before use, combine in order:
5) Orcein 0.2% - 125 mL
6) Alcoholic Hematoxylin 5% - 40 mL
7) Ferric Chloride 10% - 25 mL
8) Lugol’s Iodine – 25 mL
2 hr
14. dH2O 2x 5 dips
15. Ferric Chloride 2% - differentiate in solution until the elastic fibers
contrast sharply with the background **protect from light**
50 mL 10% ferric chloride + 200 mL dH2O
3.5 min
16. Running tap water 3 min
17. dH2O dip
18. Woodstain Scarlet-Acid Fuschin – stains fibrin intense red and
muscle red; cytoplasm, collagen, and ground substances will all be
red after this step
1 quick dip
19. dH2O 2x 5 dips
20. Acetic Acid 0.5% 30 sec
21. Phosphotungstic Acid 5% - well-differentiated sections demonstrate 30 min
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colorless collagen and blue-green mucopolysaccharides; it removes
red stain from collagen and ground substance
22. Acetic Acid 0.5% 30 sec
23. 100% ETOH 3x 1 min
24. Alcoholic Saffron – stains collagen and reticular fibers yellow 8 min
25. 100% ETOH 2x 3 min
26. Xylene 3x 3 min
27. Mount with non-aqueous mounting media
Results:
Nuclei = black
Cytoplasm = red
Elastic fibers = purple/black
Collagen/bone = yellow
Mucopolysaccharides = blue/green
Muscle = red
B.7 Immunohistochemistry staining for frozen OCT-embedded
sections
Purpose: To determine the presence, extent, and pattern of Sox9 in frozen OCT-embedded valve
leaflets.
Materials:
Acetone
PBS (-/-) diluted to 1x in ddH2O
Ethanol (histological grade)
ddH2O
Xylene
Triton (Sigma: T8532)
Water bath
Tween (Sigma: P1379)
Methanol
Hydrogen Peroxide
Oven
Humidity Chamber
PAP Pen
Cover slips
VectaStain Standard Elite ABC Kit
(Vector Labs: PK-6100)
Vector NovaRED (Vector Labs: SK-
4800)
Hematoxylin (Vector Hematoxylin QS,
H-3404)
Synthetic mounting media (Harleco
Krystalon; 64969-71; EMD Millipore,
Billerica, MA)
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Antibodies and Serum
Polyclonal rabbit anti-Sox9 (Abcam: ab26414, 0.6 mg/mL)
Biotinylated goat anti-rabbit (Vector Labs: BA-1000)
Goat serum (Sigma: G9023), heat inactivated at 56°C for 30 min
Normal rabbit IgG (R&D Systems: AB-105-C, 1 mg/mL)
Protocol:
1. Warm slide box at room temperature for 30 min
a. During this time, pre-cool acetone at -20°C
2. Fixation in pre-cooled acetone at room temperature for 10 min
3. Wash – PBS/0.05% Tween 3x 3 min
4. Peroxidase block – 3% H2O2/Methanol (10.5mL 30% H2O2 + 94.5mL methanol) for 10
min at room temperature
5. Wash – PBS/0.05% Tween 2x 3 min
6. Keep sections hydrated with PBS, while outlining sections with a PAP pen
7. Serum block at room temperature in humidity chamber for 45 min
a. 1 drop (Standard kit = 75 µL) of goat serum + 5mL PBS
8. Primary antibody at room temperature in humidity chamber for 1 hr
a. Antibody is diluted to appropriate concentration in 0.3% TritonX-100 in PBS
b. 0.3% TritonX-100: 3 µL Triton + 1 mL PBS
c. Only Triton/PBS or IgG of primary on negative controls
9. Wash – PBS/0.05% Tween 4x 3 min
10. Secondary antibody at room temperature in dark humidity chamber for 30 min
a. 1 drop (Standard kit = 75 µL) of goat serum + 2.5mL PBS + 1 drop (Standard kit
= 12.5 µL) of biotinylated goat anti-rabbit secondary
11. Wash – PBS/0.05% Tween 4x 3 min
12. VectaStain ABC at room temperature in dark humidity chamber for 30 min
a. 1 drop Reagent A + 2.5 mL PBS + 1 drop Reagent B (allow to stand for 30 min at
room temperature in the dark before use)
13. Wash – PBS/0.05% Tween 2x 3 min
14. Vector NovaRED at room temperature in the dark for 5 min
a. 5 mL dH2O
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b. 50 µL Reagent #1 mix well
c. 50 µL Reagent #2 mix well
d. 45 µL Reagent #3 mix well
e. 75 µL hydrogen peroxide solution mix well
15. Wash – dH2O 1x 5 min
16. Rinse slides in tap water
17. Counterstain with Vector Hematoxylin QS on slide for 10 sec
a. Rinse hematoxylin off slides with tap water (until water becomes colourless)
18. Dehydration
a. 70% ETOH 1x 5 min
b. 95% ETOH 1x 5 min
c. 100% ETOH 2x 5 min
d. Xylene 2x 5 min
19. Mount coverslips onto slides with synthetic mounting media
B.8 Laser capture microdissection
Purpose: To capture cells from areas of interest in frozen OCT-embedded valve leaflet sections.
**ensure all surfaces, solutions, and equipment are RNase/DNA-free
Tissue Staining:
Materials
Arcturus Histogene staining solution
(Life Technologies, KIT0415)
ETOH
Xylene
dH2O (Life Technologies, 10977015)
50 mL conical tubes
Tweezers
Preparation
Set out 9 conical tubes in the following order (with 25 mL solution/tube):
1) 70% ETOH (1)
2) dH2O (1)
3) dH2O (2)
4) 70% ETOH (2)
5) 95% ETOH
6) 100% ETOH (1)
7) 100% ETOH (2)
8) Xylene (1)
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9) Xylene (2)
Set out two sets of Kimwipes: (1) to blot and (2) to air dry xylene
Loosen caps only up to 95% ETOH wash to prevent H2O from getting into 100% ETOH
Preload pipette with 100 µL HistoStain before starting protocol
Protocol *one slide at a time*
1. Fix in 70% ETOH (1) for 30 sec
2. Rinse in dH2O (1) for 30 sec – shake vigorously to get rid of OCT
a. Blot, flick, and wipe slide before next step
3. Stain with 100 µL HistoStain for 20 sec – don’t shake bottle before preloading pipette
a. Take off stain with pipette (can reuse for 2-3 slides)
4. Rinse in dH2O (2) for 30 sec
a. Blot, flick, and wipe slide before next step
5. Dehydrate
a. 70% ETOH (2) for 30 sec
b. 95% ETOH for 30 sec
c. 100% ETOH (1) for 1 min
d. 100% ETOH (2) for 1 min
6. Xylene (1) for 5 min can start next slide after current slide has been placed into this
wash *wipe tweezers before transfer to Xylene (2)
7. Xylene (2) for 5 min
8. Air dry for 10-20 min
Laser Capture Microdissection:
Materials
Arcturus PixCell II Laser Capture
System
Arcturus CapSure Macro LCM Caps
(Life Technologies, LCM0211)
Arcturus PicoPure RNA Isolation kit
(Life Technologies, KIT0204)
500 µL Eppendorf tubes
Sticky notes
Tweezers
1% SDS
70% ETOH
Blade
Thermocycler
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Preparation
1) Turning on the microscope:
a. Wipe down microscope with 1% SDS and rinse with 70% ETOH
b. Turn ON: (1) Scope system – with light intensity set to lowest setting
(2) Computer
(3) Software (ver 2.0.0)
*view screen should be black; adjust light to test that program is not frozen;
should be at “Norm” setting on start-up with knob turned fully counter-clockwise
**if frozen, turn off everything in reverse order and restart
c. Turn the key ¼-turn clockwise to actuate the laser
d. After laser interlock has been checked for continuity, turn on laser with the
“ENABLE’ button
2) Loading the cap
a. Load Arcturus CapSure Macro LCM Caps
i. Push knobs in to unlock
ii. Push loader down and slide cap cassette to far end at the “LOAD” line on
the stage
iii. Pull knobs out on both sides of the loader to lock caps in place
b. Swing the capping arm counter-clockwise until it stops over the new cap
c. Raise the capping arm to detach the new cap from the cassette and move it
clockwise to the rest position
3) Focusing the laser
a. Position joystick so it is perpendicular to the tabletop
b. Place the slide into position so that it is centered on the desired capture area and
the slide is above the H-groove
c. Turn ON vacuum to secure slide position and focus the stage on the tissue
d. Always focus the laser at the smallest spot size
e. Fire test pulse on area away from tissue
i. Power = increase to get appropriate burn spot
ii. Duration = increase to adhere to tissue
**a focused laser should have a uniformly, black thick circle of melted
plastic on the cap with a black ring
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4) Capturing cells
a. Position cells of interest under laser
b. Fire the laser on the cells, using the joystick to adjust the position of the tissue
relative to the stationary laser
c. When enough cells have been collected, raise the capping arm and move it to the
rest position
d. Observe the holes left behind in the tissue
e. To observe the captured material:
i. Release the vacuum
ii. Move the slide so that there is clear glass over the objective view
iii. Turn ON the vacuum
iv. Return the capping arm to the work position
f. Repeat until sufficient cells have been captured on the cap
5) Removing the Cap
a. Move the arm to the capping station to drop up the cap (fully up, fully counter-
clockwise, then lower)
b. Move the arm clockwise without lifting to expose the completed cap
c. Use the capping tool to lift the cap
d. Use the adhesive from a new sheet of sticky note to remove unwanted tissue from
the cap
i. View the cap under the microscope to ensure all unwanted tissue is
removed
e. Use a blade to cut off excess areas of the cap that do not contain tissue
f. Use tweezers to peel transfer film off cap and place in a 500 µL Eppendorf tube
6) RNA extraction
a. Pipette 50 µL Extraction Buffer into the 500 µL Eppendorf tube containing
isolated tissue
b. Incubate assembly for 30 min at 42°C in Thermocycler
c. Freeze cell extract at -80°C until isolation
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B.9 RNA isolation
Purpose: To isolate RNA from laser captured valve sections.
**ensure all surfaces, solutions, and equipment are RNase/DNA free
Materials:
Arcturus PicoPure RNA Isolation kit (Life Technologies: KIT0204)
RNase-free DNase I (Qiagen: 79254)
Centrifuge
Protocol:
1) Pipette 250 µL Conditioning Buffer onto purification column
a. Incubate for 5 min at room temperature
b. Centrifuge at 16,000 x g for 1 min
2) Pipette 50 µL ETOH into cell extract from RNA extraction
a. Pipette up and down to mix well
3) Pipette cell extract and ETOH mixture into purification column
a. Centrifuge at 100 x g for 2 min binds RNA to column
b. Centrifuge at 16,000 x g for 30 sec removes flowthrough
4) Pipette 100 µL Wash Buffer 1 into column
a. Centrifuge at 8,000 x g for 1 min
5) DNase treatment:
a. 5 µL DNase I Stock Solution + 35 µL Buffer RDD – invert gently to mix
b. Pipette the 40 µL DNase mix into column
c. Incubate for 15 min at room temperature
d. Pipette 40 µL Wash Buffer 1 into column
e. Centrifuge at 8,000 x g for 15 sec
6) Pipette 100 µL Wash Buffer 2 into column
a. Centrifuge at 8,000 x g for 1 min
7) Pipettte another 100 µL Wash Buffer 2 into column
a. Centrifuge at 16,000 x g for 2 min
b. If there is residual wash buffer, re-centrifuge at 16,000 x g for 1 min
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8) Transfer column to new 0.5 mL tube from the kit
9) Pipette 11 µL Elution Buffer directly onto membrane of column (gently touch)
a. Incubate column for 1 min at room temperature
b. Centrifuge column at 1,000 x g for 1 min distributes Elution Buffer in column
c. Centrifuge at 16,000 x g for 1 min elutes RNA
10) Store sample at -80°C until use
